U.S. patent application number 15/302426 was filed with the patent office on 2017-01-19 for defined composition gene modified t-cell products.
This patent application is currently assigned to Seattle Children's Hospital (Dba Seattle Children's Research Institute). The applicant listed for this patent is Seattle Children's Hospital (dba Seattle Children's Research Institute). Invention is credited to Michael C. Jensen.
Application Number | 20170015746 15/302426 |
Document ID | / |
Family ID | 54288361 |
Filed Date | 2017-01-19 |
United States Patent
Application |
20170015746 |
Kind Code |
A1 |
Jensen; Michael C. |
January 19, 2017 |
DEFINED COMPOSITION GENE MODIFIED T-CELL PRODUCTS
Abstract
Aspects of the invention described herein, concern approaches to
make genetically modified T-cells comprising a chimeric antigen
receptor for human therapy. In some alternatives, the methods
utilize a selection and/or isolation of CD4+ and/or CD8+ T-cells
from a mixed T-cell population, such as, peripheral blood or
apheresis derived mononuclear cells. Once selected/isolated, the
CD4+ and/or CD8+ T-cells are then activated, genetically modified,
and propagated, preferably, in separate or isolated cultures in the
presence of one or more cytokines, which support survival,
engraftment and/or proliferation of the cells, as well as,
preferably promoting or inducing the retention of cell surface
receptors, such as CD62L, CD28, and/or CD27. Included herein are
also methods of treatment, inhibition, amelioration, or elimination
of a cancer by administering to a subject in need thereof, one or
more types of the genetically engineered T-cells or compositions
that comprise the genetically engineered T-cell prepared as
described herein.
Inventors: |
Jensen; Michael C.;
(Bainbridge Island, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Seattle Children's Hospital (dba Seattle Children's Research
Institute) |
Seattle |
WA |
US |
|
|
Assignee: |
Seattle Children's Hospital (Dba
Seattle Children's Research Institute)
Seattle
WA
|
Family ID: |
54288361 |
Appl. No.: |
15/302426 |
Filed: |
April 8, 2015 |
PCT Filed: |
April 8, 2015 |
PCT NO: |
PCT/US2015/024866 |
371 Date: |
October 6, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61977751 |
Apr 10, 2014 |
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61986479 |
Apr 30, 2014 |
|
|
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62058973 |
Oct 2, 2014 |
|
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62088363 |
Dec 5, 2014 |
|
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|
62089730 |
Dec 9, 2014 |
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62090845 |
Dec 11, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2800/90 20130101;
A61K 2039/505 20130101; A61K 39/3955 20130101; C12N 15/85 20130101;
A61P 35/00 20180101; C07K 2319/02 20130101; A61K 2039/5156
20130101; A61K 2039/572 20130101; C07K 2317/73 20130101; C12N
2510/00 20130101; C07K 2317/524 20130101; C07K 2317/526 20130101;
C12N 9/12 20130101; C07K 2317/14 20130101; A61K 35/17 20130101;
A61P 1/18 20180101; A61P 37/06 20180101; C07K 14/7151 20130101;
C07K 2317/622 20130101; A61K 38/179 20130101; A61K 2035/124
20130101; C07K 14/70578 20130101; C07K 2317/53 20130101; A61K 35/28
20130101; A61P 1/04 20180101; A61P 25/00 20180101; C07K 14/71
20130101; A61K 38/1774 20130101; A61P 31/12 20180101; C07K 2319/03
20130101; C07K 16/2803 20130101; C07K 2319/33 20130101; C07K
14/70521 20130101; C07K 16/32 20130101; C12Y 207/10001 20130101;
A61K 2039/5158 20130101; C07K 14/7051 20130101; A61P 35/02
20180101; C07K 14/70517 20130101; A61K 38/1793 20130101; A61P 13/12
20180101; C07K 16/2818 20130101; C12N 5/0636 20130101; A61P 15/00
20180101; A61P 43/00 20180101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 14/725 20060101 C07K014/725; C07K 14/705 20060101
C07K014/705; C12N 15/85 20060101 C12N015/85 |
Claims
1. A method of making genetically modified T-cells, which have a
chimeric antigen receptor, comprising: separating or enriching a
CD8+ expressing population of T-cells and/or a CD4+ expressing
population of T-cells, such as T-cells that are derived from
thymocytes or T-cells that are derived from engineered precursors,
desirably iPS cells, from a mixed population of T-cells so as to
generate a separated or enriched population of T-cells; stimulating
the separated or enriched population of T-cells so as to generate a
stimulated population of CD8+ T-cells and/or CD4+ T-cells;
transducing the stimulated population of CD8+ T-cells and/or CD4+
T-cells with a vector, wherein the vector encodes a chimeric
antigen receptor and a marker sequence, wherein said marker
sequence encodes a cell surface selectable marker, so as to
generate a transduced population of CD8+ T-cells and/or CD4+
T-cells; contacting the transduced population of CD8+ T-cells
and/or CD4+ T-cells with at least one cytokine, which can be
provided exogenously to the T-cells, e.g., in addition to any
cytokine that may be produced by the cells or present in media,
such as for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, or 20 days or for a period that is within a range
defined by any two of the aforementioned time periods, so as to
generate a transduced, cytokine-stimulated population of CD8+
T-cells and/or CD4+ T-cells; enriching the transduced,
cytokine-stimulated population of CD8+ T-cells and/or CD4+ T-cells
by selection of the marker sequence so as to generate an enriched
population of transduced, cytokine-stimulated CD8+ T-cells and/or
CD4+ T-cells; and propagating the enriched population of
transduced, cytokine-stimulated CD8+ T-cells and/or CD4+ T-cells
for at least one day, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, or 20 days or for a period that is
within a range defined by any two of the aforementioned time
periods, so as to obtain said genetically modified T-cells, which
have a chimeric antigen receptor.
2.-73. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of priority to
U.S. Provisional Patent Application No. 61/977,751, filed Apr. 10,
2014, U.S. Provisional Patent Application No. 61/986,479, filed
Apr. 30, 2014, U.S. Provisional Patent Application No. 62/058,973,
filed Oct. 2, 2014, U.S. Provisional Patent Application No.
62/088,363, filed Dec. 5, 2014, U.S. Provisional Patent Application
No. 62/089,730 filed Dec. 9, 2014, and U.S. Provisional Patent
Application No. 62/090,845, filed Dec. 11, 2014. The entire
disclosures of the aforementioned applications are expressly
incorporated by reference in their entireties.
REFERENCE TO SEQUENCE LISTING
[0002] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled SCRI-090WO_SEQUENCE_LISTING.TXT, created Mar. 23,
2015, which is 2.92 kb in size. The information in the electronic
format of the Sequence Listing is incorporated herein by reference
in its entirety.
FIELD OF THE INVENTION
[0003] Aspects of the invention concern approaches to make
genetically modified T-cells comprising a chimeric antigen
receptor. The disclosed methods concern the selection and/or
isolation of CD4+ and/or CD8+ T-cells from a mixed T-cell
population, which are then activated, genetically modified, and
propagated in isolated cultures in the presence of one or more
cytokines, which support survival, engraftment and/or proliferation
of the cells, as well as, promoting retention of cell surface
receptors, such as CD62L, CD28, and/or CD27.
BACKGROUND OF THE INVENTION
[0004] Acute lymphoblastic leukemia (ALL) relapse following
allogeneic hematopoietic stem cell transplantation (HSCT) can
occur. Accordingly, many believe such approaches are ineffective.
The adoptive transfer of human T lymphocytes that are engineered by
gene transfer to express chimeric antigen receptors (CARs) specific
for molecules present on the surface of tumor cells or malignant B
cells has the potential to effectively treat many advanced cancers
and malignancies, as well. In order to be an effective and long
lasting treatment, however, the administered T-cells that comprise
chimeric antigen receptors desirably have a high survival and
proliferation rate after transfer to the patient. The T-cells used
for therapy are also desirably fit for engraftment. Despite the
tremendous effort in the field, the need for additional, effective
cellular therapies remains.
SUMMARY OF THE INVENTION
[0005] Aspects of the invention described herein include methods of
manufacturing genetically modified T-cells comprising a chimeric
antigen receptor for human therapy. Alternatives include methods
utilizing a selection, enrichment, and/or isolation of CD4+ and/or
CD8+ expressing T-cells, such as T-cells that are derived from
thymocytes or T-cells that are derived from engineered precursors,
desirably iPS cells, from a mixed T-cell population. Once selected,
enriched, or isolated, the CD4+ and/or CD8+ expressing T-cells are
then activated, genetically modified, and propagated, preferably,
in separated, enriched, or isolated cultures in the presence of one
or more cytokines, which can be provided exogenously to the
T-cells, e.g., in addition to any cytokine that may be produced by
the cells or present in media and, which support, promote, induce,
or contribute to survival, engraftment and/or proliferation of the
cells, as well as, preferably supporting, promoting, inducing, or
contributing to the retention of cell surface receptors, such as
CD62L, CD28, and/or CD27. Included herein are also methods of
treatment, inhibition, amelioration, or elimination of a cancer by
administering to a subject in need thereof, one or more types of
the genetically engineered T-cells or compositions that comprise
the genetically engineered T-cell prepared as described herein.
[0006] Some aspects of the invention described herein concern
methods of making genetically modified T-cells, which have a
chimeric antigen receptor. By some approaches, these methods are
practiced by separating, isolating, or enriching a CD8+ expressing
population of T-cells and/or a CD4+ expressing population of
T-cells, such as T-cells that are derived from thymocytes or
T-cells that are derived from engineered precursors, desirably iPS
cells, from a mixed population of T-cells so as to generate a
separated, isolated, or enriched population of T-cells; stimulating
these separated, enriched, or isolated populations of T-cells so as
to generate a stimulated population of CD8+ expressing T-cells
and/or CD4+ expressing T-cells; transducing the stimulated
population of CD8+ expressing T-cells and/or CD4+ expressing
T-cells with a vector, wherein the vector encodes a chimeric
antigen receptor and a marker sequence, wherein said marker
sequence encodes a cell surface selectable marker, so as to
generate a transduced population of CD8+ expressing T-cells and/or
CD4+ expressing T-cells; contacting the transduced population of
CD8+ expressing T-cells and/or CD4+ expressing T-cells with at
least one cytokine, which can be provided exogenously to the
T-cells, e.g., in addition to any cytokine that may be produced by
the cells or present in media, so as to generate a transduced,
cytokine-stimulated population of CD8+ expressing T-cells and/or
CD4+ expressing T-cells; enriching the transduced,
cytokine-stimulated population of CD8+ expressing T-cells and/or
CD4+ expressing T-cells by selection of the marker sequence so as
to generate a separated, enriched, or isolated population of
transduced, cytokine-stimulated CD8+ expressing T-cells and/or CD4+
expressing T-cells; and propagating the separated, enriched, or
isolated population of transduced, cytokine-stimulated CD8+
expressing T-cells and/or CD4+ expressing T-cells for at least two
days so as to obtain said genetically modified T-cells, which have
a chimeric antigen receptor. In some alternatives, the CD8+
expressing T-cells and/or CD4+ expressing T-cells can be propagated
for at least or equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, or 20 days or any time that is within a
range of times defined by any two of the aforementioned time
points, so as to obtain said genetically modified T-cells, which
have a chimeric antigen receptor. In some alternatives, the
separating or enriching of the CD8+ population of T-cells and/or a
CD4+ population of T-cells from a mixed population of T-cells is
performed by affinity selection for T-cells having an epitope
present on CD8 and/or CD4. In some alternatives, the separating or
enriching of the CD8+ population of T-cells and/or a CD4+
population of T-cells from a mixed population of T-cells is
performed by flow cytometry. In some alternatives, the separating
or enriching of the CD8+ population of T-cells and/or a CD4+
population of T-cells from a mixed population of T-cells is
performed by immuno-magnetic selection. In some alternatives, the
genetically modified CD8+ T-cells and/or CD4+ T-cells comprise at
least one receptor that promotes, induces, contributes to, or
enhances engraftment fitness. In some alternatives, the at least
one receptor is CD45 RA, CD45 RO, CCR7, CD25, CD127, CD57, CD137,
CD27, CD28 and/or CD62L. In some alternatives, the at least one
receptor is CD27, CD28 and/or CD62L. In some alternatives, the
stimulating of the isolated population of T-cells is performed by
contacting the CD8+ and/or CD4+ T-cells with an antibody-bound
support, such as a bead or particle. In some alternatives, the
antibody-bound support comprises anti-TCR, anti-CD2, anti-CD3,
anti-CD4 and/or anti-CD28 antibodies. In some alternatives, the
antibody-bound support comprises anti-CD3 and/or anti-CD28
antibodies. In some alternatives, the vector further comprises a
first sequence encoding a leader sequence, a second sequence
encoding a ligand binding domain, a third sequence encoding a
signaling domain and a fourth sequence encoding a selectable
marker. In some alternatives, the vector further comprises a
sequence encoding a spacer. In some alternatives, the spacer
comprises an IgG4 hinge. In some alternatives, the vector is a
viral vector. In some alternatives, the viral vector is derived
from simian virus 40, adenoviruses, adeno-associated virus (AAV),
lentivirus, or retroviruses. In some alternatives, the viral vector
is a recombinant adenovirus, adeno-associated virus, lentivirus or
retrovirus vector. In some alternatives, the viral vector is a
lentivirus vector. In some alternatives, the marker sequence
encodes a truncated epidermal growth factor receptor (EGFRt). In
some alternatives, the at least one cytokine comprises GM-CSF,
IL-7, IL-12, IL-15, IL-18, IL-2, and/or IL-21. In some
alternatives, the at least one cytokine comprises IL7, IL-15 and/or
IL-21, which can be provided at 0.1 ng/mL, 0.2 ng/mL, 0.3 ng/mL,
0.4 ng/mL, 0.5 ng/mL, 0.6 ng/mL, 0.7 ng/mL, 0.8 ng/mL, 0.9 ng/mL,
or 1.0 ng/mL or in an amount that is within a range defined by any
two of the aforementioned amounts and/or at 10 U/mL, 20 U/mL, 30
U/mL, 40 U/mL, 50 U/mL, 60 U/mL, 70 U/mL, 80 U/mL, 90 U/mL, or 100
U/mL or in an amount that is within a range defined by any two of
the aforementioned amounts. In some alternatives, the at least one
cytokine comprises IL-2, IL-15 and/or IL-21, wherein the amount of
cytokine is provided at 0.5 ng/mL and/or 50 U/mL. In some
alternatives, the contacting is performed for 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or a
period of time within a range defined by any two of these values.
