U.S. patent application number 15/434595 was filed with the patent office on 2017-08-10 for human application of engineered chimeric antigen receptor (car) t-cells.
The applicant listed for this patent is BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM. Invention is credited to Laurence JN COOPER, Helen HULS, Lenka HURTON, Janani KRISHNAMURTHY, Simon OLIVARES, Hiroki TORIKAI, Feng WANG-JOHANNING, Ling ZHANG.
Application Number | 20170224798 15/434595 |
Document ID | / |
Family ID | 51898999 |
Filed Date | 2017-08-10 |
United States Patent
Application |
20170224798 |
Kind Code |
A1 |
COOPER; Laurence JN ; et
al. |
August 10, 2017 |
HUMAN APPLICATION OF ENGINEERED CHIMERIC ANTIGEN RECEPTOR (CAR)
T-CELLS
Abstract
The present invention concerns methods and compositions for
immunotherapy employing a modified T cell comprising a chimeric
antigen receptor (CAR). In particular aspects, CAR-expressing
T-cells are producing using electroporation in conjunction with a
transposon-based integration system to produce a population of
CAR-expressing cells that require minimal ex vivo expansion or that
can be directly administered to patients for disease (e.g., cancer)
treatment.
Inventors: |
COOPER; Laurence JN;
(Houston, TX) ; TORIKAI; Hiroki; (Houston, TX)
; ZHANG; Ling; (Houston, TX) ; HULS; Helen;
(Houston, TX) ; WANG-JOHANNING; Feng; (Bastrop,
TX) ; HURTON; Lenka; (Houston, TX) ; OLIVARES;
Simon; (Houston, TX) ; KRISHNAMURTHY; Janani;
(Houston, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM |
Austin |
TX |
US |
|
|
Family ID: |
51898999 |
Appl. No.: |
15/434595 |
Filed: |
February 16, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14890977 |
Nov 13, 2015 |
9629877 |
|
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PCT/US2014/038005 |
May 14, 2014 |
|
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15434595 |
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61823253 |
May 14, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 35/17 20130101;
C07K 14/70521 20130101; A61K 39/001109 20180801; Y02A 50/467
20180101; A61K 39/001113 20180801; A61K 39/001171 20180801; C07K
2317/622 20130101; A61P 37/00 20180101; A61K 39/00119 20180801;
C07K 2319/03 20130101; A61K 2039/505 20130101; C07K 14/7051
20130101; A61K 39/0011 20130101; A61K 39/001112 20180801; A61K
39/001104 20180801; C07K 16/30 20130101; C07K 2319/02 20130101;
A61K 39/00111 20180801; C07K 14/5443 20130101; A61K 39/001124
20180801; A61K 39/001129 20180801; A61K 39/001182 20180801; A61K
39/001106 20180801; A61K 39/001119 20180801; A61K 2039/5158
20130101; A61K 39/001164 20180801; A61K 39/001168 20180801; A61K
39/001174 20180801; A61K 39/001194 20180801; A61K 39/001151
20180801; A61K 39/001181 20180801; Y02A 50/30 20180101; A61K
39/00117 20180801; Y02A 50/411 20180101 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C07K 14/705 20060101 C07K014/705; C07K 14/725 20060101
C07K014/725; C07K 16/30 20060101 C07K016/30 |
Goverment Interests
[0002] The invention was made with government support under Grant
No. W81XWH-11-1-0002-01 awarded by the Department of Defense. The
government has certain rights in the invention.
Claims
1. A method of culturing engineered T-cells comprising: (a)
obtaining a sample of cells from a subject, the sample comprising
T-cells or T-cell progenitors; (b) transfecting the cells with a
DNA encoding a transposon-flanked chimeric antigen receptor (CAR)
and a transposase effective to integrate the DNA encoding the CAR
into the genome of the cells, to provide a population of engineered
CAR-expressing T-cells; and (c) culturing the population of
engineered CAR cells ex vivo in a medium that selectively enhances
proliferation of CAR-expressing T-cells, wherein the engineered CAR
T-cells are cultured for no more than 21 days.
2. A method of stimulating the proliferation and/or survival of
engineered T-cells comprising: (a) obtaining a sample of cells from
a the subject, the sample comprising T-cells or T-cell progenitors;
(b) transfecting the cells with a DNA encoding a transposon-flanked
chimeric antigen receptor (CAR) and a transposase effective to
integrate the DNA encoding the CAR into the genome of the cells, to
provide a population of engineered CAR-expressing T-cells; (c)
transfecting the cells with a DNA encoding a fusion protein
comprising IL-15 and IL-15R.alpha., and (d) optionally, culturing
the population of engineered CAR cells ex vivo for 2 days or
less.
3. The method of claim 1, further comprising purifying or enriching
T-cells in the sample prior to step (b).
4. The method of claim 3, wherein enriching T-cells in the sample
comprises collecting a mononuclear cell fraction.
5. The method of claim 1 , wherein the engineered CAR T-cells are
cultured no more than 14 days.
6-14. (canceled)
15. The method of claim 1, wherein the engineered CAR T-cells are
inactivated for expression of an endogenous T-cell receptor and/or
endogenous HLA.
16. The method of claim 1, wherein obtaining the sample of cells
comprises obtaining the cells from a 3rd party.
17. The method of claim 1, wherein transfecting the cells with DNA
comprises electroporating the cells.
18. The method of claim 2, wherein transfecting the cells with DNA
comprises electroporating the cells.
19. The method of claim 1, wherein transfecting the cells furthers
comprises transfecting the cells with a nucleic acid encoding a
membrane-bound Cy cytokine.
20. The method of claim 19, wherein the membrane-bound Cy cytokine
is a membrane bound IL-7, IL-15 or IL-21.
21. The method of claim 19, wherein the membrane-bound Cy cytokine
is a fusion protein comprising IL-15 and IL-15R.alpha..
22. The method of claim 1, wherein the DNA encoding a CAR is a
plasmid.
23. The method of claim 1, wherein the transposase is provided as a
DNA expression vector.
24. The method of claim 1, wherein the transposase is provided as
an mRNA.
25. The method of claim 24, wherein the mRNA comprises a capped
and/or PolyA tail.
26. (canceled)
27. The method of claim 1, wherein the transposase is salmonid-type
Tc1-like transposase (SB).
28. The method of claim 27, wherein the transposase is the SB11 or
SB100.times. transposase.
29. The method of claim 1, wherein culturing the engineered CAR
cells (c) may comprise culturing the engineered CAR cells in the
presence of dendritic cells or artificial antigen presenting cells
(aAPCs) that stimulate expansion of the CAR-expressing T-cells.
30. The method of claim 29, wherein the aAPCs are engineered K562
cells.
31. The method of claim 29, wherein the aAPCs comprises (i) an
antigen targeted by the CAR expressed on the engineered CAR cells;
(ii) CD64; CD86; (iii) CD 137L; and/or (v) membrane-bound IL-15,
expressed on the surface of the aAPCs.
32-37. (canceled)
38. The method of claim 29, wherein culturing the engineered CAR
cells in the presence of aAPCs comprises culturing the transgenic
CAR cells in a medium comprising IL-21 and/or IL-2.
39. The method of claim 29, wherein culturing the engineered CAR
cells in the presence of aAPCs comprises culturing the cells at a
ratio of about 10:1 to about 1:10 (CAR cells to aAPCs).
40-42. (canceled)
43. The method of claim 1, wherein the engineered cells are not
cultured ex vivo in the presence of aAPCs.
44. The method of claim 2, wherein the engineered cells are not
cultured ex vivo in the presence of aAPCs.
45-50. (canceled)
51. The method of claim 1, wherein the CAR is targeted to a
cancer-cell antigen.
52. The method of claim 51, wherein the cancer cell antigen is CD
19, CD20, ROR1, CD22 carcinoembryonic antigen, alphafetoprotein,
CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate-specific
antigen, melanoma-associated antigen, mutated p53, mutated ras,
HER2/Neu, folate binding protein, HIV-1 envelope glycoprotein
gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138,
CD23, CD30 , CD56, c-Met, meothelin, GD3, HERV-K, IL-11Ralpha,
kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, VEGFR2,
HER2-HER3 in combination or HER 1-HER2 in combination.
53. The method of claim 52, wherein the cancer cell antigen is CD
19 and the CAR is a CD19-targeted CAR.
54-59. (canceled)
60. A T-cell composition made by a method of claim 1.
61-104. (canceled)
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/890,977, filed Nov. 13, 2015, as a national
phase application under 35 U.S.C. .sctn.371 of International
Application No. PCT/US2014/038005, filed May 14, 2014, which claims
the benefit of U.S. Provisional Patent Application No. 61/823,253
filed May 14, 2013. The entire text of each of the above referenced
applications is incorporated herein by reference.
INCORPORATION OF SEQUENCE LISTING
[0003] The sequence listing that is contained in the file named
"UTSCP1222USC1_ST25.txt", which is 37 KB (as measured in Microsoft
Windows.RTM.) and was created on Feb. 16, 2017, is filed herewith
by electronic submission and is incorporated by reference
herein.
BACKGROUND OF THE INVENTION
[0004] 1. Field of the Invention
[0005] The present invention relates generally to the fields of
medicine, immunology, cell biology, and molecular biology. In
certain aspects, the field of the invention concerns immunotherapy.
More particularly, it concerns the manufacture of clinical-grade
chimeric antigen receptor (CAR) T cells and therapeutic methods
using such cells.
[0006] 2. Description of Related Art
[0007] The potency of clinical-grade T cells can be improved by
combining gene therapy with immunotherapy to engineer a biologic
product with the potential for superior (i) recognition of
tumor-associated antigens (TAAs), (ii) persistence after infusion,
(iii) potential for migration to tumor sites, and (iv) ability to
recycle effector functions within the tumor microenvironment. Such
a combination of gene therapy with immunotherapy can redirect the
specificity of T cells for B-lineage antigens and patients with
advanced B-cell malignancies benefit from infusion of such
tumor-specific T cells (Jena et al., 2010; Till et al., 2008;
Porter et al., 2011; Brentjens et al., 2011; Cooper and Bollard,
2012; Kalos et al., 2011; Kochenderfer et al., 2010; Kochenderfer
et al., 2012; Brentjens et al., 2013). Most approaches to genetic
manipulation of T cells engineered for human application have used
retrovirus and lentivirus for the stable expression of chimeric
antigen receptor (CAR) (Jena et al., 2010; Ertl et al., 2011; Kohn
et al., 2011). This approach, although compliant with current good
manufacturing practice (cGMP), can be expensive as it relies on the
manufacture and release of clinical-grade recombinant virus from a
limited number of production facilities. New methods are needed to
generate genetically-modified clinical-grade T cell products with
specificity for hematologic malignancies and solid tumors.
SUMMARY OF THE INVENTION
[0008] In a first embodiment there is provided a method of
providing a T-cell response in a human subject having a disease
comprising obtaining a sample of cells from the subject,
(comprising T-cells or T-cell progenitors); transfecting the cells
with a nucleic acid encoding chimeric T-cell receptor (CAR),
capable of integration into the genome of the cells, and
administering an effective amount of the transgenic cells to the
subject to provide a T-cell response.
[0009] Thus, in some aspects, a method of the embodiments
comprises: (a) obtaining a sample of cells from the subject, the
sample comprising T-cells or T-cell progenitors; (b) transfecting
the cells with a DNA encoding a transposon-flanked chimeric antigen
receptor (CAR) and a transposase effective to integrate the DNA
encoding the CAR into the genome of the cells, to provide a
population of transgenic CAR-expressing cells; (c) optionally,
culturing the population of transgenic CAR cells ex vivo in a
medium that selectively enhances proliferation of CAR-expressing
T-cells, wherein the transgenic CAR cells are cultured, if at all,
no more than 21 days; and (d) administering an effective amount of
the transgenic CAR cells to the subject to provide a T-cell
response. Thus, in some aspects, the transgenic CAR cells are
cultured ex vivo for less than 21 days, such as for less than 18,
17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 days or
less. In certain aspects, the CAR cells are cultured ex vivo no
more that 3 to 5 days. In still further aspects, steps (a)-(d) of
the instant method (i.e., obtaining cell samples to administering
CAR T cells) is completed in no more that 21, 20, 19, 18, 17, 16,
15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 days.
[0010] In further aspects, a method of providing a T-cell response
in a human subject having a disease according to the embodiments
comprises: (a) obtaining a sample of cells from the subject, the
sample comprising T-cells or T-cell progenitors and having an
initial volume of between about 20 and 200 mls when obtained from
the subject; (b) transfecting the cells with a DNA encoding a
transposon-flanked chimeric antigen receptor (CAR) and a
transposase effective to integrate the DNA encoding the CAR into
the genome of the cells, to provide a population of transgenic
CAR-expressing T-cells; (c) optionally, culturing the population of
transgenic CAR cells ex vivo in a medium that selectively enhances
proliferation of CAR-expressing T-cells; and (d) administering an
effective amount of the transgenic CAR T-cells to the subject to
provide a T-cell response. For example, the sample of cells from
the subject may be a sample of less than about 200 mls of a
peripheral blood or umbilical cord blood. In some aspects, the
sample may be collected by apheresis. However, in certain preferred
aspects, the sample is collected by a method that does not involved
apheresis (e.g., by venipuncture). In still further aspects, the
sample of cells has an initial volume of less than about 175, 150,
125, 100, 75, 50 or 25 mls (e.g., the sample of cells has an
initial volume of between about 50 and 200 mls, 50 and 100mls, or
100 and 200 mls when obtained from the subject).
[0011] In a further embodiment there is provided an isolated
transgenic cell comprising an expressed CAR targeted to the
envelope protein of HERV-K. In certain aspects, the cells comprise
DNA encoding the CAR integrated into the genome of the cell (e.g.,
CAR DNA flanked by transposon repeat sequences). For example, the
CAR sequence can comprise the CDR sequences (e.g., CDRs 1-6) of
monoclonal antibody 6H5 or the scFv sequence of monoclonal antibody
6H5. In some aspects, HERV-K-targeted CAR is at least 85% identical
to the amino acid sequence of SEQ ID NO: 4 (e.g., a sequence at
least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical to SEQ ID NO: 4). In certain aspects, a cell of the
embodiments (e.g., a human HERV-K-targeted CAR cell) can be used to
treat a subject (or provide an immune response in a subject) having
a HERV-K-expressing cancer.
[0012] In still a further embodiment there is provided an isolated
transgenic cell comprising an expressed CAR and an expressed
membrane-bound IL-15. For example, in some aspects, the
membrane-bound IL-15 comprises a fusion protein between IL-15 and
IL-15R.alpha.. In yet further aspects, the membrane-bound IL-15
comprises an amino acid sequence at least about 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 6
(referred to herein as mIL15). As further detailed herein, in some
cases, the membrane-bound IL-15 is encoded by a RNA or a DNA (e.g.,
an extra chromosomal or episomal vector). In certain aspects, the
cell comprises DNA encoding the membrane-bound IL-15 integrated
into the genome of the cell (e.g., coding DNA flanked by transposon
repeat sequences). In certain aspects, a cell of the embodiments
(e.g., human CAR cell, expressing a membrane-bound cytokine) can be
used to treat a subject (or provide an immune response in a
subject) having low levels of target antigen, such as a subject
with minimal residual disease (as further detailed herein).
[0013] In some aspects, methods of the embodiments concern
transfecting the cells with a DNA encoding a chimeric T-cell
receptor (CAR) and, in some cases, a transposase. Methods of
transfecting of cells are well known in the art, but in certain
aspects, highly efficient transfections methods such as
electroporation are employed. For example, nucleic acids may be
introduced into cells using a nucleofection apparatus. Preferably,
the transfection step does not involve infecting or transducing the
cells with virus, which can cause genotoxicity and/or lead to an
immune response to cells containing viral sequences in a treated
subject.
[0014] Further aspects of the embodiments concern transfecting
cells with an expression vector encoding a CAR. A wide range of CAR
constructs and expression vectors for the same are known in the art
and are further detailed herein. For example, in some aspects, the
CAR expression vector is a DNA expression vector such as a plasmid,
linear expression vector or an episome. In some aspects, the vector
comprises additional sequences, such as sequence that facilitate
expression of the CAR, such a promoter, enhancer, poly-A signal,
and/or one or more introns. In preferred aspects, the CAR coding
sequence is flanked by transposon sequences, such that the presence
of a transposase allows the coding sequence to integrate into the
genome of the transfected cell.
[0015] As detailed supra, in certain aspects, cells are further
transfected with a transposase that facilitates integration of a
CAR coding sequence into the genome of the transfected cells. In
some aspects, the transposase is provided as DNA expression
vector.
[0016] However, in preferred aspects, the transposase is provided
as an expressible RNA or a protein such that long-term expression
of the transposase does not occur in the transgenic cells. For
example, in some aspects, the transposase is provided as an mRNA
(e.g., an mRNA comprising a cap and poly-A tail). Any transposase
system may be used in accordance with the embodiments. However, in
some aspects, the transposase is salmonid-type Tc1-like transposase
(SB). For example, the transposase can be the so called "Sleeping
beauty" transposase, see e.g., U.S. Pat. No. 6,489,458,
incorporated herein by reference. In certain aspects, the
transposase is an engineered enzyme with increased enzymatic
activity. Some specific examples of transposases include, without
limitation, SB10, SB11 or SB100.times. transposase (see, e.g.,
Mates et al., 2009, incorporated herein by reference). For example,
a method can involve electroporation of cells with a mRNA encoding
a SB10, SB11 or SB100.times. transposase.
[0017] In still further aspects, a transgenic CAR cell of the
embodiments further comprises an expression vector for expression
of a membrane-bound cytokine that stimulates proliferation and/or
survival of T-cells. In particular, CAR cells comprising such
cytokines can proliferate and/or persist with little or no ex vivo
culture with activating and propagating cells (AaPCs) or artificial
antigen presenting cells (aAPCs) due the simulation provided by the
cytokine expression. Likewise, such CAR cells can proliferate in
vivo even when large amounts of antigen recognized by the CAR is
not present (e.g., as in the case of a cancer patient in remission
or a patient with minimal residual disease). In some aspects, the
CAR cells comprise a DNA or RNA expression vector for expression of
a C.gamma. cytokine and elements (e.g., a transmembrane domain) to
provide surface expression of the cytokine. For example, the CAR
cells can comprise membrane-bound versions of IL-7, IL-15 or IL-21.
In some aspects, the cytokine is tethered to the membrane by fusion
of the cytokine coding sequence with the receptor for the cytokine.
For example, a cell can comprise a vector for expression of a
IL-15-IL-15R.alpha. fusion protein (e.g., a protein comprising the
sequence of SEQ ID NO: 6). In still further aspects, a vector
encoding a membrane-bound C.gamma. cytokine is a DNA expression
vector, such as vector integrated into the genome of the CAR cells
or an extra-chromosomal vector (e.g., and episomal vector). In
still further aspects, expression of the membrane-bound C.gamma.
cytokine is under the control of an inducible promoter (e.g., a
drug inducible promoter) such that the expression of the cytokine
in the CAR cells (and thereby the proliferation of the CAR cells)
can be controlled by inducing or suppressing promoter activity.
[0018] Aspects of the embodiments concern obtaining a sample from a
patient comprising NK cells, NKT cells, T-cells or T-cell
progenitor cells. For example, in some cases, the sample is an
umbilical cord blood sample, a peripheral blood sample (e.g., a
mononuclear cell fraction) or a sample from the subject comprising
pluripotent cells. In some aspects, a sample from the subject can
be cultured to generate induced pluripotent stem (iPS) cells and
these cells used to produce NK cells, NKT cells or T-cells. Cell
samples may be cultured directly from the subject or may be
cryopreserved prior to use. In some aspects, obtaining a cell
sample comprises collecting a cell sample. In other aspects, the
sample is obtained by a third party. In still further aspects, a
sample from a subject can be treated to purify or enrich the
T-cells or T-cell progenitors in the sample. For example, the
sample can be subjected to gradient purification, cell culture
selection and/or cell sorting (e.g., via fluorescence-activated
cell sorting (FACS)).
[0019] In some aspects, a method of the embodiments further
comprises obtaining, producing or using antigen presenting cells.
For example, the antigen presenting cells can be dendritic cells,
activating and propagating cells (AaPCs), or inactivated (e.g.,
irradiated) artificial antigen presenting cells (aAPCs). Methods
for producing such aAPCs are know in the art and further detailed
herein. Thus, in some aspects, transgenic CAR cells are co-cultured
with inactivated aAPCs ex vivo for a limited period of time in
order to expand the CAR cell population. The step of co-culturing
CAR cells with aAPCs can be done in a medium that comprises, for
example, interleukin-21 (IL-21) and/or interleukin-2 (IL-2). In
some aspects, the co-culturing is performed at a ratio of CAR cells
to inactivated aAPCs of about 10:1 to about 1:10; about 3:1 to
about 1:5; or about 1:1 to about 1:3. For example, the co-culture
of CAR cells and aAPCs can be at a ratio of about 1:1, about 1:2 or
about 1:3.
[0020] In some aspects, cells for culture of CAR cells such as
AaPCs or aAPCs are engineered to express specific polypeptide to
enhance growth of the CAR cells. For example, the cells can
comprise (i) an antigen targeted by the CAR expressed on the
transgenic CAR cells; (ii) CD64; (ii) CD86; (iii) CD137L; and/or
(v) membrane-bound IL-15, expressed on the surface of the aAPCs. In
some aspects, the AaPCs or aAPCS comprise a CAR-binding antibody or
fragment thereof expressed on the surface of the AaPCs or aAPCs.
Preferably, AaPCs or aAPCs for use in the instant methods are
tested for, and confirmed to be free of, infectious material and/or
are tested and confirmed to be inactivated and
non-proliferating.
[0021] While expansion on AaPCs or aAPCs can increase the number or
concentration of CAR cells in a culture, this proceed is labor
intensive and expensive. Moreover, in some aspects, a subject in
need of therapy should be re-infused with transgenic CAR cells in
as short a time as possible. Thus, in some aspects, ex vivo
culturing the transgenic CAR cells (c) is for no more than 14 days,
no more than 7 days or no more than 3 days. For example, the ex
vivo culture (e.g., culture in the presence of AaPCs or aAPCs) can
be performed for less than one population doubling of the
transgenic CAR cells. In still further aspects, the transgenic
cells are not cultured ex vivo in the presence of AaPCs or
aAPCs.
[0022] In still further aspects, a method of the embodiments
comprises a step for enriching the cell population for
CAR-expressing T-cells after transfection of the cells (step (b))
or after ex vivo expansion of the cells (step (c)). For example,
the enrichment step can comprise sorting of the cell (e.g., via
FACS), for example, by using an antigen bound by the CAR or a
CAR-binding antibody. In still further aspects, the enrichment step
comprises depletion of the non-T-cells or depletion of cells that
lack CAR expression. For example, CD56.sup.+ cells can be depleted
from a culture population. In yet further aspects, a sample of CAR
cells is preserved (or maintained in culture) when the cells are
administered to the subject. For example, a sample may be
cryopreserved for later expansion or analysis.
[0023] In certain aspects, transgenic CAR cells of the embodiments
are inactivated for expression of an endogenous T-cell receptor
and/or endogenous HLA. For example, T cells can be engineered to
eliminate expression of endogenous alpha/beta T-cell receptor
(TCR). In specific embodiments, CAR.sup.+ T cells are genetically
modified to eliminate expression of TCR. In some aspects, there is
a disruption of the T-cell receptor .alpha./.beta. in
CAR-expressing T cells using zinc finger nucleases (ZFNs). In
certain aspects, the T-cell receptor .alpha..beta.-chain in
CAR-expressing T cells is knocked-out, for example, by using zinc
finger nucleases.
[0024] As further detailed herein, CAR cells of the embodiments can
be used to treat a wide range of diseases and conditions.
Essentially any disease that involves the specific or enhanced
expression of a particular antigen can be treated by targeting CAR
cells to the antigen. For example, autoimmune diseases, infections,
and cancers can be treated with methods and/or compositions of the
invention. These include cancers, such as primary, metastatic,
recurrent, sensitive-to-therapy, refractory-to-therapy cancers
(e.g., chemo-refractory cancer). The cancer may be of the blood,
lung, brain, colon, prostate, breast, liver, kidney, stomach,
cervix, ovary, testes, pituitary gland, esophagus, spleen, skin,
bone, and so forth (e.g., B-cell lymphomas or a melanomas). In the
case of cancer treatment CAR cells typically target a cancer cell
antigen (also known as a tumor-associated antigen (TAA)).
[0025] In still further aspects, transgenic CAR cells of the
embodiments may be used to treat subject having minimal residual
disease (e.g., a subject having very low amounts of CAR-targeted
antigen present), such as cancer patients that are in apparent
remission. Using new highly sensitive diagnostic techniques,
cancer-associated antigens (or cancer cells) can be detected in
patients that do not exhibit overt cancer symptoms. Such patients
may be treated by the instant methods to eliminate residual disease
by use of antigen-targeted CAR cells. In preferred embodiments,
transgenic CAR cells for targeting of residual disease further
comprise expression of a membrane-bound proliferative cytokine, as
these cells will retain the ability to expand in vivo despite the
low amount to target antigen.
[0026] The processes of the embodiments can be utilized to
manufacture (e.g., for clinical trials) of CAR.sup.+ T cells for
various tumor antigens (e.g., CD19, ROR1, CD56, EGFR, CD123, c-met,
GD2). CAR.sup.+ T cells generated using this technology can be used
to treat patients with leukemias (AML, ALL, CML), infections and/or
solid tumors. For example, methods of the embodiments can be used
to treat cell proliferative diseases, fungal, viral, bacterial or
parasitic infections. Pathogens that may be targeted include, with
limitation, Plasmodium, trypanosome, Aspergillus, Candida, HSV,
RSV, EBV, CMV, JC virus, BK virus, or Ebola pathogens. Further
examples of antigens that can be targeted by CAR cells of the
embodiments include, without limitation, CD19, CD20,
carcinoembryonic antigen, alphafetoprotein, CA-125, 5T4, MUC-1,
epithelial tumor antigen, melanoma-associated antigen, mutated p53,
mutated ras, HER2/Neu, ERBB2, folate binding protein, HIV-1
envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2,
CD123, CD23,CD30 , CD56, c-Met, meothelin, GD3, HERV-K,
IL-11Ralpha, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, or
VEGFR2. In certain aspects, method of the embodiments concern
targeting of CD19 or HERV-K-expressing cells. For example, a HERV-K
targeted CAR cell can comprise a CAR including the scFv sequence of
monoclonal antibody 6H5. In still further aspects, a CAR of the
embodiments can be conjugated or fused with a cytokine, such as
IL-2, IL-7, IL-15, IL-21 or a combination thereof.
[0027] In some embodiments, methods are provided for treating an
individual with a medical condition comprising the step of
providing an effective amount of cells from the population of cells
described herein, including more than once in some aspects, such as
at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more
days apart. In specific embodiments, the cancer is lymphoma,
leukemia, non-Hodgkin's lymphoma, acute lymphoblastic leukemia,
chronic lymphoblastic leukemia, chronic lymphocytic leukemia, or B
cell-associated autoimmune diseases.
[0028] In still yet a further embodiment there is a provided an
isolated or recombinant polypeptide comprising the a CD19-targeted
CAR comprising an amino acid sequence at least about 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1.
In a related embodiment an isolated or recombinant polynucleotide
sequence is provided encoding a CD19-targeted CAR (e.g., encoding
an amino acid sequence 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identical to SEQ ID NO: 1). For example, in some
aspects, the polynucleotide sequence is 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3 (which
encodes the CD-19-targeted CAR) or SEQ ID NO: 4 (which encodes the
CD-19-targeted CAR, expression control sequences and flanking
transposon repeats). In still a further embodiment a host cell is
provided comprising polypeptide encoding a CD19-targeted CAR and/or
a polynucleotide encoding a CD19-targeted CAR of the embodiments.
For example, the host cell can be a T-cell or T-cell precursor.
Preferably the host cell is a human cell. A skilled artisan will
recognize that any of the forgoing polypeptides, polynucleotides or
host cells may be used in accordance with the methods detailed
herein.
[0029] In still yet a further embodiment there is a provided an
isolated or recombinant polypeptide comprising the a
HERV-K-targeted CAR comprising an amino acid sequence at least
about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical to SEQ ID NO: 4. In a related embodiment an isolated or
recombinant polynucleotide sequence is provided encoding a
HERV-K-targeted CAR (e.g., encoding an amino acid sequence 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ
ID NO: 4). For example, in some aspects, the polynucleotide
sequence is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical to SEQ ID NO: 5 (which encodes the HERV-K-targeted CAR,
expression control sequences and flanking transposon repeats). In
still a further embodiment a host cell is provided comprising
polypeptide encoding a HERV-K-targeted CAR and/or a polynucleotide
encoding a HERV-K-targeted CAR of the embodiments. For example, the
host cell can be a T-cell or T-cell precursor. Preferably the host
cell is a human cell. A skilled artisan will recognize that any of
the forgoing polypeptides, polynucleotides or host cells may be
used in accordance with the methods detailed herein.
[0030] In still yet a further embodiment there is a provided an
isolated or recombinant polypeptide comprising the a membrane-bound
IL-15 comprising an amino acid sequence at least about 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID
NO: 6. In a related embodiment an isolated or recombinant
polynucleotide sequence is provided encoding a membrane-bound IL-15
(e.g., encoding an amino acid sequence 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 6). For
example, in some aspects, the polynucleotide sequence is 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID
NO: 7 (a membrane-bound IL-15, expression control sequences and
flanking transposon repeats). In still a further embodiment a host
cell is provided comprising polypeptide encoding a membrane-bound
IL-15 and/or a polynucleotide encoding a membrane-bound IL-15 of
the embodiments. For example, the host cell can be a T-cell, T-cell
precursor or aAPC. Preferably the host cell is a human cell. A
skilled artisan will recognize that any of the forgoing
polypeptides, polynucleotides or host cells may be used in
accordance with the methods detailed herein.
[0031] As used herein the specification, "a" or "an" may mean one
or more. As used herein in the claim(s), when used in conjunction
with the word "comprising", the words "a" or "an" may mean one or
more than one.
[0032] The use of the term "or" in the claims is used to mean
"and/or" unless explicitly indicated to refer to alternatives only
or the alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or." As used herein "another" may mean at least a second or
more.
[0033] Throughout this application, the term "about" is used to
indicate that a value includes the inherent variation of error for
the device, the method being employed to determine the value, or
the variation that exists among the study subjects.
[0034] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0036] FIG. 1: Steps outlining the process to electroporate and
propagate CAR.sup.+ T cells from PB and UCB.
[0037] FIGS. 2A-D: Characterization of genetically modified T cells
from PB. (A) Expression of EGFP at Day 0 of first stimulation cycle
to assess the efficiency of gene transfer. Expression of
CD19-specific CAR (CD19RCD28) as assessed by flow cytometry on
CD3.sup.+, CD8.sup.+ and CD4.sup.+ T cells at (B) approximately 24
hours after electroporation and (C) 28 days after co-culture on
aAPC. Similar expression of CAR was observed with UCB-derived T
cells. (D) Kinetics of CAR expression.
[0038] FIG. 3: Propagation of PB-derived CAR.sup.+ T cells. Rate of
numeric expansion of CD3.sup.+ and CAR.sup.+ T cells derived from
PB by repeated co-culture on .gamma.-irradiated aAPC in presence of
recombinant human soluble IL-2 and IL-21. Upward arrows indicate
the additions of .gamma.-irradiated aAPC that mark the beginning of
each stimulation cycle. UCB-derived CAR.sup.+ T cells exhibit
similar rates of numeric expansion.
[0039] FIG. 4: Schematic of the manufacturing process using SB and
aAPC systems to genetically modify and propagate CAR.sup.+ T cells
derived from PB and UCB. CD19-specific CAR.sup.+ T cells were
generated by electro-transfer of SB-derived supercoiled DNA
plasmids and subsequent co-culture on K562-derived aAPC (clone #4)
in the presence of recombinant human soluble IL-2 and IL-21.
[0040] FIGS. 5A-C: Harvest and characterization of aAPC. (A, B)
Sepax volume reduction. aAPC clone #4 grown in VueLife bags were
harvested using CS-490.1 kit in Sepax II. The Sepax harvest (S,
n=4) was compared to manual (M, n=1) procedure. The mean
pre/post-processing cell-counts (4.9.times.10.sup.8 vs.
5.times.10.sup.8) were similar using the Sepax system. (C)
Phenotype of aAPC (clone #4). Flow cytometry analysis showing
expression of CD19, CD64, CD86, CD137L and a membrane-bound version
of IL-15 (peptide fused to modified IgG4 Fc region) co-expressed
with EGFP (mIL-15-EGFP) on K562 aAPC and K562 parental
controls.
[0041] FIG. 6: Schematic of the process of generating clinical
grade CD19-specific T cells. A MCB (PACT) and WCB (MDACC) were
generated for K562-derived aAPC (clone #4). For the generation of
CAR.sup.+ T cells, aAPC were numerically expanded in bags,
harvested using the Sepax II system, irradiated (100 Gy), and
cryopreserved for later use. CD19-specific T cells were
manufactured as follows; PBMC were isolated from normal donor
apheresis products using the Sepax II system and cryopreserved. The
PBMC were later thawed, electroporated with the SB DNA plasmids
(CD19RCD28 CAR transposon, SB11 transposase) using the Nucleofector
System, co-cultured with thawed irradiated aAPC along with
cytokines (IL-2 and IL-21) for a culture period of 28 days and
cryopreserved.
[0042] FIGS. 7A-D: Phenotype of CAR.sup.+ T cell. (A) Expression of
CD19RCD28 CAR on T cells day after electroporation (culture day 1)
and after 28 days of co-culture on aAPC clone #4 along with lack of
CD19.sup.+ aAPC. (B) CAR expression by western blot analysis using
CD3-.zeta. specific antibody. Whole cell lysates were run on
SDS-PAGE under reducing conditions. Molecular weight marker (M),
Parental Jurkat cells (Lane 1), CD19RCD28.sup.+ Jurkat cells (Lane
2), CAR.sup.neg control primary T cells (Lane 3) and
CD19RCD28.sup.+ T cells (Lane 4). (C) Percent expression of
CD3.sup.+, CD4.sup.+CAR.sup.+ and CD8.sup.+CAR.sup.+ T cells with
in a lymphocyte gate in cultures over time. Each symbol represents
a separate experiment; the solid lines are mean of the three
validation experiments. (D) Immunophenotype of memory/naive,
adhesion, activation, cytolytic and exhaustion markers on CAR.sup.+
T cells at the end (d28) of co-culture.
[0043] FIGS. 8A-D: Expansion kinetics and redirected specificity of
CAR.sup.+ T cells. Genetically modified T cells were co-cultured
with aAPC clone #4 for 28 days. At the end of each stimulation
cycle (7 days), cells were counted and stained for expression of
CAR and CD3. Three validation runs (V1, V2, and V3) were performed
and the graphs represent inferred (A) CAR.sup.+ T cells, (B)
CD3.sup.+ T cells, (C) Total viable cells over time. Arrows
indicate addition of aAPC to the culture. (D) Lysis of CD19.sup.+
targets (Daudi.beta..sub.2m, NALM-6, CD19.sup.+ EL-4) as compared
to background lysis of CD19.sup.neg EL-4 using 4-hr chromium
release assay by CAR.sup.+ T cells. Mean.+-.SD of three validation
runs is represented.
[0044] FIGS. 9A-E: Safety profile associated with the SB system.
(A) Telomere length of cells was measured using fluorescence in
situ hybridization and flow cytometry (Flow-FISH) assay.