In some alternatives, the method is performed with isolated,
purified, enriched or separated CD4+ cells in the absence of,
substantially depleted of, or enriched over CD8+ cells. In some
alternatives, the method is performed with isolated, purified,
enriched or separated CD8+ in the absence of, substantially
depleted of, or enriched over CD4+ cells. In some alternatives, the
CD4+ expressing T-cells are propagated for at least 1 day, such as
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 days or for a period that is within a range defined by any
two of the aforementioned time periods. In some alternatives, the
CD8+ T-cells are propagated at least 1 day, such as 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or
for a period that is within a range defined by any two of the
aforementioned time periods. In some alternatives, the method
further comprises removing the antibody-bound support, such as
beads or particles. In some alternatives, the ligand binding domain
of the chimeric antigen receptor comprises an antibody, or a
binding portion thereof. In some alternatives, the ligand binding
domain of the chimeric antigen receptor comprises a single chain
variable fragment (scFv), or a binding portion thereof. In some
alternatives, the ligand binding domain of the chimeric antigen
receptor comprises FMC63, or a binding portion thereof. In some
alternatives, the ligand binding domain of the chimeric antigen
receptor is specific for CD19. In some alternatives, the method
further comprises cryopreserving the genetically modified CD8+
and/or CD4+ T-cells.
[0007] In some alternatives, the separating, enriching, or
isolating of the CD8+ expressing population of T-cells and/or a
CD4+ expressing population of T-cells, such as T-cells that are
derived from thymocytes or T-cells that are derived from engineered
precursors, desirably iPS cells, from a mixed population of T-cells
is performed by affinity selection for T-cells having an epitope
present on CD8 and/or CD4. In some alternatives, the separating,
enriching, or isolating of the CD8+ expressing population of
T-cells and/or a CD4+ expressing population of T-cells from a mixed
population of T-cells is performed by flow cytometry. In some
alternatives, the separating, enriching, or isolating of the CD8+
expressing population of T-cells and/or a CD4+ expressing
population of T-cells from a mixed population of T-cells is
performed by immuno-magnetic selection. In some alternatives, the
genetically modified CD8+ expressing T-cells and/or CD4+ expressing
T-cells comprise at least one receptor that promotes, induces, or
contributes to engraftment fitness. In some alternatives, the at
least one receptor that promotes, induces, or contributes to
engraftment fitness is CD45 RA, CD45 RO, CCR7, CD25, CD127, CD57,
CD137, CD27, CD28 and/or CD62L. In preferred alternatives, the at
least one receptor that promotes, induces, or contributes to
engraftment fitness is CD27, CD28 and/or CD62L. In some
alternatives, the stimulating of the isolated, enriched, or
separated population of T-cells is performed by contacting the CD8+
and/or CD4+ expressing T-cells with an antibody-bound support, such
as a bead or particle. In some of these alternatives, the
antibody-bound support comprises anti-TCR, anti-CD2, anti-CD3,
anti-CD4 and/or anti-CD28 antibodies. In preferred alternatives,
the antibody-bound support comprises anti-CD3 and/or anti-CD28
antibodies.
[0008] In many of the aforementioned alternatives, the vector
further comprises a first sequence encoding a leader sequence, a
second sequence encoding a ligand binding domain, a third sequence
encoding a signaling domain and a fourth sequence encoding a
selectable marker sequence. In some of these alternatives, the
vector further comprises a sequence encoding a spacer, which in
some alternatives may comprise an IgG4 hinge. In many of the
aforementioned alternatives, the vector is a viral vector or a
mini-circle.
[0009] In many of the aforementioned alternatives, the viral vector
is derived from simian virus 40, adenoviruses, adeno-associated
virus (AAV), lentivirus, or retroviruses. In some alternatives, the
viral vector is a recombinant adenovirus, adeno-associated virus,
lentivirus or retrovirus vector. Preferably, the viral vector is a
lentivirus vector. In many of the aforementioned alternatives, the
marker sequence encodes a truncated epidermal growth factor
receptor (EGFRt). In many of the aforementioned alternatives, the
at least one cytokine that is utilized comprises GM-CSF, IL-7,
IL-12, IL-15, IL-18, IL-2, and/or IL-21 and said cytokine is
provided exogenously to the T-cells e.g., in addition to any
cytokine that may be produced by the cells or present in media.
[0010] In desirable alternatives, the at least one cytokine
comprises IL-7, IL-15 and/or IL-21. In many of the aforementioned
alternatives, the at least one cytokine comprises IL-2, IL-15
and/or IL-21. In preferred alternatives, the contacting period is
performed for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, or 20 days or a period of time within a
range defined by any two of these time points. In many of the
aforementioned alternatives, the methods are performed with
isolated, separated, or enriched populations of CD4+ expressing
T-cells, such as T-cells that are derived from thymocytes or
T-cells that are derived from engineered precursors, desirably iPS
cells, in the absence of or having a reduced amount CD8+ expressing
T-cells, as compared to a native population of unseparated,
non-enriched, or non-isolated population of T-cells. In many of the
aforementioned alternatives, these methods are performed with
isolated, separated, or enriched populations of CD8+ expressing
T-cells, such as T-cells that are derived from thymocytes or
T-cells that are derived from engineered precursors, desirably iPS
cells, in the absence of or having a reduced amount of CD4+
expressing T-cells, as compared to a native population of
unseparated, non-enriched, or non-isolated population of T-cells.
In many of the aforementioned alternatives, the CD4+ expressing
T-cells are propagated for at least 1 day, such as 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or
any time that is within a range of times defined by any two of the
aforementioned time points. In many of the aforementioned
alternatives, the CD8+ expressing T-cells are propagated for at
least 1 day, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, or 20 days or any time that is within a range
of times defined by any two of the aforementioned time points.
[0011] In many of the aforementioned alternatives, the methods
further comprise removing the antibody-bound support, such as beads
or particles. In many of the aforementioned alternatives, the
ligand binding domain of the chimeric antigen receptor comprises an
antibody, or a binding portion thereof. In many of the
aforementioned alternatives, the ligand binding domain of the
chimeric antigen receptor comprises a single chain variable
fragment (scFv), or a binding portion thereof. In many of the
aforementioned alternatives, the ligand binding domain of the
chimeric antigen receptor comprises FMC63, or a binding portion
thereof, such as is available in U.S. Pat. No. 7,446,179, herein
expressly incorporated by reference in its entirety. In many of the
aforementioned alternatives, the ligand binding domain of the
chimeric antigen receptor is specific for CD19. In many of the
aforementioned alternatives, the method further comprises
cryopreserving the genetically modified CD8+ and/or CD4+
T-cells.
[0012] Additional aspects of the invention concern a population of
genetically modified T-cells, such as T-cells that are derived from
thymocytes or T-cells that are derived from engineered precursors,
comprising a plurality of affinity selected CD8+ and/or CD4+
expressing T-cells, in an enriched form, such as enriched from, in
the absence of, or isolated from CD8- and/or CD4- expressing
T-cells, wherein said plurality of affinity selected CD8+ and/or
CD4+ expressing T-cells have stimulated CD2, CD3, CD4 and/or CD28
receptors, wherein said plurality of affinity selected or enriched
CD8+ and/or CD4+ expressing T-cells further comprise a gene
encoding a chimeric antigen receptor and a gene encoding a cell
surface selectable marker and, wherein said plurality of affinity
selected or enriched CD8+ and/or CD4+ expressing T-cells have been
re-stimulated with at least one cytokine, which can be provided
exogenously to the T-cells, e.g., in addition to any cytokine that
may be produced by the cells or present in media, such as,
contacting the cells with an exogenously added cytokine for at
least one day, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, or 20 days or any time that is within a
range of times defined by any two of the aforementioned time
points. In some alternatives, said plurality of affinity selected
CD8+ and/or CD4+ expressing T-cells further comprise at least one
receptor that promotes, induces, or contributes to engraftment
fitness. In some alternatives, the at least one receptor that
promotes, induces, improves, or contributes to engraftment fitness
is CD45 RA, CD45 RO, CCR7, CD25, CD127, CD57, CD137, CD27, CD28
and/or CD62L. In some alternatives, the at least one receptor that
promotes, induces, improves, or contributes to engraftment fitness
is CD27, CD28 and/or CD62L. In some alternatives, the plurality of
affinity selected CD8+ and/or CD4+ expressing T-cells further
comprises a vector having a first sequence encoding a leader
sequence, a second sequence encoding a ligand binding domain, a
third sequence encoding a signaling domain and a fourth sequence
encoding a selectable marker sequence.
[0013] Some alternatives relate to a population of genetically
modified T-cells. In some alternatives, the population of
genetically modified T-cells comprises a plurality of affinity
selected CD8+ and/or CD4+ T-cells, absence of, substantially
depleted of, or enriched over CD8- and/or CD4- T-cells, wherein
said plurality of affinity selected CD8+ and/or CD4+ T-cells have
stimulated CD2, CD3, CD4 and/or CD28 receptors, wherein said
plurality of affinity selected CD8+ and/or CD4+ T-cells further
comprise a gene encoding a chimeric antigen receptor and a cell
surface selectable marker and, wherein said plurality of affinity
selected CD8+ and/or CD4+ T-cells have been re-stimulated with at
least one cytokine. In some alternatives, said plurality of
affinity selected CD8+ and/or CD4+ T-cells further comprise at
least one receptor that promotes, enhances, improves or contributes
to engraftment fitness. In some alternatives, the at least one
receptor that promotes, enhances, improves or contributes to
engraftment fitness is CD45 RA, CD45 RO, CCR7, CD25, CD127, CD57,
CD137, CD27, CD28 and/or CD62L. In some alternatives, the at least
one receptor that promotes, enhances, improves or contributes to
engraftment fitness is CD27, CD28 and/or CD62L. In some
alternatives, the plurality of affinity selected CD8+ and/or CD4+
T-cells further comprises a vector having a first sequence encoding
a leader sequence, a second sequence encoding a ligand binding
domain, a third sequence encoding a signaling domain and a fourth
sequence encoding a selectable marker. In some alternatives, the
vector further comprises a sequence encoding a spacer. In some
alternatives, the spacer comprises an IgG4 hinge. In some
alternatives, the vector is a viral vector. In some alternatives,
the viral vector is derived from simian virus 40, adenoviruses,
adeno-associated virus (AAV), lentivirus, or retroviruses. In some
alternatives, the viral vector is a recombinant adenovirus,
adeno-associated virus, lentivirus or retrovirus vector. In some
alternatives, the viral vector is a lentivirus vector. In some
alternatives, the cell surface selectable marker encodes for a
truncated epidermal growth factor receptor (EGFRt). In some
alternatives, the ligand binding domain comprises an antibody, or a
binding portion thereof. In some alternatives, the ligand binding
domain comprises a single chain variable fragment (scFv), or a
binding portion thereof. In some alternatives, the ligand binding
domain comprises FMC63, or a binding portion thereof. In some
alternatives, the ligand binding domain is specific for CD19. In
some alternatives, the population comprises isolated, purified,
separated or enriched CD8+ T-cells in the absence of, substantially
depleted of, or enriched over CD4+ T-cells. In some alternatives,
the population comprises isolated, purified, separated or enriched
CD4+ T-cells in the absence of, substantially depleted of, or
enriched over CD8+ T-cells. In some alternatives, the T cell is a
precursor T cell. In some alternatives, the precursor T cell is a
hematopoietic stem cell. In some alternatives, the vector further
comprises a sequence encoding a spacer, such as a spacer that
comprises an IgG4 hinge. In some alternatives, the vector is a
viral vector. In some alternatives, the viral vector is derived
from simian virus 40, adenoviruses, adeno-associated virus (AAV),
lentivirus, or retroviruses. In some alternatives, the viral vector
is a recombinant adenovirus, adeno-associated virus, lentivirus or
retrovirus vector. In some alternatives, the viral vector is a
lentivirus vector or a mini-circle. In some alternatives, the cell
surface selectable marker encodes for a truncated epidermal growth
factor receptor (EGFRt). In some alternatives, the ligand binding
domain comprises an antibody, or a binding portion thereof. In some
alternatives, the ligand binding domain comprises a single chain
variable fragment (scFv), or a binding portion thereof. In some
alternatives, the ligand binding domain comprises FMC63, or a
binding portion thereof, such as is available in U.S. Pat. No.
7,446,179, herein expressly incorporated by reference in its
entirety. In some alternatives, the ligand binding domain is
specific for CD19. In some alternatives, the population of
genetically modified T-cells, such as T-cells that are derived from
thymocytes or T-cells that are derived from engineered precursors,
comprises isolated CD8+ expressing T-cells in the absence of or
having a reduced amount of CD4+ expressing T-cells, as compared to
a native population of unseparated, non-enriched, or non-isolated
population of T-cells. In some alternatives, the population of
genetically modified T-cells, such as T-cells that are derived from
thymocytes or T-cells that are derived from engineered precursors,
comprises isolated CD4+ expressing T-cells in the absence of or
having a reduced amount of CD8+ expressing T-cells, as compared to
a native population of unseparated, non-enriched, or non-isolated
population of T-cells.
[0014] Additional aspects of the invention relate to a composition
or product combination for human therapy, comprising a
pharmaceutical excipient; and at least one population of the
genetically modified T-cells, as set forth in the preceding
paragraph. In some alternatives, the composition or product
combination comprises a population of genetically modified CD8+
expressing T-cells. In some alternatives, the composition or
product combination comprises a population of genetically modified
CD4+ expressing T-cells. In some alternatives, the composition or
product combination comprises the population of genetically
modified CD8+ expressing T-cells, as set forth above, and the
population of genetically modified CD4+ expressing T-cells, as set
forth above, in a mixed population or co-administered in a 1:1,
1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 2:1, 3:1, 4:1, 5:1,
6:1, 7:1, 8:1, 9:1, or 10:1 ratio or a ratio that is within a range
defined by any two of the aforementioned ratios.
[0015] Additional aspects of the invention concern methods of
treating, inhibiting, or ameliorating a disease in a subject in
need thereof comprising administering to the subject at least one
composition or product combination set forth above. In some
alternatives, the methods comprise administering a composition or
product combination comprising CD4+ expressing T-cells, such as
T-cells that are derived from thymocytes or T-cells that are
derived from engineered precursors, in advance or prior to
administration of the CD8+ expressing T-cells and in other
alternatives, the CD8+ expressing cells, such as T-cells that are
derived from thymocytes or T-cells that are derived from engineered
precursors, are administered before the CD4+ expressing T-cells. In
many alternatives, the subject is identified or selected to receive
an anti-cancer therapy. In many of the aforementioned methods, the
approach also involves measuring or evaluating an inhibition of a
disease. In many of the aforementioned methods, the approach also
involves providing said subject an additional anti-cancer therapy
before, during, or after administration of a composition or product
combination set forth above.
[0016] In many of the aforementioned methods, a composition or
product combination is administered to said subject by adoptive
cell transfer. In some alternatives, a composition or product
combination are administered to said subject after said subject has
received another form of anti-cancer therapy. In many of the
aforementioned methods, the subject is suffering from leukemia. In
many of the aforementioned methods, the subject has recurrent
and/or chemotherapy refractory CD19+ childhood acute lymphoblastic
leukemia (ALL). In many of the aforementioned methods, the subject
has recurrent and/or chemotherapy refractory CD19+ acute
lymphoblastic leukemia (ALL). In many of the aforementioned
methods, the subject is suffering from an autoimmune disease. In
many of the aforementioned methods, the subject is suffering from a
post-HSCT relapse.