Predominant T cell population at day 28 (V1 and V2, CD8.sup.+ T
cells; V3, CD4.sup.+ T cells) was compared to respective miltenyi
column purified subset of T cells from day 0. (B) Genomic DNA from
CAR.sup.+ T cells at day 28 was amplified using primers and probes
specific for CD19RCD28 CAR. Relative Quantity (RQ) analyses of the
CD19RCD28 target copy number was determined using CD19RCD28
CAR-transduced Jurkat cells, which are known to have one
integration of CD19RCD28 CAR per genome from FISH analysis, as a
reference and endogenous RNaseP as a normalizer. (C) TCR V.beta.
analysis of day 28 and day 35 CAR.sup.+ T cells. Data shows
mean.+-.SD of three validation run CAR.sup.+ T cells as compared to
day 0 unmanipulated controls. (D) Genomic PCR showing lack of SB11
transposase integration. Genomic DNA (20 ng) was amplified using
SB11 or GAPDH primers. CAR.sup.neg control T cells (lane 5) and
CAR.sup.+ T cells (lane 7) amplified using SB11 primers;
CAR.sup.neg control T cells (lane 6), CAR.sup.+ T cells (lane 8)
and Jurkat stably expressing SB11 (lane 4) amplified using GAPDH
primers. Jurkat stably expressing SB11 (Jurkat/SB11-IRES2-EGFP)
(lane 3) and the linearized plasmid, pKan-CMV-SB11 (lane 2)
amplified using SB11 primers was used as positive control. (E)
G-banded karyotypes of CAR.sup.+ T cells from the three validation
runs reveal no structural or numeric alteration. A representative
spread from validation 2 is shown.
[0045] FIG. 10: Generation of CD19RCD28 CAR transposon. The
CD19RCD28mz (CoOp)/pEK vector containing a codon optimized chimeric
antigen receptor (CAR) and SB DNA plasmid pT-MNDU3-EGFP (Singh et
al., 2008; Hollis et al., 2006) were digested with SpeI & NheI
and SpeI & NruI to release CAR and EGFP fragments respectively.
The EGFP-deleted pT-MNDU3 vector was then ligated with CAR fragment
to generate CD19RCD28mz (CoOp)/pT-MNDU3 vector. Further the
Kanamycin resistance gene and the ColE1 origin of replication
obtained by AseI & PacI digestion of pEK vector was ligated
into SalI & ZraI digested CD19RCD28mz (CoOp)pT-MNDU3 vector to
create Pre-CD19RCD28mz (CoOp)/pSBSO. In the last step, MNDU3
promoter from Pre-CD19RCD28mz(CoOp)/pSBSO was released using
digestion with NheI & NsiI and replaced with hEF-1a promoter
fragment obtained from pVitro4 vector using XhoI & NheI, to
generate the final vector CD19RCD29mz(CoOp)/pSBSO.
[0046] FIG. 11: Generation of SB11 transposase. SB transposase
vector pCMV-SB11 was digested with PvuII to release the fragment
containing CMV promoter/enhancer and SB transposase encoding gene,
which was ligated to fragment containing Kanamycin resistance gene
and ColE1 origin of replication from pEK vector to generate
pKan-CMV-SB11 vector.
[0047] FIG. 12: Schematic of CD19 expression plasmid,
.DELTA.CD19CoOp-F2A-Neo/pSBSO. The DNA fragment encoding CD19RCD28
CAR from the plasmid CoOpCD19RCD28/pSBSO was swapped with DNA
fragment encoding neomycin resistance gene (NeoR) [PCR cloned from
pSelect-Neo (InvivoGen)] fused to codon-optimized (GENEART)
truncated CD19 (.DELTA.CD19, [Serrano et al., 2006; Mahmoud et al.,
1999]) via a F2A linker (amino acid, VKQTLNFDLLKLAGDVESNPGP;
[Szymczak et al., 2004; Yang et al., 2008; Kim et al., 2011]) to
generate .DELTA.CD19CoOp-F2A-Neo/pSBSO. EF1.alpha. promoter,
Elongation factor-1.alpha. promoter; NeoR, Neomycin resistance
gene; bGHpAn, polyadenylation signal from bovine growth hormone;
ColE1, ori; KanR, Kanamycin resistance gene; IR, SB-inverted/direct
repeats.
[0048] FIG. 13: Rate of numeric expansion of CD19-specific
CAR.sup.+ T cells. Genetically modified T cells were co-cultured
with aAPC in a 7-day stimulation cycle, and weekly fold-expansion
rate from each validation run at the end of each stimulation cycle
for total, CD3.sup.+ and CAR.sup.+ T cells was calculated. Mean
fold-expansion is shown (n=3).
[0049] FIG. 14: Redirected specificity of CD19-specific CAR.sup.+ T
cells. CD19-specific lysis of CD19.sup.+ tumor targets
(Daudi.beta..sub.2m, NALM-6, CD19.sup.+ EL-4) by CAR+ T cells
generated in three validation runs (V1, V2, V3) in a standard 4-hr
chromium assay. Background autologous lysis against the CAR.sup.neg
control was 1.5%.
[0050] FIG. 15: Safety regarding chromosomal aberration. G-banded
karyotypes of CAR.sup.+ T cells generated from validation runs (V1
and V3) reveal no structural or numeric alteration.
[0051] FIGS. 16A-D: (A) A representative picture of tumor cells
(200.times.) with varying intensity (Scored 0-3) of HERV-K
expression (top panel) when compared to isotype IgG2a control
staining (bottom panel). (B) A picture of tumor cells (400.times.)
showing HERV-K staining that is punctate and bordering the cell
membrane (solid arrow) or more diffuse cytoplasmic staining (dotted
arrow). (C) A dot plot representing H-index of each patient showed
a significant difference (p<0.0267) between the benign and tumor
tissues. (D) No significant difference was seen between malignant
and metastatic tumor. And significant difference was seen between
the benign tumor and malignant or metastatic tumor.
[0052] FIGS. 17A-F: (A) HERV-K-specific CAR encoding SB plasmid.
(B) Flow plot representing CAR (Fc) expression on Day 1 and Day 35
of CD3.sup.+ HERV-K-specific CAR.sup.+ T cells. Quadrant
percentages of flow plots are in upper right corner. CAR, chimeric
antigen receptor. (C) No significant difference in total cell
growth between the HERV-K-specific CAR.sup.+ T cells and
non-specific CD-19 CAR.sup.+ T cells. (D) By Day 21 all
HERV-K-specific CAR cells are CD3.sup.+ T cells. (E) The CAR
integration analysis shows that the HERV-K-specific CAR.sup.+ T
cells have less than 2 integrations per cell. Data represents mean
of two independent experiments with 3 different donors performed in
triplicate. (F) The phenotype of HERV-K-specific CAR.sup.+ T cells
are CD3.sup.+CD56.sup.+CD45RO.sup.hi
CD45RA.sup.loCD27.sup.+CD62L.sup.+ T cells that produce high levels
of granzyme B. All data represent average of four donors.
[0053] FIGS. 18A-C: (A) A histogram representation on HERV-K
antigen expression (in red) on tumor cell surface compared to
isotype control (in blue). (B) A standard 4-h CRA of melanoma tumor
targets with varying dilution of HERV-K-specific CAR.sup.- T cells
(in solid line) compared to No DNA control T cells (in dotted
lines). Data are mean.+-.SD from four healthy donors (average of
triplicate measurements for each donor) that were pooled from two
independent experiments. Two-way ANOVA with Bonferroni post-test
was performed on (B) and (C) between the HERV-K-specific CAR.sup.+
T cells and No DNA control cells. CAR, chimeric antigen receptor;
CRA, chromium release assay; E:T, effector to target ratio. (D)
IFN-.gamma. production by CAR.sup.+ T cells upon incubation with
targets. PMA-ionomycin is used as a positive control.
[0054] FIGS. 19A-D: Specificity of HERV-K-specific CAR.sup.+ T
cells. (A) Histogram of EL4 cells artificially expressing HERV-K
antigen (in black) was plotted along with HERV-K.sup.neg EL4
parental (blue) and isotype control staining (orange). (B) A four
hour CRA showed a significant increase (p<0.001) in killing EL4
cells expressing the antigen compared to the parental by the
HERV-K-specific CAR.sup.+ T cells at varying E:T ratios. (C)
Immunoblot assay was performed to show HERV-K env-specific
shRNA-mediated knockdown in A888 cells when compared to A888 parent
or A888 treated with scrambled shRNA. Lower panel shows actin
protein expression as control. (D) CRA of HERV-K-specific CAR.sup.+
T cells with the A888 HERV-K KD cells, A888 parental (A888P) and
A888 scrambled control (A888 scra) showed significant
antigen-specific killing by the T cells. All data represent the
mean of two independent experiments by three donors preformed in
triplicate. Two-way ANOVA with Bonferroni post-test was used for
(B) to compare EL4 parental to HERV-K.sup.+ EL4 and one-way ANOVA
with Newman-Keuls multiple comparison test for (C) to compare
A888KD to A888 P and A888 scra.
[0055] FIGS. 20A-E: To determine the activity of HERV-K-specific
CAR.sup.+ T cells over 15 h period target and effector cells were
plated in 1:5 ratio with Sytox.RTM. (Invitrogen, dead cell stain)
in the media. Fifty images of each target with effector cells were
recorded every 7 min for this period. (A) Picture representing
HERV-K.sup.+ melanoma cells (A888 and A375) and HERV-Kneg control
(HEK293 parent) cells with CAR.sup.+ T cells are various time
points. Cells that turned green were recorded as dead cells and the
intensity of the fluorescence was measured. (B, C, D) Represents
the mean fluorescent intensity of the target cell. Upper lines
represent the dead cells while the lower lines represent the basal
intensity of live cells. (E) A plot representing a significant
difference (*p<0.05) in the mean fluorescent intensity at 15 h
time point compared to HEK293 parental cells. Data represents
average of 2 independent experiments with 50 images each. A one-way
ANOVA with Tukey's post-test was performed.
[0056] FIGS. 21A-E: In vivo antitumor activity of HERV-K-specific
CAR.sup.+ T cells. A) Schematic of experiment. (B) Representative
images of mice from day 3 to day 25. (C) BLI derived from
mKate.sup.+rRLuc.sup.+HERV-K.sup.+ A375-SM tumor and (D) postmortem
analysis of liver tissues with the red dots representing
mKate.sup.+tumor metastatic foci. Data are mean.+-.SD (n=5-6 mice
per group). Statistics performed with (in D) two-way ANOVA with
Bonferroni's post-tests and between treated and untreated mice.
**P<0.01 and ***P<0.001. ANOVA, analysis of variance; BLI,
bioluminescent imaging; CAR, chimeric antigen receptor; IL,
interleukin.
[0057] FIGS. 22A-B: (A) Representative pictures (200.times.) of
HERV-K antigen expression on tissues sections from 29 normal organs
are shown. The H-index was calculated as zero since no staining was
observed in any of these tissues. (B) H-index of malignant tissue
from various organs and different patient are shown in a dot
plot.
[0058] FIGS. 23A-C: (A) Growth of CD4.sup.+ versus CD8.sup.+
HERV-K-specific CAR.sup.+ T cells is shown. (B) nCounter analysis
representing expression of various genes in HERV-K-specific
CAR.sup.+ T cells versus No DNA control cells. Red indicates high
expression while green indicates low mRNA levels. (C) Ingenuity
pathway analysis of genes highly expressed in HERV-K-specific
CAR.sup.+ T cells.
[0059] FIG. 24: A 4 h standard CRA was performed with melanoma and
CD19-specific tumor targets and CD19 CAR.sup.+ T cells. All data
represent two independent experiments performed with an average of
6 donors and analyzed using 2-way ANOVA with Bonferroni
post-test.
[0060] FIG. 25: Bi-directional SB plasmid encoding HERV-K antigen
under hEF-1.alpha. promoter and neomycin resistance gene under CMV
promoter.
[0061] FIG. 26: Figure representing HERV-K-specific CAR (in green)
engagement with HERV-K antigen (in red) on tumor cell surface.
[0062] FIGS. 27A-E: (A) SB Plasmid encoding myc-ffLuc with Neomycin
resistance gene. (B) Total cell growth of HERV-K-specific CAR.sup.+
T cells and HERV-K-specific CAR-ffLuc.sup.+ T cells. (C) Four-hour
CRA of A375SM and EL4 parental cells with HERV-K-specific
CAR-ffLuc.sup.+ T cells. (D) Mouse image representing ffLuc
activity. (E) Lentiviral plasmid encoding RLuc and mKate for tumor
cell imaging.
[0063] FIGS. 28A-B: Schematics of mIL15. A) The mIL15 construct
flanked by inverted repeats which are components of the Sleeping
Beauty expression plasmid. The mIL15 mutein is a fusion of IL-15
with the full-length IL-15R.alpha. by a flexible serine-glycine
linker. B) A schematic representing the expressed protein structure
of mIL15.
[0064] FIGS. 29A-B: CAR and mIL15 expression in genetically
modified T cells after ex vivo expansion on aAPC after five
stimulation cycles. A) Expression of a representative sample of
five donors. B) Expression of the denoted marker (CAR and/or mIL15)
in genetically modified T cells.
[0065] FIG. 30: Validation of the functionality of mIL15 via
phosflow of pSTAT5. A five hour incubation of cells in serum and
cytokine-free conditions, unless otherwise noted, to obtain basal
and IL-15-mediated phosphorylation. Representative plot (n=6).
[0066] FIG. 31: Inferred counts of CAR.sup.+ T cells with or
without co-expression of mIL15 after four stimulation cycles on
aAPC. CAR.sup.+ T cells were cultured with soluble IL-2 and IL-21
(the standard culture condition) or IL-15 and IL-21 (the soluble
cytokine control), and mIL15.sup.+CAR.sup.+ T cells with IL-21.
Data are mean.+-.SD, n=4.
[0067] FIGS. 32A-B: Phenotype and specific lysis capacity of ex
vivo expanded mIL15.sup.+CAR.sup.+ T cells. A) Percent surface
expression of certain T cell, activation, and
differentiation-associated markers after 4 stimulations on aAPC.
Horizontal line indicates the mean value. *P=0.047, paired t test,
n>4. B) CAR.sup.+ T cell (left panel) and mIL15.sup.+CAR.sup.+ T
cell (right panel) specific lysis after 5 stimulations on aAPC of
CD19.sup.+ or CD19.sup.neg targets from a four-hour chromium
release assay. Data are represented as mean.+-.SD, n=3.
[0068] FIGS. 33A-C: In vitro long-term persistence of
mIL15.sup.+CAR.sup.+ T cells that remain functionally competent and
resistant to AICD. A) Ex vivo expanded mIL15.sup.+/-CAR.sup.+ T
cells after four aAPC stimulations underwent withdrawal from
antigen re-stimulation to assess long-term in vitro persistence and
observe expansion kinetics over 60+ days. The mIL15.sup.+CAR.sup.+
T cells did not receive any exogenous cytokine support, whereas
CAR.sup.+ T cells received no cytokines, IL-2, or IL-15. Data are
log of mean.+-.SD, ****P<0.0001, RM ANOVA, n=3. B) The surviving
T cells at greater than 75 days post-antigen exposure were tested
for antigen responsiveness by incubation with: no target,
CD19.sup.+/- EL4, CD19.sup.+ Nalm-6, or LAC for 6 hours. Analysis
was by flow cytometry of IFN.gamma. intracellular staining.
Representative flow plots shown, n=3. C) The surviving T cells
after 75 days post-withdrawal were tested stimulated 1:1 with aAPC
as previously described and media was supplemented with the
cytokine (if any) used during the withdrawal culture maintenance
plus the addition of IL-21. After eight days, T cells were stained
with Annexin V to determine the proportion of live versus
apoptotic/necrotic cells in the stimulated culture. Representative
flow plot shown, n=3.
[0069] FIGS. 34A-C: Long-term persisting mIL15.sup.+CAR.sup.+ T
cells take on dichotomous CD45RA.sup.+CCR7.sup.+/- phenotypes. A)
Representative flow plots of CD45RA and CCR7 populations of
mIL15.sup.+CAR.sup.+ T cells from stimulation 4 and those
persisting 75 days after last antigen stimulation, n=7. B)
Frequencies of populations subsets from (A). ***P<0.001 and
****P<0.0001, RM ANOVA, n=7. C) A representative histogram
showing Annexin V levels in CCR7.sup.neg and CCR7.sup.+
mIL15.sup.+CAR.sup.+ T cells from stimulation 4 and those
persisting 75 days after last antigen stimulation, n=3. Histograms
are gated on the lymphocyte population to avoid non-specific CCR7
staining
[0070] FIGS. 35A-E: Graphs show additional characterization of
mIL15.sup.+CAR.sup.+ T cells by flow cytometry analysis. FIG. 35A,
Homeostatic proliferation level of WD-mIL15.sup.+CAR.sup.+ T cells
from three normal donors (PKH dilution at 10 days after staining)
that have been in culture without antigen re-stimulation for 1-2
years (top panel) and proliferative capacity of these cells upon
antigen re-stimulation with aAPC (bottom panel). FIG. 35B,
Phenotype of long-term withdrawal mIL15.sup.+CAR.sup.+ T cells (CD3
and mIL15 surface expression) that were submitted for karyotyping.
Withdrawal T cells were first re-stimulated with aAPC prior to
phenotyping and submission for karyotyping. FIG. 35C, Normal
karyotype (G-banding) result of mIL15.sup.+CAR.sup.+ T cells
persisting long-term (1.5-2.46 years) in vitro in the absence of
antigen re-stimulation and exogenous cytokines. A representative
metaphase spread is shown from four normal donors. FIG. 35D, Memory
kinetics after stimulation of cells using K562 aAPCs. FIG. 35E,
Memory kinetics with 1 versus 2 stimulations of CAR and
mIL15.sup.-CAR.sup.+ T cells.
[0071] FIG. 36: Molecular profiling indicates long-term persisting
mIL15.sup.+CAR.sup.+ T cells exhibit characteristics associated
with less differentiated T cell subsets. All genes significantly
differentially expressed between mIL15.sup.+CAR.sup.+ T cells from
stimulation four and those persisting through withdrawal conditions
are functionally classified under broad categories based on gene
ontology information. Genes within categories are partitioned based
on up- or down-regulation in the persisting withdrawal
mIL15.sup.+CAR.sup.+ T cells.
[0072] FIG. 37: Validation of transcription factors associated with
T cell differentiation states indicates long-term persisting
mIL15.sup.+CAR.sup.+ T cells exhibit a low differentiation state.
Top panel: Selected differentially expressed genes (Tcf-7, Blimp-1,
and T-bet) were validated by intracellular staining and analyzed by
flow cytometry. Representative flow plots shown, n=5. Bottom panel:
Normalized mRNA copy number from the nCounter Analysis System
output.
[0073] FIGS. 38A-D: Validation of surface markers associated with T
cell differentiation states indicates long-term persisting
mIL15.sup.+CAR.sup.+ T cells exhibit less differentiation. (A, B):
Selected differentially expressed genes, IL-7Ra and CCR7, were
validated by staining and analyzed by flow cytometry. *P=0.0156 and
***P<0.001, 1-tailed Wilcoxon matched-pairs signed rank test and
paired 1-tailed T test, respectively. Representative flow plots
shown, n=5-7. (C, D): Normalized mRNA copy number from the nCounter
Analysis System output, n=3.
[0074] FIGS. 39A-B: Acquisition of IL-2 production capability by
mIL15.sup.+CAR.sup.+ T cells. A) Representative histograms of IL-2
intracellular staining of stimulation 4 and persisting antigen
withdrawal mIL15.sup.+CAR.sup.+ T cells (WD-mIL15-CAR) that were
either unstimulated or activated with lymphocyte activation
cocktail (LAC) for 6 hours, followed by intracellular IL-2 staining
and analysis by flow cytometry. B) Frequency of LAC-stimulated T
cells producing IL-2 from (A). ****P<0.0001, n=6, paired t
test.
[0075] FIGS. 40A-D: In vivo persistence and anti-tumor activity of
mIL15.sup.+CAR.sup.30 T cells in an environment with abundant tumor
antigen. A) Schematic of experiment. B) BLI derived from
mIL15.sup.+/-CAR.sup.+ffluc.sup.+ T cells adoptively transferred
into mice after Nalm-6 tumor introduction (n=5). C) Analysis of
spleen (top panel) and bone marrow (bottom panel) at day 14 for the
presence of human T cells by staining with human CD3 and detection
by flow cytometry. Representative flow plot (n=5). D) Analysis of
peripheral blood by flow cytometry for the presence of CD3.sup.+
and CD19.sup.+ cells. Frequencies obtained after gating out murine
CD45.sup.+ cells. Data depicts individual mice and mean.+-.SD.
***P<0.001, 1-way ANOVA, n=3-5.
[0076] FIGS. 41A-F: In vivo model assessing mIL15.sup.-CAR.sup.+ T
cell persistence and anti-tumor efficacy in a low antigen
environment. A) Schematic of experiment. B) T cell (ffLuc.sup.+)
BLI of mice receiving adoptive transfer of either CAR.sup.+ T cells
or mIL15.sup.+CAR.sup.+ T cells followed by CD19.sup.+ Nalm-6 tumor
injection after 6 days of T cell engraftment. C) Longitudinal BLI
monitoring Nalm-6 burden. Images represent photon flux from Nalm-6
cell-derived rLuc activity. D) Tumor flux (rLuc) over time of mice
treated with either CAR.sup.+ T cells, mIL15.sup.+CAR.sup.+ T
cells, or no T cells. Data are mean.+-.SD. ****P<0.0001, one-way
ANOVA, n=4-5. E) Analysis of harvested tissues and blood where
human T cells (human CD3.sup.+) and Nalm-6 tumor cells (human
CD19.sup.+) were detected by flow cytometry. F) Using the low tumor
model, long-term survival of mice was evaluated out to day 98.
Experimental conditions were carried out similarly as previously
described for the low tumor model where mice were engrafted with
mIL15-CAR T cells (n=7), CAR T cells (n=8), or no T cells (n=8)
followed by NALM-6 tumor challenge. Fractions in parentheses
represent the proportion of mice surviving to day 98. *P=0.045
(mIL15-CAR versus CAR T cell treatment), log-rank (Mantel-Cox).
[0077] FIGS. 42A-E: Persistence and retained function of mIL15+CAR+
T cells independent of antigen. A) Schematic of experiment. B) BLI
of ffLuc+ T cells in mice treated with CAR+ and mIL15+CAR+ T cells
in the absence of tumor antigen. C) Analysis of bone marrow,
spleen, and peripheral blood for human CD3+ T cells and CD19+ tumor
cells, as detected by flow cytometry. Representative flow plots are
shown (left panels) and plotted frequencies of human CD3+ T cells
(right panel). Data are represented as mean.+-.SD, n=5. **P=0.0027
(bone marrow), **P=0.0081 (spleen), ns=not significant, unpaired t
test. D) Longitudinal plotting of T cell flux (ffluc). Background
luminescence (gray shaded) was defined by flux obtained from mice
not receiving ffluc+ T cells. Data are represented as mean.+-.SD,
n=5. *P=0.0128, **P+0.00231, unpaired t test. E) Cells isolated
from spleen, liver, or bone marrow were ex vivo expanded on aAPC to
generate sufficient cell numbers for assessing intracellular
IFN.gamma. production in response to CD19- and CD19+ targets (as
previously described), n=7 from three tissue sources and four mice.
Histograms were gated on CD3.
[0078] FIG. 43: A schematic showing an example protocol for CAR T
cell production using SB transposase provided as a mRNA. Effective
quantities of active CAR T cells could be produced in two weeks or
less (e.g., 16 days).
[0079] FIGS. 44A-D: FIG. 44A, graph (left panel) and flow cytometry
histograms (right panel) show the percentage of T-cells stably
expressing CAR is increasing significantly from day 9 (8.3%) to day
16 (66.2%). FIG. 44B, graph shows that T-cells grow quickly and
amplify by 20-folds to day 16 after electroporation. FIG. 44C,
graphs show results from chromium release assays using day 16
T-cells. The CAR T-cell produced provided CD-19-specific
cytotoxicity against target cells (left panel). Essentially no
cytotoxic activity was seen for unmodified T-cells (right panel).
FIG. 44D, Flow cytometry histograms showing the number of central
memory T-cells (Tcm) at day 9 (left panel) and day 17 (right panel)
post electroporation. Cells for these studies were from a donor
designated as #O and SB11 mRNA was used in the electroporation.
[0080] FIGS. 45A-B: FIG. 45A, graphs show results from chromium
release assays using day 9 (upper panels) and day 15 (lower panels)
T-cells. The CAR T-cell produced provided CD-19-specific
cytotoxicity against target cells (left panels). Essentially no
cytotoxic activity was seen for unmodified T-cells (right panels).
The modified T-cells kill CD19-positive target cells on day 9 and
day 15 with similar efficiency despite the different number of
CAR-positive cells. FIG. 45B, Graphs show CAR copy number (left
panel) and CAR expression (right panel) in electroporated cells.
Results show that CAR DNA copy number decreases from 1.5 to 0.9
from day 15 to day 22 and stays stable after that time point. Cells
for these studies were from a donor designated as #1 and
SB100.times. mRNA was used in the electroporation.
[0081] FIG. 46: Flow cytometry data showing cell viability
following electroporation. After electroporation with DNA/mRNA the
total number of cells decreases first (day 1 and 2) and then cells
start to grow. According to the cellometer counts the cell number
decrease by 59%-76% on day 2 after electroporation (viability
24-41%).
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0082] Clinical trials have demonstrated anti-tumor effects in
patients that have received T cells genetically modified to have
desired specificity. Herein, a new approach to manufacturing
antigen-specific CAR.sup.+ T cells in compliance with cGMP is
detailed. The system employs a highly efficient transfection system
in conjunction with a transposon and transposase system CAR gene
integration. These non-viral approaches have significant advantages
as an alternative to viral-mediated transduction since clinical
grade CAR.sup.+ T cells preferably should not include added viral
sequences. High quality cGMP CAR T-cells were achieved by
electro-transfer of DNA plasmids derived from a transposon system,
such as from Sleeping Beauty or piggyBac, and propagation of the
genetically modified T cells on aAPC. The approach resulted in
outgrowth of clinically appealing numbers of CAR.sup.+ T cells that
demonstrated specificity for their target antigen (e.g., CD19).
Importantly, the cells met release criteria established by the FDA
for use in clinical trials. These methods avoid genotoxicity due to
virus-mediated transduction and immunogenicity due to use of
virus.
[0083] A chimeric antigen receptor (CAR) recognizes cell-surface
tumor-associated antigen independent of human leukocyte antigen
(HLA) and employs one or more signaling molecules to activate
genetically modified T cells for killing, proliferation, and
cytokine production (Jena et al., 2010). Adoptive transfer of T
cells expressing CAR has shown promise in multiple clinical trials.
It is now possible to use a modular approach to manufacture
clinical grade genetically modified T cells. In certain
embodiments, the platform technologies disclosed herein comprise
(i) non-viral gene transfer using an electroporation device (e.g.,
a nucleofector), (ii) transposition (e.g., an SB transposon, see,
U.S. Pat. No. 6,489,458, incorporated herein by reference), (iii)
CARs that signal through endodomains (e.g., CD28CD3-.zeta.,
CD137/CD3-.zeta., or other combinations), (iv) CARs with variable
lengths of extracellular domains connecting the antigen-recognition
domain to the cell surface, and, in some cases, (v) artificial
antigen presenting cells (aAPC) derived from K562 to be able to
robustly and numerically expand CAR.sup.+ T cells (Singh et al.,
2008; Singh et al., 2011; Huang et al., 2012).
[0084] In some embodiments, the presently disclosed process can be
used to genetically modify T cells derived from peripheral blood
and/or umbilical cord blood to express CAR(s) that can be
numerically expanded in vitro using aAPC (Singh et al., 2008; Singh
et al., 2011). The process has implications for cell and gene
therapy, due to the relative ease of DNA plasmid production,
electroporation, use of thawed .gamma.-irradiated master-bank aAPC,
and can be readily transferred to facilities operating in
compliance with current good manufacturing practice (cGMP) for
Phase I/II trials. The disclosed method of manufacturing T cells is
unique at least in that it does not use (i) viral-transduction or
(ii) naked DNA electroporation followed by rapid expansion on a
PBMC/LCL feeder layer. As an example, methods are disclosed for
targeting CD19 through the enforced expression of a CAR that
recognizes CD19 independent of HLA. These T cells meet release
criteria defined by sterility, phenotype, viability, and cell
number. In-process testing revealed that the
electroporated/propagated T cells express CAR in a memory/naive
population, have a normal karyotype, preserved TCR V.beta.
repertoire, and are able to recognize and lyse CD19.sup.+ tumor
targets in a CAR-dependent manner.
[0085] The electro-transfer of non-viral plasmids is an appealing
alternative to transduction since DNA species can be produced to
clinical grade at approximately 1/10.sup.th the cost of recombinant
GMP-grade virus. To improve the efficiency of integration, the
inventors adapted Sleeping Beauty (SB) transposon and transposase
for human application (Aronovich et al., 2011; Hackett et al.,
2010; Izsvak et al., 2010; Kebriaei et al., 2012; Williams, 2008).
Additionally, they have used the piggyBac transposon/transposase
system to enforce expression of CAR (Manuri et al., 2010). The
inventor's SB system uses two DNA plasmids that comprise a
transposon coding for a gene of interest (e.g., 2.sup.nd generation
CD19-specific CAR transgene, such as designated CD19RCD28) and a
transposase (e.g., SB11), which inserts the transgene into TA
dinucleotide repeats in the target cell genome (Geurts et al.,
2006; Ivies et al., 1997; Izsvak and Ivies, 1997). To improve
therapeutic potential, the inventor's 2.sup.nd generation CAR
(Kowolik et al., 2006) signals through CD28 and CD3-.zeta. with the
expectation that this will sustain T-cell proliferation and recycle
effector functions in vivo. In addition, CARs with varying
extracellular lengths and different endodomain signaling motifs can
be expressed using the SB system.
[0086] To retrieve T-cell integrants stably expressing the CAR,
K562 aAPC (clone #4) were developed, expressing the desired antigen
(e.g., CD19) along with costimulatory molecules, such as CD86,
CD137L, a membrane-bound version of interleukin (IL)-15 (peptide
fused to modified IgG4 Fc region or cytokine peptide fused to IL-15
receptor alpha), and CD64 (Fc-.gamma. receptor 1), to select for T
cells in vitro that are capable of sustained CAR-mediated
propagation. This powerful technology has allowed the manufacture
of clinically relevant numbers (up to 10.sup.10) of CAR.sup.+ T
cells suitable for human application. As needed, additional
stimulation cycles can be undertaken to generate larger numbers of
genetically modified T cells. Furthermore, if fewer CAR.sup.30 T
cells are needed, the approach of electroporation and propagation
can be scaled back employing fewer cuvettes and carrying forward
just a sub-set of the numerically expanded T cells for 0, 1, or
more rounds of proliferation on aAPC (added at the beginning of
each stimulation cycle). Typically, at least 90% of the propagated
T cells express CAR and are cryopreserved for infusion.
Furthermore, this approach can be harnessed to generate T cells to
diverse tumor types by pairing the specificity of the introduced
CAR with expression of the tumor-associated antigen (TAA)
recognized by the CAR on the aAPC. The ex vivo expansion platform
has also been adapted to manufacture NK cells, NK T cells, and
.gamma..delta. T cells.
[0087] The outgrowth of CD4.sup.+ and CD8.sup.+ T cells expressing
the 2.sup.nd generation CAR include cells with a
stem-cell/memory/naive phenotype and exhibit three hallmarks of
re-directed specificity. First, the genetically modified T cells
specifically lyse CD19.sup.+ targets. Second, they produce cytokine
(e.g., IFN-.gamma.) in response to CD19.sup.+ stimulator cells.
Third, they proliferate in response to CD19.sup.+ stimulation, all
in a CAR-dependent manner (Singh et al., 2011; Singh et al. 2008).
The aAPC and tissue culture environment (e.g., the addition of
IL-21) have been modified to generate patient- and donor-derived
CD19-specific T cells for infusion after hematopoietic stem-cell
transplantation (Singh et al., 2011; Singh et al. 2008). The
inventors can produce CAR.sup.+ T cells from peripheral blood
simply obtained by venipuncture, which avoids the cost, discomfort,
and inconvenience of obtaining mononuclear cells by apheresis. The
ability to derive large numbers of CAR.sup.+ T cells from small
numbers of mononuclear cells is particularly appealing for infusing
T cells after allogeneic umbilical cord blood transplantation. The
small size and anonymity of the neonatal donor precludes
re-accessing this individual at a later time point and only limited
numbers of harvested mononuclear cells are available as starting
material for T cell manufacture to avoid interfering with
hematopoiesis. Further advances to the manufacturing process
include a high throughput electroporation device coupled with a
fully closed WAVE bioreactor to minimize handling.
[0088] The present approach to manufacturing includes the
implementation of processing and culturing systems to reduce work
load and safeguard against a breach in sterility. To this end, the
inventors co-cultured T cells with .gamma.-irradiated aAPC in
bioreactors and/or bags rather than flasks. This transition
typically occurs after day 14 following electroporation. In
addition, the aAPC as source material are numerically expanded in
bioreactors and/or bags, and the inventors adapted the Sepax device
to process the aAPC for cryopreservation. The Sepax harvest
procedure has additional advantages beyond automation when using
large volumes of culture media (>900 mL) as it reduces
additional centrifuging steps required by the manual process.
I. DEFINITIONS
[0089] The term "chimeric antigen receptors (CARs)," as used
herein, may refer to artificial T-cell receptors, chimeric T-cell
receptors, or chimeric immunoreceptors, for example, and encompass
engineered receptors that graft an artificial specificity onto a
particular immune effector cell. CARs may be employed to impart the
specificity of a monoclonal antibody onto a T cell, thereby
allowing a large number of specific T cells to be generated, for
example, for use in adoptive cell therapy. In specific embodiments,
CARs direct specificity of the cell to a tumor associated antigen,
for example. In some embodiments, CARs comprise an intracellular
activation domain, a transmembrane domain, and an extracellular
domain comprising a tumor associated antigen binding region. In
particular aspects, CARs comprise fusions of single-chain variable
fragments (scFv) derived from monoclonal antibodies, fused to
CD3-zeta a transmembrane domain and endodomain. The specificity of
other CAR designs may be derived from ligands of receptors (e.g.,
peptides) or from pattern-recognition receptors, such as Dectins.
In particular embodiments, one can target malignant B cells by
redirecting the specificity of T cells by using a CAR specific for
the B-lineage molecule, CD19. In certain cases, the spacing of the
antigen-recognition domain can be modified to reduce
activation-induced cell death. In certain cases, CARs comprise
domains for additional co-stimulatory signaling, such as CD3-zeta,
FcR, CD27, CD28, CD137, DAP10, and/or OX40. In some cases,
molecules can be co-expressed with the CAR, including
co-stimulatory molecules, reporter genes for imaging (e.g., for
positron emission tomography), gene products that conditionally
ablate the T cells upon addition of a pro-drug, homing receptors,
chemokines, chemokine receptors, cytokines, and cytokine
receptors.
[0090] The term "T-cell receptor (TCR)" as used herein refers to a
protein receptor on T cells that is composed of a heterodimer of an
alpha (.alpha.) and beta (.beta.) chain, although in some cells the
TCR consists of gamma and delta (.gamma./.delta.) chains. In
embodiments of the invention, the TCR may be modified on any cell
comprising a TCR, including a helper T cell, a cytotoxic T cell, a
memory T cell, regulatory T cell, natural killer T cell, and gamma
delta T cell, for example.
[0091] The terms "tumor-associated antigen" and "cancer cell
antigen" are used interchangeably herein. In each case, the terms
refer to proteins, glycoproteins or carbohydrates that are
specifically or preferentially expressed by cancer cells.
II. CHIMERIC ANTIGEN RECEPTORS
[0092] As used herein, the term "antigen" is a molecule capable of
being bound by an antibody or T-cell receptor. An antigen is
additionally capable of inducing a humoral immune response and/or
cellular immune response leading to the production of B and/or T
lymphocytes.
[0093] Embodiments of the present invention involve nucleic acids,
including nucleic acids encoding an antigen-specific chimeric
antigen receptor (CAR) polypeptide, including a CAR that has been
humanized to reduce immunogenicity (hCAR), comprising an
intracellular signaling domain, a transmembrane domain, and an
extracellular domain comprising one or more signaling motifs. In
certain embodiments, the CAR may recognize an epitope comprised of
the shared space between one or more antigens. Pattern recognition
receptors, such as Dectin-1, may be used to derive specificity to a
carbohydrate antigen. In certain embodiments, the binding region
can comprise complementary determining regions of a monoclonal
antibody, variable regions of a monoclonal antibody, and/or antigen
binding fragments thereof. In another embodiment, that specificity
is derived from a peptide (e.g., cytokine) that binds to a
receptor. A complementarity determining region (CDR) is a short
amino acid sequence found in the variable domains of antigen
receptor (e.g., immunoglobulin and T-cell receptor) proteins that
complements an antigen and therefore provides the receptor with its
specificity for that particular antigen. Each polypeptide chain of
an antigen receptor contains three CDRs (CDR1, CDR2, and CDR3).
Since the antigen receptors are typically composed of two
polypeptide chains, there are six CDRs for each antigen receptor
that can come into contact with the antigen--each heavy and light
chain contains three CDRs. Because most sequence variation
associated with immunoglobulins and T-cell receptors are found in
the CDRs, these regions are sometimes referred to as hypervariable
domains. Among these, CDR3 shows the greatest variability as it is
encoded by a recombination of the VJ (VDJ in the case of heavy
chain and TCR .alpha..beta. chain) regions.
[0094] It is contemplated that the human CAR nucleic acids are
human genes to enhance cellular immunotherapy for human patients.
In a specific embodiment, the invention includes a full length CAR
cDNA or coding region. The antigen binding regions or domain can
comprise a fragment of the V.sub.H and V.sub.L chains of a
single-chain variable fragment (scFv) derived from a particular
human monoclonal antibody, such as those described in U.S. Pat. No.
7,109,304, incorporated herein by reference. The fragment can also
be any number of different antigen binding domains of a human
antigen-specific antibody. In a more specific embodiment, the
fragment is an antigen-specific scFv encoded by a sequence that is
optimized for human codon usage for expression in human cells.
[0095] The arrangement could be multimeric, such as a diabody or
multimers. The multimers are most likely formed by cross pairing of
the variable portion of the light and heavy chains into what has
been referred to by Winters as a diabody. The hinge portion of the
construct can have multiple alternatives from being totally
deleted, to having the first cysteine maintained, to a proline
rather than a serine substitution, to being truncated up to the
first cysteine. The Fc portion can be deleted. Any protein that is
stable and/or dimerizes can serve this purpose. One could use just
one of the Fc domains, e.g., either the CH2 or CH3 domain from
human immunoglobulin. One could also use the hinge, CH2 and CH3
region of a human immunoglobulin that has been modified to improve
dimerization. One could also use just the hinge portion of an
immunoglobulin. One could also use portions of CD8alpha.
[0096] The intracellular signaling domain of the chimeric receptor
of the invention is responsible for activation of at least one of
the normal effector functions of the immune cell in which the
chimeric receptor has been placed. The term "effector function"
refers to a specialized function of a differentiated cell. Effector
function of a T cell, for example, may be cytolytic activity or
helper activity including the secretion of cytokines. Effector
function in a naive, memory, or memory-type T cell includes
antigen-dependent proliferation. Thus the term "intracellular
signaling domain" refers to the portion of a protein that
transduces the effector function signal and directs the cell to
perform a specialized function. While usually the entire
intracellular signaling domain will be employed, in many cases it
will not be necessary to use the entire intracellular polypeptide.
To the extent that a truncated portion of the intracellular
signaling domain may find use, such truncated portion may be used
in place of the intact chain as long as it still transduces the
effector function signal. The term intracellular signaling domain
is thus meant to include any truncated portion of the intracellular
signaling domain sufficient to transduce the effector function
signal. Examples include the zeta chain of the T-cell receptor or
any of its homologs (e.g., eta, delta, gamma, or epsilon), MB1
chain, B29, Fc RIII, Fc RI, and combinations of signaling
molecules, such as CD3.zeta. and CD28, CD27, 4-1BB, DAP-10, OX40,
and combinations thereof, as well as other similar molecules and
fragments. Intracellular signaling portions of other members of the
families of activating proteins can be used, such as Fc.gamma.RIII
and Fc.epsilon.RI. See Gross et al. (1992), Stancovski et al.
(1993), Moritz et al. (1994), Hwu et al. (1995), Weijtens et al.
(1996), and Hekele et al. (1996) for disclosures of cTCR's using
these alternative transmembrane and intracellular domains. In a
preferred embodiment, the human CD3 .zeta. intracellular domain was
taken for activation.
[0097] The antigen-specific extracellular domain and the
intracellular signaling-domain may be linked by a transmembrane
domain, such as the human IgG.sub.4Fc hinge and Fc regions.
Alternatives include the human CD4 transmembrane domain, the human
CD28 transmembrane domain, the transmembrane human CD3.zeta.
domain, or a cysteine mutated human CD3.zeta. domain, or other
transmembrane domains from other human transmembrane signaling
proteins, such as CD16 and CD8 and erythropoietin receptor.
[0098] In some embodiments, the CAR nucleic acid comprises a
sequence encoding other costimulatory receptors, such as a
transmembrane domain and a modified CD28 intracellular signaling
domain. Other costimulatory receptors include, but are not limited
to one or more of CD28, CD27, OX-40 (CD134), DAP10, and 4-1BB
(CD137). In addition to a primary signal initiated by CD3 .zeta.,
an additional signal provided by a human costimulatory receptor
inserted in a human CAR is important for full activation of T cells
and could help improve in vivo persistence and the therapeutic
success of the adoptive immunotherapy.
[0099] In particular embodiments, the invention concerns isolated
nucleic acid segments and expression cassettes incorporating DNA
sequences that encode the CAR. Vectors of the present invention are
designed, primarily, to deliver desired genes to immune cells,
preferably T cells under the control of regulated eukaryotic
promoters, for example, MNDU3 promoter, CMV promoter, EF1alpha
promoter, or Ubiquitin promoter. Also, the vectors may contain a
selectable marker, if for no other reason, to facilitate their
manipulation in vitro. In other embodiments, the CAR can be
expressed from mRNA in vitro transcribed from a DNA template.
[0100] Chimeric antigen receptor molecules are recombinant and are
distinguished by their ability to both bind antigen and transduce
activation signals via immunoreceptor activation motifs (ITAM's)
present in their cytoplasmic tails. Receptor constructs utilizing
an antigen-binding moiety (for example, generated from single chain
antibodies (scFv)) afford the additional advantage of being
"universal" in that they bind native antigen on the target cell
surface in an HLA-independent fashion. For example, several
laboratories have reported on scFv constructs fused to sequences
coding for the intracellular portion of the CD3 complex's zeta
chain (.zeta.), the Fc receptor gamma chain, and sky tyrosine
kinase (Eshhar et al., 1993; Fitzer-Attas et al., 1998).
Re-directed T cell effector mechanisms including tumor recognition
and lysis by CTL have been documented in several murine and human
antigen-scFv: .zeta. systems (Eshhar, 1997; Altenschmidt et al.,
1997; Brocker et al., 1998).
[0101] To date non-human antigen binding regions are typically used
in constructing a chimeric antigen receptor. A potential problem
with using non-human antigen binding regions, such as murine
monoclonal antibodies, is the lack of human effector functionality
and inability to penetrate into tumor masses. In other words, such
antibodies may be unable to mediate complement-dependent lysis or
lyse human target cells through antibody-dependent cellular
toxicity or Fc-receptor mediated phagocytosis to destroy cells
expressing CAR. Furthermore, non-human monoclonal antibodies can be
recognized by the human host as a foreign protein, and therefore,
repeated injections of such foreign antibodies can lead to the
induction of immune responses leading to harmful hypersensitivity
reactions. For murine-based monoclonal antibodies, this is often
referred to as a Human Anti-Mouse Antibody (HAMA) response.
Therefore, the use of human antibodies is more preferred because
they do not elicit as strong a HAMA response as murine antibodies.
Similarly, the use of human sequences in the CAR can avoid
immune-mediated recognition and therefore elimination by endogenous
T cells that reside in the recipient and recognize processed
antigen in the context of HLA.
[0102] In some embodiments, the chimeric antigen receptor
comprises: a) an intracellular signaling domain, b) a transmembrane
domain, and c) an extracellular domain comprising an antigen
binding region.
[0103] In specific embodiments, intracellular receptor signaling
domains in the CAR include those of the T cell antigen receptor
complex, such as the zeta chain of CD3, also Fc.gamma. RIII
costimulatory signaling domains, CD28, CD27, DAP10, CD137, OX40,
CD2, alone or in a series with CD3zeta, for example. In specific
embodiments, the intracellular domain (which may be referred to as
the cytoplasmic domain) comprises part or all of one or more of TCR
zeta chain, CD28, CD27, OX40/CD134, 4-1BB/CD137, FccRI.gamma.,
ICOS/CD278, IL-2Rbeta/CD122, IL-2Ralpha/CD132, DAP10, DAP12, and
CD40. In some embodiments, one employs any part of the endogenous T
cell receptor complex in the intracellular domain. One or multiple
cytoplasmic domains may be employed, as so-called third generation
CARs have at least two or three signaling domains fused together
for additive or synergistic effect, for example.
[0104] In certain embodiments of the chimeric antigen receptor, the
antigen-specific portion of the receptor (which may be referred to
as an extracellular domain comprising an antigen binding region)
comprises a tumor associated antigen or a pathogen-specific antigen
binding domain including carbohydrate antigen recognized by
pattern-recognition receptors, such as Dectin-1. A tumor associated
antigen may be of any kind so long as it is expressed on the cell
surface of tumor cells. Exemplary embodiments of tumor associated
antigens include CD19, CD20, carcinoembryonic antigen,
alphafetoprotein, CA-125, MUC-1, CD56, EGFR, c-Met, AKT, Her2,
Her3, epithelial tumor antigen, melanoma-associated antigen,
mutated p53, mutated ras, and so forth. In certain embodiments, the
CAR can be co-expressed with a membrane-bound cytokine to improve
persistence when there is a low amount of tumor-associated antigen.
For example, CAR can be co-expressed with membrane-bound IL-15.
[0105] In certain embodiments intracellular tumor associated
antigens may be targeted, such as HA-1, survivin, WT1, and p53.
This can be achieved by a CAR expressed on a universal T cell that
recognizes the processed peptide described from the intracellular
tumor associated antigen in the context of HLA. In addition, the
universal T cell may be genetically modified to express a T-cell
receptor pairing that recognizes the intracellular processed tumor
associated antigen in the context of HLA.
[0106] The pathogen may be of any kind, but in specific embodiments
the pathogen is a fungus, bacteria, or virus, for example.
Exemplary viral pathogens include those of the families of
Adenoviridae, Epstein-Barr virus (EBV), Cytomegalovirus (CMV),
Respiratory Syncytial Virus (RSV), JC virus, BK virus, HSV, HHV
family of viruses, Picornaviridae, Herpesviridae, Hepadnaviridae,
Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae,
Papovaviridae, Polyomavirus, Rhabdoviridae, and Togaviridae.
Exemplary pathogenic viruses cause smallpox, influenza, mumps,
measles, chickenpox, ebola, and rubella. Exemplary pathogenic fungi
include Candida, Aspergillus, Cryptococcus, Histoplasma,
Pneumocystis, and Stachybotrys. Exemplary pathogenic bacteria
include Streptococcus, Pseudomonas, Shigella, Campylobacter,
Staphylococcus, Helicobacter, E. coli, Rickettsia, Bacillus,
Bordetella, Chlamydia, Spirochetes, and Salmonella. In one
embodiment the pathogen receptor Dectin-1 can be used to generate a
CAR that recognizes the carbohydrate structure on the cell wall of
fungi. T cells genetically modified to express the CAR based on the
specificity of Dectin-1 can recognize Aspergillus and target hyphal
growth. In another embodiment, CARs can be made based on an
antibody recognizing viral determinants (e.g., the glycoproteins
from CMV and Ebola) to interrupt viral infections and
pathology.
[0107] In some embodiments, the pathogenic antigen is an
Aspergillus carbohydrate antigen for which the extracellular domain
in the CAR recognizes patterns of carbohydrates of the fungal cell
wall, such as via Dectin-1.
[0108] A chimeric immunoreceptor according to the present invention
can be produced by any means known in the art, though preferably it
is produced using recombinant DNA techniques. A nucleic acid
sequence encoding the several regions of the chimeric receptor can
be prepared and assembled into a complete coding sequence by
standard techniques of molecular cloning (genomic library
screening, PCR, primer-assisted ligation, scFv libraries from yeast
and bacteria, site-directed mutagenesis, etc.). The resulting
coding region can be inserted into an expression vector and used to
transform a suitable expression host allogeneic T-cell line.
[0109] As used herein, a nucleic acid construct or nucleic acid
sequence or polynucleotide is intended to mean a DNA molecule that
can be transformed or introduced into a T cell and be transcribed
and translated to produce a product (e.g., a chimeric antigen
receptor).
[0110] In an exemplary nucleic acid construct (polynucleotide)
employed in the present invention, the promoter is operably linked
to the nucleic acid sequence encoding the chimeric receptor of the
present invention, i.e., they are positioned so as to promote
transcription of the messenger RNA from the DNA encoding the
chimeric receptor. The promoter can be of genomic origin or
synthetically generated. A variety of promoters for use in T cells
are well-known in the art (e.g., the CD4 promoter disclosed by
Marodon et al. (2003)). The promoter can be constitutive or
inducible, where induction is associated with the specific cell
type or a specific level of maturation, for example. Alternatively,
a number of well-known viral promoters are also suitable. Promoters
of interest include the (.beta.-actin promoter, SV40 early and late
promoters, immunoglobulin promoter, human cytomegalovirus promoter,
retrovirus promoter, and the Friend spleen focus-forming virus
promoter. The promoters may or may not be associated with
enhancers, wherein the enhancers may be naturally associated with
the particular promoter or associated with a different
promoter.
[0111] The sequence of the open reading frame encoding the chimeric
receptor can be obtained from a genomic DNA source, a cDNA source,
or can be synthesized (e.g., via PCR), or combinations thereof.
Depending upon the size of the genomic DNA and the number of
introns, it may be desirable to use cDNA or a combination thereof
as it is found that introns stabilize the mRNA or provide T
cell-specific expression (Barthel and Goldfeld, 2003). Also, it may
be further advantageous to use endogenous or exogenous non-coding
regions to stabilize the mRNA.
[0112] For expression of a chimeric antigen receptor of the present
invention, the naturally occurring or endogenous transcriptional
initiation region of the nucleic acid sequence encoding N-terminal
components of the chimeric receptor can be used to generate the
chimeric receptor in the target host. Alternatively, an exogenous
transcriptional initiation region can be used that allows for
constitutive or inducible expression, wherein expression can be
controlled depending upon the target host, the level of expression
desired, the nature of the target host, and the like.
[0113] Likewise, a signal sequence directing the chimeric receptor
to the surface membrane can be the endogenous signal sequence of
N-terminal component of the chimeric receptor. Optionally, in some
instances, it may be desirable to exchange this sequence for a
different signal sequence. However, the signal sequence selected
should be compatible with the secretory pathway of T cells so that
the chimeric receptor is presented on the surface of the T
cell.
[0114] Similarly, a termination region may be provided by the
naturally occurring or endogenous transcriptional termination
region of the nucleic acid sequence encoding the C-terminal
component of the chimeric receptor. Alternatively, the termination
region may be derived from a different source. For the most part,
the source of the termination region is generally not considered to
be critical to the expression of a recombinant protein and a wide
variety of termination regions can be employed without adversely
affecting expression.
[0115] As will be appreciated by one of skill in the art that, in
some instances, a few amino acids at the ends of the antigen
binding domain in the CAR can be deleted, usually not more than 10,
more usually not more than 5 residues, for example. Also, it may be
desirable to introduce a small number of amino acids at the
borders, usually not more than 10, more usually not more than 5
residues. The deletion or insertion of amino acids may be as a
result of the needs of the construction, providing for convenient
restriction sites, ease of manipulation, improvement in levels of
expression, or the like. In addition, the substitute of one or more
amino acids with a different amino acid can occur for similar
reasons, usually not substituting more than about five amino acids
in any one domain.
[0116] The chimeric construct that encodes the chimeric receptor
according to the invention can be prepared in conventional ways.
Because, for the most part, natural sequences may be employed, the
natural genes may be isolated and manipulated, as appropriate, so
as to allow for the proper joining of the various components. Thus,
the nucleic acid sequences encoding for the N-terminal and
C-terminal proteins of the chimeric receptor can be isolated by
employing the polymerase chain reaction (PCR), using appropriate
primers that result in deletion of the undesired portions of the
gene. Alternatively, restriction digests of cloned genes can be
used to generate the chimeric construct. In either case, the
sequences can be selected to provide for restriction sites that are
blunt-ended, or have complementary overlaps.
[0117] The various manipulations for preparing the chimeric
construct can be carried out in vitro and in particular embodiments
the chimeric construct is introduced into vectors for cloning and
expression in an appropriate host using standard transformation or
transfection methods. Thus, after each manipulation, the resulting
construct from joining of the DNA sequences is cloned, the vector
isolated, and the sequence screened to ensure that the sequence
encodes the desired chimeric receptor. The sequence can be screened
by restriction analysis, sequencing, or the like.
[0118] The chimeric constructs of the present invention find
application in subjects having or suspected of having cancer by
reducing the size of a tumor or preventing the growth or re-growth
of a tumor in these subjects. Accordingly, the present invention
further relates to a method for reducing growth or preventing tumor
formation in a subject by introducing a chimeric construct of the
present invention into an isolated T cell of the subject and
reintroducing into the subject the transformed T cell, thereby
effecting anti-tumor responses to reduce or eliminate tumors in the
subject. Suitable T cells that can be used include cytotoxic
lymphocytes (CTL) or any cell having a T cell receptor in need of
disruption. As is well-known to one of skill in the art, various
methods are readily available for isolating these cells from a
subject. For example, using cell surface marker expression or using
commercially available kits (e.g., ISOCELL.TM. from Pierce,
Rockford, Ill.).
[0119] It is contemplated that the chimeric construct can be
introduced into the subject's own T cells as naked DNA or in a
suitable vector. Methods of stably transfecting T cells by
electroporation using naked DNA are known in the art. See, e.g.,
U.S. Pat. No. 6,410,319. Naked DNA generally refers to the DNA
encoding a chimeric receptor of the present invention contained in
a plasmid expression vector in proper orientation for expression.
Advantageously, the use of naked DNA reduces the time required to
produce T cells expressing the chimeric receptor of the present
invention.
[0120] Alternatively, a viral vector (e.g., a retroviral vector,
adenoviral vector, adeno-associated viral vector, or lentiviral
vector) can be used to introduce the chimeric construct into T
cells. Suitable vectors for use in accordance with the method of
the present invention are non-replicating in the subject's T cells.
A large number of vectors are known that are based on viruses,
where the copy number of the virus maintained in the cell is low
enough to maintain the viability of the cell. Illustrative vectors
include the pFB-neo vectors (STRATAGENE.RTM.) disclosed herein as
well as vectors based on HIV, SV40, EBV, HSV, or BPV.
[0121] Once it is established that the transfected or transduced T
cell is capable of expressing the chimeric receptor as a surface
membrane protein with the desired regulation and at a desired
level, it can be determined whether the chimeric receptor is
functional in the host cell to provide for the desired signal
induction. Subsequently, the transduced T cells are reintroduced or
administered to the subject to activate anti-tumor responses in the
subject. To facilitate administration, the transduced T cells
according to the invention can be made into a pharmaceutical
composition or made into an implant appropriate for administration
in vivo, with appropriate carriers or diluents, which further can
be pharmaceutically acceptable. The means of making such a
composition or an implant have been described in the art (see, for
instance, Remington's Pharmaceutical Sciences, 16th Ed., Mack, ed.
(1980)). Where appropriate, the transduced T cells can be
formulated into a preparation in semisolid or liquid form, such as
a capsule, solution, injection, inhalant, or aerosol, in the usual
ways for their respective route of administration. Means known in
the art can be utilized to prevent or minimize release and
absorption of the composition until it reaches the target tissue or
organ, or to ensure timed-release of the composition. Desirably,
however, a pharmaceutically acceptable form is employed that does
not ineffectuate the cells expressing the chimeric receptor. Thus,
desirably the transduced T cells can be made into a pharmaceutical
composition containing a balanced salt solution, preferably Hanks'
balanced salt solution, or normal saline.
III. METHODS AND COMPOSITIONS RELATED TO THE EMBODIMENTS
[0122] In certain aspects, the invention includes a method of
making and/or expanding the antigen-specific redirected T cells
that comprises transfecting T cells with an expression vector
containing a DNA construct encoding the hCAR, then, optionally,
stimulating the cells with antigen positive cells, recombinant
antigen, or an antibody to the receptor to cause the cells to
proliferate.
[0123] In another aspect, a method is provided of stably
transfecting and re-directing T cells by electroporation, or other
non-viral gene transfer (such as, but not limited to sonoporation)
using naked DNA. Most investigators have used viral vectors to
carry heterologous genes into T cells. By using naked DNA, the time
required to produce redirected T cells can be reduced. "Naked DNA"
means DNA encoding a chimeric T-cell receptor (cTCR) contained in
an expression cassette or vector in proper orientation for
expression. The electroporation method of this invention produces
stable transfectants that express and carry on their surfaces the
chimeric TCR (cTCR).
[0124] "Chimeric TCR" means a receptor that is expressed by T cells
and that comprises intracellular signaling, transmembrane, and
extracellular domains, where the extracellular domain is capable of
specifically binding in an MHC unrestricted manner an antigen that
is not normally bound by a T-cell receptor in that manner.
Stimulation of the T cells by the antigen under proper conditions
results in proliferation (expansion) of the cells and/or production
of IL-2. The exemplary CD19- and HERV-K specific chimeric receptors
of the instant application are examples of a chimeric TCR. However,
the method is applicable to transfection with chimeric TCRs that
are specific for other target antigens, such as chimeric TCRs that
are specific for HER2/Neu (Stancovski et al., 1993), ERBB2 (Moritz
et al., 1994), folate binding protein (Hwu et al., 1995), renal
cell carcinoma (Weitjens et al., 1996), and HIV-1 envelope
glycoproteins gp120 and gp41 (Roberts et al., 1994). Other
cell-surface target antigens include, but are not limited to, CD20,
carcinoembryonic antigen, mesothelin, ROR1, c-Met, CD56, GD2, GD3,
alphafetoprotein, CD23, CD30, CD123, IL-11Ralpha, kappa chain,
lambda chain, CD70, CA-125, MUC-1, EGFR and variants, epithelial
tumor antigen, and so forth.
[0125] In certain aspects, the T cells are primary human T cells,
such as T cells derived from human peripheral blood mononuclear
cells (PBMC), PBMC collected after stimulation with G-CSF, bone
marrow, or umbilical cord blood. Conditions include the use of mRNA
and DNA and electroporation. Following transfection the cells may
be immediately infused or may be stored. In certain aspects,
following transfection, the cells may be propagated for days,
weeks, or months ex vivo as a bulk population within about 1, 2, 3,
4, 5 days or more following gene transfer into cells. In a further
aspect, following transfection, the transfectants are cloned and a
clone demonstrating presence of a single integrated or episomally
maintained expression cassette or plasmid, and expression of the
chimeric receptor is expanded ex vivo. The clone selected for
expansion demonstrates the capacity to specifically recognize and
lyse CD19 expressing target cells. The recombinant T cells may be
expanded by stimulation with IL-2, or other cytokines that bind the
common gamma-chain (e.g., IL-7, IL-12, IL-15, IL-21, and others).
The recombinant T cells may be expanded by stimulation with
artificial antigen presenting cells. The recombinant T cells may be
expanded on artificial antigen presenting cell or with an antibody,
such as OKT3, which cross links CD3 on the T cell surface. Subsets
of the recombinant T cells may be deleted on artificial antigen
presenting cell or with an antibody, such as Campath, which binds
CD52 on the T cell surface. In a further aspect, the genetically
modified cells may be cryopreserved.
[0126] T-cell propagation (survival) after infusion may be assessed
by: (i) q-PCR using primers specific for the CAR; (ii) flow
cytometry using an antibody specific for the CAR; and/or (iii)
soluble TAA.
[0127] Embodiments of the invention also concern the targeting of a
B-cell malignancy or disorder including B cells, with the
cell-surface epitope being CD19-specific using a redirected immune
T cell. Malignant B cells are an excellent target for redirected T
cells, as B cells can serve as immunostimulatory antigen-presenting
cells for T cells. Preclinical studies that support the anti-tumor
activity of adoptive therapy with donor-derived CD19-specific
T-cells bearing a human or humanized CAR include (i) redirected
killing of CD19.sup.+ targets, (ii) redirected secretion/expression
of cytokines after incubation with CD19.sup.+ targets/stimulator
cells, and (iii) sustained proliferation after incubation with
CD19.sup.+ targets/stimulator cells.
[0128] In certain embodiments of the invention, the CAR cells are
delivered to an individual in need thereof, such as an individual
that has cancer or an infection. The cells then enhance the
individual's immune system to attack the respective cancer or
pathogenic cells. In some cases, the individual is provided with
one or more doses of the antigen-specific CAR T-cells. In cases
where the individual is provided with two or more doses of the
antigen-specific CAR T-cells, the duration between the
administrations should be sufficient to allow time for propagation
in the individual, and in specific embodiments the duration between
doses is 1, 2, 3, 4, 5, 6, 7, or more days.
[0129] The source of the allogeneic T cells that are modified to
include both a chimeric antigen receptor and that lack functional
TCR may be of any kind, but in specific embodiments the cells are
obtained from a bank of umbilical cord blood, peripheral blood,
human embryonic stem cells, or induced pluripotent stem cells, for
example. Suitable doses for a therapeutic effect would be at least
10.sup.5 or between about 10.sup.5 and about 10.sup.10 cells per
dose, for example, preferably in a series of dosing cycles. An
exemplary dosing regimen consists of four one-week dosing cycles of
escalating doses, starting at least at about 10.sup.5 cells on Day
0, for example increasing incrementally up to a target dose of
about 10.sup.10 cells within several weeks of initiating an
intra-patient dose escalation scheme. Suitable modes of
administration include intravenous, subcutaneous, intracavitary
(for example by reservoir-access device), intraperitoneal, and
direct injection into a tumor mass.
[0130] A pharmaceutical composition of the present invention can be
used alone or in combination with other well-established agents
useful for treating cancer. Whether delivered alone or in
combination with other agents, the pharmaceutical composition of
the present invention can be delivered via various routes and to
various sites in a mammalian, particularly human, body to achieve a
particular effect. One skilled in the art will recognize that,
although more than one route can be used for administration, a
particular route can provide a more immediate and more effective
reaction than another route. For example, intradermal delivery may
be advantageously used over inhalation for the treatment of
melanoma. Local or systemic delivery can be accomplished by
administration comprising application or instillation of the
formulation into body cavities, inhalation or insufflation of an
aerosol, or by parenteral introduction, comprising intramuscular,
intravenous, intraportal, intrahepatic, peritoneal, subcutaneous,
or intradermal administration.
[0131] A composition of the present invention can be provided in
unit dosage form wherein each dosage unit, e.g., an injection,
contains a predetermined amount of the composition, alone or in
appropriate combination with other active agents. The term unit
dosage form as used herein refers to physically discrete units
suitable as unitary dosages for human and animal subjects, each
unit containing a predetermined quantity of the composition of the
present invention, alone or in combination with other active
agents, calculated in an amount sufficient to produce the desired
effect, in association with a pharmaceutically acceptable diluent,
carrier, or vehicle, where appropriate. The specifications for the
novel unit dosage forms of the present invention depend on the
particular pharmacodynamics associated with the pharmaceutical
composition in the particular subject.
[0132] Desirably an effective amount or sufficient number of the
isolated transduced T cells is present in the composition and
introduced into the subject such that long-term, specific,
anti-tumor responses are established to reduce the size of a tumor
or eliminate tumor growth or regrowth than would otherwise result
in the absence of such treatment. Desirably, the amount of
transduced T cells reintroduced into the subject causes a 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% decrease in
tumor size when compared to otherwise same conditions wherein the
transduced T cells are not present.
[0133] Accordingly, the amount of transduced T cells administered
should take into account the route of administration and should be
such that a sufficient number of the transduced T cells will be
introduced so as to achieve the desired therapeutic response.
Furthermore, the amounts of each active agent included in the
compositions described herein (e.g., the amount per each cell to be
contacted or the amount per certain body weight) can vary in
different applications. In general, the concentration of transduced
T cells desirably should be sufficient to provide in the subject
being treated at least from about 1.times.10.sup.6 to about
1.times.10.sup.9 transduced T cells, even more desirably, from
about 1.times.10.sup.7 to about 5.times.10.sup.8 transduced T
cells, although any suitable amount can be utilized either above,
e.g., greater than 5.times.10.sup.8 cells, or below, e.g., less
than 1.times.10.sup.7 cells. The dosing schedule can be based on
well-established cell-based therapies (see, e.g., Topalian and
Rosenberg, 1987; U.S. Pat. No. 4,690,915), or an alternate
continuous infusion strategy can be employed.
[0134] These values provide general guidance of the range of
transduced T cells to be utilized by the practitioner upon
optimizing the method of the present invention for practice of the
invention. The recitation herein of such ranges by no means
precludes the use of a higher or lower amount of a component, as
might be warranted in a particular application. For example, the
actual dose and schedule can vary depending on whether the
compositions are administered in combination with other
pharmaceutical compositions, or depending on interindividual
differences in pharmacokinetics, drug disposition, and metabolism.
One skilled in the art readily can make any necessary adjustments
in accordance with the exigencies of the particular situation.
IV. EXEMPLARY HUMAN CD19-SPECIFIC CHIMERIC ANTIGEN RECEPTOR T
CELLS
[0135] CD19, a cell surface glycoprotein of the immunoglobulin
superfamily, is a potentially attractive target for antibody
therapy of B cell-associated malignancies. This antigen is absent
from hematopoietic stem cells, and in healthy individuals its
presence is exclusively restricted to the B-lineage and possibly
some follicular dendritic cells (Scheuermann et al., 1995). In
fact, it is present on B cells from the earliest recognizable
B-lineage cells during development to B-cell blasts but is lost on
maturation to plasma cells. Furthermore, CD19 is not shed from the
cell surface and rarely lost during neoplastic transformation
(Scheuermann et al., 1995). The protein is expressed on most
malignant B-lineage cells, including cells from patients with
chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma (NHL), and
acute lymphoblastic leukemia (ALL) (Uckun et al., 1988). CD19
primarily acts as a B cell co-receptor in conjunction with CD21 and
CD81. Upon activation, the cytoplasmic tail of CD19 becomes
phosphorylated, which leads to binding by Src-family kinases and
recruitment of PI-3 kinase.
[0136] In one aspect compositions and methods of the embodiments
concern human CD19-specific chimeric T cell receptor (or chimeric
antigen receptor, CAR) polypeptide (designated hCD19CAR) comprising
an intracellular signaling domain, a transmembrane domain, and an
extracellular domain, the extracellular domain comprising a human
CD19 binding region. In another aspect, the CD19 binding region is
a F(ab')2, Fab', Fab, Fv, or scFv. The binding region may comprise
an amino acid sequence that is at least, at most, or about 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to
the wild-type amino acid sequence. The intracellular domain may
comprise an intracellular signaling domain of human CD3.zeta. and
may further comprise human CD28 intracellular segment. In certain
aspects the transmembrane domain is a CD28 transmembrane
domain.
[0137] In a further aspect compositions of the invention include a
nucleic acid encoding the polypeptide described above. In certain
aspects the nucleic acid sequence is optimized for human codon
usage.
[0138] In still a further aspect compositions of the invention
include cells expressing the polypeptide described herein. The T
cell may comprise an expression cassette encoding hCD19CAR
polypeptide. The expression cassette can be comprised in a
non-viral vector, such as a transposon, or a human transposon, or
recombinant variant thereof. The expression cassette can be
comprised in a viral vector or recombinant variant thereof. The
expression cassette can be genomically integrated or episomally
maintained or expressed from mRNA.
[0139] In yet a further aspect the invention includes a method of
making a T cell expressing a human CD19-specific CAR comprising
introducing an expression cassette into the cell, wherein the
expression cassette encodes a polypeptide comprising a human
extracellular CD19 binding domain, a transmembrane domain, and one
or more intracellular signaling domain(s). The method may further
comprise stimulating the cells with CD19.sup.+ cells, recombinant
CD19, or an antibody to the receptor to cause the cells to
proliferate, kill, and/or make cytokines; for example, the cells
may be stimulated to proliferate or expand with CD19.sup.+
artificial antigen presenting cells.
[0140] In certain aspects the invention includes methods of
treating a human disease condition associated with a cell
expressing endogenous CD19 comprising infusing a patient with an
amount of a recombinant cell expressing a human CD19-specific CAR
sufficient to treat the condition, wherein the human CD19-specific
CAR comprises a human CD19 extracellular binding domain, a
transmembrane domain, and an intracellular signaling domain. The
condition can be lymphoma, leukemia, Non-Hodgkin's lymphoma, acute
lymphoblastic leukemia, chronic lymphoblastic leukemia, chronic
lymphocytic leukemia, or B cell-associated autoimmune diseases, for
example.
[0141] The invention relates to the generation of a human
CD19-specific chimeric antigen receptor (hCD19RCD28 or hCAR). In
certain aspects recombinant cells expressing hCAR have improved in
vivo persistence and anti-tumor efficacy. The human hCAR has a
reduced immunogenicity compared to murine hCAR, which comprises a
scFv segment derived from a murine CD19-specific monoclonal
antibody (mAb). Anti-tumor effects can be augmented by genetically
modified cells, rendered specific for CD19. Typically, T cell
specificity is achieved by electrotransfer of an expression
cassette encoding hCAR.
[0142] The hCAR may be a chimeric receptor comprising one or more
activation endodomain(s), such as a CD3-.zeta.-derived activation
domain. Additional T-cell activation motifs include, but are not
limited to, CD28, CD27, OX-40, DAP10, and 4-1BB. In certain aspects
the activation domain can also include a CD28 transmembrane and/or
activation domain. In a further aspect the hCAR encoding region
and/or expression cassette codon optimized for expression in human
cells and subjects, e.g., in one embodiment the scFv region
obtained from VH and VL sequences of a CD19-specific human
antibodies are incorporated into the CD19 binding segment of the
hCAR (for example see U.S. Pat. No. 7,109,304, which is
incorporated herein by reference in its entirety). In another
embodiment, the hCAR expression cassette is episomally maintained
or integrated into the genome of the recombinant cell. In certain
aspects the expression cassette is comprised in a nucleic acid
capable of integration by using an integrase mechanism, a viral
vector, such as a retroviral vector, or a nonviral vector, such as
a transposon mechanism. In a further embodiment the expression
cassette is included in a transposon based nucleic acid. In a
particular embodiment, the expression cassette is part of a two
component Sleeping Beauty (SB) or piggyBac system that utilizes a
transposon and transposase for enhanced non-viral gene
transfer.
[0143] Recombinant hCAR expressing cells can be numerically
expanded to clinically-meaningful numbers. One example of such
expansion uses artificial antigen presenting cells (aAPC).
Recombinant hCAR expressing cells can be verified and identified by
flow cytometry and western blot analyses. Recombinant hCAR
expressing T cells, expressing a CD19-specific CAR can recognize
and kill CD19 expressing target cells. In a further aspect, hCAR
can be expressed into Universal cells that can be infused across
transplantation barriers to help prevent immunogenicity. The hCAR
can be used along with human genes for imaging (such as by positron
emission tomography, PET) and conditional ablation of T cells, in
the event of cytotoxicity. The recombinant cells of the invention
can be used in CD19-specific cellular therapies.
V. EXEMPLARY HERV-TARGETING CHIMERIC ANTIGEN RECEPTOR T CELLS
[0144] During the human genome project, a set of ancient retrovirus
named human endogenous retrovirus (HERVs) was discovered to be
stably integrated into the human genome forming 8.5% of the total
human genome. Among the HERVs, HERV-K was found as an oncogenic
allelic variant involved in melanoma, breast cancer, ovarian
cancer, teratocarcinoma and prostate cancer along with various
autoimmune diseases like multiple sclerosis and rheumatoid
arthritis (Buscher et al., 2005; Dreyfus, 2011; Seifarth et al.,
1995). The oncogenic potential of HERV-K is contributed by the
envelope (env) and GAG protein (Rec). Recent studies have shown
that the expression of the HERV-K env protein exclusively on tumor
cell surface and not on normal skin cells (Wang-Johanning et al.,
2008; Li et al., 2010). The expression of HERV-K env protein was
found to increase with more aggressive and metastatic type III and
type IV melanoma than less aggressive and localized type I melanoma
(Buscher et al., 2005; Hahn et al., 2008; Serafino et al., 2009).