[0017] Accordingly, some aspects of the present invention relate to
the following alternatives:
[0018] 1. A method of making genetically modified T-cells, which
have a chimeric antigen receptor, comprising:
[0019] separating or enriching a CD8+ expressing population of
T-cells and/or a CD4+ expressing population of T-cells, such as
T-cells that are derived from thymocytes or T-cells that are
derived from engineered precursors, desirably iPS cells, from a
mixed population of T-cells so as to generate a separated or
enriched population of T-cells;
[0020] stimulating the separated or enriched population of T-cells
so as to generate a stimulated population of CD8+ T-cells and/or
CD4+ T-cells;
[0021] transducing the stimulated population of CD8+ T-cells and/or
CD4+ T-cells with a vector, wherein the vector encodes a chimeric
antigen receptor and a marker sequence, wherein said marker
sequence encodes a cell surface selectable marker, so as to
generate a transduced population of CD8+ T-cells and/or CD4+
T-cells;
[0022] contacting the transduced population of CD8+ T-cells and/or
CD4+ T-cells with at least one cytokine, which can be provided
exogenously to the T-cells, e.g., in addition to any cytokine that
may be produced by the cells or present in media, such as for 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20
days or for a period that is within a range defined by any two of
the aforementioned time periods, so as to generate a transduced,
cytokine-stimulated population of CD8+ T-cells and/or CD4+
T-cells;
[0023] enriching the transduced, cytokine-stimulated population of
CD8+ T-cells and/or CD4+ T-cells by selection of the marker
sequence so as to generate an enriched population of transduced,
cytokine-stimulated CD8+ T-cells and/or CD4+ T-cells; and
[0024] propagating the enriched population of transduced,
cytokine-stimulated CD8+ T-cells and/or CD4+ T-cells for at least
one day, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, or 20 days or for a period that is within a range
defined by any two of the aforementioned time periods, so as to
obtain said genetically modified T-cells, which have a chimeric
antigen receptor.
[0025] 2. The method of alternative 1, wherein the separating or
enriching of the CD8+ population of T-cells and/or a CD4+
population of T-cells from a mixed population of T-cells is
performed by affinity selection for T-cells having an epitope
present on CD8 and/or CD4.
[0026] 3. The method of alternative 1 or 2, wherein the separating
or enriching of the CD8+ population of T-cells and/or a CD4+
population of T-cells from a mixed population of T-cells is
performed by flow cytometry.
[0027] 4. The method of alternative 1 or 2, wherein the separating
or enriching of the CD8+ population of T-cells and/or a CD4+
population of T-cells from a mixed population of T-cells is
performed by immuno-magnetic selection.
[0028] 5. The method of any one of alternatives 1-4, wherein the
genetically modified CD8+ T-cells and/or CD4+ T-cells comprise at
least one receptor that promotes, induces, contributes to, or
enhances engraftment fitness.
[0029] 6. The method of alternative 5, wherein the at least one
receptor is CD45 RA, CD45 RO, CCR7, CD25, CD127, CD57, CD137, CD27,
CD28 and/or CD62L.
[0030] 7. The method of alternative 5 or 6, wherein the at least
one receptor is CD27, CD28 and/or CD62L.
[0031] 8. The method of any one of alternatives 1-7, wherein the
stimulating of the isolated population of T-cells is performed by
contacting the CD8+ and/or CD4+ T-cells with an antibody-bound
support, such as a bead or particle.
[0032] 9. The method of alternative 8, wherein the antibody-bound
support comprises anti-TCR, anti-CD2, anti-CD3, anti-CD4 and/or
anti-CD28 antibodies.
[0033] 10. The method of alternative 8 or 9, wherein the
antibody-bound support comprises anti-CD3 and/or anti-CD28
antibodies.
[0034] 11. The method of any one of alternatives 1-10, wherein the
vector further comprises a first sequence encoding a leader
sequence, a second sequence encoding a ligand binding domain, a
third sequence encoding a signaling domain and a fourth sequence
encoding a selectable marker.
[0035] 12. The method of any one of alternatives 1-11, wherein the
vector further comprises a sequence encoding a spacer.
[0036] 13. The method of alternative 12, wherein the spacer
comprises an IgG4 hinge.
[0037] 14. The method of any one of alternatives 1-13, wherein the
vector is a viral vector.
[0038] 15. The method of alternative 14, wherein the viral vector
is derived from simian virus 40, adenoviruses, adeno-associated
virus (AAV), lentivirus, or retroviruses.
[0039] 16. The method of alternative 14 or 15, wherein the viral
vector is a recombinant adenovirus, adeno-associated virus,
lentivirus or retrovirus vector.
[0040] 17. The method of any one of alternatives 14-16, wherein the
viral vector is a lentivirus vector.
[0041] 18. The method of any one of alternatives 1-17, wherein the
marker sequence encodes a truncated epidermal growth factor
receptor (EGFRt).
[0042] 19. The method of any one of alternatives 1-18, wherein the
at least one cytokine comprises GM-CSF, IL-7, IL-12, IL-15, IL-18,
IL-2 and/or IL-21.
[0043] 20. The method of any one of alternatives 1-19, wherein the
at least one cytokine comprises IL7, IL-15 and/or IL-21, which can
be provided at 0.1 ng/mL, 0.2 ng/mL, 0.3 ng/mL, 0.4 ng/mL, 0.5
ng/mL, 0.6 ng/mL, 0.7 ng/mL, 0.8 ng/mL, 0.9 ng/mL, or 1.0 ng/mL or
in an amount that is within a range defined by any two of the
aforementioned amounts and/or at 10 U/mL, 20 U/mL, 30 U/mL, 40
U/mL, 50 U/mL, 60 U/mL, 70 U/mL, 80 U/mL, 90 U/mL, or 100 U/mL or
in an amount that is within a range defined by any two of the
aforementioned amounts.
[0044] 21. The method of any one of alternatives 1-19, wherein the
at least one cytokine comprises IL-2, IL-15 and/or IL-21, wherein
the amount of cytokine is provided at 0.5 ng/mL and/or 50 U/mL.
[0045] 22. The method of any one of alternatives 1-21, wherein the
contacting is performed for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, or 20 days or a period of time within a
range defined by any two of these values.
[0046] 23. The method of any one of alternatives 1-20 or 22,
wherein the method is performed with isolated, purified, enriched
or separated CD4+ cells in the absence of, substantially depleted
of, or enriched over CD8+ cells.
[0047] 24. The method of any one of alternatives 1-19, 21 or 22,
wherein the method is performed with isolated, purified, enriched
or separated CD8+ in the absence of, substantially depleted of, or
enriched over CD4+ cells.
[0048] 25. The method of any one of alternatives 1-20, 22 or 23,
wherein the CD4+ expressing T-cells are propagated for at least 1
day, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, or 20 days or for a period that is within a range
defined by any two of the aforementioned time periods.
[0049] 26. The method of any one of alternatives 1-19, 21, 22 or
24, wherein the CD8+ T-cells are propagated at least 1 day, such as
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 days or for a period that is within a range defined by any
two of the aforementioned time periods.
[0050] 27. The method of any one of alternatives 8-26, wherein the
method further comprises removing the antibody-bound support, such
as beads or particles.
[0051] 28. The method of any one of alternatives 1-27, wherein the
ligand binding domain of the chimeric antigen receptor comprises an
antibody, or a binding portion thereof.
[0052] 29. The method of any one of alternatives 1-28, wherein the
ligand binding domain of the chimeric antigen receptor comprises a
single chain variable fragment (scFv), or a binding portion
thereof.
[0053] 30. The method of any one of alternatives 1-29, wherein the
ligand binding domain of the chimeric antigen receptor comprises
FMC63, or a binding portion thereof.
[0054] 31. The method of any one of alternatives 1-30, wherein the
ligand binding domain of the chimeric antigen receptor is specific
for CD19.
[0055] 32. The method of any one of alternatives 1-31, wherein the
method further comprises cryopreserving the genetically modified
CD8+ and/or CD4+ T-cells.
[0056] 33. A population of genetically modified T-cells comprising:
[0057] a plurality of affinity selected CD8+ and/or CD4+ T-cells,
absence of, substantially depleted of, or enriched over CD8- and/or
CD4- T-cells, wherein said plurality of affinity selected CD8+
and/or CD4+ T-cells have stimulated CD2, CD3, CD4 and/or CD28
receptors, wherein said plurality of affinity selected CD8+ and/or
CD4+ T-cells further comprise a gene encoding a chimeric antigen
receptor and a cell surface selectable marker and, wherein said
plurality of affinity selected CD8+ and/or CD4+ T-cells have been
re-stimulated with at least one cytokine.
[0058] 34. The population of genetically modified T-cells of
alternative 33, wherein said plurality of affinity selected CD8+
and/or CD4+ T-cells further comprise at least one receptor that
promotes, enhances, improves or contributes to engraftment
fitness.
[0059] 35. The population of genetically modified T-cells of
alternative 33 or 34, wherein the at least one receptor that
promotes, enhances, improves or contributes to engraftment fitness
is CD45 RA, CD45 RO, CCR7, CD25, CD127, CD57, CD137, CD27, CD28
and/or CD62L.
[0060] 36. The population of genetically modified T-cells of
alternative 34 or 35, wherein the at least one receptor that
promotes, enhances, improves or contributes to engraftment fitness
is CD27, CD28 and/or CD62L.
[0061] 37. The population of genetically modified T-cells of any
one of alternatives 33-36, wherein the plurality of affinity
selected CD8+ and/or CD4+ T-cells further comprises a vector having
a first sequence encoding a leader sequence, a second sequence
encoding a ligand binding domain, a third sequence encoding a
signaling domain and a fourth sequence encoding a selectable
marker.
[0062] 38. The population of genetically modified T-cells of
alternative 37, wherein the vector further comprises a sequence
encoding a spacer.
[0063] 39. The population of genetically modified T-cells of
alternative 38, wherein the spacer comprises an IgG4 hinge.
[0064] 40. The population of genetically modified T-cells of any
one of alternatives 33-39, wherein the vector is a viral
vector.
[0065] 41. The population of genetically modified T-cells of
alternative 40, wherein the viral vector is derived from simian
virus 40, adenoviruses, adeno-associated virus (AAV), lentivirus,
or retroviruses.
[0066] 42. The population of genetically modified T-cells of
alternative 40 or 41, wherein the viral vector is a recombinant
adenovirus, adeno-associated virus, lentivirus or retrovirus
vector.
[0067] 43. The population of genetically modified T-cells of any
one of alternatives 40-42, wherein the viral vector is a lentivirus
vector.
[0068] 44. The population of genetically modified T-cells of any
one of alternatives 33-43, wherein the cell surface selectable
marker encodes for a truncated epidermal growth factor receptor
(EGFRt).
[0069] 45. The population of genetically modified T-cells of any
one of alternatives 37-44, wherein the ligand binding domain
comprises an antibody, or a binding portion thereof.
[0070] 46. The population of genetically modified T-cells of any
one of alternatives 37-45, wherein the ligand binding domain
comprises a single chain variable fragment (scFv), or a binding
portion thereof.
[0071] 47. The population of genetically modified T-cells of any
one of alternatives 37-46, wherein the ligand binding domain
comprises FMC63, or a binding portion thereof.
[0072] 48. The population of genetically modified T-cells of any
one of alternatives 37-47, wherein the ligand binding domain is
specific for CD19.
[0073] 49. The population of genetically modified T-cells of any
one of alternatives 33-48, wherein the population comprises
isolated, purified, separated or enriched CD8+ T-cells in the
absence of, substantially depleted of, or enriched over CD4+
T-cells.
[0074] 50. The population of genetically modified T-cells of any
one of alternatives 33-48, wherein the population comprises
isolated, purified, separated or enriched CD4+ T-cells in the
absence of, substantially depleted of, or enriched over CD8+
T-cells.
[0075] 51. A composition or product combination for human therapy,
comprising: [0076] a pharmaceutical excipient; and [0077] at least
one population of genetically modified T-cells according to any one
or more of alternatives 33-50.
[0078] 52. The composition or product combination of alternative
51, wherein the composition or product combination comprises the
population of genetically modified T-cells of alternative 49.
[0079] 53. The composition or product combination of alternative
47, wherein the composition or product combination comprises the
population of genetically modified T-cells of alternative 50.
[0080] 54. The composition or product combination of alternative
47, wherein the composition or product combination comprises the
population of genetically modified T-cells of alternative 49 and
the population of genetically modified T-cells of alternative 50,
mixed or co-administered in a 1:1 ratio.
[0081] 55. A method of treating, inhibiting, or ameliorating a
disease in a subject in need thereof comprising:
[0082] administering to the subject at least one composition or
product combination of any one or more of alternatives 51-54.
[0083] 56. The method of alternative 55, wherein the method
comprises administering the composition or product combination of
alternative 52.
[0084] 57. The method of alternative 55, wherein the method
comprises administering the composition or product combination of
alternative 53.
[0085] 58. The method of alternative 56, wherein the method further
comprises administering the composition or product combination of
alternative 53.
[0086] 59. The method of alternative 57, wherein the method further
comprises administering the composition or product combination of
alternative 52.
[0087] 60. The method of alternative 55, wherein the method
comprises administering the composition or product combination of
alternative 54, such as by an approach, wherein the CD8+ expressing
T-cells are administered in advance of the CD4+ expressing T-cells,
such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 60
minutes before the CD4+ T-cells are administered or a time period
that is within a range defined by any two of the aforementioned
times.
[0088] 61. The method of any one or more of alternatives 55-60,
wherein the subject is identified or selected to receive an
anti-cancer therapy.
[0089] 62. The method of any one or more of alternatives 55-61,
further comprising measuring or evaluating an inhibition of a
disease.
[0090] 63. The method of any one or more of alternatives 55-62,
further comprising providing said subject an additional anti-cancer
therapy before, during, or after administration of the composition
or product combination of any one or more of alternatives
51-54.
[0091] 64. The method of any one or more of alternatives 55-63,
wherein the composition or product combination of any one or more
of alternatives 51-54 are administered to said subject by adoptive
cell transfer.
[0092] 65. The method of any one or more of alternatives 55-64,
wherein the composition or product combination of any one or more
of alternatives 51-54 are administered to said subject after said
subject has received another form of anti-cancer therapy.
[0093] 66. The method of any one or more of alternatives 55-65,
wherein the composition or product combination of any one or more
of alternatives 51-54 are administered to said subject after said
subject has received another form of anti-cancer therapy.
[0094] 67. The method of any one or more of alternatives 55-66,
wherein the subject is suffering from leukemia.
[0095] 68. The method of any one or more of alternatives 55-67,
wherein the subject has recurrent and/or chemotherapy refractory
CD19+ childhood acute lymphoblastic leukemia (ALL).
[0096] 69. The method of any one or more of alternatives 55-68,
wherein the subject has recurrent and/or chemotherapy refractory
CD19+ acute lymphoblastic leukemia (ALL).
[0097] 70. The method of any one or more of alternatives 55-69,
wherein the subject is suffering from an autoimmune disease.
[0098] 71. The method of any one or more of alternatives 55-70,
wherein the subject is suffering from a post-HSCT relapse.
BRIEF DESCRIPTION OF THE DRAWINGS
[0099] FIG. 1A shows the absolute number of anti-CD19 CAR bearing
T-cells in 11 patients following adoptive transfer of the CAR
bearing T-cells from Day 0 to Day 65.
[0100] FIG. 1B shows the absolute number of anti-CD19 CAR bearing
T-cells following three doses of treatment.
[0101] FIG. 2A shows the persistence of anti-CD19 CAR bearing
T-cells in peripheral blood of patients following adoptive transfer
of the CAR bearing T-cells from Day 0 to Day 65. FIG. 2B shows the
persistence of anti-CD19 car bearing T-cells following three doses
of treatment.
[0102] FIG. 3A shows the persistence of anti-CD19 CAR bearing
T-cells in bone marrow of patients following adoptive transfer of
the CAR bearing T-cells from Day 0 to Day 65. FIG. 3B shows the
persistence of anti-CD19 CAR bearing T-cells in bone marrow
patients following three doses of treatment.
[0103] FIG. 4 shows the percent acute lymphoblastic leukemia (ALL)
in patient X following three doses.
[0104] FIG. 5 shows a table of the patient characteristics that
were used in the studies.
[0105] FIG. 6 shows the development of CNS lymphoma, as related to
disease and dosage. As shown, two bar graphs indicate the number of
patients subjected to the treatment comprising anti-CD19 CAR
bearing T-cells and their severity of disease following 3
treatments.
[0106] FIG. 7 shows grade 4 encephalopathy in an abnormal MRI of a
patient suffering from ALL. The first panel indicates the results
following treatment using anti-CD19 CAR bearing T-cells.
[0107] FIG. 8 shows acute Skin Graft-versus-host disease (GVHD)
following treatment with anti-CD19 CAR bearing T-cells. As shown in
panel A, S03 developed de novo grade 2 acute skin GVHD d17 post CAR
T-cell engraftment. In panel B, a skin biopsy revealed that only 9%
of skin localized T-cells marked EGFRt+ while 79% of circulating
T-cells marked EGFRt+. In panel C, peripheral blood at the same
time showed the majority of the T-cells were CAR+. This subject was
treated with a 2 week course of 1 mg/kg of prednisone, followed by
a rapid taper over a six week period and resolution of the GVHD.
Despite the prednisone, the subject has ongoing persistent CAR+
T-cells.
[0108] FIG. 9 shows a table of patients experiencing Ig or TCR as a
marker of minimal residual disease (MRD) following treatment using
anti-CD19 CAR bearing T-cells.
[0109] FIG. 10 shows a table of patient profiles after allogeneic
hematopoietic stem cell transplantation of anti-CD19CAR
T-cells.
[0110] FIG. 11 shows the FACS scatter analysis of the cells
isolated from the peripheral blood and CSF following hematopoietic
stem cell transplantation (HSCT) of anti-CD19 CAR T-cells.
[0111] FIG. 12 shows the tumor burden vs the response after three
doses of hematopoietic stem cell transplantation of anti-CD19 CAR
T-cells.
[0112] FIG. 13 shows the remission duration of patients following
three doses of hematopoietic stem cell transplantation.
[0113] FIG. 14 shows the tumor burden vs the response after two
doses of hematopoietic stem cell transplantation of anti-CD19CAR
T-cells at day 7.
[0114] FIG. 15 shows the magnitude and duration of CAR/EGFRt+
T-cell persistence in a patient after three doses of hematopoietic
stem cell transplantation of anti-CD19 CAR T-cells.
[0115] FIG. 16 shows the duration of B cell aplasia after three
doses of hematopoietic stem cell transplantation of anti-CD19 CAR
T-cells.
[0116] FIG. 17 shows the CRS profile of a patient after three doses
of hematopoietic stem cell transplantation of anti-CD19 CAR
T-cells.
[0117] FIG. 18 shows two flow diagrams illustrating the
manufacturing of genetically modified T-cells. As depicted, for the
initial expansion methodology bulk processing was not always
initiated immediately. A portion of the apheresis product was
always taken for Ficoll processing. If no bulk cultures were to be
initiated then that product separated by Ficoll was
cryopreserved.
[0118] FIG. 19 shows a flow diagram of comparisons of different
methods of making genetically modified T-cells that have chimeric
antigen receptors.
[0119] FIG. 20 shows the initial comparisons in cell growth using
different concentrations of cells in the starter culture. As shown,
PD0064 are the PD0063 donor which are enriched CD8+ expressing
T-cells were used to test starting cell density in T25 flask on
initiation. The experiments tested two cell densities and both
showed greater viability and expansion when the volume was
increased early during the experiment.
[0120] FIG. 21 shows an initiation comparison of cell growth. This
was the full scale data used to transition to the update
manufacturing process currently being used.
[0121] FIG. 22 shows a flow diagram illustrating the variations of
cocktails of cytokines that were used to test CD4+ and CD8+
expressing T-cells for growth.
[0122] FIGS. 23A-F illustrate the comparison of cytokines that were
tested during the growth of CD4+ and CD8+ expressing T-cells.
[0123] FIGS. 24A-F illustrate the comparison of cytokines that were
tested during the growth of CD4+ and CD8+ expressing T-cells.
[0124] FIGS. 25A-B shows a cytokine comparisons for cell growth.
For PD0059 donor sample the same cytokine testing experiment was
performed as in PD0051 except testing a new CD4 and CD8 donor
(PD0057).
[0125] FIG. 26 shows a flow diagram illustrating the initial
expansion methodology and the current expansion methodology derived
from the experimentation of testing different cytokine
mixtures.
[0126] FIG. 27 shows expansion comparisons of samples PD0080, PD084
and PD0085. These experiments were repeats of the "early expansion"
methodology.
[0127] FIG. 28 shows the tests on PD0044 for producing the
genetically modified cells. For the first scale up PLAT-02 (Phase I
and Phase II trial) "pre-qual" from cryopreserved selected cells
were used.
[0128] FIG. 29 shows the tests on PD0046 for producing the
genetically modified cells from the scale up of PLAT-02 "Qual run
#1".
[0129] FIG. 30 shows the tests on PD0063 for producing the
genetically modified cells. From the scale up or PLAT-02 "Qual run
#2". Growth curves show TNC from both V-197 bags of cells together.
Bead removal and EGFRt enrichment occurred afterward. D+14 for CD4+
expressing T-cells and D+15 for CD8+ expressing T-cells. CD8+
expressing T-cells grown in IL-2 (50 U/mL)/IL-15 (0.5 ng/mL), CD4+
expressing T-cells grown in IL-7 (5 ng/mL)/IL-15 (0.5 ng/mL). Bead
removal and EGFRt enrichment occurred at D12 for CD4+ expressing
T-cells and D13 for CD8+ expressing T-cells.
[0130] FIGS. 31A-B show the expansion of cells from bulk PBMC
cultures when grown in the presence of cytokines. As shown, CD4+
expressing T-cells ( ) were grown in the presence of IL-2 and
IL-15. CD8+ expressing T-cells (.box-solid.) were grown in the
presence of IL-7 and IL-15.
[0131] FIGS. 32A-B show the expansion of enriched CD8+ and CD4+
expressing T-cells in cytokine mixtures. As shown in sample PD0044,
enriched CD8+ expressing T-cells were expanded in the presence of
IL-2 and IL-15. In sample PD0044, enriched CD4+ expressing T-cells
were expanded in the presence of IL-7 and IL-15 for over 20
days.
[0132] FIG. 33 shows expansion of enriched CD4+ and CD8+ expressing
T-cells using an earlier methodology as shown in the flow chart of
FIG. 18. Shown are experiments of cells in sample 14602-S01,
14602-S02, and 14602-S03/14602-S03-02.
[0133] FIG. 34 shows expansion of enriched CD4+ and CD8+ expressing
T-cells using an earlier methodology as shown in the flow chart of
FIG. 18. Shown are experiments of cells in sample
14602-S04/14602-S04-02, 14602-S05, 14602-S06 and
14602-S06-2/14602-S06-04.
[0134] FIG. 35 shows the expansion of enriched CD4+ and CD8+
expressing T-cells using the "Early Expansion methodology as shown
in the flow chart of FIG. 18. Shown are experiments of cells in
sample 14602-S07, 14602-S08, and 14602-S09.
[0135] FIG. 36 shows the expansion of cells in samples 14602-S10,
14602-S11, 14602-S12, and 14602-S13.
[0136] FIG. 37 shows the expansions of cells in samples 14602-S14,
14602-S15, and 14602-S16.
[0137] FIG. 38 shows the enriched CD4+ and CD8+ expressing T-cells
extended phenotypes after cell expansion.
[0138] FIGS. 39A-B shows the survival of a mouse injected with
cells from the sample PD00044 and PD00046. PD00046 cells were noted
for having expression of engraftment fitness markers (CD27, CD28,
CD127, and CD62L).
[0139] FIG. 40 shows the average tumor progression in mice treated
with cells from the PD0044 and PD0046 cell batches.
[0140] FIG. 41 shows the experimental set up of testing tumor
progression in mice treated with T-cells that are manufactured for
engraftment fitness.
[0141] FIG. 42 shows a comparison between CD4+ and CD8+ expressing
T-cells from the samples PD0051 and PD00055 on the day of
administration into animals and the cytokine growth conditions.
[0142] FIG. 43, shows a comparison of the three groups of mice that
were treated with PBS, PD0051 (normal expansion cells) and PD00055
(cells expanded in the presence of cytokine combinations)
[0143] FIG. 44, shows the T-cell persistence in peripheral blood of
the mice by detection of the EGFRt CAR marker.
[0144] FIG. 45 shows the experimental set up for testing three
groups of mice in order to determine an in vivo difference in
killing ability between cells that have been grown in various
cytokine conditions in vitro with repeat antigen encounters.
[0145] FIG. 46 shows the tumor progression of PD0051 and PD0055
treated mice following treatment from day 0 to day 120.
[0146] FIG. 47 shows PD0051 and PD0055 cells challenged with repeat
antigen exposure.
[0147] FIG. 48 shows JME13-29 repeated Raji Tumor challenge of
"normal expansion cells" versus T-cells pulsed with cytokine
combinations.
[0148] FIG. 49 shows the experimental set up to determine if there
is an in vivo difference in killing ability between cells that have
been grown under the same conditions as PLAT-01 (Phase I clinical
trial) and PLAT-02 (Phase I and Phase II clinical trial), as well
as, "in between" protocols.
[0149] FIG. 50 shows a table that indicates the cell products and
the levels of EGFRt from the CD8+ and CD4+ expressing T-cells.
[0150] FIG. 51 shows the average tumor progression after treatment
with T-cells at specific dose titrations.
[0151] FIG. 52 shows the PLAT comparisons within dosing groups of
T-cell treatments.
[0152] FIGS. 53A-E show the comparison of T-cells from the products
of PLAT-1.00, PLAT-1.33, PLAT-1.67 and PLAT-2.00 at similar dosing
concentrations. The figure illustrates the tumor progression of
individual animals. The start of each row shows the grouped for
each PLAT group with the various dose titrations, followed by the
individual groups teased out.
[0153] FIG. 54 shows the dose titration survival of mice treated
with T-cells from the products of PLAT-1.00, PLAT-1.33, PLAT-1.67
and PLAT-2.00. The figure illustrates the PLAT comparison within
dosing groups of cell treatments.
[0154] FIG. 55 shows the PLAT comparison of survival curves. The
figure illustrates the average tumor progression after treatment
with T-cells at specific doses.
[0155] FIG. 56 shows the survival of the mice in response to T-cell
treatment in which the T-cells were grown in normal expansion
methods or grown in the presence of cytokines. The figure also
shows if the mice developed xenoGraft Host disease after T-cell
treatment
[0156] FIG. 57 shows the survival of the mice in response to T-cell
treatment in which the T-cells were grown in normal expansion
methods or grown in the presence of cytokines. The figure also
shows if the mice developed xenoGraft Host disease after T-cell
treatment
[0157] FIGS. 58A-C show the survival of the mice in response to
T-cell treatment in which the T-cells were grown in normal
expansion methods or grown in the presence of cytokines. The
figures also show if the mice developed xenoGraft Host disease
after T-cell treatment
[0158] FIGS. 59A-H show the survival of the mice in response to
T-cell treatment in which the T-cells were grown in normal
expansion methods or grown in the presence of cytokines. The
figures also show if the mice developed xenoGraft Host disease
after T-cell treatment
[0159] FIGS. 60A-D show the survival of the mice in response to
T-cell treatment in which the T-cells were grown in normal
expansion methods or grown in the presence of cytokines. The
figures also show if the mice developed xenoGraft Host disease
after T-cell treatment.
[0160] FIG. 61 shows a table of JME14-03 Group N, at Day 51 post-T
cell treatment at 51 days and at 90 days.
DETAILED DESCRIPTION
[0161] The following definitions are provided to facilitate
understanding of several of the alternatives described herein.
[0162] As used herein, "a" or "an" can mean one or more than
one.
[0163] As used herein, "nucleic acid" or "nucleic acid molecule"
refers to polynucleotides or oligonucleotides such as
deoxyribonucleic acid (DNA) or ribonucleic acid (RNA),
oligonucleotides, fragments generated by the polymerase chain
reaction (PCR), and fragments generated by any of ligation,
scission, endonuclease action, exonuclease action, and by synthetic
generation. Nucleic acid molecules can be composed of monomers that
are naturally-occurring nucleotides (such as DNA and RNA), or
analogs of naturally-occurring nucleotides (e.g., enantiomeric
forms of naturally-occurring nucleotides), or a combination of
both. Modified nucleotides can have alterations in sugar moieties
and/or in pyrimidine or purine base moieties. Sugar modifications
include, for example, replacement of one or more hydroxyl groups
with halogens, alkyl groups, amines, and azido groups, or sugars
can be functionalized as ethers or esters. Moreover, the entire
sugar moiety can be replaced with sterically and electronically
similar structures, such as aza-sugars and carbocyclic sugar
analogs. Examples of modifications in a base moiety include
alkylated purines and pyrimidines, acylated purines or pyrimidines,
or other well-known heterocyclic substitutes. Nucleic acid monomers
can be linked by phosphodiester bonds or analogs of such linkages.
Analogs of phosphodiester linkages include phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the
like. The term "nucleic acid molecule" also includes so-called
"peptide nucleic acids," which comprise naturally-occurring or
modified nucleic acid bases attached to a polyamide backbone.
Nucleic acids can be either single stranded or double stranded.
[0164] "Genetically modify," as used herein, refers to a process
for modifying an organism or a cell such as a bacterium, T-cell,
bacterial cell, eukaryotic cell, insect, plant or mammal with
genetic material, such as nucleic acid, that has been altered using
genetic engineering techniques. For example, nucleic acid such as
DNA can be inserted in the host genome by first isolating and
copying the genetic material of interest using molecular cloning
methods to generate a DNA sequence, or by synthesizing the DNA, and
then inserting this construct into the host organism. Genes can
also be removed, or "knocked out", using a nuclease. Gene targeting
is a different technique that uses homologous recombination to
change an endogenous gene, and can be used to delete a gene, remove
exons, add a gene, or introduce point mutations.