This selective expression of HERV-K env protein on melanoma cells
can be harnessed as a treatment strategy for patients with
refractory or metastatic melanoma. Patients with metastatic
melanoma have a poor prognosis due to resistance to conventional
therapies such as chemotherapy, radiation and surgery (Bhatia et
al., 2009). Thus, new targeted treatment strategies are needed to
improve therapeutic outcome.
[0145] To generate T-cell therapy for melanoma, the inventors
targeted a TAA derived from HERV, whose genome stably integrated
into humans millions of years ago. To target HERK-K, T cells were
engineered to express a CAR specific for the env protein by
replacing the antigen-binding exodomain of CD19-specific CAR with
the single chain antibody (scFv) sequence of an anti-HERV-K
env-specific monoclonal antibody. This new CAR was cloned as a
transposon into the SB system. DNA plasmids coding for the HERV-K
env-specific CAR and SB transposase were electro-transferred into
primary human T cells and genetically modified CAR.sup.+ T cells
were selectively propagated on irradiated artificial antigen
presenting cells (aAPC) expressing HERV-K env and desired T-cell
co-stimulatory molecules. After co-culture on .gamma.-irradiated
aAPC, 95% of CD3.sup.+ T cells expressed the CAR and these
CAR.sup.+ T cells were able to specifically kill HERV-K env.sup.+,
but not HERV-K env-, tumor targets in vitro in contrast to
CAR.sup.+ T cells. Specificity of these CAR.sup.+ T cells was
proven by expressing the HERV-K env protein in antigen negative EL4
mouse cells which were preferentially killed compared to HERV-K
env- EL4 parental cells and HERV-K knockdown by shRNA (specific
targeting for HERV-K env RNA) on A888-mel cells resulted in reduced
killing compared to parental. The CAR.sup.+ T cells were also
successful in reducing tumor growth and metastasis of A375-super
metastatic (SM) tumor cells from lungs to liver in vivo. The mice
with tumor receiving the CAR.sup.+ T cells lived longer and
appeared healthier than the tumor alone mice group. Thus, T cells
targeting an active retrovirus can be used as an immunotherapy for
melanoma, using an approach that has translational appeal for
clinical trials.
[0146] Clonal evolution of melanoma cells can render the TCR
therapy ineffective due to its dependency on MHC complex for tumor
recognition. A CAR can overcome this clonal evolution of melanoma
cells by mediated cell death in a MHN independent manner, unlike
TCR-based T-cell therapy. The HERV-K antigen is expressed only on
the tumor cell surface and not on normal cells. Therefore,
HERV-K-specific CAR.sup.+ T cells can specifically target and
abolish tumor cells without any adverse side effects. They may be
infused to treat HERV-K.sup.+ malignancies at many stages along the
cancer spectrum, from minimal residual disease, to bulk tumors, to
disease that is refractory to conventional therapies.
[0147] HERV-K is expressed on many tumor types, including, but not
limited to, melanoma (Muster et al., 2003; Buscher et al., 2005; Li
et al., 2010; Reiche et al., 2010; Serafino et al., 2009), breast
cancer (Patience et al., 1996; Wang-Johanning et al., 2003;
Seifarth et al., 1995), ovarian cancer (Wang-Johanning et al.,
2007), lymphoma (Contreras-Galindo et al., 2008), and
teratocarcinoma (Bieda et al., 2001; Lower et al., 1993).
Furthermore, infected cells, including those infected by HIV (Jones
et al., 2012), also express HERV-K. This provides an attractive
opportunity that one CAR design targeting HERV-K may be used to
treat a variety of cancers and infections.
VI. EXEMPLARY MEMBRANE-BOUND IL-15 CO-EXPRESSING CHIMERIC ANTIGEN
RECEPTOR T CELLS FOR TARGETING MINIMALLY RESIDUAL DISEASE
[0148] Chemotherapeutic treatment of adult and pediatric B-lineage
acute lymphoblastic leukemia (B-ALL) have disease relapse rates of
65% and 20%, respectively, due to drug-resistant residual disease.
The high incidence of B-ALL relapse, especially in poor prognostic
groups, has prompted the use of immune-based therapies using
allogeneic hematopoietic stem cell transplantation (HSCT). This
therapy is dependent on alloreactive cells present in the donor
graft for the eradication of remaining leukemic cells, or minimal
residual disease, to improve disease-free survival. Donor
lymphocyte infusions have been used to enhance the ability of
engrafted T cells to target residual B-ALL after allogeneic HSCT,
but this treatment approach for such patients achieves less than a
10% remission rate and is associated with a high degree of
morbidity and mortality from the frequency and severity of
graft-versus-host disease (GVHD). With relapse a common and lethal
problem in these refractory malignancies, adoptive therapy using
peripheral blood mononuclear cells (PBMC)-derived T cells after
HSCT may be used to increase the anti-tumor effect, or
graft-versus-leukemia (GVL) effect, by retargeting the specificity
of donor T cells to a tumor-associated antigen (TAA).
[0149] Numerous formulations of CARs specific for target antigens
have been developed, with CD19-specific CAR targeting the CD19
antigen on the cell surface of B-ALL. Observing long-term
persistence of CAR.sup.+ cells and achieving durable responses in
patients across different clinical protocols remains a critical
issue hampering the therapeutic efficacy of CAR-based
therapies.
[0150] Currently, CAR-modified T cells are reliant on obtaining
survival signaling through the CAR which occurs only upon encounter
with the tumor antigen. In clinical situations where these
CAR-modified T cells are infused into patients with bulky disease,
there is ample tumor antigen present to provide sufficient
activation and survival signaling via the CAR. However, patients
with relapsed B-ALL are often conditioned with myeloablative
chemotherapy followed by HSCT and present with minimal residual
disease (MRD). In this case, patients have a low tumor load and the
minute level of TAA severely restricts the CAR-mediated signaling
necessary for supporting the infused T cells consequently
compromising therapeutic potential. An alternate CAR-independent
means for improving T cell persistence would be anticipated to
improve the engraftment of CAR-modified T cells.
[0151] Cytokines in the common gamma chain receptor family
(.gamma.C) are important costimulatory molecules for T cells that
are critical to lymphoid function, survival, and proliferation.
IL-15 possesses several attributes that are desirable for adoptive
therapy. IL-15 is a homeostatic cytokine that supports the survival
of long-lived memory cytotoxic T cells, promotes the eradication of
established tumors via alleviating functional suppression of
tumor-resident cells, and inhibits AICD.
[0152] IL-15 is tissue restricted and only under pathologic
conditions is it observed at any level in the serum, or
systemically. Unlike other .gamma.C cytokines that are secreted
into the surrounding milieu, IL-15 is trans-presented by the
producing cell to T cells in the context of IL-15 receptor alpha
(IL-15R.alpha.). The unique delivery mechanism of this cytokine to
T cells and other responding cells: (i) is highly targeted and
localized, (ii) increases the stability and half-life of IL-15, and
(iii) yields qualitatively different signaling than is achieved by
soluble IL-15.
[0153] In one embodiment, the invention provides a method of
generating chimeric antigen receptor (CAR)-modified T cells with
long-lived in vivo potential for the purpose of treating, for
example, leukemia patients exhibiting minimal residual disease
(MRD). In aggregate, this method describes how soluble molecules
such as cytokines can be fused to the cell surface to augment
therapeutic potential. The core of this method relies on
co-modifying CAR T cells with a human cytokine mutein of
interleukin-15 (IL-15), henceforth referred to as mIL15. The mIL15
fusion protein is comprised of codon-optimized cDNA sequence of
IL-15 fused to the full length IL15 receptor alpha via a flexible
serine-glycine linker. This IL-15 mutein was designed in such a
fashion so as to: (i) restrict the mIL15 expression to the surface
of the CAR.sup.+ T cells to limit diffusion of the cytokine to
non-target in vivo environments, thereby potentially improving its
safety profile as exogenous soluble cytokine administration has led
to toxicities; and (ii) present IL-15 in the context of
IL-15R.alpha. to mimic physiologically relevant and qualitative
signaling as well as stabilization and recycling of the
IL-15/IL-15Ra complex for a longer cytokine half-life. T cells
expressing mIL15 are capable of continued supportive cytokine
signaling, which is critical to their survival post-infusion. The
mIL15.sup.+CAR.sup.+ T cells generated by non-viral Sleeping Beauty
System genetic modification and subsequent ex vivo expansion on a
clinically applicable platform yielded a T cell infusion product
with enhanced persistence after infusion in murine models with
high, low, or no tumor burden. Moreover, the mIL15.sup.+CAR.sup.+ T
cells also demonstrated improved anti-tumor efficacy in both the
high or low tumor burden models.
[0154] The improved persistence and anti-tumor activity of
mIL15.sup.+CAR.sup.+ T cells over CAR.sup.+ T cells in the high
tumor burden model indicates that mIL15.sup.+CAR.sup.+ T cells may
be more efficacious than CAR.sup.+ T cells in treating leukemia
patients with active disease where tumor burden is prevalent. Thus,
mIL15.sup.+CAR.sup.+ T cells could supplant CAR.sup.+ T cells in
adoptive therapy in the broadest of applications. The capability of
mIL15.sup.+CAR.sup.+ T cells to survive independent of survival
signaling via the CAR enables these modified T cells to persist
post-infusion despite the lack of tumor antigen. Consequently, this
is anticipated to generate the greatest impact in therapeutic
efficacy in a MRD treatment setting, especially in patients who
have had myeloablative chemotherapy and hematopoietic stem cell
transplantation. These patients would receive adoptive T cell
transfer with mIL15.sup.+CAR.sup.+ T cells to treat their MRD and
prevent relapse.
[0155] Membrane-bound cytokines, such as mIL15, have broad
implications. In addition to membrane-bound IL-15, other
membrane-bound cytokines are envisioned. The membrane-bound
cytokines can also be extended to cell surface expression of other
molecules associated with activating and propagating cells used for
human application. These include, but are not limited to cytokines,
chemokines, and other molecules that contribute to the activation
and proliferation of cells used for human application.
[0156] Membrane-bound cytokine, such as mIL15 can be used ex vivo
to prepare cells for human application(s) and can be on infused
cells (e.g., T cells) used for human application. For example,
membrane-bound IL-15 can be expressed on artificial antigen
presenting cells (aAPC), such as derived from K562, to stimulate T
cells and NK cells (as well as other cells) for activation and/or
proliferation. The population of T cells activated/propagated by
mIL15 on aAPC includes genetically modified lymphocytes, but also
tumor-infiltrating lymphocytes, and other immune cells. These aAPC
are not infused. In contrast, mIL15 (and other membrane-bound
molecules) can be expressed on T cells, and other cells, that are
infused.
[0157] Therapeutic efficacy of MRD treatment with CAR-modified T
cells is hampered by a lack of persistence after adoptive transfer
of the T cells. The capability of mIL15.sup.+CAR.sup.+ T cells to
survive long-term in vivo independently of tumor antigen indicates
great potential for treating patients with MRD. In this case, mIL15
and the persisting T cells that it supports would address a need,
as current approaches for MRD patients are insufficient. The
persistence of T cells and other lymphocytes that are infused in
patients with MRD applies beyond CAR.sup.+ T cells. Any immune cell
that is used to treat and prevent malignancy, infection or
auto-immune disease must be able to persist over the long term if
continued therapeutic impact is to be achieved. Thus, activating T
cells for persistence beyond the signal derived from endogenous
T-cell receptor or an introduced immunoreceptor is important to
many aspects of adoptive immunotherapy. The expression of
membrane-bound cytokine(s) thus can be used to augment the
therapeutic potential and persistence of T cells and other immune
cells infused for a variety of pathologic conditions.
[0158] The inventors have generated a mutein of IL-15 that is
expressed as a membrane-bound fusion protein of IL-15 and
IL-15R.alpha. (mIL15) on CAR.sup.+ T cells. The mIL15 construct was
co-electro-transferred with a CD19-specific CAR (on Day 0) into
primary human T cells as two Sleeping Beauty DNA transposon
plasmids. Clinically relevant numbers of mIL15.sup.+CAR.sup.+ T
cells were generated by co-culture on CD19.sup.+ artificial antigen
presenting cells and supplemented IL-21. Signaling through the
IL-15 receptor complex in genetically modified T cells was
validated by phosphorylation of STATS (pSTATS) and these T cells
demonstrated redirected specific lysis of CD19.sup.+ tumor targets
equivalent to CAR.sup.+ T cells. Furthermore, after antigen
withdrawal, signaling generated by mIL15 increased the prevalence
of T cells with a less differentiated/younger phenotype that
possessed memory-associated attributes including specific cell
surface markers, transcription factors, and the capacity to secrete
IL-2. These characteristics are desirable traits in T cells used in
adoptive transfer as they are correlated with T cell subsets with
demonstrated capability to persist long-term in vivo. In
immunocompromised NSG mice bearing a disseminated CD19.sup.+
leukemia, the mIL15.sup.+CAR.sup.+ T cells demonstrated both
persistence and an anti-tumor effect whereas its CAR.sup.+ T cell
counterpart could not maintain significant persistence despite the
presence of TAA. In a preventative mouse (NSG) model where
mIL15.sup.+/- CAR.sup.+ T cells were first engrafted for six days
followed by the introduction of a disseminated CD19.sup.+ leukemia,
only the mIL15.sup.+CAR.sup.+ T cells were found to persist as well
as prevent tumor engraftment. To test whether mIL15.sup.+CAR.sup.+
T cells were capable of persistence independent of stimulation from
TAA, mIL15.sup.+/-CAR.sup.+ T cells were adoptively transferred
into NSG mice with no tumor. Only mIL15.sup.+CAR.sup.+ T cells were
capable of persisting in this in vivo environment without exogenous
cytokine support or the presence of CD19 TAA. These data
demonstrate that mIL15 can be co-expressed on CAR.sup.+ T cells
resulting in enhanced in vivo persistence without the need for TAA
or exogenous cytokine support. In summary, this cytokine fusion
molecule: (i) provides stimulatory signals via pSTAT5 leading to
augmented in vivo T-cell persistence while maintaining
tumor-specific functionality, (ii) maintains T-cell subsets that
promotes a memory-like phenotype, (iii) eliminates the need and
cost for clinical-grade IL-2 for in vitro and in vivo T-cell
expansion and persistence, and (iv) mitigates the need for
clinical-grade soluble IL-15.
VII. IMMUNE SYSTEM AND IMMUNOTHERAPY
[0159] In some embodiments, a medical disorder is treated by
transfer of a redirected T cell that elicits a specific immune
response. In one embodiment of the present invention, B-cell
lineage malignancy or disorder is treated by transfer of a
redirected T cell that elicits a specific immune response. Thus, a
basic understanding of the immunologic responses is necessary.
[0160] The cells of the adaptive immune system are a type of
leukocyte, called a lymphocyte. B cells and T cells are the major
types of lymphocytes. B cells and T cells are derived from the same
pluripotent hematopoietic stem cells, and are indistinguishable
from one another until after they are activated. B cells play a
large role in the humoral immune response, whereas T cells are
intimately involved in cell-mediated immune responses. They can be
distinguished from other lymphocyte types, such as B cells and NK
cells by the presence of a special receptor on their cell surface
called the T-cell receptor (TCR). In nearly all other vertebrates,
B cells and T cells are produced by stem cells in the bone marrow.
T cells travel to and develop in the thymus, from which they derive
their name. In humans, approximately 1%-2% of the lymphocyte pool
recirculates each hour to optimize the opportunities for
antigen-specific lymphocytes to find their specific antigen within
the secondary lymphoid tissues.
[0161] T lymphocytes arise from hematopoietic stem cells in the
bone marrow, and typically migrate to the thymus gland to mature. T
cells express a unique antigen binding receptor on their membrane
(T-cell receptor), which can only recognize antigen in association
with major histocompatibility complex (MHC) molecules on the
surface of other cells. There are at least two populations of T
cells, known as T helper cells and T cytotoxic cells. T helper
cells and T cytotoxic cells are primarily distinguished by their
display of the membrane bound glycoproteins CD4 and CD8,
respectively. T helper cells secret various lymphokines that are
crucial for the activation of B cells, T cytotoxic cells,
macrophages, and other cells of the immune system. In contrast, T
cytotoxic cells that recognize an antigen-MHC complex proliferate
and differentiate into effector cell called cytotoxic T lymphocytes
(CTLs). CTLs eliminate cells of the body displaying antigen, such
as virus infected cells and tumor cells, by producing substances
that result in cell lysis. Natural killer cells (or NK cells) are a
type of cytotoxic lymphocyte that constitutes a major component of
the innate immune system. NK cells play a major role in the
rejection of tumors and cells infected by viruses. The cells kill
by releasing small cytoplasmic granules of proteins called perforin
and granzyme that cause the target cell to die by apoptosis.
[0162] Antigen-presenting cells, which include macrophages, B
lymphocytes, and dendritic cells, are distinguished by their
expression of a particular MHC molecule. APCs internalize antigen
and re-express a part of that antigen, together with the MHC
molecule on their outer cell membrane. The major histocompatibility
complex (MHC) is a large genetic complex with multiple loci. The
MHC loci encode two major classes of MHC membrane molecules,
referred to as class I and class II MHCs. T helper lymphocytes
generally recognize antigen associated with MHC class II molecules,
and T cytotoxic lymphocytes recognize antigen associated with MHC
class I molecules. In humans the MHC is referred to as the HLA
complex and in mice the H-2 complex.
[0163] The T-cell receptor, or TCR, is a molecule found on the
surface of T lymphocytes (or T cells) that is generally responsible
for recognizing antigens bound to major histocompatibility complex
(MHC) molecules. It is a heterodimer consisting of an alpha and
beta chain in 95% of T cells, while 5% of T cells have TCRs
consisting of gamma and delta chains. Engagement of the TCR with
antigen and MHC results in activation of its T lymphocyte through a
series of biochemical events mediated by associated enzymes,
co-receptors, and specialized accessory molecules. In immunology,
the CD3 antigen (CD stands for cluster of differentiation) is a
protein complex composed of four distinct chains (CD3.gamma.,
CD3.delta., and two times CD3.epsilon.) in mammals, that associate
with molecules known as the T-cell receptor (TCR) and the
.zeta.-chain to generate an activation signal in T lymphocytes. The
TCR, .zeta.-chain, and CD3 molecules together comprise the TCR
complex. The CD3.gamma., CD3.delta., and CD3.epsilon. chains are
highly related cell surface proteins of the immunoglobulin
superfamily containing a single extracellular immunoglobulin
domain. The transmembrane region of the CD3 chains is negatively
charged, a characteristic that allows these chains to associate
with the positively charged TCR chains (TCR.alpha. and TCR.beta..
The intracellular tails of the CD3 molecules contain a single
conserved motif known as an immunoreceptor tyrosine-based
activation motif or ITAM for short, which is essential for the
signaling capacity of the TCR.
[0164] CD28 is one of the molecules expressed on T cells that
provide co-stimulatory signals, which are required for T cell
activation. CD28 is the receptor for B7.1 (CD80) and B7.2 (CD86).
When activated by Toll-like receptor ligands, the B7.1 expression
is upregulated in antigen presenting cells (APCs). The B7.2
expression on antigen presenting cells is constitutive. CD28 is the
only B7 receptor constitutively expressed on naive T cells.
Stimulation through CD28 in addition to the TCR can provide a
potent co-stimulatory signal to T cells for the production of
various interleukins (IL-2 and IL-6 in particular).
[0165] The strategy of isolating and expanding antigen-specific T
cells as a therapeutic intervention for human disease has been
validated in clinical trials (Riddell et al., 1992; Walter et al.,
1995; Heslop et al., 1996).
[0166] Malignant B cells appear to be an excellent targets for
redirected T cells, as B cells can serve as immunostimulatory
antigen-presenting cells for T cells (Glimcher et al., 1982).
Lymphoma, by virtue of its lymph node tropism, is anatomically
ideally situated for T cell-mediated recognition and elimination.
The localization of infused T cells to lymph node in large numbers
has been documented in HIV patients receiving infusions of
HIV-specific CD8.sup.+ CTL clones. In these patients, evaluation of
lymph node biopsy material revealed that infused clones constituted
approximately 2%-8% of CD8.sup.+ cells of lymph nodes. Lymph node
homing might be further improved by co-transfecting T cells with a
cDNA construct encoding the L-selection molecule under a
constitutive promoter since this adhesion molecule directs
circulating T cells back to lymph nodes and is down-regulated by in
vitro expansion (Chao et al., 1997). The present invention may
provide a method of treating a human disease condition associated
with a cell expressing endogenous CD19 comprising infusing a
patient with a therapeutically effective dose of the recombinant
human CD19-specific CAR expressing cell as described above. The
human disease condition associated with a cell expressing
endogenous CD19 may be selected from the group consisting of
lymphoma, leukemia, non-Hodgkin's lymphoma, acute lymphoblastic
leukemia, chronic lymphoblastic leukemia, chronic lymphocytic
leukemia, and B cell-associated autoimmune diseases.
[0167] Leukemia is a cancer of the blood or bone marrow and is
characterized by an abnormal proliferation (production by
multiplication) of blood cells, usually white blood cells
(leukocytes). It is part of the broad group of diseases called
hematological neoplasms. Leukemia is a broad term covering a
spectrum of diseases. Leukemia is clinically and pathologically
split into its acute and chronic forms.
[0168] Acute leukemia is characterized by the rapid proliferation
of immature blood cells. This crowding makes the bone marrow unable
to produce healthy blood cells. Acute forms of leukemia can occur
in children and young adults. In fact, it is a more common cause of
death for children in the U.S. than any other type of malignant
disease. Immediate treatment is required in acute leukemia due to
the rapid progression and accumulation of the malignant cells,
which then spill over into the bloodstream and spread to other
organs of the body. Central nervous system (CNS) involvement is
uncommon, although the disease can occasionally cause cranial nerve
palsies. Chronic leukemia is distinguished by the excessive build
up of relatively mature, but still abnormal, blood cells. Typically
taking months to years to progress, the cells are produced at a
much higher rate than normal cells, resulting in many abnormal
white blood cells in the blood. Chronic leukemia mostly occurs in
older people, but can theoretically occur in any age group. Whereas
acute leukemia must be treated immediately, chronic forms are
sometimes monitored for some time before treatment to ensure
maximum effectiveness of therapy.
[0169] Furthermore, the diseases are classified into lymphocytic or
lymphoblastic, which indicate that the cancerous change took place
in a type of marrow cell that normally goes on to form lymphocytes,
and myelogenous or myeloid, which indicate that the cancerous
change took place in a type of marrow cell that normally goes on to
form red cells, some types of white cells, and platelets (see
lymphoid cells vs. myeloid cells).
[0170] Acute lymphocytic leukemia (also known as acute
lymphoblastic leukemia, or ALL) is the most common type of leukemia
in young children. This disease also affects adults, especially
those aged 65 and older. Chronic lymphocytic leukemia (CLL) most
often affects adults over the age of 55. It sometimes occurs in
younger adults, but it almost never affects children. Acute
myelogenous leukemia (also known as acute myeloid leukemia, or AML)
occurs more commonly in adults than in children. This type of
leukemia was previously called "acute nonlymphocytic leukemia."
Chronic myelogenous leukemia (CML) occurs mainly in adults. A very
small number of children also develop this disease.
[0171] Lymphoma is a type of cancer that originates in lymphocytes
(a type of white blood cell in the vertebrate immune system). There
are many types of lymphoma. According to the U.S. National
Institutes of Health, lymphomas account for about five percent of
all cases of cancer in the United States, and Hodgkin's lymphoma in
particular accounts for less than one percent of all cases of
cancer in the United States. Because the lymphatic system is part
of the body's immune system, patients with a weakened immune
system, such as from HIV infection or from certain drugs or
medication, also have a higher incidence of lymphoma.
[0172] In the 19th and 20th centuries the affliction was called
Hodgkin's Disease, as it was discovered by Thomas Hodgkin in 1832.
Colloquially, lymphoma is broadly categorized as Hodgkin's lymphoma
and non-Hodgkin lymphoma (all other types of lymphoma). Scientific
classification of the types of lymphoma is more detailed. Although
older classifications referred to histiocytic lymphomas, these are
recognized in newer classifications as of B, T, or NK cell
lineage.
[0173] Autoimmune disease, or autoimmunity, is the failure of an
organism to recognize its own constituent parts (down to the
sub-molecular levels) as "self," which results in an immune
response against its own cells and tissues. Any disease that
results from such an aberrant immune response is termed an
autoimmune disease. Prominent examples include Coeliac disease,
diabetes mellitus type 1 (IDDM), systemic lupus erythematosus
(SLE), Sjogren's syndrome, multiple sclerosis (MS), Hashimoto's
thyroiditis, Graves' disease, idiopathic thrombocytopenic purpura,
and rheumatoid arthritis (RA).
[0174] Inflammatory diseases, including autoimmune diseases are
also a class of diseases associated with B-cell disorders. Examples
of autoimmune diseases include, but are not limited to, acute
idiopathic thrombocytopenic purpura, chronic idiopathic
thrombocytopenic purpura, dermatomyositis, Sydenham's chorea,
myasthenia gravis, systemic lupus erythematosus, lupus nephritis,
rheumatic fever, polyglandular syndromes, bullous pemphigoid,
diabetes mellitus, Henoch-Schonlein purpura,
post-streptococcalnephritis, erythema nodosurn, Takayasu's
arteritis, Addison's disease, rheumatoid arthritis, multiple
sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme,
IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis,
Goodpasture's syndrome, thromboangitisubiterans, Sjogren's
syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,
thyrotoxicosis, scleroderma, chronic active hepatitis,
polymyositis/dermatomyositis, polychondritis, pamphigus vulgaris,
Wegener's granulomatosis, membranous nephropathy, amyotrophic
lateral sclerosis, tabes dorsalis, giant cell
arteritis/polymyalgia, perniciousanemia, rapidly progressive
glomerulonephritis, psoriasis, and fibrosing alveolitis. The most
common treatments are corticosteroids and cytotoxic drugs, which
can be very toxic. These drugs also suppress the entire immune
system, can result in serious infection, and have adverse affects
on the bone marrow, liver, and kidneys. Other therapeutics that has
been used to treat Class III autoimmune diseases to date have been
directed against T cells and macrophages. There is a need for more
effective methods of treating autoimmune diseases, particularly
Class III autoimmune diseases.
VIII. ARTIFICIAL ANTIGEN PRESENTING CELLS
[0175] In some cases, aAPCs are useful in preparing therapeutic
compositions and cell therapy products of the embodiments. For
general guidance regarding the preparation and use of
antigen-presenting systems, see, e.g., U.S. Pat. Nos. 6,225,042,
6,355,479, 6,362,001 and 6,790,662; U.S. Patent Application
Publication Nos. 2009/0017000 and 2009/0004142; and International
Publication No. WO2007/103009).
[0176] aAPCs are typically incubated with a peptide of an optimal
length that allows for direct binding of the peptide to the MHC
molecule without additional processing. Alternatively, the cells
can express and antigen of interest (i.e., in the case of
MHC-independent antigen recognition). In addition to peptide-MHC
molecules or antigens of interest, the aAPC systems may also
comprise at least one exogenous assisting molecule. Any suitable
number and combination of assisting molecules may be employed. The
assisting molecule may be selected from assisting molecules such as
co-stimulatory molecules and adhesion molecules. Exemplary
co-stimulatory molecules include CD70 and B7.1 (B7.1 was previously
known as B7 and also known as CD80), which among other things, bind
to CD28 and/or CTLA-4 molecules on the surface of T cells, thereby
affecting, for example, T-cell expansion, Thl differentiation,
short-term T-cell survival, and cytokine secretion such as
interleukin (IL)-2 (see Kim et al., 2004, Nature, Vol. 22(4), pp.
403-410). Adhesion molecules may include carbohydrate-binding
glycoproteins such as selectins, transmembrane binding
glycoproteins such as integrins, calcium-dependent proteins such as
cadherins, and single-pass transmembrane immunoglobulin (Ig)
superfamily proteins, such as intercellular adhesion molecules
(ICAMs), that promote, for example, cell-to-cell or cell-to-matrix
contact. Exemplary adhesion molecules include LFA-3 and ICAMs, such
as ICAM-1. Techniques, methods, and reagents useful for selection,
cloning, preparation, and expression of exemplary assisting
molecules, including co-stimulatory molecules and adhesion
molecules, are exemplified in, e.g., U.S. Pat. Nos. 6,225,042,
6,355,479, and 6,362,001.
[0177] Cells selected to become aAPCs, preferably have deficiencies
in intracellular antigen-processing, intracellular peptide
trafficking, and/or intracellular MHC Class I or Class II
molecule-peptide loading, or are poikilothermic (i.e., less
sensitive to temperature challenge than mammalian cell lines), or
possess both deficiencies and poikilothermic properties.
Preferably, cells selected to become aAPCs also lack the ability to
express at least one endogenous counterpart (e.g., endogenous MHC
Class I or Class II molecule and/or endogenous assisting molecules
as described above) to the exogenous MHC Class I or Class II
molecule and assisting molecule components that are introduced into
the cells. Furthermore, aAPCs preferably retain the deficiencies
and poikilothermic properties that were possessed by the cells
prior to their modification to generate the aAPCs. Exemplary aAPCs
either constitute or are derived from a transporter associated with
antigen processing (TAP)-deficient cell line, such as an insect
cell line. An exemplary poikilothermic insect cells line is a
Drosophila cell line, such as a Schneider 2 cell line (see, e.g.
Schneider, J. Embryol. Exp. Morph. 1972 Vol 27, pp. 353-365).
Illustrative methods for the preparation, growth, and culture of
Schneider 2 cells, are provided in U.S. Pat. Nos. 6,225,042,
6,355,479, and 6,362,001.
[0178] In one embodiment, aAPCs are also subjected to a freeze-thaw
cycle. In an exemplary freeze-thaw cycle, the aAPCs may be frozen
by contacting a suitable receptacle containing the aAPCs with an
appropriate amount of liquid nitrogen, solid carbon dioxide (i.e.,
dry ice), or similar low-temperature material, such that freezing
occurs rapidly. The frozen aAPCs are then thawed, either by removal
of the aAPCs from the low-temperature material and exposure to
ambient room temperature conditions, or by a facilitated thawing
process in which a lukewarm water bath or warm hand is employed to
facilitate a shorter thawing time. Additionally, aAPCs may be
frozen and stored for an extended period of time prior to thawing.
Frozen aAPCs may also be thawed and then lyophilized before further
use. Preferably, preservatives that might detrimentally impact the
freeze-thaw procedures, such as dimethyl sulfoxide (DMSO),
polyethylene glycols (PEGs), and other preservatives, are absent
from media containing aAPCs that undergo the freeze-thaw cycle, or
are essentially removed, such as by transfer of aAPCs to media that
is essentially devoid of such preservatives.
[0179] In other preferred embodiments, xenogenic nucleic acid and
nucleic acid endogenous to the aAPCs, may be inactivated by
crosslinking, so that essentially no cell growth, replication or
expression of nucleic acid occurs after the inactivation. In one
embodiment, aAPCs are inactivated at a point subsequent to the
expression of exogenous MHC and assisting molecules, presentation
of such molecules on the surface of the aAPCs, and loading of
presented MHC molecules with selected peptide or peptides.
Accordingly, such inactivated and selected peptide loaded aAPCs,
while rendered essentially incapable of proliferating or
replicating, retain selected peptide presentation function.
Preferably, the crosslinking also yields aAPCS that are essentially
free of contaminating microorganisms, such as bacteria and viruses,
without substantially decreasing the antigen-presenting cell
function of the aAPCs. Thus crosslinking maintains the important
APC functions of aAPCs while helping to alleviate concerns about
safety of a cell therapy product developed using the aAPCs. For
methods related to crosslinking and aAPCs, see for example, U.S.
Patent Application Publication No. 20090017000, which is
incorporated herein by reference.
IX. KITS OF THE INVENTION
[0180] Any of the compositions described herein may be comprised in
a kit. In some embodiments, allogeneic CAR T-cells are provided in
the kit, which also may include reagents suitable for expanding the
cells, such as media, aAPCs, growth factors, antibodies (e.g., for
sorting or characterizing CAR T-cells) and/or plasmids encoding
CARs or transposase.
[0181] In a non-limiting example, a chimeric receptor expression
construct, one or more reagents to generate a chimeric receptor
expression construct, cells for transfection of the expression
construct, and/or one or more instruments to obtain allogeneic
cells for transfection of the expression construct (such an
instrument may be a syringe, pipette, forceps, and/or any such
medically approved apparatus).
[0182] In some embodiments, an expression construct for eliminating
endogenous TCR .alpha./.beta. expression, one or more reagents to
generate the construct, and/or CAR.sup.+ T cells are provided in
the kit. In some embodiments, there includes expression constructs
that encode zinc finger nuclease(s).
[0183] In some aspects, the kit comprises reagents or apparatuses
for electroporation of cells.
[0184] The kits may comprise one or more suitably aliquoted
compositions of the present invention or reagents to generate
compositions of the invention. The components of the kits may be
packaged either in aqueous media or in lyophilized form. The
container means of the kits may include at least one vial, test
tube, flask, bottle, syringe, or other container means, into which
a component may be placed, and preferably, suitably aliquoted.
Where there is more than one component in the kit, the kit also
will generally contain a second, third, or other additional
container into which the additional components may be separately
placed. However, various combinations of components may be
comprised in a vial. The kits of the present invention also will
typically include a means for containing the chimeric receptor
construct and any other reagent containers in close confinement for
commercial sale. Such containers may include injection or blow
molded plastic containers into which the desired vials are
retained, for example.
X. EXAMPLES
[0185] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
Clinical Application of Sleeping Beauty and Artificial Antigen
Presenting Cells to Genetically Modify T Cells from Peripheral and
Umbilical Cord Blood--Materials and Methods
[0186] Isolation of Mononuclear Cells (MNC) from PB and UCB. On or
before Day 0, dilute PB with an equal volume and UCB with four
volumes of PBS-EDTA. Slowly layer diluted blood (25 mL) onto Ficoll
(12 mL) in a 50 mL centrifuge tube(s) and centrifuge at 400.times.g
for 30-40 min (no brake). Collect and transfer the mononuclear cell
fraction (interface) using a transfer pipette to a fresh 50 mL
centrifuge tube. Bring the volume up to 50 mL with PBS-EDTA and
centrifuge at 450.times.g for 10 min. Aspirate the supernatant and
gently re-suspend the cell pellet(s) in 50 mL of Complete Culture
Media (CCM). Centrifuge at 400.times.g for 10 min. Gently
re-suspend and pool the cell pellets in CCM and perform a cell
count using Trypan blue exclusion (Cellometer, PBMC program). MNC
can be used for electroporation (Nucleofection) or cryopreserved
for future use.
[0187] Preparation of T Cells for Electroporation on Day 0. If
using cryopreserved MNC, quickly thaw sufficient cells for a full
scale electroporation (2.times.10.sup.8 adding .about.20% to
account for cell loss during centrifugation and 2 hr incubation) in
a 37.degree. C. water bath. Gently re-suspend and transfer cells to
an appropriately sized centrifuge tube containing pre-warmed
Complete Phenol-Free RPMI culture media (PF-RPMI), centrifuge at
200.times.g for 10 min (no brake), and aspirate the supernatant.
Next, and if using freshly isolated MNC, re-suspend the MNC in
PF-RPMI, perform a cell count (Cellometer) and transfer cells to an
appropriately sized cell culture vessel at a concentration of
10.sup.6 cells/ml. Incubate the cells in a humidified 37.degree.
C./5% CO.sub.2 incubator for 2 h.+-.30 min. Transfer the MNC to a
sterile centrifuge tube, spin at 200.times.g for 5 min (no brakes),
aspirate the supernatant, and gently re-suspend and combine the
cell pellets in PF-RPMI. Perform a cell count (Cellometer) and
calculate the volume of the cell suspension required
(2.times.10.sup.8 MNC). Transfer the calculated volume to a sterile
50 mL centrifuge tube and spin at 200.times.g for 10 min (no
brake). Aspirate the supernatant so that no residual media remains
and gently re-suspend by tapping side of tube.