[0165] Genetic modification performed by transduction is described
herein. "Transduction" refers to methods of transferring genetic
material, such as, for example, DNA or RNA, to a cell by way of a
vector. Common techniques use viral vectors, electroporation, and
chemical reagents to increase cell permeability. The DNA can be
transferred by a virus, or via a viral vector. As described herein,
methods are provided for modifying immune CD4+ and/or CD8+ T-cells.
In order to achieve high expression of therapeutic genes and/or
increase the amount of chimeric antigen receptors on a cell
surface, for example, T-cells can be transduced with genetic
material encoding a chimeric antigen receptor. T-cells can be
genetically modified using a virus. Viruses commonly used for gene
therapy are adenovirus, adeno-associated virus (AAV), retroviruses
and lentiviruses.
[0166] Various transduction techniques have been developed, which
utilize recombinant infectious virus particles for delivery of the
nucleic acid encoding a chimeric antigen receptor. This represents
a currently preferred approach to the transduction of T
lymphocytes. As described herein, the viral vectors used for
transduction can include virus vectors derived from simian virus
40, adenoviruses, adeno-associated virus (AAV), lentiviral vectors,
and retroviruses. Thus, gene transfer and expression methods are
numerous but essentially function to introduce and express genetic
material in mammalian cells. Several of the above techniques can be
used to transduce hematopoietic or lymphoid cells, including
calcium phosphate transfection, protoplast fusion, electroporation,
and infection with recombinant adenovirus, adeno-associated virus,
lentivirus, or retrovirus vectors. Primary T lymphocytes have been
successfully transduced by electroporation and by retroviral or
lentiviral infection. As such, retroviral and lentiviral vectors
can provide a highly efficient method for gene transfer in
eukaryotic cells. Retroviral and lentiviral vectors provide highly
efficient methods for gene transfer into T-cells. Moreover,
retroviral or lentiviral integration takes place in a controlled
fashion and results in the stable integration of one or a few
copies of the new genetic information per cell.
[0167] An "expression vector" or a vector, as described herein, is
a nucleic acid molecule encoding a gene that is expressed in a
host-cell. Typically, an expression vector comprises a
transcription promoter, a gene, and a transcription terminator.
Gene expression is usually placed under the control of a promoter,
and such a gene is said to be "operably linked to" the promoter.
Similarly, a regulatory element and a core promoter are operably
linked if the regulatory element modulates the activity of the core
promoter.
[0168] In some alternatives, a method of making genetically
modified T-cells, such as T-cells that are derived from thymocytes
or T-cells that are derived from engineered precursors, which have
a chimeric antigen receptor is provided, wherein the method
comprises separating, isolating, or enriching a CD8+ expressing
population of T-cells and/or a CD4+ expressing population of
T-cells from a mixed population of T-cells so as to generate an
isolated, separated, or enriched population of T-cells, stimulating
the isolated, separated or enriched population of T-cells so as to
generate a stimulated population of CD8+ expressing T-cells and/or
CD4+ expressing T-cells, transducing the stimulated population of
CD8+ expressing T-cells and/or CD4+ expressing T-cells with a
vector, wherein the vector encodes a chimeric antigen receptor and
a marker sequence, wherein said marker sequence encodes a cell
surface selectable marker, so as to generate a transduced
population of CD8+ expressing T-cells and/or CD4+ expressing
T-cells, contacting the transduced population of CD8+ expressing
T-cells and/or CD4+ expressing T-cells, for at least one day, such
as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, or 20 days or any time that is within a range of times defined
by any two of the aforementioned time points, with at least one
cytokine so as to generate a transduced, cytokine-stimulated
population of CD8+ expressing T-cells and/or CD4+ expressing
T-cells, enriching, separating, or isolating the transduced,
cytokine-stimulated population of CD8+ expressing T-cells and/or
CD4+ expressing T-cells by selection of the marker encoded by the
marker sequence so as to generate an enriched, separated or
isolated population of transduced, cytokine-stimulated CD8+
expressing T-cells and/or CD4+ expressing T-cells and propagating
the enriched, separated, or isolated population of transduced,
cytokine-stimulated CD8+ expressing T-cells and/or CD4+ expressing
T-cells for at least one day, such as 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or any time that
is within a range of times defined by any two of the aforementioned
time points, so as to obtain said genetically modified T-cells,
which have a chimeric antigen receptor. In some alternatives of the
method, the vector is a viral vector. In some alternatives of the
method, the viral vector is derived from simian virus 40,
adenoviruses, adeno-associated virus (AAV), lentivirus, or
retroviruses. In some alternatives of the method, the viral vector
is a recombinant adenovirus, adeno-associated virus, lentivirus or
retrovirus vector. In some alternatives of the method, the viral
vector is a lentivirus vector.
[0169] A "leader sequence" also known as the "5' untranslated
region (5' UTR), is the region of mRNA that is located upstream
from the initiation codon and is important in regulating the
translation of an mRNA transcript. In some alternatives of the
method of making genetically modified T-cells, which have a
chimeric antigen receptor, the vector encoding the chimeric antigen
receptor comprises a sequence encoding a leader sequence.
[0170] A "ligand" as described herein refers to a small molecule
that can form a complex with another molecule or biomolecule for a
biological purpose such as, for example, signal triggering. Binding
can occur through intermolecular forces, for example ionic bonds,
hydrogen bonds, and van der walls interactions. Ligand binding to a
receptor protein can alter the three dimensional structure and
determine its functional state.
[0171] By way of example and not of limitation, ligands can include
substrates, proteins, small molecules, inhibitors, activators and
neurotransmitters. The strength of binding of a ligand is referred
to as the binding affinity and can be determined by direct
interactions and solvent effects. A ligand can be bound by a
"ligand binding domain." A ligand binding domain can refer to a
conserved sequence in a structure that can bind a specific ligand.
Without being limiting, a ligand binding domain can be a specific
protein domain that is specific for a ligand or ligands.
[0172] Specific" or "Specificity" can refer to the characteristic
of a ligand for the binding partner or alternatively, the binding
partner for the ligand, and can include complementary shape, charge
and hydrophobic specificity for binding. Specificity for binding
can include stereospecificity, regioselectivity and
chemoselectivity.
[0173] A "signaling domain," also known as a "Co-stimulatory
domain" is an intracellular or cytoplasmic domain of a protein or a
receptor protein that interacts with the interior of the cells and
functions by relaying a signal. The portion of the protein that
resides in the intracellular portion of the cell is also referred
to as the "endodomain." This interaction can occur through the
intracellular domain communicating via specific protein-protein or
protein-ligand interactions with an effector molecule or an
effector protein, which in turn can send the signal along a signal
chain to its destination.
[0174] The signaling or co-stimulatory domain also refers to a
signaling moiety that provides to T-cells a signal which, in
addition to the primary signal provided by for instance the CD3
zeta chain of the TCR/CD3 complex, mediates a T-cell response, such
as, for example, an immune response, activation, proliferation,
differentiation, cytokine secretion, cytolytic activity, perforin
and/or granzyme activity and the like. A signaling or
co-stimulatory domain can include all or a portion of, but is not
limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, lymphocyte
function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C,
B7-H3, and/or a ligand that specifically binds with CD83.
[0175] "Selectable marker sequence," is a gene introduced into a
vector or a cell that confers a trait for artificial selection. A
selectable marker sequence or marker sequence can be a screenable
marker to allow a researcher to distinguish between wanted and
unwanted cells, or to enrich for a specific cell type. In some
alternatives of the method of making genetically modified T-cells,
which have a chimeric antigen receptor, a vector is provided
wherein the vector encodes a chimeric antigen receptor comprising a
marker sequence, wherein said marker sequence encodes a cell
surface selectable marker.
[0176] In some alternatives, a method of making genetically
modified T-cells, which have a chimeric antigen receptor is
provided, wherein the method comprises separating, isolating or
enriching a CD8+ expressing population of T-cells and/or a CD4+
expressing population of T-cells, such as T-cells that are derived
from thymocytes or T-cells that are derived from engineered
precursors, desirably iPS cells, from a mixed population of T-cells
so as to generate an isolated, separated, or enriched population of
T-cells, stimulating the isolated, separated or enriched population
of T-cells so as to generate a stimulated population of CD8+
expressing T-cells and/or CD4+ expressing T-cells, transducing the
stimulated population of CD8+ expressing T-cells and/or CD4+
expressing T-cells with a vector, wherein the vector encodes a
chimeric antigen receptor and a marker sequence, wherein said
marker sequence encodes a cell surface selectable marker, so as to
generate a transduced population of CD8+ expressing T-cells and/or
CD4+ expressing T-cells, contacting the transduced population of
CD8+ expressing T-cells and/or CD4+ expressing T-cells, for at
least one day, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, or 20 days or any time that is within a
range of times defined by any two of the aforementioned time
points, with at least one cytokine, which is preferably an
exogenously added cytokine e.g., in addition to any cytokine either
produced by the T-cells or present in the media, so as to generate
a transduced, cytokine-stimulated population of CD8+ expressing
T-cells and/or CD4+ expressing T-cells, separating, enriching, or
isolating the transduced, cytokine-stimulated population of CD8+
expressing T-cells and/or CD4+ expressing T-cells by selection of
the marker encoded by the marker sequence so as to generate an
enriched, separated, or isolated population of transduced,
cytokine-stimulated CD8+ expressing T-cells and/or CD4+ expressing
T-cells and propagating the enriched, separated, or isolated
population of transduced, cytokine-stimulated CD8+ expressing
T-cells and/or CD4+ expressing T-cells for at least one day, such
as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, or 20 days or any time that is within a range of times defined
by any two of the aforementioned time points, so as to obtain said
genetically modified T-cells, which have a chimeric antigen
receptor.
[0177] In some alternatives of the method, the vector further
comprises a first sequence encoding a leader sequence, a second
sequence encoding a ligand binding domain, a third sequence
encoding a signaling domain and a fourth sequence encoding a
selectable marker sequence. In some alternatives of the method, the
vector further comprises a sequence encoding a spacer. In some
alternatives of the method, the spacer comprises an IgG4 hinge. In
some alternatives of the method, the vector is a viral vector. In
some alternatives of the method, the viral vector is derived from
simian virus 40, adenoviruses, adeno-associated virus (AAV),
lentivirus, or retroviruses. In some alternatives of the method,
the viral vector is a recombinant adenovirus, adeno-associated
virus, lentivirus or retrovirus vector. In some alternatives of the
method, the viral vector is a lentivirus vector. In some
alternatives of the method, the marker sequence encodes a truncated
epidermal growth factor receptor (EGFRt). In some alternatives of
the method, the ligand binding domain of the chimeric antigen
receptor comprises an antibody, or a binding portion thereof. In
some alternatives of the method, the ligand binding domain of the
chimeric antigen receptor comprises a single chain variable
fragment (scFv), or a binding portion thereof. In some alternatives
of the method, the ligand binding domain of the chimeric antigen
receptor comprises FMC63, or a binding portion thereof. In some
alternatives of the method, the ligand binding domain of the
chimeric antigen receptor is specific for CD19. In some
alternatives of the method, the vector comprises a sequence
encoding a maker sequence. In some of these alternatives, the
marker sequence is a truncated epidermal growth factor receptor
(EGFRt).
[0178] "Codon optimization" as described herein, refers to the
design process of altering codons to codons known to increase
maximum protein expression efficiency. In some alternatives, codon
optimization for expression in human is described, wherein codon
optimization can be performed by using algorithms that are known to
those skilled in the art so as to create synthetic genetic
transcripts optimized for high mRNA and protein yield in humans.
Programs containing algorithms for codon optimization in humans are
readily available. Such programs can include, for example,
OptimumGene.TM. or GeneGPS.RTM. algorithms. Additionally human
codon optimized sequences can be obtained commercially, for
example, from Integrated DNA Technologies.
[0179] In some alternatives, a method of making genetically
modified T-cells, which have a chimeric antigen receptor is
provided, wherein the method comprises separating, isolating, or
enriching a CD8+ expressing population of T-cells and/or a CD4+
expressing population of T-cells, such as T-cells that are derived
from thymocytes or T-cells that are derived from engineered
precursors, desirably iPS cells, from a mixed population of T-cells
so as to generate an isolated, separated, or enriched population of
T-cells, stimulating the isolated, separated, or enriched
population of T-cells so as to generate a stimulated population of
CD8+ expressing T-cells and/or CD4+ expressing T-cells, transducing
the stimulated population of CD8+ expressing T-cells and/or CD4+
expressing T-cells with a vector, wherein the vector encodes a
chimeric antigen receptor and a marker sequence, wherein said
marker sequence encodes a cell surface selectable marker, so as to
generate a transduced population of CD8+ expressing T-cells and/or
CD4+ expressing T-cells, contacting the transduced population of
CD8+ expressing T-cells and/or CD4+ expressing T-cells, for at
least one day, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, or 20 days or any time that is within a
range of times defined by any two of the aforementioned time
points, with at least one cytokine, which is preferably an
exogenously added cytokine e.g., in addition to any cytokine either
produced by the T-cells or present in the media, so as to generate
a transduced, cytokine-stimulated population of CD8+ expressing
T-cells and/or CD4+ expressing T-cells, enriching, isolating, or
separating the transduced, cytokine-stimulated population of CD8+
expressing T-cells and/or CD4+ expressing T-cells by selection of
the marker encoded by the marker sequence so as to generate an
enriched, isolated, or separated population of transduced,
cytokine-stimulated CD8+ expressing T-cells and/or CD4+ expressing
T-cells and propagating the enriched, separated, or isolated
population of transduced, cytokine-stimulated CD8+ expressing
T-cells and/or CD4+ expressing T-cells for at least one day, such
as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, or 20 days or any time that is within a range of times defined
by any two of the aforementioned time points, so as to obtain said
genetically modified T-cells, which have a chimeric antigen
receptor. In some alternatives of the method, the vector further
comprises a first sequence encoding a leader sequence, a second
sequence encoding a ligand binding domain, a third sequence
encoding a signaling domain and a fourth sequence encoding a
selectable marker sequence. In some alternatives of the method, the
sequences of the vector are codon optimized for expression in
humans. In some alternatives, the sequences of the vector are
optimized to have selected codons specifically for maximal protein
expression in human cells, which can increase the concentration of
proteins or CARs of a T-cell.
[0180] Optimization can also be performed to reduce the occurrence
of secondary structure in a polynucleotide. In some alternatives of
the method, optimization of the sequences in the vector can also be
performed to reduce the total GC/AT ratio. Strict codon
optimization can lead to unwanted secondary structure or an
undesirably high GC content that leads to secondary structure. As
such, the secondary structures affect transcriptional efficiency.
Programs such as GeneOptimizer can be used after codon usage
optimization, for secondary structure avoidance and GC content
optimization. These additional programs can be used for further
optimization and troubleshooting after an initial codon
optimization to limit secondary structures that can occur after the
first round of optimization. Alternative programs for optimization
are readily available. In some alternatives of the method, the
vector comprises sequences that are optimized for secondary
structure avoidance and/or the sequences are optimized to reduce
the total GC/AT ratio and/or the sequences are optimized for
expression in humans.
[0181] Marker domains can also provide important aspects to
alternatives described herein. Utilization of a marker domain on
the cell surface can allow for transduced lymphocyte ablation in
the event of a toxic event associated with administration or the
presence of transduced T-cells. For example, in the case of an
EGFRt marker sequence, full length antibodies to EGFR can be
utilized to bind to cells expressing the EGFR and kill them via
antibody dependent cell mediated cytotoxicity (ADCC). In some
alternatives, an antibody specific for a marker domain can be
linked to a cytotoxic agent, such as a radionuclide or a toxin,
which results in a therapeutic capable of ablation or killing of
the transduced cells in vivo. In some alternatives, a marker
sequence is provided for enrichment and cell selection. In an
exemplary alternative described herein, an EGFRt marker sequence is
used in a purification and enrichment method allowing for
immunomagnetic selection against the EGFRt marker.
[0182] A "spacer" as described herein can refer to a polypeptide
chain that can range in length from a length of 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,
66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,
83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,
113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,
126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,
139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,
152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,
165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,
178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190,
191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,
204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,
217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,
230, 231, 232, 233, 234, 235, 236, 237, 238, 239 or 240 amino acids
or a length within a range defined by any two of the aforementioned
lengths. A spacer can comprise any 20 amino acids, for example, in
any order to create a desirable length of polypeptide chain in a
chimeric antigen receptor, which includes the amino acids arginine,
histidine, lysine, aspartic acid, glutamic acid, serine, threonine,
asparagine, glutamine, cysteine, glycine, proline, alanine, valine,
isoleucine, methionine, phenylalanine, tyrosine and/or tryptophan.
A spacer sequence can be a linker between the scFv and the
transmembrane domain of the chimeric antigen receptor. In some
alternatives of the method of making genetically modified T-cells,
which have a chimeric antigen receptor, the vector further
comprises a sequence encoding a spacer. In some alternatives of the
method, the spacer comprises a sequence with a length of 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,
63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,
80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,
111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,
124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,
137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,
150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162,
163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,
176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,
189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201,
202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214,
215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,
228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239 or 240
amino acids or a length within a range defined by any two of the
aforementioned lengths. In some alternatives of the method, the
spacer resides between the scFv and the transmembrane region of the
chimeric antigen receptor.
[0183] "IgG4 hinge" as described herein, refers to the polypeptide
domains that are between the heavy chain and the light chains of
the IgG4 antibody. In some alternatives, a method of making
genetically modified T-cells, which have a chimeric antigen
receptor is provided, wherein the method comprises separating,
enriching, or isolating a CD8+ expressing population of T-cells
and/or a CD4+ expressing population of T-cells, such as T-cells
that are derived from thymocytes or T-cells that are derived from
engineered precursors, desirably iPS cells, from a mixed population
of T-cells so as to generate an isolated, separated, or enriched
population of T-cells, stimulating the isolated, separated, or
enriched population of T-cells so as to generate a stimulated
population of CD8+ expressing T-cells and/or CD4+ expressing
T-cells, transducing the stimulated population of CD8+ expressing
T-cells and/or CD4+ expressing T-cells with a vector, wherein the
vector encodes a chimeric antigen receptor and a marker sequence,
wherein said marker sequence encodes a cell surface selectable
marker, so as to generate a transduced population of CD8+
expressing T-cells and/or CD4+ expressing T-cells, contacting the
transduced population of CD8+ expressing T-cells and/or CD4+
expressing T-cells, for at least one day, such as 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or any
time that is within a range of times defined by any two of the
aforementioned time points, with at least one cytokine, which is
preferably an exogenously added cytokine e.g., in addition to any
cytokine either produced by the T-cells or present in the media, so
as to generate a transduced, cytokine-stimulated population of CD8+
expressing T-cells and/or CD4+ expressing T-cells, enriching the
transduced, cytokine-stimulated population of CD8+ expressing
T-cells and/or CD4+ expressing T-cells by selection of the marker
encoded by the marker sequence so as to generate an enriched,
separated, or isolated population of transduced,
cytokine-stimulated CD8+ expressing T-cells and/or CD4+ expressing
T-cells and propagating the enriched population of transduced,
cytokine-stimulated CD8+ expressing T-cells and/or CD4+ expressing
T-cells for at least one day, such as 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or any time that
is within a range of times defined by any two of the aforementioned
time points, so as to obtain said genetically modified T-cells,
which have a chimeric antigen receptor.
[0184] In some alternatives of the method, the vector further
comprises a first sequence encoding a leader sequence, a second
sequence encoding a ligand binding domain, a third sequence
encoding a signaling domain and a fourth sequence encoding a
selectable marker. In some alternatives of the method, the vector
further comprises a sequence encoding a spacer. In some
alternatives, the spacer comprises a hinge region of a human
antibody. In some alternatives of the method, the spacer comprises
an IgG4 hinge. In some alternatives, the IgG4 hinge region is a
modified IgG4 hinge. A "modified IgG4 hinge" as described herein
can refer to a hinge region that can have at least 90%, 92%, 95%,
or 100% sequence identity or a sequence identity within a range
defined by any two of the aforementioned percentages, with a hinge
region amino acid sequence as set forth in SEQ ID NO: 1 (SEQ ID NO:
1; ESKYGPPCPPCP), SEQ ID NO: 2 (SEQ ID NO: 2; YGPPCPPCP), SEQ ID
NO: 3 (SEQ ID NO: 3; KYGPPCPPCP), or SEQ ID NO: 4 (SEQ ID NO: 4;
EVVKYGPPCPPCP). As mentioned above, any one or more of these
sequences can be codon optimized for expression in humans and any
one or more of these sequences can be optimized to reduce secondary
structure or GC/AT ratio and any one or more of these sequences can
be consensus sequences generated from at least two isotype
genes.
[0185] A "transmembrane domain" is a region of a protein that is
hydrophobic that can reside in the bilayer of a cell to anchor a
protein that is embedded to the biological membrane. Without being
limiting, the topology of the transmembrane domain can be a
transmembrane alpha helix. In some alternatives of the method of
making genetically modified T-cells, which have a chimeric antigen
receptor, the vector comprises a sequence encoding a transmembrane
domain. In some alternatives of the method, the transmembrane
domain comprises a CD28 transmembrane sequence or a fragment
thereof that is a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, or 28 amino acids or a length
within a range defined by any two of the aforementioned lengths. In
some alternatives of the method, the CD28 transmembrane sequence or
fragment thereof comprise 28 amino acids in length. In some
alternatives of the method, the transmembrane domain comprises the
sequence set forth in SEQ ID NO: 5 (SEQ ID NO: 5;
MFWVLVVVGGVLACYSLLVTVAFIIFWV).
[0186] A "signaling domain," also known as a "Co-stimulatory
domain" is an intracellular or cytoplasmic domain of a protein or a
receptor protein that interacts with the interior of the cells and
functions by relaying a signal. The portion of the protein that
resides in the intracellular portion of the cell is also referred
to as the "endodomain." This interaction can occur through the
intracellular domain communicating via specific protein-protein or
protein-ligand interactions with an effector molecule or an
effector protein, which in turn can send the signal along a signal
chain to its destination. The signaling or co-stimulatory domain
also refers to a signaling moiety that provides to T-cells a signal
which, in addition to the primary signal provided by for instance
the CD3 zeta chain of the TCR/CD3 complex, mediates a T-cell
response, such as, for example, an immune response, activation,
proliferation, differentiation, cytokine secretion, cytolytic
activity, perforin and/or granzyme activity and the like. A
signaling or co-stimulatory domain can include all or a portion of,
but is not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS,
lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,
NKG2C, and/or B7-H3, and/or a ligand that specifically binds with
CD83.
[0187] In some alternatives of the method of making genetically
modified T-cells, which have a chimeric antigen receptor, the
vector encoding the chimeric antigen receptor further comprises a
sequence for a co-stimulatory domain, wherein the co-stimulatory
domain is an intracellular signaling domain that interacts with
other intracellular mediators to mediate a cell response including
an immune response, activation, proliferation, differentiation,
cytokine secretion, cytolytic activity, perforin and/or granzyme
activity and the like. In some alternatives of the method, the
vector comprises a sequence encoding a signaling domain. In some
alternatives of the method, the signaling domain comprises a 4-1BB
domain and/or CD3-zeta domain. In some alternatives, the 4-1BB
domain comprises the sequence set forth in SEQ ID NO: 6 (SEQ ID NO:
6; KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL). In some
alternatives, the CD3-zeta domain comprises the sequence set forth
in SEQ ID NO: 7 (SEQ ID NO: 7;
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ
EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR).
[0188] A "chimeric antigen receptor" (CAR) described herein, also
known as chimeric T-cell receptor, refers to an artificial T-cell
receptor or a genetically engineered receptor, which grafts a
desired specificity onto an immune effector cell. These receptors
can be used to graft the specificity of a monoclonal antibody or a
binding portion thereof onto a T-cell, for example; with transfer
of the coding sequence for the CAR to a recipient cell being
facilitated by a retroviral vector. The structure of the CAR can
comprise single-chain variable fragments (scFv) derived from
monoclonal antibodies, fused to CD3-zeta transmembrane and
endodomain. Such molecules result in the transmission of a zeta
signal in response to recognition by the scFv of its target.
[0189] In some alternatives, a method of making genetically
modified T-cells, which have a chimeric antigen receptor is
provided, wherein the method comprises separating, isolating, or
enriching a CD8+ expressing population of T-cells and/or a CD4+
expressing population of T-cells, such as T-cells that are derived
from thymocytes or T-cells that are derived from engineered
precursors, desirably iPS cells, from a mixed population of
T-cells, from a mixed population of T-cells, so as to generate an
isolated, separated, or enriched population of T-cells, stimulating
the isolated, separated, or enriched population of T-cells so as to
generate a stimulated population of CD8+ expressing T-cells and/or
CD4+ expressing T-cells, transducing the stimulated population of
CD8+ expressing T-cells and/or CD4+ expressing T-cells with a
vector, wherein the vector encodes a chimeric antigen receptor and
a marker sequence, wherein said marker sequence encodes a cell
surface selectable marker, so as to generate a transduced
population of CD8+ expressing T-cells and/or CD4+ expressing
T-cells, contacting the transduced population of CD8+ expressing
T-cells and/or CD4+ expressing T-cells with at least one cytokine,
such as for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, or 20 days or any time that is within a range
of times defined by any two of the aforementioned time points, so
as to generate a transduced, cytokine-stimulated population of CD8+
expressing T-cells and/or CD4+ expressing T-cells, enriching,
separating or isolating the transduced, cytokine-stimulated
population of CD8+ expressing T-cells and/or CD4+ expressing
T-cells by selection of the marker encoded by the marker sequence
so as to generate an enriched, separated, or isolated population of
transduced, cytokine-stimulated CD8+ expressing T-cells and/or CD4+
expressing T-cells and propagating the enriched, separated, or
isolated population of transduced, cytokine-stimulated CD8+
expressing T-cells and/or CD4+ expressing T-cells for at least one
day, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, or 20 days or any time that is within a range of times
defined by any two of the aforementioned time points, so as to
obtain said genetically modified T-cells, which have a chimeric
antigen receptor. In some alternatives of the method, the chimeric
antigen receptor is specific for CD19.
[0190] These artificial T-cell receptors, or CARs, can be used as a
therapy for cancer or a viral infection using a technique called
"adoptive cell transfer." T-cells are removed from a patient and
modified so that they express receptors specific for a molecule
displayed on a cancer cell or a virus, or a virus-infected cell.
The genetically engineered T-cells, which can then recognize and
kill the cancer cells or the virus infected cells or promote
clearance of the virus, are reintroduced into the patient. In some
alternatives, a method of treating, inhibiting, or ameliorating a
disease in a subject in need thereof is provided.
[0191] In some aspects, the method can comprise administering to
the at least one composition or product combination, wherein the at
least one composition or product combination comprises a population
of genetically modified T-cells, wherein the population of
genetically modified T-cells comprises a plurality of affinity
selected CD8+ and/or CD4+ expressing T-cells, in the absence of,
enriched from, substantially separated from or substantially
isolated from CD8- and/or CD4- expressing T-cells, wherein said
plurality of affinity selected CD8+ and/or CD4+ expressing T-cells
have stimulated CD2, CD3, CD4 and/or CD28 receptors, wherein said
plurality of affinity selected CD8+ and/or CD4+ expressing T-cells
further comprise a gene encoding a chimeric antigen receptor and a
cell surface selectable marker and, wherein said plurality of
affinity selected CD8+ and/or CD4+ expressing T-cells have been
re-stimulated with at least one cytokine, such as for at least 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or
20 days or any time that is within a range of times defined by any
two of the aforementioned time points. In some alternatives, the at
least one composition or product combination is administered to
said subject by adoptive cell transfer.
[0192] An "antibody" as described herein refers to a large Y-shape
protein produced by plasma cells that is used by the immune system
to identify and neutralize foreign objects such as bacteria and
viruses. The antibody protein can comprise four polypeptide chains;
two identical heavy chains and two identical light chains connected
by disulfide bonds. Each chain is composed of structural domains
called immunoglobulin domains. These domains can contain 70-110
amino acids and are classified into different categories according
to their size and function. In some alternatives, the ligand
binding domain is an antibody fragment, desirably, a binding
portion thereof. In some alternatives, the antibody fragment or
binding portion thereof present on a CAR is specific for CD19.
[0193] A "single chain variable fragment" or scFv is a fusion
protein that comprises the variable regions of the heavy chain (VH)
and the light chains (VL) of an immunoglobulin that is connected
with a short linker peptide. Without being limiting, the linker can
comprise glycine for flexibility and hydrophilic amino acids, for
example serine or threonine for solubility. The linker can connect
the N-terminus of the VH with the C-terminus of the VL or it can
connect the C-terminus of the VH with the N-terminus of the VL. In
some alternatives, the ligand binding domain present on a CAR is a
single chain variable fragment (scFv). In some alternatives, the
scFv domain present on a CAR is specific for a CD19 present on a
tumor cell.
[0194] "FMC63" is a CD19 specific monoclonal antibody. CD19 is a
protein that is found on the surface of white blood cells and can
assemble with the antigen receptor of B lymphocytes in order to
decrease the threshold for antigen receptor-dependent stimulation.
CD19 is expressed on follicular dendritic cells and B cells. CD19
is present on B cells from earliest recognizable B-lineage cells
during development to B-cell blasts but is lost on maturation to
plasma cells. 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. As on T-cells,
several surface molecules form the antigen receptor and form a
complex on B lymphocytes.