[0188] Electroporation (Nucleofection) of MNC (Full Scale Process
Using 10 Cuvettes) on Day 0. Pre-incubate a sterile 12-well plate
with 10 wells containing 4 mL of warm PF-RPMI in a humidified
37.degree. C./5% CO.sub.2 incubator. Prepare and pre-warm the Lonza
Nucleofector Solution Human T cell kit (reconstituted per
manufacturer's instructions, www.lonza.com) to ambient temperature
in a Biosafety Cabinet (BSC). Prepare Nucleofector solution/DNA
master mix by adding 100 .mu.L of supplemented Nucleofector
solution, 15 .mu.g of transposon (supercoiled DNA plasmid
designated as CD19RCD28/pSBSO), and 5 .mu.g of transposase
(supercoiled DNA plasmid designated as pCMV-SB11) per
reaction/cuvette. Disperse the MCN cell pellet by gently tapping
the side of the centrifuge tube and re-suspend in Nucleofection
solution/DNA master mix at a final cell concentration of
2.times.10.sup.7 cells/100 .mu.L. Carefully transfer 100 .mu.L of
the cell suspension to each of ten Lonza Nucleofection cuvettes,
being careful to avoid bubbles. Tap the cuvette once, and
electroporate using program U-014 (for unstimulated T cells).
Transfer the cuvettes and the 12-well plate to the BSC. Harvest the
electroporated cells from each cuvette using an Amaxa fine tip
transfer pipette by adding .about.500 .mu.L of the pre-warmed
culture medium from the corresponding well and return the plate to
a humidified 37.degree. C./5% CO.sub.2 incubator for 2 h.+-.30 min.
Following the 2-hr incubation, harvest and transfer the cells from
all wells to a sterile centrifuge tube. Wash cells by
centrifugation at 140.times.g for 8 min, ambient temperature, no
brake and aspirate and discard the supernatant so that no residual
medium covers the cell pellet. Disperse the cell pellet by gently
tapping the side of the centrifuge tube and gently re-suspend in
CCM to achieve a single cell suspension. Perform cell count and
adjust cell concentration to 10.sup.6 cells/mL in CCM. Transfer the
cell suspension to cell culture flask(s) and place in the incubator
overnight. The same process may be used for control
EGFP-transfected cells (5.times.10.sup.6 cells/cuvette with 5 .mu.g
Amaxa control EGFP supercoiled plasmid, pmaxGFP).
[0189] Analysis of CAR Expression by Flow Cytometry on Day 1 of the
1st and Subsequence Stimulation Cycles. Harvest the electroporated
cells and perform a cell count using Trypan blue exclusion
(Hemocytometer). Stain cells (1-2.times.10.sup.6) with antibody
specific for CD3, CD4, CD8, and human IgG Fcy as a measurement of
CAR expression. Acquire cells on the FACS Calibur and analyze the
data using FCS Express software to calculate expression of CAR.
Calculate CAR.sup.+ cells in culture by the formula: (No. of Total
viable cells).times.(% CAR.sup.+ cells)=No. of CAR.sup.+ cells.
[0190] Preparation of aAPC (clone #4) on Day 1 of the 1st and
Subsequent Stimulation Cycles. aAPC (clone #4) was derived from
K562 cells (parental line obtained from American Type Culture
Collection) to co-express the desired T cell co-stimulatory
molecule. Thaw an aliquot of frozen 100 Gy irradiated aAPC in a
37.degree. C. water bath. Cells are washed twice by centrifugation
at 400.times.g, 10 min in CCM and counted using Cellometer (Trypan
blue exclusion). Calculate number of viable aAPC required for
stimulation: (No. of CAR.sup.+ cells).times.2=No. of irradiated
aAPC required
[0191] aAPC-mediated Stimulation of CAR.sup.+ T Cells on Day 1
Beginning of 1st and Subsequent Stimulation Cycles. Mix the
electroporated cells (expressing CAR) and .gamma.-irradiated aAPC
(clone #4) in a sterile container at a ratio of 1:2 (CAR.sup.+
cell: viable aAPC) in CCM. Note that the aAPC ratio is adjusted for
the expression of CAR based on flow cytometry the day after
electroporation. Add IL-21 (30 ng/mL) to the cell suspension.
Aliquot in T-75 cm.sup.2 flask(s) and/or VueLife Culture bags at a
concentration of 10.sup.6 cells/mL and return to the incubator.
[0192] Continued Culture of CAR.sup.+ T Cells on Days 3 and 5.
Perform a half-media change, replenish IL-21, and maintain T cells
at a concentration of 10.sup.6 cells/mL.
[0193] End of First aAPC-mediated Stimulation Cycle on Day 7.
Harvest cells, count, and stain for CD3, CD4, CD8, and Fc.gamma.
(CAR).
[0194] Depletion of CD56.sup.+ Cells between 7 and 14 days after
electroporation. Perform a CD56 depletion using paramagnetic beads
if CD56.sup.-CD3.sup.neg lymphocytes.gtoreq.10%.
[0195] Recursive Addition of aAPC to Propagate T Cells to
Clinically-sufficient Numbers During Stimulation Cycles #2, #3,
& #4 Corresponding to Days 8.fwdarw.14, Days 15.fwdarw.21,
& Days 22.fwdarw.28. Repeat the stimulation process up to 4
times. Add IL-2 (50 U/mL) to the cultures beginning on Days 7, 14
and 21, and then at each media change (three times a week, on a
Monday-Wednesday-Friday schedule). Cryopreserve (archive) excess T
cells as needed using a controlled rate freezer for release testing
and infusion.
Example 2
Clinical Application of Sleeping Beauty and Artificial Antigen
Presenting Cells to Genetically Modify T Cells from Peripheral and
Umbilical Cord Blood--Results
[0196] Electro-transfer of DNA plasmids and propagation of T cells
on .gamma.-irradiated aAPC can be used to generate
clinically-appealing numbers of T cells derived from PB and UCB for
human applications. These genetically modified T cells express an
introduced CAR that recognizes the TAA CD19, independent of major
histocompatibility complex. The SB-derived DNA plasmids to express
the (i) transposon, a 2.sup.nd generation CAR (CD19RCD28) that
signals through CD28 and CD3-.epsilon..sup.14, and (ii)
transposase, SB11 (Jin et al., 2011), have been previously
described (Singh et al., 2011; Davies et al., 2010; Kebriaei et
al., 2012). The plasmids used in the current study were produced
commercially by Waisman Clinical Biomanufacturing Facility
(Madison, Wis.). The aAPC (clone #4), derived from K562 cells
(parental line obtained from American Type Culture Collection),
co-express desired T cell co-stimulatory molecules (each introduced
molecule at 90% on cell surface of aAPC), as previously described
(Manuri et al., 2010). Here, CD19-specific T cells could be
generated from mononuclear cells (MNC) derived from PB or UCB using
SB transposition to introduce the CAR followed by addition of aAPC
to numerically expand the T cells in a CAR-dependent manner (FIGS.
1 and 4) (Singh et al., 2011; Singh et al., 2008). Ten cuvettes
(2.times.10.sup.7 MNC/cuvette) were electroporated for each
recipient using 15 .mu.g of DNA plasmid (CD19RCD28/pSBSO) coding
for transposon (CAR) and 5 .mu.g of DNA plasmid (pCMV-SB11) coding
for transposase (SB11). The number of cuvettes can be reduced if
MNC are limiting or scaled back for laboratory work. The day of
electroporation is defined as "Day 0" of Stimulation cycle #1. As
controls for flow cytometry and culture conditions, autologous T
cells are mock electroporated (without DNA plasmid) and numerically
expanded on .gamma.-irradiated aAPC (clone #4) that had been
pre-loaded with OKT3 to cross-link CD3 to sustain T cell
proliferation. The efficiency of electrotransfer and viability of
the T cells the day after electroporation was routinely assessed
(FIG. 2B). The expression of EGFP from control DNA plasmid
(designated pmaxGFP) and CAR at this initial time point reflects
protein expression from the integrated and episomal plasmid.
Typically, EGFP expression was measured at 60% the day after
electroporation and CAR expression at .about.40% (FIG. 2A) with T
cell viability between 40%-50%. Recursive additions of
.gamma.-irradiated aAPC in the presence of soluble recombinant
human IL-2 and IL-21 retrieve T cells stably expressing CAR
(CD19RCD28). CD3.sup.negCD56.sup.+ NK cells are depleted from the
culture using CD56-specific paramagnetic beads if the percentage of
these NK cells is .gtoreq.10% and especially if the percentage of
CAR expressed on the T cells is low. This depletion prevents the
rapid overgrowth of NK cells which interferes with the ability of
aAPC to sustain the proliferation of CAR.sup.+ T cells. On
occasion, depletion of NK cells from CAR.sup.+ T cells is
undertaken during the last two stimulation cycles, but this
introduces a loss of desired cells due to co-expression of CD56 on
some CAR.sup.+ T cells. The T cells were grown in a functionally
closed system using VueLife culture bags past Day 14. A subset of
the genetically modified and propagated T cells are typically
cryopreserved at Day 14 or Day 21 (end of Stimulation cycles #2 or
#3) of co-culture on aAPC to serve as a source of archived material
for future analyses and to be thawed if unanticipated problems
subsequently occur during the manufacturing process. T cells are
typically harvested on or about Day 28 of culture (FIG. 3) that
routinely express >90% CAR and are >80% viable (FIGS. 2C, D).
After four weeks of co-culture on aAPC, the average fold-expansion
of CD3.sup.+ T cells is 19,800.+-.11,313 with CAR.sup.+ expression
being 90%.+-.7.5% (Singh et al., 2011). These T cells are
cryopreserved and undergo in-process and release testing that
informs on the safety and therapeutic potential of the manufactured
product. Release testing is undertaken in compliance with clinical
laboratory improvement amendments (CLIA) to generate a certificate
of analysis prior to infusion into recipients on clinical
trials.
Example 3
Manufacture of Clinical-Grade CD19-Specific T Cells Stably
Expressing Chimeric Antigen Receptor Using Sleeping Beauty and
Artificial Antigen Presenting Cells--Materials and Methods
[0197] Generation of clinical-grade DNA plasmids. The SB
transposon, .sub.CoOpCD19RCD28pSBSO, expresses the human codon
optimized (CoOp) 2.sup.nd generation .sub.CoOpCD19RCD28 CAR (SEQ ID
NO: 1) under EF-1/HTLV hybrid composite promoter (InvivoGen)
comprised of Elongation Factor-1.alpha. (EF-1.alpha.) (Kim et al.,
1990) and 5' untranslated region of the Human T-Cell Leukemia Virus
(HTLV) (Singh et al., 2011; Davies et al., 2010). The derivation of
this DNA plasmid is described in FIG. 10. The SB transposase, SB11,
under the cytomegalovirus (CMV) promoter was expressed in cis from
the DNA plasmid pCMV-SB11 (Singh et al., 2011). The derivation of
this DNA plasmid is described in FIG. 11. Both plasmids were
sequenced in their entirety and manufactured by Waisman Clinical
Biomanufacturing Facility (Madison, Wis.) using kanamycin for
selection of the bacterial strain E. coli DH5.alpha.. The release
criteria for the DNA plasmids are shown in Table 1. CD19 was
expressed using the DNA plasmid .DELTA.CD19.sub.CoOp-F2A-Neo/pSBSO
(FIG. 12).
TABLE-US-00001 TABLE 1 Release criteria for DNA plasmids coding for
SB transposon and transposase. Test Specification Appearance Clear
colorless liquid Restriction Mapping & Digest with Nde I;
AflIII & NheI; HindIII Agarose Gel & AvrII; Xba & NcoI
for transposon. Digest with NdeI; XhoI & PvuII; AflIII; NcoI
for transposase Sequencing Sequence conforms to original coding
Concentration via 2.0 .+-. 0.2 mg/mL absorbance A260/A280
absorbance 1.8-2.0 ratio Kinetic LAL test for <50 EU/mg
Bacterial Endotoxin Plasmid Form (% >90% supercoiled supercoiled
via HPLC) Sterility Test No growth observed E. coli host protein
via <0.3% ELISA E. coli RNA via HPLC <10%
[0198] Cell counting. Trypan-blue exclusion was used to distinguish
live from dead cells and counted using Cellometer (Nexecelom
Bioscience) (Singh et al., 2011).
[0199] Isolation of PBMC. Leukapheresis products from two male
volunteer healthy donors were purchased from Key Biologics LLC
(Memphis, Tenn.). The peripheral blood mononuclear cells (PBMC)
were isolated by adapting the Biosafe Sepax system (Eysins,
Switzerland) for work in compliance with cGMP. Briefly, after
closing all the clamps on the CS-900 kit, 100 mL Ficoll (GE
Healthcare) was aseptically transferred via 60 mL syringes to a
density gradient media bag ("ficoll bag") via Luer-lock connector
and the tubing was heat sealed using a hand held sealer (Sebra,
Model #2380). The kit was spike-connected to a 1,000 mL bag
containing CliniMACS buffer (PBS/EDTA, Miltenyi, Cat #70026) with
20 mL 25% Human Serum Albumin (HSA) (Baxter) (2% v/v, wash buffer)
for washes, a final product bag [300 mL Transfer Pack with Coupler
(Baxter/Fenwal 4R2014)] and a reagent/blood bag. Using the density
gradient-based separation protocol (v126), the syringe piston was
loaded into the centrifuge chamber and the cover of the Sepax unit
closed. The reagent/blood bag and product bags were hung and all
stopcocks were seated on the rotary pins in the `T` position. After
connecting the pressure-sensor line, the kit was validated by
automatic single-use test and then manually primed using gravity
flow. After completion of the cycle, the final product was
aseptically transferred into a centrifuge tube and washed once each
with wash buffer and phosphate buffered saline (PBS) at 400 g for
10 minutes. After counting, cells were cryopreserved using
cryopreservation media (50% HSA, 40% Plasmalyte, 10% DMSO) in
CryoMACS Freeze bags (Miltenyi) and vials (Nunc) using BM5 program
(4.degree. C. to -4.degree. C. at rate -2.degree. C./min,
-4.degree. C. to -60.degree. C. at rate -35.degree. C./min,
-60.degree. C. to -20.degree. C. at rate 8.degree. C./min,
-20.degree. C. to -45.degree. C. at rate -2.5.degree. C./min,
-45.degree. C. to -80.degree. C. at rate -10.degree. C./min) in a
controlled-rate freezer (Planer Kryo 750).
[0200] Manufacture of aAPC (clone #4) master and working cell
banks. K562 were transduced by lentivirus at the University of
Pennsylvania to generate aAPC (clone #4, designated
CJK64.86.41BBL.GFP.IL-15.CD19) that co-express (i) CD19, (ii) CD64,
(iii) CD86, (iv) CD137L, and (v) membrane bound IL-15 (mIL15) as a
bi-cistronic vector with
[0201] EGFP. The aAPC were numerically expanded in HyQ RPMI 1640
(Hyclone) containing 10% heat-inactivated defined FBS (Hyclone) and
2 mM Glutamax-1 (Life Technologies-Invitrogen) culture media (CM)
maintaining the cells at 5.times.10.sup.5 cells/mL. A master cell
bank (MCB) of 320 vials was produced through Production Assistance
of Cellular Therapies (PACT) (Table 2). A 200 vial working cell
bank (WCB) of Clone 4 aAPC derived from the MCB was then generated
at MDACC and tested (Table 3).
TABLE-US-00002 TABLE 2 Release criteria for K562-derived aAPC
(clone #4) master cell bank. Test Specification
Replication-competent Lentivirus Negative Product Enhanced Reverse
Transcriptase Negative for Detection of Retrovirus Endotoxin LAL
Negative Agar Cultivable and Non-Agar Negative Cultivable
Mycoplasma HIV-1/2 Proviral DNA by PCR Negative HBV DNA by PCR
Negative HCV RNA by RT-PCR Negative CMV DNA by PCR Negative
Parvovirus B19 Negative HTLV-I/II Proviral DNA by PCR Negative EBV
DNA by PCR Negative HHV-6 DNA by PCR Negative HHV-7 DNA by PCR
Negative HHV-8 DNA by PCR Negative Adeno-associated Virus Negative
In vivo Inapparent Virus Negative Negative Bovine Virus by 9CFR
Negative Porcine Virus by modified 9CFR PT-1 Negative
Adeno-associated Virus Negative Isoenzyme analysis Human Origin
Morphology by Transmission Electron No identifiable virus-like
Microscopy particles nor any other microbial agents
Immunophenotyping: CD19 90% CD64 90% CD86 90% CD137L 90% EGFP-mIL15
90% Bacteriostasis & Fungistasis Negative Sterility by
21CFR610.2 Negative
TABLE-US-00003 TABLE 3 Release criteria for K562-derived aAPC
(clone #4) working cell bank. TEST LABORATORY SPECIFICATION
Bacteriostasis and Fungistasis AppTec Negative Laboratory Services
Sterility by 21CFR610.12 AppTec Negative Laboratory Services Agar
Cultivable and Non-Agar BioReliance Negative Cultivable Mycoplasma
In vitro Adventitious Virus BioReliance Negative testing Identity
Isoenzyme analysis BioReliance Human Origin Phenotype MDACC GMP
.gtoreq.80% GFP.sup.+ Flow Cytometry .gtoreq.80% CD19.sup.+
Laboratory .gtoreq.80% CD86.sup.+ .gtoreq.80% CD137L.sup.+
.gtoreq.80% CD64.sup.+ .gtoreq.80% CD32.sup.+ Endotoxin (LAL)
EndoSafe MDACC GMP .ltoreq.5 EU/mL Quality Control Laboratory
Viability by Trypan Blue MDACC GMP .gtoreq.60% Quality Control
Laboratory
[0202] aAPC (clone #4) to selectively propagate CAR.sup.+ T cells.
The .gamma.-irradiated aAPC were used to numerically expand the
genetically modified T cells. Thawed aAPC from WCB were propagated
in CM for up to 60 days in VueLife cell culture bags and harvested
using Biosafe Sepax II harvest procedure. Briefly, a CS-490.1 kit
was connected to a 300 mL output bag (transfer pack) via Luer lock
connection. The separation chamber was installed in the pit and the
tubing was inserted into the optical sensor and stopcocks aligned
in the T position. After connecting the pressure sensor line, the
product bag and supernatant/plasma bags were hung on the holder.
The modified protocol PBSCv302 was selected from the Sepax menu and
the volume of input product to be processed (initial volume) was
set to .ltoreq.840 mL. After validation and kit test, the procedure
was started. Following completion, the bags were removed, clamps
closed and the kit was removed. The cells from the final product
bag were aseptically removed, washed twice with wash media (10% HSA
in Plasmalyte) and counted. aAPC were irradiated (100 Gy) using a
CIS BIO International radiator (IBL-437 C#09433) and cryopreserved
for later use in cryopreservation media using a controlled-rate
freezer (Planer Kryo 750).
[0203] OKT3-loading of aAPC. The OKT3-loaded (via CD64) aAPC
(clone#4) were used to propagate control (CAR.sup.neg) autologous
control T cells that had not undergone genetic modification. The
aAPC, obtained from culture, were incubated overnight in serum-free
X-Vivo 15 (cat #04-744Q, Lonza) containing 0.2% acetyl cysteine
(Acetadote, Cumberland Pharmaceuticals) termed Loading Medium (LM).
The next day cells were washed, irradiated (100 Gy) using a Gamma
Cell 1000 Elite Cs-137 radiator (MDS Nordion), resuspended in LM at
a concentration of 10.sup.6 cells/mL along with 1 .mu.g/10.sup.6
cells of functional grade purified anti-human CD3 (clone-OKT3,
16-0037-85, eBioscience) and incubated with gentle agitation on a
3-D rotator (Lab-Line) at 4.degree. C. for 30 minutes. Following
three washes with LM the cells were used in experiments or frozen
in aliquots in liquid nitrogen in the vapor layer for later
use.
[0204] Manufacture of CAR.sup.+ T cells. Thawed PBMC were
resuspended in (i) Human T-cell kit (cat #VPA-1002, Lonza; 100
.mu.L for 2.times.10.sup.7 cells in one cuvette), with (ii) the DNA
plasmid (coopCD19RCD28/pSBSO) coding for CD19RCD28 CAR transposon
(15 .mu.g supercoiled DNA per 2.times.10.sup.7 PBMC per cuvette),
and (iii) the DNA plasmid (pCMV-SB11) coding for SB11 transposase
(5 .mu.g supercoiled DNA per 2.times.10.sup.7 PBMC per cuvette).
This mixture was immediately transferred to a cuvette (Lonza),
electroporated (defining culture day 0) using Nucleofector II
(Amaxa/Lonza), rested in 10% RPMI complete media for 2 to 3 hours,
and after a half-media change, incubated overnight at 37.degree.
C., 5% CO.sub.2. The following day, cells were harvested, counted,
phenotyped by flow cytometry, and co-cultured with
.gamma.-irradiated aAPC at a ratio of 1:2 (CAR.sup.+ T cell:aAPC),
which marked culture day 1 and the beginning of a 7-day stimulation
cycle. IL-21 (cat #AF-200-21, PeproTech) and IL-2 (cat #NDC
65483-116-07, Novartis) were added on a Monday-Wednesday-Friday
schedule onwards of day 1 and day 7, respectively. NK cells can
prevent the numeric expansion of CAR.sup.+ T cells, especially if
their overgrowth occurs early in the tissue culturing process.
Therefore, CD56-depletion was performed if CD3.sup.negCD56.sup.+
cells.gtoreq.10% using CD56 beads (cat #70206, Miltenyi Biotech, 20
.mu.L beads/10.sup.7 cells) on LS columns (cat #130-042-401,
Miltenyi Biotech) in CliniMACS buffer containing 25% HSA (80
.mu.L/10.sup.7 cells). T cells were cryopreserved as backup on
culture day 21 after electroporation and the end of the 3.sup.rd
stimulation cycle using a controlled-rate freezer (Planer Kryo 750)
as described above, and stored in liquid nitrogen (vapor-layer).
The cell counts for total, CD3.sup.+, and CAR.sup.+ T cells were
plotted over time and slopes determined using linear regression.
The fold-expansion results were compared using a Student's t-test.
CD4/CD8 ratios were calculated for each time point and validation
runs and averaged.
[0205] Generation of CAR.sup.neg control T cells. As a control,
5.times.10.sup.6 mock transfected PBMC were co-cultured with
irradiated and anti-CD3 (OKT3) loaded K562-derived aAPC clone #4 at
a ratio of 1:1 in a 7-day stimulation cycle. All the cultures were
supplemented with IL-21 (30 ng/mL) from culture day 1 onwards, and
IL-2 (50 U/mL) starting 7 days after the start of the culture. All
cytokines were subsequently added on a Monday-Wednesday-Friday
schedule.
[0206] Cell lines. CD19.sup.+ Daudif.beta..sub.2m [Burkitt
lymphoma, co-expressing .beta..sub.2 microglobulin, (Rabinovich et
al., 2008)] and CD19.sup.+ NALM-6 (pre-B cell) were cultured as
described previously (Singh et al., 2011). EL-4 cells (mouse T-cell
lymphoma line) from ATCC were modified to express CD19 using the
construct .DELTA.CD19coop-F2A-Neo/pSBSO. Briefly, 5.times.10.sup.6
EL-4 cells were resuspended in 100 .mu.L of Amaxa Mouse T cell
Nucleofector kit (Catalogue #VPA-1006) with SB transposon
(.DELTA.CD19.sub.coOp-F2A-Neo/pSBSO, 3 .mu.g) and SB transposase
(pCMV-SB11, 1 .mu.g) and electroporated (program X-001) using
Nucleofector II (Lonza). The transfectants were cultured in a
cytocidal concentration of G418 (0.8 mg/mL) and underwent
fluorescent activated cell sorting (FACS) for homogeneous
expression of CD19 to obtain a clone (clone #17). Jurkat cell were
obtained from ATCC and electroporated (Program T-14, Nucleofector
II, Lonza) with .sub.coOpCD19RCD28mz (CoOp)/pSBSO using the
Amaxa/Lonza Nucleofector solution (Kit V). Two weeks after
electroporation, Jurkat cells stably expressing CAR underwent FACS
for homogeneous expression of CAR to obtain a clone (clone #12)
(Maiti et al., 2013). Cell lines were maintained in HyQ RPMI 1640
(Hyclone) supplemented with 2 mM Glutamax-1 (Invitrogen) and 10%
heat-inactivated Fetal Calf Serum (FCS) (Hyclone; 10% RPMI). All
cell lines were validated using STR profiling or karyotyping
according to institutional cell line authentication policy.
[0207] Immunophenotype of cells. Cells were stained using
antibodies (Table 4) in 100 .mu.L FACS Buffer (2% FBS, 0.1% Sodium
Azide) for 30 minutes at 4.degree. C. For intracellular staining,
after fixing/permeabilization for 20 minutes, cells were stained in
perm wash buffer with appropriate antibodies for 30 minutes at
4.degree. C. Acquisition was performed using FACSCalibur (BD
Bioscience) and analyzed using Cell Quest BD Bioscience) or FCS
Express 3.00.0612 (De Novo Software, Thornhill, Ontario,
Canada).
TABLE-US-00004 TABLE 4 Antibodies used for flow cytometry.
Catalogue Cells Antibody/Fluorochrome Vendor No. T cells CD3-PE BD
Biosciences 347347 CD4-APC BD Biosciences 340443 CD8-PerCPCy5.5 BD
Biosciences 341051 CD16-PE BD Biosciences 347616 CD25-APC BD
Biosciences 555434 CD28-PerCPCy5.5 BD Biosciences 337181 CD32-FITC
BD Biosciences 555448 CD39-FITC eBioscience 11-0399-42 CD45TA-APC
BD Biosciences 550855 CD45RO-PE BD Biosciences 555489 CD57-FITC BD
Biosciences 555619 CD56-APC BD Biosciences 555518 CD62L-PE BD
Biosciences 555544 CD69-PE BD Biosciences 555531 CD127
(IL-7Ra)-Alexa BD Biosciences 558598 Fluor 647 CD150-PE BD
Biosciences 559592 CD279 (PD-1)-PE BD Biosciences 557946
CCR7-PerCPCy5.5 BioLegend 335605 Granzyme B-Alexa BD Biosciences
560212 Fluor 647 HLA-DR-PerCPCy5.5 BD Biosciences 339205
Perforin-FITC BD Biosciences 556577 Anti-human Fc.gamma.-PE
Invitrogen H10104 K562 aAPC CD19-PE BD Biosciences 555413 (Clone
#4) CD19-APC BD Biosciences 555415 CD64-PE BD Biosciences 558592
CD86-PE BD Biosciences 555658 CD137L-PE BD Biosciences 559446
F(ab')2 fragment of Jackson 115-116-072 Goat anti-Mouse IgG,
Immunoresearch F(ab')2 fragment specific-PE (For OKT3 loading)
[0208] Western Blot. Protein expression of the chimeric CD3-.zeta.
(73-kDa) derived from CD19RCD28 was assessed as described
previously (Singh et al., 2008). Briefly, protein lysates were
transferred using iBlot Dry Blotting System (Invitrogen) onto
nitrocellulose membrane, incubated with mouse anti-human CD3-.zeta.
monoclonal antibody (cat #551033, 0.5 .mu.g/mL, BD Biosciences, CA)
followed by horseradish peroxidase (HRP)-conjugated goat anti-mouse
IgG (cat #1858413, 1:10,000; Pierce, Ill.), developed using
SuperSignal West Femto Maximum Sensitivity substrate (Pierce, Ill.)
and chemiluminescence captured using VersaDoc.TM. 4000 gel
documentation system (BioRad, CA).
[0209] Telomere Length Analysis by Fluorescence in situ
Hybridization and Flow Cytometry (Flow-FISH). Telomere length of
the T cells was measured by using the Telomere PNA Kit/FITC for
Flow Cytometry (DAKO) according to the manufacturer's instructions.
Briefly, isolated cells (CD4 or CD8) and control cells (cat
#85112105, CEM-1301 cell line; ECACC) were mixed in equal measure
in hybridization solution with or without FITC-labeled telomere PNA
probe for 10 minutes at 82.degree. C.; hybridized overnight in the
dark at room temperature; washed twice with a wash solution at
40.degree. C.; resuspended in PBS containing 2% FCS and propidium
iodide (1 .mu.g/mL); and analyzed on a FACSCalibur (BD
Biosciences). FITC-labeled fluorescent calibration beads (cat
#824A, Quantum.TM. FITC MESF, Bangs Laboratories) were used for
calibration of the flow cytometry machine. Relative telomere length
(RTL) was determined by comparing T cells with a CEM-1301 cell line
per:
R T L = ( Mean FL 1 sample cells with probe - Mean FL 1 sample
cells without probe ) .times. 2 .times. 100 Mean FL 1 reference
cells with probe - Mean FL 1 reference cells without probe
##EQU00001##
[0210] Chromium Release Assay. T cells were evaluated for their
cytotoxicity in a standard 4-hour chromium release assay using
.sup.51Cr-labeled target cells. T cells were plated in triplicate
at 1.times.10.sup.5, 0.5.times.10.sup.5, 0.25.times.10.sup.5,
0.125.times.10.sup.5 and 0.0625.times.10.sup.5 cells/well with
5.times.10.sup.3 target cells in a 96-well V-bottom plate (Costar).
After incubation, 50 .mu.L of supernatant was harvested onto a
LumaPlate (Perkin Elmer), read in TopCount NXT (Perkin Elmer), and
percent specific lysis was calculated per:
Experimental 51 Cr released - Spontaneous 51 Cr released Maximum 51
Cr released - Spontaneous 51 Cr released .times. 100
##EQU00002##
Spontaneous and maximum release was determined by measuring
chromium in the conditioned supernatant from target cells incubated
with CM or 0.1% Triton X-100 (Sigma), respectively.
[0211] Endotoxin Testing. Endotoxin level in final products was
determined using Endosafe.RTM.-PTS Portable Test System (Charles
River Laboratories) as per the manufacturer's guidelines. The test
has a detection limit of 0.01-10 EU/mL, which can be converted to
EU/patient weight.
[0212] Mycoplasma testing. Mycoplasma detection by PCR was
performed using the TaKaRa Mycoplasma Detection Kit (Clontech)
according to manufacturer's instructions.
[0213] T-cell receptor V.beta. repertoire. T-cell receptor
(TCR)-V.beta. usage of culture day 28 and day 35 CAR.sup.+ T cells
was determined using a panel of 24 TCR V.beta.-specific mAbs (cat
#IM3497, IO TEST Beta Mark TCR-V.beta. repertoire kit, Beckman
Coulter) used in association with CD3-specific mAb (cat #340949, BD
Biosciences, 10 .mu.L) and isotype-matched control mAbs (cat
#552834, BD Biosciences).
[0214] Real-time PCR to determine copy number of integrated CAR. To
determine the copy number of integrated CD19RCD28 CAR in
genetically modified T cells, 50-100 ng genomic DNA (cat #80204,
AllPrep DNA/RNA Mini Kit, Qiagen) was amplified using Steponeplus
real-time PCR system (Applied Biosystems) in a PCR reaction (2 min
at 50.degree. C., 10 min at 95.degree. C., followed by 40 cycles of
15 sec at 95.degree. C. and 1 min at 60.degree. C.) using the
following primers: forward (5'-CAGCGACGGCAGCTTCTT-3'; SEQ ID NO:
8), reverse (5'-TGCATCACGGAGCTAAA-3'; SEQ ID NO: 9) and probe
(5'-AGAGCCGGTGGCAGG-3'; SEQ ID NO: 10). Primers (Cat #4316844,
Applied Biosystems) for RNAse P gene was used as an internal
control. Serially-diluted genomic DNA from a genetically modified
Jurkat-cell (clone #12) containing 1 copy of CAR from CD19RCD28mz
(CoOp)/pSBSO DNA plasmid was used to generate a standard curve
(Maiti et al., 2013). All the primers, probes and TaqMan Gene
Expression Master Mix were purchased from Applied Biosystems.
[0215] PCR for SB11 transposase. DNA (20 ng) (AllPrep DNA/RNA Mini
Kit, Qiagen) isolated from CAR.sup.+ T cells was amplified using a
thermal cycler (PTC-200, DNA Engine, BioRad) using forward
(5'-ATGGGAAAATCAAAAGAAATC-3'; SEQ ID NO: 11) and reverse
(5'-CTAGTATTTGGTAGCATTGC-3'; SEQ ID NO: 12) primers in a PCR
reaction (95.degree. C. for 5 min; 25 cycles of 95.degree. C. for
15 sec, 58.degree. C. for 40sec, 72.degree. C. for 60 sec; followed
by a final extension at 72.degree. C. for 7 min). GAPDH was used as
the housekeeping gene and was amplified in the same PCR reaction
using the primers, forward (5'-TCTCCAGAACATCATCCCTGCCAC-3'; SEQ ID
NO: 13) and reverse (5'-TGGGCCATGAGGTCCACCACCCTG-3'; SEQ ID NO:
14). Linearized pCMV-SB11 plasmid DNA (1 ng) and genomic DNA (20
ng) from genetically modified Jurkat cells stably expressing SB11
and EGFP (expressed from DNA plasmid SB11-IRES2-EGFP) (Maiti et
al., 2013) were used as a positive control. Mock electroporated (no
DNA) and OKT3-aAPC-propagated T cells were used as a negative
control.
[0216] Assay to assess for unwanted autonomous T-cell growth. To
monitor aberrant T-cell growth, 2.times.10.sup.5 CAR.sup.+ T cells,
harvested after 4 aAPC-mediated stimulation cycles (28 days after
electroporation) were cultured in triplicate in a 24-well tissue
culture plate for an additional 18 days. (i) Positive control: the
presence of aAPC and cytokines (50 U/mL IL-2 and 30 ng/mL IL-21).
(ii) Test: the absence of aAPC and cytokines. The genetically
modified T cells passed the assay when total viable cells at day 18
were (i) >2.times.10.sup.5 cells for CAR.sup.+ T cells cultured
with aAPC and cytokines and (ii) <2.times.10.sup.4 cells for
CAR.sup.+ T cells cultured without aAPC and cytokines.
[0217] G-band karyotyping. CAR.sup.+ T cells at the end of
co-culture were harvested and the slides were stained using Giemsa
stain using standard procedure. A total of 20 G-banded metaphases
were analyzed.
Example 4
Manufacture of Clinical-Grade CD19-Specific T Cells Stably
Expressing Chimeric Antigen Receptor Using Sleeping Beauty and
Artificial Antigen Presenting Cells--Results
[0218] aAPC (clone #4). K562 functioning as aAPC (clone #4) were
employed to selectively propagate CAR.sup.+ T cells. The cultured
aAPC were harvested from VueLife bags by a Sepax II device using
the volume reduction procedure, which took .about.40 minutes. The
mean preprocessing volume and aAPC counts were 575 mL (range
500-700mL) and 4.9.times.10.sup.8 (range 3.2.times.10.sup.8 to
7.7.times.10.sup.8), respectively. After processing with Sepax II,
mean recovery of cells was 108% (range 75% to 136%), with an output
volume of 125 mL (range 50-200 mL) resulting in mean volume
reduction of 78.3% (range 60% to 91.6%, FIGS. 5A, B). The automated
cell recovery was similar to a manual volume reduction procedure
(82%), which took 45 minutes of sustained operator time and
resulted in 91% volume reduction. aAPC were regularly monitored by
flow cytometry for >80% expression of the introduced transgenes
coding for CD19, CD64, CD86, CD137L, and EGFP (as a marker for
expression of mIL15). The immunophenotyping was undertaken upon
generating the MCB and WCB and upon each addition of
.gamma.-irradiated aAPC to T-cell cultures (that marked the
beginning of each stimulation cycle, FIG. 5C). MCB and WCB for
Clone 4 aAPC tested negative for sterility and mycoplasma on cells
and cell supernatant. In the biosafety testing, no virus was
detected by adventitious virus testing, replication competent
retrovirus testing, and screening for a range of human pathogenic
viruses. Testing validated that the aAPC (clone #4) was derived
from K562 based on finger printing (Table 5).
TABLE-US-00005 TABLE 5 STR fingerprinting of K562 aAPC (Clone #4).