[0195] Mutations in CD19 are associated with severe
immunodeficiency syndromes characterized by diminished antibody
production. For example, aberrant expression of CD19 is a marker of
monocytic lineage in acute myelogenous leukemia. Since CD19 is a
hallmark of B-cells, the protein can be used to diagnose cancers
that arise from this type of cell, notably B-cell lymphomas. Since
2011, treatments targeting CD19 have begun to enter trials. Most
current experimental anti-CD19 drugs in development work by
exploiting the presence of CD19 to direct treatment specifically
towards B-cell cancers. However, it is now emerging that the
protein plays an active role in driving the growth of these
cancers, by stabilizing the concentrations of the MYC oncoprotein.
Thus, CD19 and its downstream signaling can be attractive
therapeutic targets.
[0196] In some alternatives, a method of making genetically
modified T-cells, which have a chimeric antigen receptor is
provided, wherein the method comprises separating, enriching,
isolating, or purifying a population of CD8+ expressing T-cells
and/or a population of CD4+ expressing T-cells, such as T-cells
that are derived from thymocytes or T-cells that are derived from
engineered precursors, desirably iPS cells, from a mixed population
of T-cells, from a mixed population of T-cells, from a mixed
population of T-cells so as to generate an isolated, separated,
enriched, or purified population of T-cells, stimulating the
isolated, separated, enriched, or purified population of T-cells so
as to generate a stimulated population of CD8+ expressing T-cells
and/or CD4+ expressing T-cells, transducing the stimulated
population of CD8+ expressing T-cells and/or CD4+ expressing
T-cells with a vector, wherein the vector encodes a chimeric
antigen receptor and a marker sequence, wherein said marker
sequence encodes a cell surface selectable marker, so as to
generate a transduced population of CD8+ expressing T-cells and/or
CD4+ expressing T-cells, contacting the transduced population of
CD8+ expressing T-cells and/or CD4+ expressing T-cells with at
least one cytokine, such as for at least 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or any time that
is within a range of times defined by any two of the aforementioned
time points, so as to generate a transduced, cytokine-stimulated
population of CD8+ expressing T-cells and/or CD4+ expressing
T-cells, enriching the transduced, cytokine-stimulated population
of CD8+ expressing T-cells and/or CD4+ expressing T-cells by
selection of the marker so as to generate an enriched population of
transduced, cytokine-stimulated CD8+ expressing T-cells and/or CD4+
expressing T-cells and propagating the enriched population of
transduced, cytokine-stimulated CD8+ expressing T-cells and/or CD4+
expressing T-cells for at least one day, such as 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or any
time that is within a range of times defined by any two of the
aforementioned time points, so as to obtain said genetically
modified T-cells, which have a chimeric antigen receptor.
[0197] In some alternatives of the method, the vector further
comprises a first sequence encoding a leader sequence, a second
sequence encoding a ligand binding domain, a third sequence
encoding a signaling domain and a fourth sequence encoding a
selectable marker sequence. In some alternatives of the method, the
vector further comprises a sequence encoding a spacer. In some
alternatives of the method, the ligand binding domain of the
chimeric antigen receptor comprises an antibody, or a binding
portion thereof. In some alternatives of the method, the ligand
binding domain of the chimeric antigen receptor comprises a single
chain variable fragment (scFv), or a binding portion thereof. In
some alternatives of the method, the ligand binding domain of the
chimeric antigen receptor comprises FMC63, or a binding portion
thereof. In some alternatives of the method, the ligand binding
domain of the chimeric antigen receptor is specific for CD19.
[0198] "Purification" of T-cells as described herein, refers to the
isolation of highly purified, functional T-cells for research or
for methods for generating T-cells for therapy. "Isolation" of
T-cells, or "separating" of T-cells, as described herein, refers to
a procedure of isolating or separating the desired T-cells, T-cell
populations, or subpopulations from contaminating cell populations
or other components, for example by using techniques, such as
indirect panning. In this method, cells can be isolated, separated,
or selected by their capacity to bind to an antibody that is
attached to a support, such as a plastic or poly carbonate surface,
bead, particle, plate, or well. Cells can bind on the basis of
particular cell surface markers. In some cases the desired T-cell
populations are "enriched" meaning that the population of the
desired T-cells is greater after enrichment than that of the native
population from which the T-cells originated.
[0199] In some alternatives of the method of making a genetically
modified T-cell, the CD8+ expressing population of T-cells and/or
CD4+ expressing population of T-cells are isolated, enriched,
purified or separated from undesired components using CD8 specific
antibodies and/or CD4 specific antibodies. In some alternatives of
the method, the CD8+ expressing population of T-cells and/or CD4+
expressing population of T-cells are isolated, enriched, or
purified using flow cytometry. In some alternatives, the CD8+
expressing population of T-cells and/or CD4+ expressing population
of T-cells are isolated, enriched, purified, or separated using
immunomagnetic selection. In some alternatives of the method, the
CD8+ expressing population of T-cells and/or CD4+ expressing
population of T-cells are isolated, enriched, purified, or
separated using antibodies against CD45 RA, CD45 RO, CCR7, CD25,
CD127, CD57, CD137, CD27, CD28, CD8, CD4, and/or CD62L. In some
alternatives of the method, the CD8+ expressing population of
T-cells and/or CD4+ expressing population of T-cells are isolated,
enriched, purified, or separated by using antibodies against CD27,
CD28 and/or CD62L. In some alternatives, the method is performed
with isolated, enriched, or separated CD4+ expressing T-cells in
the absence of or in greater abundance than the amount of CD8+
expressing cells in the mixed population of T-cells used for the
isolation, enrichment, or purification. In some alternatives, the
method is performed with isolated, enriched, or separated CD8+ in
the absence of or in greater abundance than the amount of CD4+
expressing cells in the mixed population of T-cells used for the
isolation, enrichment, or purification.
[0200] "Stimulation" or activation of T-cells refers to the method
of inducing a T-cell to initiate a response, such as a signal
transduction response, e.g., proliferation, while preserving T-cell
viability and immune function. For example, simultaneous signaling
to TCR/CD3 and CD8 can trigger a physiological activation and
expansion of human T-cells. Following appropriate stimulation,
T-cells may proliferate extensively in vitro. In some alternatives,
a method of making genetically modified T-cells, which have a
chimeric antigen receptor is provided wherein the method comprises
isolating, separating, enriching, or purifying a CD8+ expressing
population of T-cells and/or a CD4+ expressing population of
T-cells, such as T-cells that are derived from thymocytes or
T-cells that are derived from engineered precursors, desirably iPS
cells, from a mixed population of T-cells, so as to generate an
isolated, separated, purified, or enriched population of T-cells,
stimulating the isolated, separated, purified, or enriched
population of T-cells so as to generate a stimulated population of
CD8+ expressing T-cells and/or CD4+ expressing T-cells, transducing
the stimulated population of CD8+ expressing T-cells and/or CD4+
expressing T-cells with a vector, wherein the vector encodes a
chimeric antigen receptor and a marker sequence, wherein said
marker sequence encodes a cell surface selectable marker, so as to
generate a transduced population of CD8+ expressing T-cells and/or
CD4+ expressing T-cells, contacting the transduced population of
CD8+ expressing T-cells and/or CD4+ expressing T-cells with at
least one cytokine, such as for at least 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or any time that
is within a range of times defined by any two of the aforementioned
time points, so as to generate a transduced, cytokine-stimulated
population of CD8+ expressing T-cells and/or CD4+ expressing
T-cells, enriching, separating, or isolating the transduced,
cytokine-stimulated population of CD8+ expressing T-cells and/or
CD4+ expressing T-cells by selection of the marker encoded by the
marker sequence so as to generate an enriched population of
transduced, cytokine-stimulated CD8+ T-cells and/or CD4+ T-cells
and propagating the enriched, isolated, or separated population of
transduced, cytokine-stimulated CD8+ expressing T-cells and/or CD4+
expressing T-cells for at least one day, such as for at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20
days or any time that is within a range of times defined by any two
of the aforementioned time points, so as to obtain said genetically
modified T-cells, which have a chimeric antigen receptor. In some
alternatives, the stimulating is performed with an antibody-bound
support, such as a bead or particle. In some alternatives, the
stimulating is performed with an antibody-bound support comprising
anti-TCR, anti-CD2, anti-CD3, anti-CD4 and/or anti-CD28 antibodies.
In some alternatives, the stimulating is performed with an
antibody-bound support comprising anti-CD3 and/or anti-CD28
antibodies. In some alternatives, the method further comprises
removing the antibody-bound support, such as beads or
particles.
[0201] "T-cells" or "T lymphocytes" as used herein can be from any
mammal, preferably a primate, including monkeys or humans, a
companion animal such as a dog, cat, or horse, or a domestic
animal, such as a sheep, goat, or cattle. In some alternatives the
T-cells are allogeneic (from the same species but different donor)
as the recipient subject; in some alternatives the T-cells are
autologous (the donor and the recipient are the same); in some
alternatives the T-cells arc syngeneic (the donor and the
recipients are different but are identical twins).
[0202] "CD8+ expressing T-cell" or "CD8+ T-cell," used synonymously
throughout, is also known as a TC, cytotoxic T lymphocyte, CTL,
T-killer cell, cytolytic T-cell or killer T-cell. As described
herein, CD8+ T-cells are T-lymphocytes that can kill cancer cells,
virally infected cells, or damaged cells. CD8+ T-cells express
T-cell receptors (TCRs) that can recognize a specific antigen. CD8+
T-cells express CD8 on the surface. CD8+ expressing T-cells have
the ability to make some cytokines, however the amounts of
cytokines made by CD8+ T-cells are not at a concentration that
promotes, improves, contributes to, or induces engraftment
fitness.
[0203] "CD4+ expressing T-cell," or "CD4+ T-cell," used
synonymously throughout, is also known as T helper cells, which
play an important role in the immune system, and in the adaptive
immune system. CD4+ T-cells also help the activity of other immune
cells by releasing T-cell cytokines. These cells help, suppress or
regulate immune responses. They are essential in B cell antibody
class switching, in the activation and growth of cytotoxic T-cells,
and in maximizing bactericidal activity of phagocytes, such as
macrophages. CD4+ expressing T-cells have the ability to make some
cytokines, however the amounts of cytokines made by CD4+ T-cells
are not at a concentration that promotes, improves, contributes to,
or induces engraftment fitness.
[0204] Mature T cells express the surface protein CD4 and are
referred to as CD4+ T-cells. CD4+ T-cells are generally treated as
having a pre-defined role as helper T-cells within the immune
system. For example, when an antigen-presenting cell expresses an
antigen on MHC class II, a CD4+ cell will aid those cells through a
combination of cell to cell interactions (e.g. CD40 and CD40L) and
through cytokines Nevertheless, there are rare exceptions; for
example, sub-groups of regulatory T-cells, natural killer cells,
and cytotoxic T-cells express CD4. All of the latter CD4+
expressing T-cell groups are not considered T helper cells.
[0205] "Central memory" T-cell (or "T.sub.CM") as used herein
refers to an antigen experienced CTL that expresses CD62L or CCR-7
and CD45RO on the surface thereof, and does not express or has
decreased expression of CD45RA as compared to naive cells. In some
alternatives, central memory cells are positive for expression of
CD62L, CCR7, CD28, CD127, CD45RO, and/or CD95, and have decreased
expression of CD54RA as compared to naive cells.
[0206] "Effector memory" T-cell (or "T.sub.EM") as used herein
refers to an antigen experienced T-cell that does not express or
has decreased expression of CD62L on the surface thereof as
compared to central memory cells, and does not express or has
decreased expression of CD45RA as compared to naive cell. In some
alternatives, effector memory cells are negative for expression of
CD62L and/or CCR7, as compared to naive cells or central memory
cells, and have variable expression of CD28 and/or CD45RA.
[0207] "Naive" T-cells as used herein refers to a non-antigen
experienced T lymphocyte that expresses CD62L and/or CD45RA, and/or
does not express CD45RO- as compared to central or effector memory
cells. In some alternatives, naive CD8+ T lymphocytes are
characterized by the expression of phenotypic markers of naive
T-cells including CD62L, CCR7, CD28, CD127, and/or CD45RA.
[0208] "Effector" "T.sub.E" T-cells as used herein refers to a
antigen experienced cytotoxic T lymphocyte cells that do not
express or have decreased expression of CD62L, CCR7, CD28, and are
positive for granzyme B and/or perforin, as compared to central
memory or naive T-cells.
[0209] "Engraftment fitness" as described herein, refers to the
ability of a cell to grow and proliferate after the cells have
entered the body, e.g., blood stream, through adoptive transfer.
Engraftment can usually occur within two to four weeks after the
transfer. Engraftment can be monitored by checking blood counts for
a specific cell on a frequent basis. In some alternatives of the
method of treating, inhibiting, or ameliorating a disease in a
subject is provided, the method can comprise administering a
composition or product combination comprising the genetically
modified T-cells, as described herein. In some alternatives, the
method can further comprise monitoring the subject by checking the
blood counts for the genetically modified T-cells that expresses a
chimeric antigen receptor e.g., by identifying the presence or
absence of a marker associated with the transferred T-cells.
[0210] T-cells with improved engraftment fitness may have specific
markers on the cell surface that confer generation and long term
maintenance of T-cell immunity. There are several proteins that are
known for T-cell activation and survival. CD28 is a protein
expressed on T-cells that provide co-stimulatory signals required
for T-cell activation and survival. CD27 is required for generation
and long-term maintenance of T-cell immunity. It binds to ligand
CD70, and plays a key role in regulating B-cell activation and
immunoglobulin synthesis. L-selectin, also known as CD62L is a cell
adhesion molecule found on lymphocytes. L-selectin functions as a
"homing receptor" for lymphocytes or T-cells to enter secondary
lymphoid tissues via high endothelial venues. Ligands present on
endothelial cells will bind to lymphocytes expressing L-selectin,
slowing lymphocyte trafficking through the blood, and can
facilitate entry into a secondary lymphoid organ at that point. In
some alternatives, of the method of making genetically modified
T-cells, which have a chimeric antigen receptor, the T-cells
comprise at least one receptor that promotes, induces, improves, or
contributes to engraftment fitness. In some alternatives, the at
least one receptor is CD45 RA, CD45 RO, CCR7, CD25, CD127, CD57,
CD137, CD27, CD28 and/or CD62L. In some alternatives, the at least
one receptor is CD27, CD28 and/or CD62L. In some alternatives
provided herein, methods are described in which CD4+ and CD8+
expressing T-cells are genetically modified by transduction of
genetic material in the form of a vector such that the genetically
modified CD4+ and CD8+ expressing T-cells expresses a specific
chimeric antigen receptor. In some alternatives of the genetically
modified CD4+ and CD8+ T-cells, the genetically modified CD4+ and
CD8+ T-cells are further modified to improve or enhance engraftment
fitness.
[0211] "Cytokines" as described herein, refers to small proteins
(5-25 kDa) that are important in cell signaling. Cytokines are
released by cells and affect the behavior of other cells, and
sometimes the releasing cell itself, such as a T-cell. Cytokines
can include, for example, chemokines, interferons, interleukins,
lymphokines, and tumor necrosis factor. Cytokines can be produced
by a broad range of cells, which can include, for example, immune
cells like macrophages, B lymphocytes, T lymphocytes and mast
cells, as well as, endothelial cells, fibroblasts, and various
stromal cells.