STR K562 aAPC (Clone 4) AMEL X CSF1PO 9, 10 D13S317 8 D16S539 11,
12 D18S51 15, 16 D19S433 14, 14.2 D21S11 29, 30, 31 D2S1338 17
D3S1358 16 D5S818 11 D7S820 9, 11 D8S1179 12 FGA 21 TH01 9.3 TPOX
8, 9 vWA 16
[0219] Manufacture of CAR.sup.+ T cells. Validation studies were
undertaken for large scale production of CD19-specific CAR.sup.+ T
cells to establish that PBMC can be electroporated and propagated
to clinically meaningful numbers (Huls et al., 2013) (FIG. 6) and
meet pre-established release criteria (Table 6). Two normal donor
apheresis products were processed to isolate mononuclear cells
(MNC) using the Sepax cell-processing system. The apheresis
products 201 mL (donor 1) and 202 mL (donor 2) were processed in
two batches (-100 mL/batch) generating a 50 mL output product. The
preprocessing counts were similar to post-processing counts of the
apheresis products. A total of 5.3.times.10.sup.9 and
7.1.times.10.sup.9 cells were isolated containing 40.5% and 51%
CD3.sup.+ T cells respectively from donor 1 and donor 2. Cells were
then cryopreserved in aliquots (4.times.10.sup.7 cells/mL) in
CryoMACS freeze bags (10 mL) and reference cryovials (1 mL) for
later use. Three separate validation experiments were performed and
are summarized in Table 6. For validation run 1 and 2 (V1, V2)
cells from donor 1 and for validation 3 (V3) cells from donor 2
were used. For each run freshly-thawed PBMC were electroporated and
ex vivo numerically expanded in separate culturing procedures
(Table 7). On culture day 0, 3.times.10.sup.8 (V1) and
8.times.10.sup.8 (V2, V3) cells were thawed (viability, 88.9% to
97.6%) and rested for 2 hours prior to electroporation. 2 to
3.times.10.sup.8 cells (V1=2.times.10.sup.8; V2 and
V3=3.times.10.sup.8) were electroporated at 2.times.10.sup.7 cells
per cuvette with CD19RCD28mz(CoOp)/pSBSO transposon and pCMV-SB11
transposase DNA plasmids and the following day (culture day 1)
co-cultured with aAPC clone #4 based on CAR expression. The
electroporation efficiency for the three validation runs was
assessed on culture day 1 as measured by expression of CAR (33.7%,
25.5% and 47.1%). CAR expression at the end of co-culture (culture
day 28) was 92%, 99.2%, and 96.7%, and the cultures contained mean
95%.+-.5.3% CD3.sup.+ T cells with negligible amounts of
contaminating CD19.sup.+ cells (mean=0.7%.+-.0.15%) and CD32.sup.+
aAPC (mean=0.6%.+-.0.6%, FIG. 7A, Table 6). The inventors further
confirmed CAR expression by Western blot of whole-cell lysates of
electroporated/propagated T cells using CD3-.zeta. chain-specific
mAb revealing an expected 73-kDa chimeric .zeta. chain (Singh et
al., 2008) (FIG. 7B). Upon inspection of the kinetics of T-cell
growth on aAPC, the inventors observed an accelerated rate of
T-cell propagation at the end of the second week of culture (end of
stimulation cycle 2), which is consistent with increased
fold-expansion of total (p=0.01) and CD3.sup.+ (p=0.01) T cells as
compared to the fold-expansion in the first week of stimulation.
The weekly fold-expansion at the end of the third and fourth week
for total (p=0.01, p<0.001), CD3.sup.+ (p=0.03, p<0.001),
CAR.sup.+ (p=0.02, p<0.001) T cells was consistently higher than
that of week-one, respectively (FIG. 13).
TABLE-US-00006 TABLE 6 Acceptance criteria for releasing
electroporated and propagated T cells Results Validation Validation
Validation Test Specification Run #1 Run #2 Run #3 Sterility-
Negative (No No growth at No growth at No growth at Bacteria and
growth at 14 14 days 14 days 14 days Fungi days) Mycoplasma
Negative by PCR Negative Negative Negative Visual No evidence of No
evidence of No evidence of No evidence of Inspection contamination
contamination contamination contamination Viability .gtoreq.70% 99%
99% 99% (Trypan Blue or 7AAD) Gated .gtoreq.80% CD3.sup.+ 89%
CD3.sup.+ 97% CD3.sup.+ 99% CD3.sup.+ Immuno- .gtoreq.10% CAR.sup.+
92% CAR.sup.+ 99% CAR.sup.+ 96% CAR.sup.+ phenotyping <5%
CD32.sup.+ 1.3% CD32.sup.+ 0.04% CD32.sup.+ 0.51% CD32.sup.+ <5%
CD19.sup.+ 0.75% CD19.sup.+ 0.49% CD19.sup.+ 0.78% CD19.sup.+
Endotoxin Endotoxin lebel <0.004 EU/mL <0.667 EU/mL <0.089
EU/mL LAL <5 EU/recipient weight (for validation, per mL) Screen
for <2 .times. 10.sup.4 cells/mL <2 .times. 10.sup.4 cells/mL
<2 .times. 10.sup.4 cells/mL <2 .times. 10.sup.4 cells/mL
Unwanted for the cells without Autonomous cytokines or aAPC Growth
at Day 18
TABLE-US-00007 TABLE 7 Characterization of T cells before and after
co-culture on .gamma.-irradiated aAPC. Aphersis Day 1 after
electroporation Day 28 of co-cul CD4: CD3.sup.+ CD3.sup.+ CD4:
CD3.sup.+ CD4.sup.+ CD8.sup.+ CD4: CD3.sup.+ CD4.sup.+ Expt. CD3
CD8 CD4.sup.+ CD8.sup.+ CD3 CD8 CAR CAR.sup.+ CAR.sup.+ CAR.sup.+
CD3 CD8 CAR CAR.sup.+ CAR.sup.+ V1 40.5 1.65 18.5 11.2 64.7 2.4
33.7 41.0 28.0 11.1 88.9 0.02 92.0 97.7 2.46 V2 59.8 4.1 25.5 17.1
25.9 3.8 97.1 0.01 99.2 97.4 1.77 V3 51.2 1.59 29.1 18.3 87.7 2.3
47.1 45.0 20.1 31.5 99.2 0.8 96.0 96.0 43.4 * Total (inferred)
viable cells indicates data missing or illegible when filed
[0220] The inventors observed similar weekly fold-expansion for
CD3.sup.+ and CAR.sup.+ T cells past week one of stimulation. After
4 weeks of co-culture on .gamma.-irradiated aAPC there was an
average 545-fold numeric expansion of CD3.sup.+ T cells with a
1,111-fold expansion of CAR.sup.+ T cells. The ex vivo expansion
(culture day 28) resulted in an average 2.86.times.10.sup.10
CD3.sup.+ T cells, almost all of which were CAR.sup.+
(2.65.times.10.sup.10). The propagation kinetics of total (p=0.18),
CD3.sup.+ (p=0.17) and CAR.sup.+ (p=0.2) T cells for the three
validation runs were similar (FIGS. 8A, B, C). These data support
the recursive addition of aAPC for the selective outgrowth of
CD19-specific T cells.
[0221] Immunophenotype of electroporated and propagated CAR.sup.+ T
cells. Two of the three validation runs resulted in a preferential
growth of CD8.sup.+CAR.sup.+ T cells (mean 76%.+-.22%) as compared
to CD4.sup.+CAR.sup.+ T cells (mean 16%.+-.24%) (FIG. 7C) which was
predicted by inclusion of IL-21 (Singh et al., 2011). Mean CD4/CD8
ratios for CAR.sup.+ T cells modulated during the course of the
co-culture as there was a predominance of CD4.sup.+CAR.sup.+ T
cells at the start of the co-culture on aAPC (culture day 1,
ratio=3.3) leading to equal amounts of CD4.sup.+CAR.sup.+ and
CD8.sup.+CAR.sup.+ T cells at culture day 14 (ratio=0.9), after
which the ratio declined (culture day 21, ratio=0.4; culture day
28, ratio=0.3). The total CD4/CD8 ratio followed a similar trend
and declined over time (Table 7). The CAR.sup.+ T cells at the end
of the propagation period (culture day 28) were activated (CD69
expression, mean 43.6%; HLA-DR expression, mean 61.2%), capable of
cytolysis (Granzyme B expression, mean 92.3%) and expressed markers
of memory/naive T cells (CD62L expression, mean 45.6%; CD28
expression, mean 66.4%; CD27 expression, mean 77.5%, CD45RA
expression, mean 99.7%). The inventors were not able to detect
cell-surface markers of exhaustion/senescence (PD-1 expression,
mean 2.7%; CD57 expression, mean 3.3%) (FIG. 7D). These data are
consistent with the aAPC supporting the outgrowth of a
heterogeneous population of CAR.sup.+ T cells.
[0222] Redirected specificity of CAR.sup.+ T cells. T cells
generated from all three validation runs were able to specifically
lyse CD19.sup.+ tumor targets. An average of 57%.+-.4% (mean.+-.SD,
range, 61.2% to 53.8%) Daudi.beta..sub.2m and 49%.+-.7%
(mean.+-.SD, range, 41% to 54%) NALM-6 was lysed at an effector to
target ratio of 20:1. CD19-specific killing was demonstrated by
average 6.2.+-.2.6 (mean.+-.SD)-fold higher killing of CD19.sup.+
EL-4 (range, 4.2 to 9.2 fold) as compared to the CD19.sup.neg
parental EL-4 cells at effector/target ratio of 20:1 with a
1.4.+-.1 (mean SD)-fold background CD19-specific lysis by
CAR.sup.neg (mock electroporated) controls (FIGS. 8D, 14). This
implies that the CAR in the electroporated and propagated T cells
redirected killing to CD19.
[0223] Lack of unwanted autonomous proliferation by genetically
modified and propagated T cells. The inventors evaluated growth of
CAR.sup.+ T cells in the presence/absence of K562 aAPC and
cytokines (IL-2 and IL-21) to rule out aberrant T-cell growth due
to potential genotoxicity caused by SB transposition. At the end of
18 days of culture the inventors observed <2.times.10.sup.4
cells (average 2,800, range 0.0-5.6.times.10.sup.3) in the
genetically modified T cells receiving no cytokines and aAPC, while
the control group receiving cytokines and aAPC numerically expanded
to an average of 7.6.times.10.sup.7 cells, range
4.12.times.10.sup.7 to 12.8.times.10.sup.7 (Table 8). These data
indicate CAR.sup.+ T cells cannot sustain proliferation upon
withdrawal of growth factors and antigenic stimulation.
TABLE-US-00008 TABLE 8 Lack of autonomous cell growth by
genetically modified T cells. No. of T cells (day 18) % fold- No.
of Without With change Day of T cells cytokines cytokines of T
Experiment culture.sup.a seeded.sup.b and aAPC.sup.c and aAPC.sup.d
cells.sup.e V1 28 2 .times. 10.sup.5 0 12.80 .times. 10.sup.7
0.000% V2 28 2 .times. 10.sup.5 5.6 .times. 10.sup.3 5.88 .times.
10.sup.7 0.009% V3 28 2 .times. 10.sup.5 2.8 .times. 10.sup.3 4.12
.times. 10.sup.7 0.007% .sup.aDays of culture when T cells were
seeded .sup.bTotal number of T cells seeded in culture at the start
of the experiments .sup.cTotal number of T cells counted in the
absence of cytokines and aAPC .sup.dTotal (inferred) number of T
cells counted in the presence of cytokines and aAPC (positive
control) .sup.ePerfect fold-change = [(c/b) / (d/b)]*100
TABLE-US-00009 TABLE 9 In-process testing for electroporated and
propagated T cells. Expression Cell Surface CAR Expression Flow
Cytometry Total CAR Expression Western Blot Analysis Functionality
Cytotoxicity Chromium Release Assay Persistence Memory/Naive
Phenotype Flow Cytometry Telomere Length Flow-FISH Safety CAR Copy
Number Q-PCR SB11 Detection PCR TCR V.beta. Repertoire Flow
Cytometry Karyotyping G-banding
[0224] Telomere length in CAR.sup.+ T cells. Telomere length is an
important measure of cellular differentiation and progression to
senescence. Therefore to evaluate the effect of SB transposition
and ex vivo numeric expansion of CAR.sup.+ T cells on telomere
length, the inventors compared telomere lengths from CAR.sup.+ T
cells (culture day 28) to their respective matched unmanipulated
controls (prior to electroporation) using Flow-FISH assay. Due to
the generation of predominately CD8.sup.+CAR.sup.+ T cells in V1
and V2 and CD4.sup.+ CAR.sup.+ T cells in V3, the inventors
compared telomere lengths of CD8.sup.+T cells for V1 and V2 and
CD4.sup.+ T cells for V3 to CD8.sup.+ and CD4.sup.- T cells,
respectively, from prior to propagation (FIG. 9A). The average
telomere length of T cells after ex vivo numeric expansion (culture
day 28, 8.93%.+-.1.33%) was similar to that of unmanipulated
control T cells (day 0, 7.77%.+-.1.21%). These results indicate
that electroporation and propagation of CAR.sup.+ T cells does not
result in erosion of telomere lengths.
[0225] Copy number of CAR transgene. The copy number of integrated
CD19RCD28 transgenes was determined using CD19RCD28.sup.+ Jurkat
cell clone #12 as reference and endogenous RNase P as a normalizer
(Maiti et al., 2013). The average transgene copy per T cells
generated in the validation runs was 0.96.+-.0.09 (range, 0.75 to
1.07, FIG. 9B). These data indicate that SB transposition resulted
in approximately one integrated copy of CAR per T-cell genome.
[0226] TCR V.beta. usage. The electroporation and propagation of T
cells may lead to emergence of oligoclonal or clonal population of
T cells that could be indicative of preferential growth and thus an
indicator of a genotoxic event. Therefore, the inventors evaluated
TCR VP usage by flow cytometry as a means to assess repertoire
diversity. All 24 TCR families tested were preserved in T cells
after 28 days of co-culture on aAPC and similar to the
pre-electroporation repertoire. Further, the TCR VP families were
preserved upon prolong culture time (culture day 35, FIG. 9C).
These data suggest maintenance of a broad TCR diversity in cultured
CAR.sup.+ T cells and do not reveal an imbalance in the use of TCR
sequences.
[0227] Lack of SB11 transposase in CAR.sup.+ T cells. Continued
expression of SB11 transposase may lead to remobilization of the
integrated CAR transgene. Therefore, the inventors performed a
genomic PCR to rule out illegitimate homologous recombination of
the DNA plasmid coding for SB11 in CAR.sup.+ T cells. Within the
limits of the assay the inventors were unable to detect a band
(.about.1 kb) in PCR reactions containing DNA from CAR.sup.+ T
cells cultured for 28 days and amplified using SB11-specific
primers (FIG. 9D). These data indicate a lack of integration of
SB11 transposase in the electroporated and propagated T cells.
[0228] Karyotype of CAR.sup.+ T cells. The integrity of chromosome
structure was evaluated to rule out global genotoxicity associated
with SB transposition. G-banding of CAR+ T cells (harvested 28 days
after electroporation) from all the three validation runs revealed
a normal (male) karyotype in all analyzed metaphase spreads (FIGS.
9E, 15).
Example 5
Targeting an Ancient Retrovirus Expressed in Cancers and Infections
Using Adoptive T Cells Engineered to Express Chimeric Antigen
Receptor--Methods
[0229] Immunohistochemistry. Tissue microarray (#ME1004a, ME2082b,
FDA998t) obtained from U.S. Biomax (Rockville, Md.) was hydrated
with DiH.sub.2O. Antigen retrieval using citrate buffer pH 6
without EDTA was performed. Slides were blocked with 3% hydrogen
peroxide (Biocare Medical, Concord, Calif.), avidin (Biocare
Medical), biotin (Biocare Medical) and a polyvalent whole serum
(Biocare Medical). Slides were incubated for 30 minute each with
HERV-K mAb (0.6 mg/ml) at 1:20 dilution followed by biotinylated
anti-mouse IgG (Biocare Medical) and strepavidin-HRP
(Sigma-Aldrich, St Louis, Mo.) and visualized with a Mayer's
Hematoxylin counterstain (Sigma-Aldrich). Similar staining
procedures were performed on the slides with isotype control mouse
IgG2a (0.25 mg/ml) antibody (BD Pharmingen, Franklin Lakes,
N.J.).
[0230] Plasmids. The scFv sequence (from mAb clone 6H5) against
HERV-K envelope protein was codon optimized (CoOp) (Invitrogen,
Carlsbad, Calif.) and cloned into the SB transposon under control
of the human elongation factor-1.alpha. (hEF-1.alpha.) promoter,
flanked by SB inverted repeats forming .sub.coOp6H5CD28/pSBSO.
[0231] HERV-K antigen was expressed from SB plasmid containing
bidirectional promoters hEF-1.alpha. and cytomegalovirus (CMV). The
codon optimized full length antigen sequence with the viral
transmembrane domain was cloned under the control of the
hEF-1.alpha. promoter and the neomycin gene was transcribed under
the control of the CMV promoter in a bi-directional vector. The
transposase (SB11) was expressed in cis from the plasmid pCMV-SB11
(Davies et al., 2010).
[0232] To generate the in vivo imaging SB plasmid for T cells,
codon optimized firefly luciferase was fused to a myc tag and was
expressed under the control of the CMV promoter. A lentiviral
vector encoding mKate-renilla luciferase under the control of the
eEF1.alpha. promoter was used as an imaging vector for melanoma
tumor cells in vivo.
[0233] Cells lines and their propagation. A375-mel and A888-mel
were a kind gift from Dr. Lazlo Radvanyi, The University of Texas
MD Anderson Cancer Center (Houston, Tex.). A624-mel and EL4
parental were obtained from American Type Culture Collection
(Rockville, MD). A375-SM (super-metastatic melanoma cell line) was
received from CCGS core facility at the University of Texas MD
Anderson Cancer Center (Houston, Tex.). All cell lines were
cultured in RPMI (Thermo Scientific, Rockford, Ill.) with 10% FBS
(Thermo Scientific) and 5% glutamax (Gibco Life technologies, Grand
Island, N.Y.). All cell lines were verified by morphology, cell
finger printing and/or flow cytometry. They were tested for
Mycoplasma and conserved in research cell banks.
[0234] Generation and expansion of HERV-K-specific CAR expressing T
cells. PBMCs from healthy donors were isolated by Ficoll-Paque
density gradient centrifugation (GE Healthcare Bio-Sciences,
Piscataway, N.J.), and electroporated with the HERV-K SB transposon
and SB11 transposase using the Amaxa electroporation system.
Briefly, 2.times.10.sup.7 PBMC cells were washed and incubated in
complete RPMI supplemented with 10% fetal bovine serum (Thermo
Scientific) and 1% glutamax (Gibco Life technologies) for 2 h.
These cells were then resuspended in 100 .mu.l of Amaxa
Nucleofector solution (Human T-cell Kit), along with HERV-K CAR
transposon (6H5CD28/pSBSO, 15 .mu.g) and SB transposase (pCMV-SB11,
5 .mu.g), transferred to a single cuvette, and electroporated using
the U-14 program in the Amaxa electroporator (Lonza, USA). The
electroporated T cells were incubated for 4 h at 37.degree. C. in
complete phenol-free RPMI (Thermo Scientific) after which a
half-media change was performed.
[0235] K562 express endogenous HERV-K antigen and thus serve as
aAPC to propagate HERV-K-specific CAR.sup.+ T cells. The
electroporated T cells cultured in RPMI containing 10% FBS were
supplemented with .gamma.-irradiated (100 Gy) K562-aAPC at a 1:2 T
cell:aAPC ratio. Irradiated aAPCs were added at the end of every
week for T cell stimulation at the same ratio. Soluble IL-21
(eBioscience) and IL-2 (Chiron) cytokines were supplemented at a
concentration of 30 ng/ml and 50 U/ml, respectively, to complete
RPMI media every other day in the culture after electroporation.
Mock transfected No DNA control T cells grown in the presence of
OKT3 loaded K562 cells served as a negative control. CD19CAR.sup.+
T cells electroporated with .sub.coOpCD19CARCD28/pSBSO and SB11
transposase were grown under the same culture conditions as in
CAR.sup.+ T cells and served as a non-specific CAR.sup.+ T cell
control.
[0236] Every week the T cell cultures were monitored for the
presence of CD3.sup.negCD56.sup.+ cells and were depleted if the
population exceeded 10% of the total population. This depletion
usually occured between 10 and 14 days of initial co-culture with
aAPCs. The depletion was carried out using CD56 beads (Miltenyi
Biotech Inc, Auburn, Calif.) on Automax (Miltenyi Biotech) using
the positive selection "possel" according to manufacturer's
instruction.
[0237] T-cell viability was assessed based on trypan blue exclusion
using a Cellometer automated cell counter (Auto T4 Cell Counter,
Nexcelom Bioscience, Lawrence, Mass.). The viability was analyzed
using the program "PBMC_human_frozen" and "activated T cell,"
during electroporation and co-culture period. The fold expansion
(compared to day 1) of total, CD3.sup.+, CD4.sup.+, CD8.sup.+ and
CAR.sup.+ cells at the end of 7, 14, 28, 32 days of co-culture for
individual donors was calculated and the average of 3 donors were
compared between the CD19CAR.sup.+ T cells and No DNA control cells
using a Student's t test.
[0238] Flow cytometry. All reagents were obtained from BD
Biosciences (Franklin Lakes, N.J.) unless mentioned otherwise. One
million cells were stained with antibody conjugated with
fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin
chlorophyll protein conjugated to cyanine dye (PerCPCy5.5), or
allophycocyanin (APC). The antibodies used include anti-CD3 FITC (5
.mu.l), anti-CD3 PerCPCy5.5 (2.5 .mu.l), anti-CD3 PE (2 .mu.l)
anti-CD4 APC (2.5 .mu.l), anti-CD8 PerCPCy5.5 (4 .mu.l), anti-CD56
APC (2.5 .mu.l), AnnexinV (5 .mu.l), anti-CD32 FITC (5 .mu.l),
anti-CD45RA FITC (5 .mu.l), anti-CD45RO APC (2.5 .mu.l),
anti-Granzyme B FITC (5 .mu.l), anti-CD62L APC (2.5 .mu.l),
anti-IFN-.gamma. APC (2 .mu.l), anti-CD27 PE (2 .mu.l),
anti-.alpha..beta.TCR FITC (5 .mu.l), anti-.gamma..delta.TCR PE (2
.mu.l), and anti-CCR7 PerCPCy5.5 (2.5 .mu.l, Biolegend).
FITC-conjugated (3 .mu.l, Invitrogen) and PE-conjugated (2.5 .mu.l,
Invitrogen) F(ab').sub.2 fragment of goat anti-human Fc.gamma. was
used to detect cell surface expression of the HERV-K-specific CAR.
Blocking of nonspecific antibody binding was achieved using FACS
wash buffer (2% FBS and 0.1% sodium azide in PBS). Data acquisition
was on a FACSCalibur (BD Biosciences) using CellQuest version 3.3
(BD Biosciences). Analyses and calculation of median fluorescence
intensity (MFI) was undertaken using FlowJo version 7.5.5.
[0239] nCounter analysis digital gene expression system. Difference
in gene expression between HERV-K-specific CAR.sup.+ T cells and No
DNA control T cells were evaluated using the nCounter Analysis
System (model no. NCT-SYST-120, NanoString Technologies; Geiss et
al., 2008). Briefly, 10.sup.4 HERV-K-specific CAR.sup.+ T cells or
No DNA T cells were lysed in RNeasy lysis buffer (RLT; 5 .mu.l,
Qiagen, Gaithersburg, Md.) and the mRNA were hybridized with a
reported code set and a capture code set custom designed using the
nCounter Gene Expression Assay Kit for 12 h at 65.degree. C. An
nCounter prep station was used for the post-hybridiztion processes.
Nanomir software was used to normalize the mRNA levels with an
internal control. R-program, tree-view and clustal view were used
to output the data with statistical analysis. The normalized
results were expressed as the relative mRNA level. Ingenuity
Pathway Analysis (IPA) (Ingenuity Systems, www.ingenuity.com) was
performed on statistically significant genes to understand their
biological interaction based on a database derived from literature
sources.
[0240] Integration Analysis. Genomic DNA from HERV-K-specific
CAR.sup.+ T cells was isolated using a QIAamp DNA mini kit (Qiagen)
and real-time PCR was performed as previously described (Maiti et
al., 2013). Genomic DNA from Jurkat cells (Clone #14) bearing a
single integration of the CAR copy previously described was used as
a positive control (Maiti et al., 2013). The experiment was done in
triplicate with 100 ng of genomic DNA mixed with 10 .mu.l of TaqMan
Gene Expression Master Mix (Applied Biosystems, Foster City,
Calif.), 1 .mu.l (1.times. probe at 250 nM and 1.times. primer at
900 nM) of 20.times. FAM-labeled CAR-specific TaqMan probe primer
set specific for IgG4Fc [forward (5'-GAGGGCAACGTCTTTAGCTG-3'; SEQ
ID NO: 15) and reverse (5'-GATGATGAAGGCCACTGTCA-3'; SEQ ID NO: 16)
primers and carboxyfluorescein (FAM)-labeled probe
(5'-AGATGTTCTGGGTGCTGGTC-3'; SEQ ID NO: 17)] and 1 .mu.l (1.times.
primer at 900 nM and lx probe at 250 nM) of 20.times. VIC labeled
TaqMan RNaseP Probe Primer set (Applied Biosystems) in a total
reaction volume of 20 .mu.l. The primer hybridization occurred at
the IgGFc4 portion of the CAR. The amplification cycle included 2
minutes at 50.degree. C., 10 minutes at 95.degree. C., and forty
cycles of 15 seconds at 95.degree. C. and 1 minute at 60.degree. C.
Detection was performed with a StepOnePlus Real-Time PCR System
(Applied Biosystems). The autosomal RNaseP gene, present at 2
copies per diploid cell, was used as an endogenous reference for
normalization (Jin et al., 2011). The .DELTA..DELTA.CT method
(Applied Biosystems, Calif.) was used to calculate the number of
integrations with reference to RNaseP and Jurkat Clone as
normalization controls.
[0241] Chromium Release Assay (CRA). The CRA was performed as
previously described (Maiti et al., 2013; Jin et al., 2011).
Briefly, HERV-K.sup.+ve targets were treated with .sup.51Cr for 2 h
and incubated with day 35 HERV-K-specific CAR.sup.+ T-cells or No
DNA control T cells and the percentage of .sup.51Cr release was
calculated using the following formula:
% 51 Cr release = Experimental release - Background release Maximum
release - Background release .times. 100 ##EQU00003##
[0242] Time-lapse Bio-imaging. (A) To visualize CAR engagement with
the antigen on the tumor cells, time lapse imaging was performed
using a BioStation IM Cell-S1/Cell-S1-P system (Nikon, Melville,
N.Y.). K562 parental cells were stained with anti-HERV-K APC
antibody (1 .mu.g) and the HERV-K-specific CAR.sup.+ T cells were
stained for CAR surface expression using FITC labeled F(ab')2
fragment of goat anti-human Fc.gamma. antibody (5 .mu.l, BD
Biosciences). The T cells and target cells were mixed at a ratio of
5:1 and plated in complete RPMI culture medium on a T-35 mm glass
bottom plate (Fisher Scientific, Hampton, N.H.). The cells were
immediately imaged every 2 minute at 37.degree. C. for up to 8 h.
Each image was recorded at 1600.times.1200 pixels with a 20X
objective, using phase-contrast, fluorescence channel 2 to observe
green HERV-K-specific CAR.sup.+ T cells, and fluorescence channel 3
to observe red K562 cells with an exposure time of 1/125 and 1/5
sec, respectively. CAR engagement with antigen was seen when the
APC-labeled antigen overlapped with the green-labeled CAR.
[0243] (B) To visualize and quantify the time taken for HERV-K
specific CAR.sup.+ T cells to kill a tumor cell, the tumor cells
were plated with HERV-K-specific CAR.sup.+ T cells in a T-35 mm
glass bottom plate with complete RPMI containing 1 ng/mg Sytox.RTM.
(Invitrogen). Tumor cell death was recorded as the time when the
tumor cell wall was punctured and the cells turned green using
BiostationIM cell-S1-P system (Nikon). The intensity of green
fluorescence in each tumor cell was recorded using live cell
imaging software (Nikon) over a period of 15 h.
[0244] Intracellular IFN-.gamma. release assay. HERV-K-specific
CAR.sup.+ T cells were co-cultured with tumor cells at a 1:10 ratio
in a round-bottom 96-well plate with 200 .mu.l of complete RPMI
culture medium. Protein transport inhibitor (BD Golgi Plug
containing Brefeldin A) was added in all wells to trap the
IFN-.gamma. inside the cell. The co-culture was incubated for 4 h
at 37.degree. C. and then stained for HERV-K-specific CAR
expression for 20 min at 4.degree. C. The cells were then washed,
fixed, and permeabilized with 100 .mu.l of Cytofix/Cytoperm buffer
(catalogue no. 555028) for 20 min at 4.degree. C. The permeabilized
cells were then stained for the cytokine with anti-IFN-.gamma. APC
conjugated antibody. The cells were washed and analyzed by
FACSCalibur. PMA (phorbol 12 myristate 13 acetate)--and
ionomycin--(BD Biosciences) treated T cells were used as positive
controls for this assay. Similar assays were performed with No DNA
control T cells.
[0245] Development of HERV-K.sup.+ve EL4 cell line. Two million EL4
cells were suspended in AMAXA mouse T cell nuclofector solution
(Amaxa, USA) with HERV-K antigen-expressing SB transposon and SB 11
transposase (2 .mu.g of total DNA) to a final volume of 100 ml.
This suspension was transferred to a single cuvette and
electroporated using the C-09 program in Amexa electroporator. The
cells were incubated for 4 h at 37.degree. C. in electroporation
media supplied with the kit supplemented with 10% FBS (Thermo
Scientific Pierce). The cells were then transferred to DMEM, 10%
FBS (Thermo Scientific Pierce) and 5% glutamax (Gibco Life
Technologies). These cells were then grown in the presence of 0.8
mg/ml of G418 and neomycin-resistant HERVK.sup.+ve EL4 cells were
sorted using HERV-K antibody and grown to obtain a pure population
of cells.
[0246] shRNA-mediated HERV-K knockdown in A888-mel cells. A888-mel
cells were grown to 90% confluence in a 6-well plate. The media was
later replaced using 100 .mu.l of either HERV-K-specific shRNA or
scrambled shRNA lentivirus and polybrene (5 .mu.g/ml) and
transduced for 4 h at 37.degree. C. and then replaced with regular
RPMI media. The cells were then sorted based on GFP expression and
grown. Scrambled shRNA-transduced A888-mel cells were used as
control. An immunoblot assay of the cell line lysates was performed
to determine the extent of HERV-K knockdown. The 6H5 HERV-K
antibody was used to detect the HERV-K antigen expression on
A888-mel cells with HERV-K shRNA or scrambled shRNA or parental
cells. Ten million cells were lysed with RIPA buffer containing
protease inhibitor (Roche Applied Science, San Francisco, Calif.).
BCA assay was performed to detect protein concentration (Thermo
Scientific Pierce). A 4%-20% gradient gel (Biorad, Hercules,
Calif.) was used to run 10 .mu.g of protein boiled in SDS loading
buffer. The protein was then transferred to a nitrocellulose
membrane and blocked with 5% milk in PBST and incubated with 6H5
HERV-K antibody (mg/ml). Binding was detected by goat anti-mouse
Fc-HRP (Sigma-Aldrich) and developed using ECL Westfemto.TM.
substrate (Thermo Scientific Pierce). The blot was imaged using
Versa doc Quantityone.TM. software (Biorad) and blot quantified
using Image J software.
[0247] In vivo Analysis. Metastatic melanoma model: 5-week-old
female NOD.Cg-Prkdc.sub.scidIl2rg.sub.tmIwjl/Szj (NSG) mice
(Jackson Laboratories, Bar Harbor, Me.) were intravenously injected
with 10.sup.6 A375-SM cells on Day 0. A375-SM cells were previously
stably transduced with mKate-rRLuc and cell sorted for homogenous
population. Mice in the treatment cohorts (n=7) started receiving
2.times.10.sup.6 HERV-K-specific CAR-FfLuc.sup.+ T cells starting
on Days 7, 14 and 21. IL-2 (600 U; eBioscience) was injected
intraperitonealy (i.p.) three times a week during the treatment
period. One cohort of mice (n=6) bearing the tumor received no
treatment while a control group of mice (n=3) without tumor
received a similar number of CAR.sup.+ T cells as in treatment
group.
[0248] Flux quantification: Bioluminescence imaging (BLI) was
performed every week to image the tumor and T cell activity in
vivo. Mice were anesthetized and placed in anterior-posterior
position for BLI using a Xeno IVIS 100 series system (Caliper Life
Sciences) as previously descreibed (Singh et al., 2007). To image
the HERV-K-specific CAR-ffLuc.sup.+ T cell activity, 150 .mu.l (200
.mu.g/mouse) of D-Luciferin potassium salt (Caliper Life Sciences)
was injected intraperitoneal (i.p.). Ten minutes after injection
emitted photons were quantified using the Living Image 2.50.1
(Caliper Life Sciences) program. To image the tumor cell activity,
100 .mu.l of Endurin (Promega, Fitchburg, Wash.) was injected i.p.
Twenty minutes after injection the tumor activity was quantified
similar to ffLuc. Unpaired student's t-tests were performed to
establish statistical significance of the flux.
[0249] Statistical Analysis. For analyzing statistical differences
in antigen expression between various grades and stages of melanoma
and normal tissue, student's t-tests and ANOVA were used. For
analyzing the differences between control versus HERV-K-specific
CAR.sup.+ T cell expansion, phenotype analysis, and functional
assays, student's t-tests, means, standard deviations, and 95%
confidence intervals (CIs) were calculated. For the metastatic
melanoma model, in vivo, student's t-tests were used to analyze the
significance in the flux data between the tumor and treatment
group. All statistical tests were two-sided and performed using
Graph Pad Prism software (GraphPad Software Inc, San Diego,
Calif.). All P values less than 0.05 were considered statistically
significant.
Example 6
Targeting an Ancient Retrovirus Expressed in Cancers and Infections
Using Adoptive T Cells Engineered to Express Chimeric Antigen
Receptor--Results
[0250] Expression of HERV-K in melanoma patient samples. To
elucidate the physiological relevance of HERV-K during melanoma
invasion and metastasis in vivo, the inventors sought to assess the
expression of the tumor antigen HERV-K envelope protein in biopsies
obtained from patients with various stages of melanoma. Malignant
tumor tissues from two hundred sixty-eight patients and benign skin
and breast tissue from forty patients were analyzed using IHC. The
tumor tissue staining was graded based on the percentage of tumor
cells positive for HERV-K and the intensity of staining and their
product was calculated to obtain the H-index. Tumor tissues showed
varied level of staining intensity and were scored 0, 1, 2 or 3
(FIG. 16A) when compared to the isotype control staining on the
same tissue. The antigen was expressed on the cells either in a
punctuate form along the cell surface as shown with a solid arrow
or as diffuse cytoplasmic staining as shown with dotted arrow (FIG.
16B). This may suggest the circulation and accumulation of the
antigen onto the cell surface for the purpose of shedding the viral
protein (REF). Tumor cells express significantly higher H-index of
HERV-K antigen compared to benign tumor (FIG. 16C). Though there is
no difference in H-index between the malignant and metastatic
tumor, a significant difference can be seen between the tumors in
stage I and II compared to tumors in stage III and IV (FIGS. 16D,
E). In order to further show the specificity of HERV-K expression
on tumor cells and not on normal cells, tissue from thirty-three
types of normal organ each obtained from three normal donors were
analyzed. No significant expression of HERV-K was observed in any
of the normal organ tissues (FIG. 22). These findings suggest that
HERV-K up-regulation specifically in tumor cells can serve as a
unique target marker for immunotherapy.
[0251] Propagation and Characterization of HERV-K-specific
CAR.sup.+ T cells. The HERV-K env-specific monoclonal antibody
(mAb) was developed in mouse and the 6H5 mAb clone was found most
sensitive in detecting antigen in vitro (Wang-Johanning et al.,
2003). The scFv sequence of the 6H5 mAb clone was used to construct
the CAR. The scFv cassette was fused to IgG4Fc region by a flexible
linker, followed by the CD28 transmembrane and CD28 and CD3z
intracellular domains. This was then cloned into the SB transposon
vector (FIG. 17A).
[0252] To generate CAR.sup.+ T cells specific to HERV-K env
antigen, the inventors electroporated peripheral blood mononuclear
cells (PBMCs) with SB transposon along with SB11 transposase and
propagated the cells on endogenously-derived HERV-K.sup.+ve K562
aAPC. To selectively propagate T cells with stable expression of
CAR, these aAPC, which endogenously express the HERV-K antigen,
were genetically modified to co-express desired T-cell
co-stimulatory molecules CD86, 4-1BBL, and membrane bound IL-15
(co-expressed with enhanced green fluorescent protein, EGFP) (Singh
et al., 2008). PBMCs without any transposon electroporated, grown
on OKT3-loaded aAPCs under the same culture conditions served as a
negative No DNA control.
[0253] The expression of CAR was detected using a polyclonal Fc
antibody specific for the IgG4Fc region. The CAR.sup.+ T cells were
stained with Fc antibody every seventh day before supplementing the
culture with irradiated aAPCs. The flow data revealed that 95% of T
cells express CAR on its surface, which could be detected for up to
35 days of culture (FIG. 17B). No significant difference was seen
in the growth kinetics of HERV-K-specific CAR.sup.+ T cells when
compared to No DNA control cells, and all the HERV-K-specific
CAR.sup.+ T cells were CD3.sup.+ T cells by day 14 of culture
(FIGS. 17C, D). The percent average number of CAR.sup.+ CD4
increases while CAR.sup.+ CD8 cells decreases along the culture
period (FIG. 23A). Real-time PCR analysis on the genomic DNA of
HERV-K-specific CAR.sup.+ T cells compared to No DNA control cells
showed that there are less than two integration of the CAR in the
CAR.sup.+ T cell genome (FIG. 17D). The cultured CAR.sup.+ T cells
have an effector memory phenotype, which includes
CD3.sup.+CD56.sup.-CD45RO.sup.+TCR.alpha..beta..sup.+CD27.sup.negCCR7.sup-
.neg cells with substantial lytic potential observed by Granzyme B
levels (FIG. 17F).
[0254] The mRNA levels of HERV-K-specific CAR.sup.+ T cells from
three normal donors were measured and compared to mRNA levels from
No DNA control T cells on Day 28 of culture using nCounter
analysis. HERV-K-specific CAR.sup.+ T cells had significantly
higher levels of chemoattractants, transcriptional regulators and
activators. Increased levels of Perforinl and Granzyme H in
CAR.sup.+ T cells shows the higher lytic potential of these cells
(FIG. 23B). Ingenuity Pathway Analysis (IPA) (p<0.05) suggested
several of these upregulated genes in HERV-K-specific CAR.sup.+ T
cells were involved in NF-.kappa.B activation. The chemoattractant
and the cytokines were involved in IL-10, IFN-.gamma. and IL-12
regulation (FIG. 23C). These data strengthen the previous
observation that HERV-K-specific CAR.sup.+ T cells have a central
effector phenotype.
[0255] Characterization of HERV-K CAR.sup.+ T cell functionality.
The antigen expression on melanoma cell lines, such as A888, A375,
A375-SM, A624 were analyzed using the monoclonal 6H5 antibody
directed against the HERV-K env protein (FIG. 18A). The HERV-K
antigen expression on melanoma cells were compared with the isotype
control (mouse IgG2a). In order to analyze the functionality of
HERV-K-specific CAR.sup.+ T cells, HERV-K.sup.+ tumor cells were
pulsed with radioactive chromium and cultured with varying ratios
of HERV-K-specific CAR.sup.+ T cells. CRA was performed with no DNA
control as a negative control. Significantly higher levels of
HERV-K.sup.+ tumor cell lysis were observed with HERV-K-specific
CAR.sup.+ T cells when compared to no DNA control T cells (FIG.
18B). An unrelated CAR, such as CD19-specific CAR.sup.+ T cells
were also used to perform CRA and basal levels of non-specific
killing were observed with HERV-K antigen positive tumor cells when
compared to CD19 antigen positive tumor cells, such as EL-4 cells
bearing CD19 antigen (FIG. 24A).
[0256] In order to further prove the functionality of these
HERV-K-specific CAR.sup.+ T cells, a four-hour IFN-.gamma. release
was performed. Melanoma tumor targets were co-cultured with
HERV-K-specific CAR.sup.+ T cells or No DNA control T cells at a
1:10 ratio and intracellular cytokine levels were analyzed using
flow cytometry. T cells cultured with PMA-Ionomycin served as a
positive control. The HERV-K-specific CAR.sup.+ T cells showed
higher levels of IFN-.gamma. release compared to the no DNA control
T cells (FIG. 18C) and this result correlates with the CRA.
[0257] Specificity of HERV-K-specific CAR.sup.+ T cells. To show
the specificity of the HER-K-specific CAR, HERV-K.sup.neg EL-4
cells were electroporated with bi-directional SB vector encoding
HERV-K antigen under the hEF-1.alpha. promoter and neomycin
resistance under the CMV promoter (FIG. 25A). The EL-4
HERV-K.sup.+ve cells were then single cell sorted and grown in the
presence of mammalian selection marker, neomycin, to obtain a pure
HERV-K env-expressing population. Interestingly, these cells lost
the HERV-K antigen expression within ten days of culture due
post-translational modification and proteolytic cleavage. A
four-hour CRA was performed on these EL-4 HERV-K.sup.+ve cells
within ten days of sorting. Chromium pulsed EL-4 HERV-K.sup.+ve
cells and HERV-K.sup.neg EL4 parental cells were co-cultured with
varying concentrations of HERV-K-specific CAR.sup.+ T cells and
graded tumor-specific lysis was observed in an antigen-specific
manner (FIG. 19B).
[0258] To further prove the specificity, HERV-K env antigen was
knocked down in A888 melanoma cells using shRNA lentivirus. An
immunoblot analysis showed about 50% knockdown in the HERV-K
protein level compared to the A888 parent and control scrambled
shRNA (FIG. 19C). A CRA of HERV-K-specific CAR.sup.+ T cells with
the HERV-K knockdown cells, A888 parental, and A888-with scrambled
shRNA showed significant reduction in killing with HERV-K knockdown
compared to the parent and control tumor cells (FIG. 19D).
[0259] Time lapse imaging was performed to visualize and quantify
the time taken for CAR.sup.+ T cells to kill the tumor target and
the number of tumor cells killed over a period of 15 h. The tumor
cells and T cells were cultured at a ratio of 1:5 in the presence
of Sytox.RTM., which turns cells green when the cell membrane is
damaged. As the melanoma target A888 and A375 cells were killed by
the HERV-K-specific CAR.sup.+ T cells, a spike in green florescence
was reported around 5 h, which gradually increased over a 15 h time
period. No killing was observed with HERVK-K.sup.neg HEK293
parental targets. About 30% of A888 cells and 35% of A375 cells
were killed by CAR.sup.+ T cells over a period of 15 h. To
visualize the antigen-specific CAR engagement, K562 cells were
stained positive for HERV-K antigen in red and HERV-K-specific CAR
was stained for Fc in green. The overlap of CAR and antigen was
seen over a period of 5 h after which the antibody-bound antigen
and CAR were internalized by the cells (FIG. 26A).
[0260] Tumor killing ability of HERV-K-specific CAR.sup.+ T cells
in vivo. PBMCs were double electroporated with SB vectors encoding
HERV-K-specific CAR and myc-FFLuc with SB11 transpsosase. The SB
vector with the myc-ffLuc gene has a neomycin resistance gene
attached through a linker (FIG. 27A). These HERV-K-specific
CAR-ffLuc.sup.+ T cells had similar growth kinetics as the
HERV-K-specific-CAR.sup.+ T cells and their tumor killing ability
and specificity was comparable (FIGS. 27B, C).
[0261] A metastatic melanoma model in which the A375-super
metastatic (A375-SM) cell line was infused though the tail vein of
NSG mice was developed. These cells engrafted in the lungs and
metastasized to the liver. In order to visualize the reduction in
tumor mass non-invasively, the tumor cells were transduced with a
lentiviral vector bearing the mKate-rRLuc gene and sorted using
mKate marker to obtain a pure population of the cells (FIG. 21A).
After a week of tumor cell engraftment, 20 million CAR.sup.+ T
cells were infused intravenously on days 8, 15, and 22 days along
with IL-2 (i.p.) twice a week for three weeks. Bioluminescence
imaging (BLI) was used to assess the rRLuc activity in each group
of mice. The mouse group with tumor cells alone had significantly
higher luciferase activity by day 25 compared to the mouse group
with tumor cells that received the HERV-K CAR.sup.+ T cells (FIGS.
21A, B). Also mice with tumor alone became moribund by day 28 due
to the high metastasis of the tumor to the liver while the mouse
group receiving the CAR.sup.+ T cells exhibited a healthy appetite
and activity. Ex vivo imaging of mKate on tumor cells showed that
the tumor alone mice had substantially more tumor colonies on the
liver than the treatment group (FIG. 21C). Pathological examination
proved this observation where the tumor group had significantly
higher metastatic colonies in the liver compared to the treatment
group suggesting the role of HERV-K CAR.sup.+ T cells in reducing
tumor growth and metastasis (FIG. 21D).
[0262] Discussion. These results show that HERV-K-specific
CAR.sup.+ T cells were able to successfully target a viral
glycoprotein and kill HERV-K.sup.+ tumor cells in an
antigen-specific manner in vitro and reduce the melanoma tumor
growth and metastasis in vivo.
[0263] HERV-K env is markedly up-regulated during melanoma
metastasis and exclusively present on tumor cells and not on
adjacent normal melanocytes. In melanoma, HERV-K env protein is
associated with MEK-ERK and p16INK4A-CDK4 pathway activation
relating to tumor progression (Li et al., 2010). HERV-K molecular
mimicry also results in reduced glutathione peroxidase levels
resulting in increased reactive oxygen species in tissue leading to
melanomogenesis (Krone et al., 2005). Abnormal levels of HERV-K env
expression appear to be a trigger factor involved in melanoma onset
resulting in morphological and cellular modifications resulting in
tumor progression (Serafino et al., 2009). Modulation of viral
transcription of HERV-K resulted in increased inflammation and
morphological differentiation in melanoma cells (Sciamanna et al.,
2005). The tissue microarray represents increased levels of antigen
expression correlating with melanoma progression while the normal
human tissue has a basal level or an absence of HERV-K
expression.
[0264] The immunogenic nature of melanoma makes it an attractive
model and target to develop potent T cell-based therapy. One of the
major limitations on T cell-based therapy is the HLA-based
restriction on TCR, which limits antigen recognition (Garrido et
al., 1997. Genetically modifying T cells to express tumor antigen
specific receptor circumvents this restriction and confers
non-HLA-based antigen recognition (Gross et al., 1989). The T cell
modifications can be brought about by using either viral or
non-viral vectors. The Sleeping Beauty vector is a non-viral
approach to introduce genes into T cells. One of the main
advantages of the Sleeping Beauty system over other viral
transduction methods is that it is genetically safe, efficient, and
less expensive than retroviral transduction (Maiti et al., 2013).
Culturing these CAR.sup.+ T cells with IL-2 and IL-21 yields enough
cells for infusion purposes. This has led to preliminary clinical
trials using CD19 CAR.sup.+ T cells against B-lineage malignancies.
HERV-K-specific CAR.sup.+ T cells were grown in a similar manner to
the clinical-grade CD19 CAR.sup.+ T cells. These HERV-K-specific
CAR.sup.+ T cells were mainly of an effector memory phenotype,
which are well adapted to target and kill tumor cells.
[0265] Multiple factors, such as cytokines, hormones and chemicals,
are known to regulate HERV-K levels during cancer (Taruscio and
Mantovani, 2004). The HERV-K env antigen is known to circulate
between the cell membrane and cytoplasm and also bud off from the
cell surface in certain tumor conditions. Hence, using adoptive T
cell therapy can be a favorable treatment strategy that only
requires transient antigen expression on the cell surface.
[0266] HERV-K env antigen is also found associated with breast
cancer, ovarian cancer, prostate cancer, lymphoma, teratocarcinoma,
autoimmune diseases, such as multiple sclerosis, and infectious
diseases, such as HIV (Wang-Johanning et al., 2003;
Contreras-Galindo et al., 2008; Jones et al., 2012; Ono, 1986;
Wang-Johanning et al., 2007). Thus, targeting the env antigen using
adoptive T cell therapy can be a treatment option for multiple
disease conditions. Consistent with this hypothesis, HERV-K env can
be successfully and selectively targeted by HERV-K CAR.sup.+ T
cells. Infusing these genetically modified CAR.sup.+ T cells in
vivo was associated with tumor regression and reduced metastasis
showing the antitumor activity of these cells.
Example 7
Using Membrane-Bound Cytokine(s) to Generate T Cells with
Long-Lived In Vivo Potential for Use in Immunotherapy of Minimal
Residual Disease
[0267] Generation and expression of mIL15. The mIL15 construct
(FIG. 28) fuses the IL-15 cDNA sequence (NM_000585.4) to the full
length IL-15R.alpha.) (NM_002189.3) with a serine-glycine linker.
The signal peptides for IL-15 and IL-15R.alpha. were omitted and
the IgE signal peptide (gb|AAB59424.1) was used for the mIL15
construct. This construct will produce an IL-15 that is
membrane-bound, but also presented in the context of IL-15R.alpha.
which is akin to the trans-presentation model described above. The
DNA plasmids were synthesized by GeneArt (Regensburg, Germany) and
subsequently subcloned into a Sleeping Beauty plasmid (a non-viral
gene transfer method). Primary human T cells were co-electroporated
with a CAR plasmid (specific for CD19), with or without the mIL15
plasmid, and the SB-11 transposon. Propagation and expansion of the
genetically modified T cells was achieved by weekly stimulation a
CD19.sup.+ K562 artificial antigen presenting cell (aAPC) variant
expressing 41BBL and CD86 co-stimulatory molecules. The CAR used is
a 2nd generation CAR containing CD3.zeta. and CD28 signaling
cytoplasmic domains. The presence of CD19 on the aAPC allows for
the selective outgrowth of antigen-specific T cells while the
costimulatory molecules improve in vitro expansion. The mIL15
molecule can be stably co-expressed with the CAR by the modified T
cells and the co-expressing T cells represent the bulk of
population (FIG. 29). Additionally, total CAR-expression in the
modified T cells reaches greater than 90% for the
mIL15-CAR-modified T cells (FIG. 29).
[0268] Functionality of mIL15. IL-15 receptor complex signaling
primarily induces phosphorylation of signal transducer and
activator of transcription 5. By looking at phosphorylated STATS
(pSTAT5) using phosflow, it can be determined whether mIL15 is
capable of inducing the cytokine signaling pathway. The pSTATS
levels were elevated in CAR.sup.+ T cells having had cytokine
supplementation with these levels abrogated under serum and
cytokine starvation. Under starvation conditions, pSTATS levels in
mIL15.sup.+CAR.sup.+ T cells were maintained (FIG. 30). These data
demonstrate that mIL15 is functional and activates the pSTATS
portion of the cytokine signaling pathway.
[0269] Propagation of clinically significant numbers of
mIL15.sup.+CAR.sup.+ T cells. In redirecting T cell specificity
with the CAR, the focus in ex vivo expansion is on driving the
CAR.sup.+ T cell population, in this case CAR with or without
mIL15. The standard CAR.sup.+ T cells are grown with soluble IL-2
and IL-21 while mIL15.sup.+CAR.sup.+ T cells were given soluble
IL-21 to capitalize on a reported synergy between IL-15 and IL-21.
The mIL15.sup.+CAR.sup.+ T cells supplemented with IL-21
demonstrated comparable expansion to the standard CAR.sup.+ T
cells, as well as the control CAR.sup.+ T cells given IL-15 and
IL-21 (P=0.53, 2-way ANOVA; FIG. 31). The ex vivo expansion of
mIL15.sup.+CAR.sup.+ T cells produces clinically significant
numbers of cells.
[0270] Assessing the phenotype and functionality of
mIL15.sup.+CAR.sup.+ T cells. The phenotype of the ex vivo expanded
mIL15.sup.+CAR.sup.+ T cells are largely similar to the CAR.sup.+ T
cells except for IL-7Ra expression. The general phenotype of the
cells, which at this time point represents the infusion product,
are predominantly CD8.sup.+ (cytotoxic) T cells with moderate to
high expression of the activation markers CD45RO and CD25. There
were variable low to moderate levels of the expression of T cell
memory-associated markers (CD62L, CCR7, CD27, and CD28) (FIG. 32A)
for both T cell groups. After ex vivo expansion of the modified T
cells, it is necessary for the T cells to retain their redirected T
cell specificity and lytic function. A chromium release assay was
conducted to assess the function of the T cells in the expansion
product. CD19.sup.+ and CD19.sup.- EL4 targets were plated with the
modified T cells at varying effector to target ratios. Specific
lysis of CD19.sup.+ tumor targets was demonstrated across all
effector to target ratios by both mIL15.sup.+CAR.sup.+ T cells and
CAR.sup.+ T cells (P<0.001, 2-way ANOVA, n=3) and did not differ
from one another (P>0.05, 2-way ANOVA). CD19.sup.+ target lysis
by mIL15.sup.+CAR.sup.+ T cells was specific and significantly
different from background lysis of CD19.sup.- targets (P<0.001,
2-way ANOVA) (FIG. 32B). The mIL15.sup.+CAR.sup.+ T cells retained
their redirected specificity and lytic capacity.
[0271] Specific mIL15.sup.+CAR.sup.+ T cell subsets persist
long-term in vitro and remain functional. To assess long-term in
vitro persistence, four aAPC stimulation expanded
mIL15.sup.+/-CAR.sup.+ T cells were cultured long-term without
further antigen re-stimulation. CAR.sup.+ T cells received IL-2,
IL-15, or no cytokine supplementation while mIL15.sup.+CAR.sup.+ T
cells did not receive exogenous cytokines. As anticipated,
CAR.sup.+ T cells receiving no cytokine supplementation did not
persist. The mIL15.sup.+CAR.sup.+ T cells as well as CAR.sup.+ T
cells receiving IL-2 or IL-15 had significantly greater relative
fold expansion than the unsupplemented CAR.sup.+ T cells
(P<0.0001, repeated measures ANOVA, n=3; FIG. 33A). The
maintenance of mIL15.sup.+CAR.sup.+ T cell relative expansion near
zero also suggests that these modified cells are not growing in an
unrestricted manner. To be a benefit for the clinical application,
CAR.sup.+ T cells must remain responsive to antigen. Hence, these T
cells at 75+ days from antigen encounter were challenged with: no
target, CD19.sup.- EL-4, CD19.sup.+ EL-4, CD19.sup.+ Nalm-6 (a
human leukemia cell line), or lymphocyte activating cocktail (LAC)
and interferon .gamma. (IFNg) production was assessed by
intracellular cytokine staining after 6 hours incubation with the
targets. Similarly to the CAR.sup.+ T cells receiving either IL-2
or IL-15, the mIL15.sup.+CAR.sup.+ T cells also produced IFNg in
response to CD19.sup.+ targets and the LAC (FIG. 33B). In another
assay, these 75-day withdrawal T cells were stimulated with aAPC
and supplemented with IL-21. Thus, CAR.sup.+ T cells cultured with
IL-2 now had IL-2 and IL-21 provided, the IL-15 culture T cells
then received IL-15 and IL-21, and the mIL15.sup.+CAR.sup.+ T cells
were supplemented with IL-21 only. T cell viability was assessed
via Annexin V staining eight days after the aAPC stimulation. The
mIL15.sup.+CAR.sup.+ T cells were shown to have the greatest live
cell population (Annexin V .sup.neg) (FIG. 33C) and indicates a
resistance to activation induced cell death.
[0272] Persisting mIL15.sup.+CAR.sup.+ T cells possess traits of
less differentiated T cells. In characterizing the long-term
persisting mIL15.sup.+CAR.sup.+ T cells, the inventors hypothesized
that persisting mIL15.sup.+CAR.sup.+ T cells would exhibit
characteristics associated with less differentiated T cell subsets
as these cell subsets are known for their long-lived potential.
With the constitutive presence, and thus possible constitutive
signaling, during the ex vivo culture of the mIL15.sup.+CAR.sup.+ T
cells, the inventors assessed if these T cells had a molecular
programming for a less differentiated state that would yield cells
with a persistence advantage. Analysis of multiplexed digital gene
profiling using the nCounter Analysis System was performed on
CAR.sup.+ and the mIL15.sup.+CAR.sup.+ T cells from stimulation 4
(withdrawal assay T cell input). Analysis of normalized mRNA counts
used a negative binomial distribution-based statistical program
(Lohse et al., Nucleic Acids Res. (2012) 40, W622-7). Genes were
considered significantly differentially expressed if greater than
two-fold differential mRNA counts, P<0.05, FDR q<0.05. Only
five genes were considered differentially expressed using these
criteria, thus indicating there is no culture or molecular
programming advantage afforded by mIL15.
[0273] The inventors then characterized the long-term persisting
mIL15.sup.+CAR.sup.+ T cells. First, their phenotype was assessed
using CD45RA and CCR7 markers to phenotypically describe their
differentiation state. These markers characterize the
differentiation state of T cells as
CD45RA.sup.+CCR7.sup.+<CD45RA.sup.-CCR7.sup.+<CD45RA.sup.-CCR7.sup.-
-<CD45RA.sup.+CCR7.sup.- representing cells from the least to
most differentiated state. In comparing the 75-day withdrawal
mIL15.sup.+CAR.sup.+ T cells to their counterparts at the
initiation of the experiment (the Stim 4 mIL15.sup.+CAR.sup.+ T
cells), it is observed that the persisting mIL15.sup.+CAR.sup.+ T
cell culture has an increased proportions of CD45RA.sup.+CCR7.sup.+
and CD45RA.sup.+CCR7.sup.- T cell subsets (***P<0.001, 2-way
repeated measures ANOVA, n=7; FIG. 34). CCR7 expression was
significantly enhanced in the withdrawal mIL15.sup.+CAR.sup.+ T
cells relative to CAR T cells. Viability of CCR7.sup.neg and
CCR7.sup.+ subsets was assessed by Annexin V staining of
mIL15.sup.+CAR.sup.+ T cells, CAR.sup.+ T cells receiving IL-2
(50U/ml), and CAR.sup.+ T cells receiving soluble IL-15 (5 ng/ml)
after antigen withdrawal. It was found that irrespective of the
type of cytokine stimulation (IL-2, IL-15 complex, or mIL15),
CCR7.sup.neg T cells showed equal frequencies of live cells. In
contrast, CCR7.sup.+ T cells exposed to mIL15 had significantly
higher viability than the CAR T cells receiving IL-2 or IL-15
complex (both P<0.05; FIG. 34). These data suggest that mIL15 is
sufficient to support the CCR7.sup.+ phenotype which thus
contributes to maintaining the less differentiated
CD45RA.sup.+CCR7.sup.+ T cell subset. The capacity for the
mIL15.sup.+CAR.sup.+ T cells to promote the persistence of less
differentiated T cells is a desired phenotype for adoptive therapy
and appears to corroborate other studies reporting that long-lived
T cells subsets possess a less differentiated phenotype.
Additionally, the survival of a highly differentiated subset was
also observed, possibly supported by constitutive IL-15
signaling.
[0274] Long-term persisting mIL15.sup.+CAR.sup.+ T cells display
some molecular markers associated with less differentiated T cell
subsets. The T cells were analyzed for their gene expression
patterns using the nCounter Analysis System and a heirarchically
clustered heat map of differentially expressed genes between
mIL15.sup.+CAR.sup.+ T cells from stimulation 4 and those surviving
to day 75 of withdrawal was produced (>2-fold cutoff, P<0.05,
FDR q<0.05). These studies identified 108 significantly
differentially expressed genes (>2-fold cutoff, P<0.05, FDR
q<0.05). Gene Ontology classification was assessed using DAVID
functional annotation. The functional classification of the
differentially expressed genes can be grouped into broad
categories: T cell activation, differentiation, proliferation, and
apoptosis. Namely, there were greater numbers of genes in
mIL15.sup.+CAR.sup.+ T cells that were down-regulated in the
positive regulation of differentiation, regulation of apoptosis,
and induction of apoptosis. Greater numbers of genes in
mIL15.sup.+CAR.sup.+ T cells were up-regulated in the negative
regulation of differentiation and the Wnt signaling pathway (FIG.
36). This suggests that the molecular signature of persisting
mIL15.sup.+CAR.sup.+ T cells is less differentiated than
mIL15.sup.+CAR.sup.+ T cells at stimulation 4 (T cells at
experiment initiation).
[0275] Expression of selected genes was validated by flow
cytometry. Expression of transcription factors associated with a
less differentiated state (Tcf-7) and acquisition of effector
function/differentiated state (Blimp-1 and Tbet) indicate that
persisting mIL15.sup.+CAR.sup.+ T cells exhibit a transcription
factor balance associated with less differentiated cells. This is
characterized by greater expression of Tcf-7 and lower expression
of Blimp-1 and Tbet (FIG. 37). Assessment of the cell surface
markers IL-7Ra and CCR7 was done as they are characteristically
expressed by less differentiated T cell subsets. The persisting
mIL15.sup.+CAR.sup.+ T cells have increased expression of these
markers associated with long-lived T cell subsets (FIG. 38). One
additional measure for distinguishing the level of T cell
differentiation is by the capacity of less differentiated T cells
to produce IL-2. The mIL15.sup.+CAR.sup.+ T cells from either
stimulation 4 or the 75-day withdrawal cultures were mock-treated
or treated with LAC for 6 hours and then assessed for IL-2
production by intracellular cytokine staining. The stimulation 4 T
cells were unable to produce IL-2 whereas the persisting 75-day
withdrawal T cells acquired the capability to produce IL-2 (FIG.
39). These results collectively suggest that while there is no
identifiable difference between ex vivo expanded CAR.sup.+ T cells
or mIL15.sup.+CAR.sup.+ T cells, the resulting long-term persisting
mIL15.sup.+CAR.sup.+ T cells exhibit characteristics associated
with less-differentiated T cells subsets which demonstrate
long-term survival in vivo.
[0276] In vivo persistence and anti-tumor efficacy of
mIL15.sup.+CAR.sup.+ T cells in a high tumor burden model. To
assess in vivo persistence, mIL15.sup.+CAR.sup.+ T cells and
CAR.sup.+ T cells were co-modified to express firefly luciferase
(ffLuc) to enable longitudinal monitoring of T cells in vivo using
bioluminescence imaging (BLI). These modified T cells were
adoptively transferred, using one T cell infusion of
20.times.10.sup.6 CAR.sup.+ T cells, into NSG mice bearing
disseminated Nalm-6 (CD19.sup.+) malignancy with a no treatment
control group. After 14 days the mice were sacrificed. While
CAR.sup.+ T cells were not found to persist, the
mIL15.sup.+CAR.sup.+ T cells were observed by BLI to persist
throughout the 11 day imaging period (FIG. 40B). Bone marrow,
spleen, and peripheral blood, were harvested and assessed by flow
cytometry for the presence of human T cells using human CD3 as a
marker and gating out murine lymphocytes. Mice infused with the
mIL15.sup.+CAR.sup.+ T cells had significant CD3.sup.+ T cells
detected in the bone marrow (0.49-2.17%, P=0.0001, unpaired t-test,
n=5), spleen (1.15-12.38%, P<0.0001, unpaired t-test, n=5) and
peripheral blood (58.39-92.60%, P<0.0001, unpaired t-test, n=5)
(FIG. 40C). There were no CD3.sup.+ cells detected in samples from
the CAR.sup.+ T cell treated group (FIG. 40C) and the no treatment
group (tumor only) in any of the assessed tissues. In this model,
mIL15.sup.+CAR.sup.+ T cells demonstrated tumor control in the
peripheral blood (FIG. 40D), but complete tumor clearance was not
observed. These data indicate that despite the prevalence of tumor
antigen, CAR.sup.+ T cells had insufficient in vivo persistence
whereas mIL15.sup.+CAR.sup.+ T cells were present at significant
levels throughout the body.
[0277] In vivo persistence and anti-tumor efficacy of
mIL15.sup.+CAR.sup.+ T cells in a low tumor burden model. The
inventors next assessed T cell engraftment and anti-tumor activity
in a preventative model with a low tumor burden. The modified T
cells (mIL15.sup.-/-CAR.sup.+ffLuc.sup.+) were infused into the NSG
mice with no exogenous cytokines and allowed to engraft for six
days before an infusion of renilla luciferase (rLuc)-modified
Nalm-6. The mice underwent BLI over the course of 30 days. In this
preventative model, the mIL15.sup.+CAR.sup.+ T cells were observed
to persist throughout the duration of the experiment and prevented
tumor engraftment, which was a significant anti-tumor effect
compared to the CAR.sup.+ T cell and no T cell treatment groups
(P<0.0001, one-way ANOVA, n=4-5) (FIG. 41C-D). Analysis of the
organs and peripheral blood by flow cytometry detected human
CD3.sup.+ T cells in the mIL15.sup.+CAR.sup.+ T cell treatment
group, but interestingly the T cells were only found in the bone
marrow (FIG. 41E) and may indicate preferential homing or survival
after tumor encounter. In a similar experiment, survival was tested
and mIL15.sup.+CAR.sup.+ T cell-treated mice exhibited
significantly improved survival compared to the no T cell treatment
or CAR.sup.+ T cell-treated mice [P=0.045 (mIL15.sup.+CAR.sup.+ T
cells versus CAR.sup.+ T cells , Log-rank Mantel-Cox test, n=7-8;
FIG. 41).
[0278] In vivo persistence of mIL15.sup.+CAR.sup.+ T cells in the
absence of CAR activation. To assess if mIL15.sup.+CAR.sup.+ T
cells can persist long-term in vivo without the need for CAR
signaling, modified T cells (mIL15.sup.+/-CAR.sup.+ffLuc.sup.+)
were infused into mice (with no tumor or exogenous cytokines) and
monitored for up to 47 days. Testing the T cells in this manner
will also elucidate whether the persisting T cells demonstrate
unrestricted growth or if they maintain their population in a
homeostatic-like manner. No CAR.sup.+ T cells were observed to
persist, whereas the presence of mIL15.sup.+CAR.sup.+ T cells
exhibited sustained persistence in the absence of exogenous
cytokines and antigen (FIG. 42B). This was further confirmed by
flow cytometry of CD3 stained cells isolated from the bone marrow,
spleen, and peripheral blood (FIG. 43C). Assessing longitudinal T
cell persistence, flux values indicate that mIL15.sup.+CAR.sup.+ T
cell levels appeared to level off over time or slowly decline, but
did not indicate the occurrence of uncontrolled expansion (FIG.
42D). These persisting T cells were harvested and ex vivo expanded
in the same manner as previously described for ex vivo expansion of
genetically modified T cells. These cells were capable of
antigen-specific activation as assayed by interferon-.gamma.
production (FIG. 42E). This in vivo data demonstrates enhanced
persistence of mIL15.sup.+CAR.sup.+ T cells in a CAR-independent
and homeostatic-like manner whilst retaining their antigen-specific
responsiveness.
[0279] Cell proliferation and memory kinetics in
mIL15.sup.+CAR.sup.+ T cells. Flow cytometry analyses were used to
further characterize mIL15.sup.+CAR.sup.+ T cells (results shown in
FIG. 35). Long-term persisting mIL15.sup.+CAR.sup.+ T cells
maintain in culture with low turnover rates as indicated by minimal
PKH dilution over 10 days in samples that have been in constant
culture for greater than one year. Moreover, dividing cells
appeared to be predominantly CCR7.sup.-. Such data indicates that
the T cells are maintained homeostatically as opposed to
unrestricted autonomous proliferation or growth (FIG. 35A). The
mIL15.sup.+CAR.sup.+ T cells in continuous long-term culture (1.5
to 2.45 years) were activated with aAPCs, phenotyped and submitted
for karyotyping. Phenotyping showed that these T cells expressed
mIL15 and karyoptyping results for all submitted donors showed
normal metaphase-spreads (FIG. 35B-C). FIG. 35D shows the memory
kinetics after stimulation of cells using K562 aAPCs: Stim 1-CAR
versus mIL15.sup.+CAR conditions during withdrawal: For these
studies CAR T cells got only IL-21 during the first 9 days of the
stimulation and then only IL2 during Ag withdrawal conditions;
mIL5.sup.+CAR T cells got IL-21 during the first 9 days and then no
exogenous cytokines during withdrawal conditions. Results indicate
that there is no difference in CCR7 and IL-7Ra expression at Day 19
post-Stim1 between CAR and mIL15.sup.+CAR T cells. CCR7 and IL-7Ra
expression increases with time away from antigen exposure (Day 29
vs Day 19) for mIL15-modified T cells only. CAR expression
increases to .about.80% without exposure to antigen (PB522 was 51%
and PB273 was 53% at 9 days post-Stiml. FIG. 35E, shows memory
kinetics 1 versus 2 stimulations of CAR and mIL15.sup.+CAR T cells.
Conditions during withdrawal: CAR T cells got only IL2 during Ag
withdrawal which was instituted 10 days after the stimulation;
mIL15.sup.+CAR T cells got no exogenous cytokines during
withdrawal. Comparing CCR7 and IL-7Ra expression at similar time
points after 1 Stim and 2 Stims indicated no memory difference
between CAR vs mIL15.sup.+CAR with the 1 Stim at 19 days. In Stim
2, at equivalent time point to Stim 1 CAR T cells having 2 Stims
have far less CCR7 and almost no IL-7Ra compared to Stim 1
counterparts. mIL15.sup.+CAR T cells having 2 Stims also have less
memory, but retain more "memory" than CAR only T cells with
mIL15.sup.+CAR T cells having some CCR7 left (but IL-7Ra is almost
completely gone).
Example 8
Generation of Minimally Manipulated T-cells Using mRNA Coding SB
Transposase
[0280] Studies were undertaken to determine if use of a SB
transposase provided as an mRNA for electroporation could further
enhance CAR T cell production using a SB transposase system. For
these studies cells were electroporated as detailed above using
both a plasmid encoding a CAR flanked by transposon repeats and a
mRNA encoding the transposase (e.g., SB11 or 100.times.). mRNA for
the studies were m7GTP capped and included a poly-A tail. A
schematic showing a protocol for CAR T cell production using a mRNA
SB transposase provided as a mRNA is shown in FIG. 43. For these
studies cells from donor #0 were electroporated with the SB11 mRNA,
while studies cells from donor #1 were electroporated with the
100.times. mRNA.
[0281] Cell produced from donor #0 were characterized by flow
cytometry analysis for CAR expression and cell proliferation. As
shown in FIG. 44A the percentage of T-cells stably expressing CAR
increases significantly from day 9 (8.3%) to day 16 (66.2%). The
T-cells grow quickly and amplify by 20-folds to day 16 after
electroporation (FIG. 44B). Further studies shown in FIG. 44D were
used to assess the number of central memory T-cells (Tcm) at day 9
(left panel) and day 17 (right panel) post electroporation. These
results show that though the number of less differentiated central
memory T-cells (Tcm) declines between day 9 and day 17, it still
remains relatively high (27%). Next, chromium release assays were
used to determine the cytotoxic activity of the cells. As shown in
FIG. 44C the CAR T-cell produced provided CD-19-specific
cytotoxicity against target cells, while essentially no cytotoxic
activity was seen for unmodified T-cells.
[0282] Cell produced from donor #1 were also studied. As shown in
FIG. 45A the CAR T-cell produced from this donor also provided
CD-19-specific cytotoxicity against target cells and essentially no
cytotoxic activity was seen for unmodified T-cells (right panels).
Moreover, the modified T-cells kill CD19-positive target cells on
day 9 and day 15 with similar efficiency despite the different
number of CAR-positive cells. In FIG. 45B the results of studies to
assess CAR copy number and expression as shown. These results
indicate that CAR DNA copy number decreases from 1.5 to 0.9 from
day 15 to day 22 and stays stable after that time point.
[0283] Finally, flow cytometry was used to assess cell viability
following electroporation. As shown in FIG. 46, after
electroporation with DNA/mRNA the total number of cells decreases
first (day 1 and 2) and then cells start to grow. According to the
cellometer counts the cell number decrease by 59%-76% on day 2
after electroporation (viability 24-41%).
[0284] Thus, the foregoing data demonstrated that by use of a CAR
production techniques that employs mRNA encoding a transposase an
effective number of target-specific cytotoxic T cells can be
produced in an extremely show period of time and with minimal
manipulation. Although the efficiency of mRNA electroporation is
donor-dependent, it appears that the SB100.times. transposase may
be more efficient in CAR production. Interestingly, even though
earlier after electroporation (day 9) the lower percentage of cells
express CAR, the total population of cells kills targeted tumor
cells almost as efficiently as one from the later stage (day 15-16)
and contains more central memory (less differentiated) cells. It
was also confirmed that SB11 and SB100.times. coding mRNA provide
integration of about one copy CAR-coding gene per cell genome under
tested conditions.
[0285] 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.
REFERENCES
[0286] The following references, to the extent that they provide
exemplary procedural or other details supplementary to those set
forth herein, are specifically incorporated herein by reference.
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Sequence CWU 1
1
171711PRTArtificial Sequencefusion protein 1Met Leu Leu Leu Val Thr
Ser Leu Leu Leu Leu Cys Glu Leu Pro His 1 5 10 15 Pro Ala Phe Leu
Leu Ile Pro Asp Ile Gln Met Thr Gln Gln Thr Thr 20 25 30 Ser Ser
Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg 35 40 45
Ala Ser Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys 50
55 60 Pro Asp Gly Thr Val Lys Leu Leu Leu Ile Tyr His Thr Ser Arg
Leu 65 70 75 80 His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Gly
Ser Gly Thr 85 90 95 Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
Glu Asp Ile Ala Thr 100 105 110 Tyr Tyr Phe Cys Gln Gln Gly Asn Thr
Leu Pro Tyr Thr Phe Gly Gly 115 120 125 Gly Thr Lys Leu Glu Ile Ile
Thr Gly Ser Thr Ser Gly Ser Gly Lys 130 135 140 Pro Gly Ser Gly Glu
Gly Ser Thr Lys Gly Glu Glu Val Lys Leu Gln 145 150 155 160 Glu Ser
Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr 165 170 175
Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp 180
185 190 Ile Arg Gln Pro Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val
Ile 195 200 205 Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Ala Leu
Lys Ser Arg 210 215 220 Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln
Val Phe Leu Lys Lys 225 230 235 240 Met Asn Ser Leu Gln Thr Asp Asp
Thr Ala Ile Tyr Tyr Cys Ala Lys 245 250 255 His Tyr Tyr Tyr Tyr Gly
Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 260 265 270 Gly Thr Ser Val
Thr Val Ser Ser Glu Glu Ser Lys Tyr Gly Pro Pro 275 280 285 Cys Pro
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Ser Val 290 295 300
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 305
310 315 320 Pro Glu Val Val Thr Cys Val Val Val Asp Val Ser Gln Glu
Asp Pro 325 330 335 Glu Val Gln Phe Asn Trp Tyr Val Val Asp Gly Val
Glu Val His Asn 340 345 350 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
Asn Ser Thr Thr Tyr Arg 355 360 365 Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys 370 375 380 Glu Tyr Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile 385 390 395 400 Glu Lys Thr
Ile Ser Lys Ala Ala Lys Gly Gln Pro Arg Glu Pro Gln 405 410 415 Val
Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Thr Lys Asn Gln 420 425
430 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
435 440 445 Val Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr 450 455 460 Thr Pro Pro Val Leu Asp Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser 465 470 475 480 Arg Leu Thr Val Asp Lys Ser Arg Trp
Gln Glu Glu Gly Asn Val Phe 485 490 495 Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys 500 505 510 Lys Ser Leu Ser Leu
Ser Leu Gly Lys Met Phe Trp Val Leu Val Val 515 520 525 Val Gly Gly
Val Leu Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala 530 535 540 Phe
Ile Ile Phe Trp Val Arg Ser Lys Arg Arg Ser Arg Gly Gly His 545 550
555 560 Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg
Arg 565 570 575 Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala
Ala Tyr Arg 580 585 590 Ser Arg Val Lys Lys Phe Ser Arg Ser Ala Asp
Ala Pro Ala Tyr Gln 595 600 605 Gln Gly Gln Asn Gln Leu Tyr Asn Glu
Glu Leu Asn Leu Gly Arg Arg 610 615 620 Glu Glu Tyr Asp Val Leu Asp
Lys Arg Arg Gly Arg Asp Pro Pro Glu 625 630 635 640 Met Gly Gly Lys
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 645 650 655 Glu Leu
Gln Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly 660 665 670
Met Lys Gly Glu Arg Arg Arg Gly Gly Lys Gly His Asp Gly Leu Tyr 675
680 685 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Ala Leu
His 690 695 700 Met Gln Ala Leu Pro Pro Arg 705 710
22034DNAArtificial SequenceSynthetic polynucleotide sequence
2atgctgctgc tggtgaccag cctgctgctg tgtgagctgc cccaccccgc ctttctgctg
60atccccgaca tccagatgac ccagaccacc tccagcctga gcgccagcct gggcgaccgg
120gtgaccatca gctgccgggc cagccaggac atcagcaagt acctgaactg
gtatcagcag 180aagcccgacg gcaccgtcaa gctgctgatc taccacacca
gccggctgca cagcggcgtg 240cccagccggt ttagcggcag cggctccggc
accgactaca gcctgaccat ctccaacctg 300gagcaggagg acatcgccac
ctacttttgc cagcagggca acacactgcc ctacaccttt 360ggcggcggaa
caaagctgga gatcaccggc agcacctccg gcagcggcaa gcctggcagc
420ggcgagggca gcaccaaggg cgaggtgaag ctgcaggaga gcggccctgg
cctggtggcc 480cccagccaga gcctgagcgt gacctgtacc gtgtccggcg
tgtccctgcc cgactacggc 540gtgtcctgga tccggcagcc ccctaggaag
ggcctggagt ggctgggcgt gatctggggc 600agcgagacca cctactacaa
cagcgccctg aagagccggc tgaccatcat caaggacaac 660agcaagagcc
aggtgttcct gaagatgaac agcctgcaga ccgacgacac cgccatctac
720tactgtgcca agcactacta ctacggcggc agctacgcca tggactactg
gggccagggc 780accagcgtga ccgtgtccag cgagagcaag tacggccctc
cctgcccccc ttgccctgcc 840cccgagttcc tgggcggacc cagcgtgttc
ctgttccccc ccaagcccaa ggacaccctg 900atgatcagcc ggacccccga
ggtgacctgt gtggtggtgg acgtgtccca ggaggacccc 960gaggtccagt
tcaactggta cgtggacggc gtggaggtgc acaacgccaa gaccaagccc
1020cgggaggagc agttcaatag cacctaccgg gtggtgtccg tgctgaccgt
gctgcaccag 1080gactggctga acggcaagga atacaagtgt aaggtgtcca
acaagggcct gcccagcagc 1140atcgagaaaa ccatcagcaa ggccaagggc
cagcctcggg agccccaggt gtacaccctg 1200ccccctagcc aagaggagat
gaccaagaat caggtgtccc tgacctgcct ggtgaagggc 1260ttctacccca
gcgacatcgc cgtggagtgg gagagcaacg gccagcccga gaacaactac
1320aagaccaccc cccctgtgct ggacagcgac ggcagcttct tcctgtacag
caggctgacc 1380gtggacaaga gccggtggca ggagggcaac gtctttagct
gctccgtgat gcacgaggcc 1440ctgcacaacc actacaccca gaagagcctg
tccctgagcc tgggcaagat gttctgggtg 1500ctggtcgtgg tgggtggcgt
gctggcctgc tacagcctgc tggtgacagt ggccttcatc 1560atcttttggg
tgaggagcaa gcggagcaga ggcggccaca gcgactacat gaacatgacc
1620ccccggaggc ctggccccac ccggaagcac taccagccct acgcccctcc
cagggacttc 1680gccgcctacc ggagccgggt gaagttcagc cggagcgccg
acgcccctgc ctaccagcag 1740ggccagaacc agctgtacaa cgagctgaac
ctgggccgga gggaggagta cgacgtgctg 1800gacaagcgga gaggccggga
ccctgagatg ggcggcaagc cccggagaaa gaaccctcag 1860gagggcctgt
ataacgaact gcagaaagac aagatggccg aggcctacag cgagatcggc
1920atgaagggcg agcggcggag gggcaagggc cacgacggcc tgtaccaggg
cctgagcacc 1980gccaccaagg atacctacga cgccctgcac atgcaggccc
tgccccccag atga 203434246DNAArtificial SequenceSynthetic
polynucleotide sequence 3agttgaagtc ggaagtttac atacacttaa
gttggagtca ttaaaactcg tttttcaact 60actccacaaa tttcttgtta acaaacaata
gttttggcaa gtcagttagg acatctactt 120tgtgcatgac acaagtcatt
tttccaacaa ttgtttacag acagattatt tcacttataa 180ttcactgtat
cacaattcca gtgggtcaga agtttacata cactaagttg actgtgcctt
240taaacagctt ggaaaattcc agaaaatgat gtcatggctt tagaagcttc
tgatagacta 300attgacatca tttgagtcaa ttggaggtgt acctgtggat
gtatttcaag gaattctgtg 360gaatgtgtgt cagttagggt gtggaaagtc
cccaggctcc ccagcaggca gaagtatgca 420aagcatcgag cggccgcaat
aaaatatctt tattttcatt acatctgtgt gttggttttt 480tgtgtgaatc
gtaactaaca tacgctctcc atcaaaacaa aacgaaacaa aacaaactag
540caaaataggc tgtccccagt gcaagtgcag gtgccagaac atttctctat
cgaaggatct 600gcgatcgctc cggtgcccgt cagtgggcag agcgcacatc
gcccacagtc cccgagaagt 660tggggggagg ggtcggcaat tgaaccggtg
cctagagaag gtggcgcggg gtaaactggg 720aaagtgatgt cgtgtactgg
ctccgccttt ttcccgaggg tgggggagaa ccgtatataa 780gtgcagtagt
cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacagctga
840agcttcgagg ggctcgcatc tctccttcac gcgcccgccg ccctacctga
ggccgccatc 900cacgccggtt gagtcgcgtt ctgccgcctc ccgcctgtgg
tgcctcctga actgcgtccg 960ccgtctaggt aagtttaaag ctcaggtcga
gaccgggcct ttgtccggcg ctcccttgga 1020gcctacctag actcagccgg
ctctccacgc tttgcctgac cctgcttgct caactctacg 1080tctttgtttc
gttttctgtt ctgcgccgtt acagatccaa gctgtgaccg gcgcctacct
1140gagatcaccg gcgaaggagg cctatcatga agatctatcg attgtacagc
tagccgccac 1200catgctgctg ctggtgacca gcctgctgct gtgtgagctg
ccccaccccg cctttctgct 1260gatccccgac atccagatga cccagaccac
ctccagcctg agcgccagcc tgggcgaccg 1320ggtgaccatc agctgccggg
ccagccagga catcagcaag tacctgaact ggtatcagca 1380gaagcccgac
ggcaccgtca agctgctgat ctaccacacc agccggctgc acagcggcgt
1440gcccagccgg tttagcggca gcggctccgg caccgactac agcctgacca
tctccaacct 1500ggagcaggag gacatcgcca cctacttttg ccagcagggc
aacacactgc cctacacctt 1560tggcggcgga acaaagctgg agatcaccgg
cagcacctcc ggcagcggca agcctggcag 1620cggcgagggc agcaccaagg
gcgaggtgaa gctgcaggag agcggccctg gcctggtggc 1680ccccagccag
agcctgagcg tgacctgtac cgtgtccggc gtgtccctgc ccgactacgg
1740cgtgtcctgg atccggcagc cccctaggaa gggcctggag tggctgggcg
tgatctgggg 1800cagcgagacc acctactaca acagcgccct gaagagccgg
ctgaccatca tcaaggacaa 1860cagcaagagc caggtgttcc tgaagatgaa
cagcctgcag accgacgaca ccgccatcta 1920ctactgtgcc aagcactact
actacggcgg cagctacgcc atggactact ggggccaggg 1980caccagcgtg
accgtgtcca gcgagagcaa gtacggccct ccctgccccc cttgccctgc
2040ccccgagttc ctgggcggac ccagcgtgtt cctgttcccc cccaagccca
aggacaccct 2100gatgatcagc cggacccccg aggtgacctg tgtggtggtg
gacgtgtccc aggaggaccc 2160cgaggtccag ttcaactggt acgtggacgg
cgtggaggtg cacaacgcca agaccaagcc 2220ccgggaggag cagttcaata
gcacctaccg ggtggtgtcc gtgctgaccg tgctgcacca 2280ggactggctg
aacggcaagg aatacaagtg taaggtgtcc aacaagggcc tgcccagcag
2340catcgagaaa accatcagca aggccaaggg ccagcctcgg gagccccagg
tgtacaccct 2400gccccctagc caagaggaga tgaccaagaa tcaggtgtcc
ctgacctgcc tggtgaaggg 2460cttctacccc agcgacatcg ccgtggagtg
ggagagcaac ggccagcccg agaacaacta 2520caagaccacc ccccctgtgc
tggacagcga cggcagcttc ttcctgtaca gcaggctgac 2580cgtggacaag
agccggtggc aggagggcaa cgtctttagc tgctccgtga tgcacgaggc
2640cctgcacaac cactacaccc agaagagcct gtccctgagc ctgggcaaga
tgttctgggt 2700gctggtcgtg gtgggtggcg tgctggcctg ctacagcctg
ctggtgacag tggccttcat 2760catcttttgg gtgaggagca agcggagcag
aggcggccac agcgactaca tgaacatgac 2820cccccggagg cctggcccca
cccggaagca ctaccagccc tacgcccctc ccagggactt 2880cgccgcctac
cggagccggg tgaagttcag ccggagcgcc gacgcccctg cctaccagca
2940gggccagaac cagctgtaca acgagctgaa cctgggccgg agggaggagt
acgacgtgct 3000ggacaagcgg agaggccggg accctgagat gggcggcaag
ccccggagaa agaaccctca 3060ggagggcctg tataacgaac tgcagaaaga
caagatggcc gaggcctaca gcgagatcgg 3120catgaagggc gagcggcgga
ggggcaaggg ccacgacggc ctgtaccagg gcctgagcac 3180cgccaccaag
gatacctacg acgccctgca catgcaggcc ctgcccccca gatgactacg
3240acccgggtga tcagcgggat ctgctgtgcc ttctagttgc cagccatctg
ttgtttgccc 3300ctcccccgtg ccttccttga ccctggaagg tgccactccc
actgtccttt cctaataaaa 3360tgaggaaatt gcatcgcatt gtctgagtag
gtgtcattct attctggggg gtggggtggg 3420gcaggacagc aagggggagg
attgggaaga caatagcagg catgctgggg atgcggtggg 3480ctctatgggt
acccaggtgc tgaagaattg acccggttcc tcctgggcca gaaagaagca
3540ggcacatccc cttctctgtg acacaccctg tccacgcccc tggttcttag
ttccagcccc 3600actcatagga cactcatagc tcaggagggc tccgccttca
atcccacccg ctaaagtact 3660tggagcggtc tctccctccc tcatcagccc
accaaaccaa acctagcctc caagagtggg 3720aagaaattaa agcaagatag
gctattaagt gcagagggag agaaaatgcc tccaacatgt 3780gaggaagtaa
tgagagaaat catagaatta tcgggccgct gcattctagt tgtggtttgt
3840ccaaactcat caatgtatct tatcatgtct ggatcccatc acaaagctct
gacctcaatc 3900ctatagaaag gaggaatgag ccaaaattca cccaacttat
tgtgggaagc ttgtggaagg 3960ctactcgaaa tgtttgaccc aagttaaaca
atttaaaggc aatgctacca aatactaatt 4020gagtgtatgt taacttctga
cccactggga atgtgatgaa agaaataaaa gctgaaatga 4080atcattctct
ctactattat tctgatattt cacattctta aaataaagtg gtgatcctaa
4140ctgaccttaa gacagggaat ctttactcgg attaaatgtc aggaattgtg
aaaaagtgag 4200tttaaatgta tttggctaag gtgtatgtaa acttccgact tcaact
42464680PRTArtificial SequenceFusion protein 4Met Leu Leu Leu Val
Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu
Leu Ile Pro Met Ala Gln Val Lys Leu Gln Gln Ser Gly 20 25 30 Pro
Asp Leu Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala 35 40
45 Ser Gly Tyr Ser Phe Thr Gly Tyr Tyr Met His Trp Val Lys Gln Ser
50 55 60 His Gly Lys Ser Leu Glu Trp Ile Gly Arg Val Asn Pro Asn
Ser Gly 65 70 75 80 Gly Thr Ser Tyr Asn Gln Lys Phe Lys Asp Lys Ala
Ile Leu Thr Val 85 90 95 Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu
Leu Arg Ser Leu Thr Ser 100 105 110 Glu Asp Ser Ala Val Tyr Tyr Cys
Ala Arg Ser Lys Gly Asn Tyr Phe 115 120 125 Tyr Ala Met Asp Tyr Trp
Gly Gln Gly Thr Thr Val Thr Val Ser Ser 130 135 140 Ser Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp 145 150 155 160 Ile
Glu Leu Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Leu Gly Gln 165 170
175 Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser His Gly
180 185 190 Thr Ser Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro
Pro Lys 195 200 205 Phe Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly
Ile Pro Ala Arg 210 215 220 Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe
Thr Leu Thr Ile Asn Pro 225 230 235 240 Val Glu Thr Asp Asp Val Ala
Ile Tyr Tyr Cys Gln Gln Ser Asn Glu 245 250 255 Asp Pro Pro Thr Phe
Gly Gly Gly Thr Lys Leu Glu Leu Lys Glu Ser 260 265 270 Lys Tyr Gly
Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly 275 280 285 Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 290 295
300 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln
305 310 315 320 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
Val Glu Val 325 330 335 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe Asn Ser Thr Tyr 340 345 350 Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly 355 360 365 Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Gly Leu Pro Ser Ser Ile 370 375 380 Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 385 390 395 400 Tyr Thr
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser 405 410 415
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 420
425 430 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro 435 440 445 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
Leu Thr Val 450 455 460 Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe
Ser Cys Ser Val Met 465 470 475 480 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 485 490 495 Leu Gly Lys Met Phe Trp
Val Leu Val Val Val Gly Gly Val Leu Ala 500 505 510 Cys Tyr Ser Leu
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg 515 520 525 Ser Lys
Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr Pro 530 535 540
Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro 545
550 555 560 Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg
Ser Ala 565 570 575 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu
Tyr Asn Glu Leu 580 585 590 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val
Leu Asp Lys Arg Arg Gly
595 600 605 Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro
Gln Glu 610 615 620 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala
Glu Ala Tyr Ser 625 630 635 640 Glu Ile Gly Met Lys Gly Glu Arg Arg
Arg Gly Lys Gly His Asp Gly 645 650 655 Leu Tyr Gln Gly Leu Ser Thr
Ala Thr Lys Asp Thr Tyr Asp Ala Leu 660 665 670 His Met Gln Ala Leu
Pro Pro Arg 675 680 54255DNAArtificial SequenceSynthetic
polynucleotide sequence 5agttgaagtc ggaagtttac atacacttaa
gttggagtca ttaaaactcg tttttcaact 60actccacaaa tttcttgtta acaaacaata
gttttggcaa gtcagttagg acatctactt 120tgtgcatgac acaagtcatt
tttccaacaa ttgtttacag acagattatt tcacttataa 180ttcactgtat
cacaattcca gtgggtcaga agtttacata cactaagttg actgtgcctt
240taaacagctt ggaaaattcc agaaaatgat gtcatggctt tagaagcttc
tgatagacta 300attgacatca tttgagtcaa ttggaggtgt acctgtggat
gtatttcaag gaattctgtg 360gaatgtgtgt cagttagggt gtggaaagtc
cccaggctcc ccagcaggca gaagtatgca 420aagcatcgag cggccgcaat
aaaatatctt tattttcatt acatctgtgt gttggttttt 480tgtgtgaatc
gtaactaaca tacgctctcc atcaaaacaa aacgaaacaa aacaaactag
540caaaataggc tgtccccagt gcaagtgcag gtgccagaac atttctctat
cgaaggatct 600gcgatcgctc cggtgcccgt cagtgggcag agcgcacatc
gcccacagtc cccgagaagt 660tggggggagg ggtcggcaat tgaaccggtg
cctagagaag gtggcgcggg gtaaactggg 720aaagtgatgt cgtgtactgg
ctccgccttt ttcccgaggg tgggggagaa ccgtatataa 780gtgcagtagt
cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacagctga
840agcttcgagg ggctcgcatc tctccttcac gcgcccgccg ccctacctga
ggccgccatc 900cacgccggtt gagtcgcgtt ctgccgcctc ccgcctgtgg
tgcctcctga actgcgtccg 960ccgtctaggt aagtttaaag ctcaggtcga
gaccgggcct ttgtccggcg ctcccttgga 1020gcctacctag actcagccgg
ctctccacgc tttgcctgac cctgcttgct caactctacg 1080tctttgtttc
gttttctgtt ctgcgccgtt acagatccaa gctgtgaccg gcgcctacct
1140gagatcaccg gcgaaggagg cctatcatga agatctatcg attgtacagc
tagccgccac 1200catgctgctg ctggtgacca gcctgctgct gtgtgagctg
ccccaccccg cctttctgct 1260gatccccatg gcccaggtga agctgcagca
gagcggccct gatctggtga agcctggcgc 1320cagcgtgaag atcagctgca
aggccagcgg ctacagcttc accggctact acatgcactg 1380ggtgaaacag
agccacggca agagcctgga atggatcggc agagtgaacc ccaatagcgg
1440cggcaccagc tacaaccaga agttcaagga caaggccatc ctgaccgtgg
acaagagcag 1500cagcaccgcc tacatggaac tgcggagcct gaccagcgag
gacagcgccg tgtactactg 1560cgcccggtcc aagggcaact acttctacgc
catggactac tggggccagg gcaccaccgt 1620gaccgtgtct agcagcggcg
gaggaagcgg agggggagga tctggcggag gcggcagcga 1680tatcgagctg
acccagagcc ctagcagcct ggccgtgtca ctgggccaga gagccaccat
1740cagctgcaga gcctccgaga gcgtggatag ccacggcacc agcctgatgc
actggtatca 1800gcagaagccc ggccagcccc ccaagttcct gatctaccgg
gccagcaacc tggaaagcgg 1860catccccgcc agattttccg gcagcggcag
cagaaccgac ttcaccctga ccatcaaccc 1920cgtggagaca gacgacgtgg
ccatctacta ctgccagcag agcaacgagg accctcccac 1980ctttggcgga
ggcaccaagc tggaactgaa ggagagcaag tacggccctc cctgcccccc
2040ttgccctgcc cccgagttcc tgggcggacc cagcgtgttc ctgttccccc
ccaagcccaa 2100ggacaccctg atgatcagcc ggacccccga ggtgacctgt
gtggtggtgg acgtgtccca 2160ggaggacccc gaggtccagt tcaactggta
cgtggacggc gtggaggtgc acaacgccaa 2220gaccaagccc cgggaggagc
agttcaatag cacctaccgg gtggtgtccg tgctgaccgt 2280gctgcaccag
gactggctga acggcaagga atacaagtgt aaggtgtcca acaagggcct
2340gcccagcagc atcgagaaaa ccatcagcaa ggccaagggc cagcctcggg
agccccaggt 2400gtacaccctg ccccctagcc aagaggagat gaccaagaat
caggtgtccc tgacctgcct 2460ggtgaagggc ttctacccca gcgacatcgc
cgtggagtgg gagagcaacg gccagcccga 2520gaacaactac aagaccaccc
cccctgtgct ggacagcgac ggcagcttct tcctgtacag 2580caggctgacc
gtggacaaga gccggtggca ggagggcaac gtctttagct gctccgtgat
2640gcacgaggcc ctgcacaacc actacaccca gaagagcctg tccctgagcc
tgggcaagat 2700gttctgggtg ctggtcgtgg tgggtggcgt gctggcctgc
tacagcctgc tggtgacagt 2760ggccttcatc atcttttggg tgaggagcaa
gcggagcaga ggcggccaca gcgactacat 2820gaacatgacc ccccggaggc
ctggccccac ccggaagcac taccagccct acgcccctcc 2880cagggacttc
gccgcctacc ggagccgggt gaagttcagc cggagcgccg acgcccctgc
2940ctaccagcag ggccagaacc agctgtacaa cgagctgaac ctgggccgga
gggaggagta 3000cgacgtgctg gacaagcgga gaggccggga ccctgagatg
ggcggcaagc cccggagaaa 3060gaaccctcag gagggcctgt ataacgaact
gcagaaagac aagatggccg aggcctacag 3120cgagatcggc atgaagggcg
agcggcggag gggcaagggc cacgacggcc tgtaccaggg 3180cctgagcacc
gccaccaagg atacctacga cgccctgcac atgcaggccc tgccccccag
3240atgactacga cccgggtgat cagcgggatc tgctgtgcct tctagttgcc
agccatctgt 3300tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
gccactccca ctgtcctttc 3360ctaataaaat gaggaaattg catcgcattg
tctgagtagg tgtcattcta ttctgggggg 3420tggggtgggg caggacagca
agggggagga ttgggaagac aatagcaggc atgctgggga 3480tgcggtgggc
tctatgggta cccaggtgct gaagaattga cccggttcct cctgggccag
3540aaagaagcag gcacatcccc ttctctgtga cacaccctgt ccacgcccct
ggttcttagt 3600tccagcccca ctcataggac actcatagct caggagggct
ccgccttcaa tcccacccgc 3660taaagtactt ggagcggtct ctccctccct
catcagccca ccaaaccaaa cctagcctcc 3720aagagtggga agaaattaaa
gcaagatagg ctattaagtg cagagggaga gaaaatgcct 3780ccaacatgtg
aggaagtaat gagagaaatc atagaattat cgggccgctg cattctagtt
3840gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatcccatca
caaagctctg 3900acctcaatcc tatagaaagg aggaatgagc caaaattcac
ccaacttatt gtgggaagct 3960tgtggaaggc tactcgaaat gtttgaccca
agttaaacaa tttaaaggca atgctaccaa 4020atactaattg agtgtatgtt
aacttctgac ccactgggaa tgtgatgaaa gaaataaaag 4080ctgaaatgaa
tcattctctc tactattatt ctgatatttc acattcttaa aataaagtgg
4140tgatcctaac tgaccttaag acagggaatc tttactcgga ttaaatgtca
ggaattgtga 4200aaaagtgagt ttaaatgtat ttggctaagg tgtatgtaaa
cttccgactt caact 42556422PRTArtificial SequenceFusion protein 6Met
Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val 1 5 10
15 His Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp
20 25 30 Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu
Ser Asp 35 40 45 Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys
Phe Leu Leu Glu 50 55 60 Leu Gln Val Ile Ser Leu Glu Ser Gly Asp
Ala Ser Ile His Asp Thr 65 70 75 80 Val Glu Asn Leu Ile Ile Leu Ala
Asn Asn Ser Leu Ser Ser Asn Gly 85 90 95 Asn Val Thr Glu Ser Gly
Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys 100 105 110 Asn Ile Lys Glu
Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe 115 120 125 Ile Asn
Thr Ser Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Leu Gln Ile Thr 145
150 155 160 Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val
Lys Ser 165 170 175 Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn
Ser Gly Phe Lys 180 185 190 Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu
Cys Val Leu Asn Lys Ala 195 200 205 Thr Asn Val Ala His Trp Thr Thr
Pro Ser Leu Lys Cys Ile Arg Asp 210 215 220 Pro Ala Leu Val His Gln
Arg Pro Ala Pro Pro Ser Thr Val Thr Thr 225 230 235 240 Ala Gly Val
Thr Pro Gln Pro Glu Ser Leu Ser Pro Ser Gly Lys Glu 245 250 255 Pro
Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr Ala Ala Thr Thr Ala 260 265
270 Ala Ile Val Pro Gly Ser Gln Leu Met Pro Ser Lys Ser Pro Ser Thr
275 280 285 Gly Thr Thr Glu Ile Ser Ser His Glu Ser Ser His Gly Thr
Pro Ser 290 295 300 Gln Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala Ser
Ala Ser His Gln 305 310 315 320 Pro Pro Gly Val Tyr Pro Gln Gly His
Ser Asp Thr Thr Val Ala Ile 325 330 335 Ser Thr Ser Thr Val Leu Leu
Cys Gly Leu Ser Ala Val Ser Leu Leu 340 345 350 Ala Cys Tyr Leu Lys
Ser Arg Gln Thr Pro Pro Leu Ala Ser Val Glu 355 360 365 Met Glu Ala
Met Glu Ala Leu Pro Val Thr Trp Gly Thr Ser Ser Arg 370 375 380 Asp
Glu Asp Leu Glu Asn Cys Ser His His Leu Ser Arg Met Asp Tyr 385 390
395 400 Lys Asp Asp Asp Asp Lys Asp Tyr Lys Asp Asp Asp Asp Lys Asp
Tyr 405 410 415 Lys Asp Asp Asp Asp Lys 420 73499DNAArtificial
SequenceSynthetic polynucleotide sequence 7agttgaagtc ggaagtttac
atacacttaa gttggagtca ttaaaactcg tttttcaact 60actccacaaa tttcttgtta
acaaacaata gttttggcaa gtcagttagg acatctactt 120tgtgcatgac
acaagtcatt tttccaacaa ttgtttacag acagattatt tcacttataa
180ttcactgtat cacaattcca gtgggtcaga agtttacata cactaagttg
actgtgcctt 240taaacagctt ggaaaattcc agaaaatgat gtcatggctt
tagaagcttc tgatagacta 300attgacatca tttgagtcaa ttggaggtgt
acctgtggat gtatttcaag gaattctgtg 360gaatgtgtgt cagttagggt
gtggaaagtc cccaggctcc ccagcaggca gaagtatgca 420aagcatcgag
cggccgcaat aaaatatctt tattttcatt acatctgtgt gttggttttt
480tgtgtgaatc gtaactaaca tacgctctcc atcaaaacaa aacgaaacaa
aacaaactag 540caaaataggc tgtccccagt gcaagtgcag gtgccagaac
atttctctat cgaaggatct 600gcgatcgctc cggtgcccgt cagtgggcag
agcgcacatc gcccacagtc cccgagaagt 660tggggggagg ggtcggcaat
tgaaccggtg cctagagaag gtggcgcggg gtaaactggg 720aaagtgatgt
cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa
780gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga
acacagctga 840agcttcgagg ggctcgcatc tctccttcac gcgcccgccg
ccctacctga ggccgccatc 900cacgccggtt gagtcgcgtt ctgccgcctc
ccgcctgtgg tgcctcctga actgcgtccg 960ccgtctaggt aagtttaaag
ctcaggtcga gaccgggcct ttgtccggcg ctcccttgga 1020gcctacctag
actcagccgg ctctccacgc tttgcctgac cctgcttgct caactctacg
1080tctttgtttc gttttctgtt ctgcgccgtt acagatccaa gctgtgaccg
gcgcctacct 1140gagatcaccg gcgaaggagg cctatcatga agatctatcg
attgtacagc tagccgccac 1200catggattgg acctggattc tgtttctggt
ggccgctgcc acaagagtgc acagcaactg 1260ggtgaatgtg atcagcgacc
tgaagaagat cgaggatctg atccagagca tgcacattga 1320tgccaccctg
tacacagaat ctgatgtgca ccctagctgt aaagtgaccg ccatgaagtg
1380ttttctgctg gagctgcagg tgatttctct ggaaagcgga gatgcctcta
tccacgacac 1440agtggagaat ctgatcatcc tggccaacaa tagcctgagc
agcaatggca atgtgacaga 1500gtctggctgt aaggagtgtg aggagctgga
ggagaagaac atcaaggagt ttctgcagag 1560ctttgtgcac atcgtgcaga
tgttcatcaa tacaagctct ggcggaggat ctggaggagg 1620cggatctgga
ggaggaggca gtggaggcgg aggatctggc ggaggatctc tgcagattac
1680atgccctcct ccaatgtctg tggagcacgc cgatatttgg gtgaagtcct
acagcctgta 1740cagcagagag agatacatct gcaacagcgg ctttaagaga
aaggccggca cctcttctct 1800gacagagtgc gtgctgaata aggccacaaa
tgtggcccac tggacaacac ctagcctgaa 1860gtgcattaga gatcctgccc
tggtccacca gaggcctgcc cctccatcta cagtgacaac 1920agccggagtg
acacctcagc ctgaatctct gagcccttct ggaaaagaac ctgccgccag
1980ctctcctagc tctaataata ccgccgccac aacagccgcc attgtgcctg
gatctcagct 2040gatgcctagc aagtctccta gcacaggcac aacagagatc
agcagccacg aatcttctca 2100cggaacacct tctcagacca ccgccaagaa
ttgggagctg acagcctctg cctctcacca 2160gcctccagga gtgtatcctc
agggccactc tgatacaaca gtggccatca gcacatctac 2220agtgctgctg
tgtggactgt ctgccgtgtc tctgctggcc tgttacctga agtctagaca
2280gacacctcct ctggcctctg tggagatgga ggccatggaa gccctgcctg
tgacatgggg 2340aacaagcagc agagatgagg acctggagaa ttgttctcac
cacctgtcgc gaatggacta 2400caaggacgat gacgacaagg attataaaga
tgatgatgat aaagattata aagacgacga 2460tgataagtcg cgatgatgat
gactcgagac tagtcccggg tgatcagcgg gatctgctgt 2520gccttctagt
tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
2580aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
attgtctgag 2640taggtgtcat tctattctgg ggggtggggt ggggcaggac
agcaaggggg aggattggga 2700agacaatagc aggcatgctg gggatgcggt
gggctctatg ggtacccagg tgctgaagaa 2760ttgacccggt tcctcctggg
ccagaaagaa gcaggcacat ccccttctct gtgacacacc 2820ctgtccacgc
ccctggttct tagttccagc cccactcata ggacactcat agctcaggag
2880ggctccgcct tcaatcccac ccgctaaagt acttggagcg gtctctccct
ccctcatcag 2940cccaccaaac caaacctagc ctccaagagt gggaagaaat
taaagcaaga taggctatta 3000agtgcagagg gagagaaaat gcctccaaca
tgtgaggaag taatgagaga aatcatagaa 3060ttatcgggcc gctgcattct
agttgtggtt tgtccaaact catcaatgta tcttatcatg 3120tctggatccc
atcacaaagc tctgacctca atcctataga aaggaggaat gagccaaaat
3180tcacccaact tattgtggga agcttgtgga aggctactcg aaatgtttga
cccaagttaa 3240acaatttaaa ggcaatgcta ccaaatacta attgagtgta
tgttaacttc tgacccactg 3300ggaatgtgat gaaagaaata aaagctgaaa
tgaatcattc tctctactat tattctgata 3360tttcacattc ttaaaataaa
gtggtgatcc taactgacct taagacaggg aatctttact 3420cggattaaat
gtcaggaatt gtgaaaaagt gagtttaaat gtatttggct aaggtgtatg
3480taaacttccg acttcaact 3499818DNAArtificial SequenceSynthetic
primer 8cagcgacggc agcttctt 18917DNAArtificial SequenceSynthetic
primer 9tgcatcacgg agctaaa 171015DNAArtificial SequenceSynthetic
primer 10agagccggtg gcagg 151121DNAArtificial SequenceSynthetic
primer 11atgggaaaat caaaagaaat c 211220DNAArtificial
SequenceSynthetic primer 12ctagtatttg gtagcattgc
201324DNAArtificial SequenceSynthetic primer 13tctccagaac
atcatccctg ccac 241424DNAArtificial SequenceSynthetic primer
14tgggccatga ggtccaccac cctg 241520DNAArtificial SequenceSynthetic
primer 15gagggcaacg tctttagctg 201620DNAArtificial
SequenceSynthetic primer 16gatgatgaag gccactgtca
201720DNAArtificial SequenceSynthetic primer 17agatgttctg
ggtgctggtc 20
* * * * *
References