[0212] Cytokines can act through receptors, and are important in
the immune system as the cytokines can modulate the balance between
humoral and cell-based immune responses, and they can regulate the
maturation, growth, and responsiveness of particular cell
populations. Some cytokines enhance or inhibit the action of other
cytokines in complex ways. Without being limiting, cytokines can
include, for example, Acylation stimulating protein, Adipokine,
Albinterferon, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16,
CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24,
CCL25, CCL26, CCL27, CCL28, CCL3, CCL5, CCL6, CCL7, CCL8, CCL9,
Chemokine, Colony-stimulating factor, CX3CL1, CX3CR1, CXCL1,
CXCL10, CXCL11, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CXCL2,
CXCL3, CXCL5, CXCL6, CXCL7, CXCL9, Erythropoietin, Gc-MAF,
Granulocyte colony-stimulating factor, Granulocyte macrophage
colony-stimulating factor, Hepatocyte growth factor, IL 10 family
of cytokines, IL 17 family of cytokines, IL1A, IL1B, Inflammasome,
Interferome, Interferon, Interferon beta 1a, Interferon beta 1b,
Interferon gamma, Interferon type I, Interferon type II, Interferon
type III, Interleukin, Interleukin 1 family, Interleukin 1 receptor
antagonist, Interleukin 10, Interleukin 12, Interleukin 12 subunit
beta, Interleukin 13, Interleukin 15, Interleukin 16, Interleukin
2, Interleukin 23, Interleukin 23 subunit alpha, Interleukin 34,
Interleukin 35, Interleukin 6, Interleukin 7, Interleukin 8,
Interleukin 36, Leukemia inhibitory factor, Leukocyte-promoting
factor, Lymphokine, Lymphotoxin, Lymphotoxin alpha, Lymphotoxin
beta, Macrophage colony-stimulating factor, Macrophage inflammatory
protein, Macrophage-activating factor, Monokine, Myokine,
Myonectin, Nicotinamide phosphoribosyltransferase, Oncostatin M,
Oprelvekin, Platelet factor 4, Proinflammatory cytokine,
Promegapoietin, RANKL, Stromal cell-derived factor 1, Talimogene
laherparepvec, Tumor necrosis factor alpha, Tumor necrosis factors,
XCL1, XCL2, GM-CSF, and/or XCR1. In some alternatives of the method
of making genetically modified T-cells, a transduced population of
CD8+ expressing T-cells and/or CD4+ expressing T-cells is contacted
with at least one cytokine so as to generate a transduced,
cytokine-stimulated population of CD8+ T-cells and/or CD4+ T-cells.
In some alternatives of the method, the at least one cytokine
utilized comprises GM-CSF, IL-7, IL-12, IL-15, IL-18, IL-2, and/or
IL-21. In some alternatives, the period of contact with the
cytokine is at least one day, such as for at least 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or
any time that is within a range of times defined by any two of the
aforementioned time points.
[0213] "Interleukins" or IL as described herein, are cytokines that
the immune system depends largely upon. Examples of interleukins,
which can be utilized herein, for example, include IL-1, IL-2,
IL-3, IL-4, IL-5, IL-6, 11-7, IL-8/CXCL8, IL-9, IL-10, IL-11,
IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20,
IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29,
IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, and/or IL-36. Contacting
T-cells with interleukins can have effects that promote, support,
induce, or improve engraftment fitness of the cells. IL-1, for
example can function in the maturation & proliferation of
T-cells. IL-2, for example, can stimulate growth and
differentiation of T-cell response. IL-3, for example, can promote
differentiation and proliferation of myeloid progenitor cells.
IL-4, for example, can promote proliferation and differentiation.
IL-7, for example, can promote differentiation and proliferation of
lymphoid progenitor cells, involved in B, T, and NK cell survival,
development, and homeostasis. IL-15, for example, can induce
production of natural killer cells. IL-21, for example,
co-stimulates activation and proliferation of CD8+ T-cells,
augments NK cytotoxicity, augments CD40-driven B cell
proliferation, differentiation and isotype switching, and promotes
differentiation of Th17 cells.
[0214] In some alternatives, a method of making a genetically
modified T-cell is provided, wherein the method comprises
purifying, separating, enriching, or isolating a CD8+ population of
T-cells and/or a CD4+ population of T-cells from a mixed population
of T-cells, such as T-cells that are derived from thymocytes or
T-cells that are derived from engineered precursors, desirably iPS
cells, from a mixed population of T-cells, so as to generate an
isolated, separated, enriched, or purified population of T-cells,
stimulating the isolated, separated, enriched, or purified
population of T-cells so as to generate a stimulated population of
CD8+ T-cells and/or CD4+ T-cells, transducing the stimulated
population of CD8+ T-cells and/or CD4+ T-cells with a vector,
wherein the vector encodes a chimeric antigen receptor and a marker
sequence, wherein said marker sequence encodes a cell surface
selectable marker, so as to generate a transduced population of
CD8+ T-cells and/or CD4+ T-cells, contacting the transduced
population of CD8+ T-cells and/or CD4+ T-cells with at least one
cytokine so as to generate a transduced, cytokine-stimulated
population of CD8+ T-cells and/or CD4+ T-cells, enriching,
isolating, or separating the transduced, cytokine-stimulated
population of CD8+ T-cells and/or CD4+ T-cells by selection of the
marker encoded by the marker sequence so as to generate an
enriched, isolated or separated population of transduced,
cytokine-stimulated CD8+ T-cells and/or CD4+ T-cells and
propagating the enriched, isolated, or separated population of
transduced, cytokine-stimulated CD8+ T-cells and/or CD4+ T-cells
for at least one day, such as for at least 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or any time
that is between a range of times defined by any two of the
aforementioned time points, so as to obtain said genetically
modified T-cells, which have a chimeric antigen receptor. In some
alternatives, the at least one cytokine comprises GM-CSF, IL-7,
IL-12, IL-18, IL-15, IL-2, and/or IL-21. In some alternatives, the
at least one cytokine comprises IL/7, IL-15 and/or IL-21. In some
alternatives, the at least one cytokine comprises IL-2, IL-15
and/or IL-21. In some alternatives of the method, the cytokine
contacting is performed for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, or 20 days or a period of time within a
range defined by any two of these time periods. In some
alternatives, the addition of the at least one cytokine improves,
enhances, promotes, or induces engraftment fitness. In some
alternatives, the enriched CD4+ expressing T-cells are contacted,
for example propagated, in 5 ng/mL recombinant human IL-7 (rhIL-7)
and/or 0.5 ng/mL recombinant human IL-15 (rhIL-15). In some
alternatives, the enriched CD8+ expressing T-cells are contacted,
for example propagated, in 50 U/mL recombinant human IL-2 (rhIL-2)
and/or 0.5 ng/mL recombinant human IL-15 (rhIL-15). In more
alternatives, the enriched CD4+ expressing T-cells are contacted,
for example propagated, in 0.1 ng/mL, 0.2 ng/mL, 0.3 ng/mL, 0.4
ng/mL, 0.5 ng/mL, 0.6 ng/mL, 0.7 ng/mL, 0.8 ng/mL, 0.9 ng/mL, or
1.0 ng/mL rhIL-7 or in an amount that is within a range defined by
any two of the aforementioned amounts of rhIL-7 and/or 0.1 ng/mL,
0.2 ng/mL, 0.3 ng/mL, 0.4 ng/mL, 0.5 ng/mL, 0.6 ng/mL, 0.7 ng/mL,
0.8 ng/mL, 0.9 ng/mL, or 1.0 ng/mL rhIL-15 or in an amount that is
within a range defined by any two of the aforementioned amounts of
rhIL-15. In more alternatives, the enriched CD8+ expressing T-cells
are contacted, for example propagated, in 10 U/mL, 20 U/mL, 30
U/mL, 40 U/mL, 50 U/mL, 60 U/mL, 70 U/mL, 80 U/mL, 90 U/mL, or 100
U/mL of rhIL-2 or in an amount that is within a range defined by
any two of the aforementioned amounts of rhIL-2 and/or 0.1 ng/mL,
0.2 ng/mL, 0.3 ng/mL, 0.4 ng/mL, 0.5 ng/mL, 0.6 ng/mL, 0.7 ng/mL,
0.8 ng/mL, 0.9 ng/mL, or 1.0 ng/mL rhIL-7 and/or 0.1 ng/mL, 0.2
ng/mL, 0.3 ng/mL, 0.4 ng/mL, 0.5 ng/mL, 0.6 ng/mL, 0.7 ng/mL, 0.8
ng/mL, 0.9 ng/mL, or 1.0 ng/mL rhIL-15 or in an amount that is
within a range defined by any two of the aforementioned amounts of
rhIL-15. In some alternatives the aforementioned cytokines and
amounts are co-administered to the T-cells, e.g., in a mixture or
added shortly after one another, and in other alternatives, the
aforementioned cytokines are contacted with the T cells separately,
e.g., separated by a time period of 1-10 minutes or 1-10 hours.
[0215] "Enriched" and "depleted" are also used herein to describe
amounts of cell types in a mixture refers to the subjecting of the
mixture of the cells to a process or step, which results in an
increase in the number of the "enriched" type and a decrease in the
number of the "depleted" cells. Thus, depending upon the source of
the original population of cells subjected to the enriching
process, a mixture or composition can contain 60, 70, 80, 90, 95,
or 99 percent or more, or any value within a range defined by any
two of these values (in number or count) of the "enriched" cells
and 40, 30, 20, 10, 5 or 1 percent or less or any value within a
range defined by any two of these values (in number or count) of
the "depleted" cells. In some alternatives of the method of making
genetically modified T-cells, enriching the transduced,
cytokine-stimulated population of CD8+ T-cells and/or CD4+ T-cells
by affinity selection of a cell surface marker is contemplated,
such that an enriched population of transduced, cytokine-stimulated
CD8+ T-cells and/or CD4+ T-cells is generated.
[0216] "Propagating cells" or propagation refers to steps to allow
proliferation, expansion, growth and reproduction of cells. For
example, cultures of CD8+ T-cells and CD4+ T-cells can typically be
incubated under conditions that are suitable for the growth and
proliferation of T lymphocytes. In some alternatives of the method
of making genetically modified T-cells, which have a chimeric
antigen receptor, the CD4+ expressing T-cells are propagated for at
least 1 day and may be propagated for 20 days, such as 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days
or for a period that is within a range defined by any two of the
aforementioned time periods. In some alternatives of the method of
making genetically modified T-cells, which have a chimeric antigen
receptor, the CD8+ expressing T-cells are propagated for at least 1
day and may be propagated for 20 days, such as 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or for a
period that is within a range defined by any two of the
aforementioned time periods.
[0217] "Affinity selection," as described herein, refers to the
selection of a specific molecule or cell having a selectable cell
surface marker by binding to the molecule or marker or an epitope
present thereon with a binding affinity agent, which allows for one
to select out the specific molecule or cell of interest. Affinity
selection can be performed by, for example, antibodies, conjugated
antibodies, lectins, aptamers, and/or peptides. In some
alternatives, of the method of making genetically modified T-cells,
the separating of the CD8+ population of T-cells and/or a CD4+
population of T-cells from a mixed population of T-cells is
performed by affinity selection for T-cells having an epitope
present on CD8 and/or CD4. In some alternatives of the method,
anti-CD8 or anti-CD4 antibodies or binding portions thereof are
used to select out the cells of interest. In some alternatives of
the method, the separating of the CD8+ population of T-cells and/or
a CD4+ population of T-cells from a mixed population of T-cells is
performed by flow cytometry. In some alternatives of the method,
the separating of the CD8+ population of T-cells and/or a CD4+
population of T-cells from a mixed population of T-cells is
performed by immuno-magnetic selection. In some alternatives of the
method, the anti-CD8 or the anti-CD4 antibodies are conjugated to a
solid support such as, for example, an inert bead or an inert
particle.
[0218] "T cell precursors" as described herein refers to lymphoid
precursor cells that can migrate to the thymus and become T cell
precursors, which do not express a T cell receptor. All T cells
originate from hematopoietic stem cells in the bone marrow.
Hematopoietic progenitors (lymphoid progenitor cells) from
hematopoietic stem cells populate the thymus and expand by cell
division to generate a large population of immature thymocytes. The
earliest thymocytes express neither CD4 nor CD8, and are therefore
classed as double-negative (CD4 CD8) cells. As they progress
through their development, they become double-positive thymocytes
(CD4.sup.+CD8.sup.+), and finally mature to single positive
(CD4.sup.+CD8.sup.- or CD4.sup.+CD8.sup.+) thymocytes that are then
released from the thymus to peripheral tissues. About 98% of
thymocytes die during the development processes in the thymus by
failing either positive selection or negative selection, whereas
the other 2% survive and leave the thymus to become mature
immunocompetent T cells.
[0219] The double negative (DN) stage of the precursor T cell is
focused on producing a functional .beta.-chain whereas the double
positive (DP) stage is focused on producing a functional
.alpha.-chain, ultimately producing a functional .alpha..beta. T
cell receptor. As the developing thymocyte progresses through the
four DN stages (DN1, DN2, DN3, and DN4), the T cell expresses an
invariant .alpha.-chain but rearranges the .beta.-chain locus. If
the rearranged .beta.-chain successfully pairs with the invariant
.alpha.-chain, signals are produced which cease rearrangement of
the .beta.-chain (and silence the alternate allele) and result in
proliferation of the cell. Although these signals require this
pre-TCR at the cell surface, they are dependent on ligand binding
to the pre-TCR. These thymocytes will then express both CD4 and CD8
and progresses to the double positive (DP) stage where selection of
the .alpha.-chain takes place. If a rearranged .beta.-chain does
not lead to any signaling (e.g. as a result of an inability to pair
with the invariant .alpha.-chain), the cell may die by neglect
(lack of signaling).
[0220] "Hematopoietic stem cells" or "HSC" as described herein, are
precursor cells that can give rise to myeloid cells such as, for
example, macrophages, monocytes, macrophages, neutrophils,
basophils, eosinophils, erythrocytes, megakaryocytes/platelets,
dendritic cells and lymphoid lineages (such as, for example,
T-cells, B-cells, NK-cells). HSCs have a heterogeneous population
in which three classes of stem cells exist, which are distinguished
by their ratio of lymphoid to myeloid progeny in the blood
(L/M).
[0221] "Cryopreservation" is a process of preserving cells, whole
tissues, or substances susceptible to damage by cooling to sub-zero
temperatures. At the low temperatures any enzymatic or chemical
activity, which can cause damage to the cells, tissue or substances
in question are effectively stopped. Cryopreservation methods seek
to reach low temperatures without causing additional damage caused
by the formation of ice during freezing. Traditional
cryopreservation has relied on coating the material to be frozen
with a class of molecules termed cryoprotectants. New methods are
constantly being investigated due to the inherent toxicity of many
cryoprotectants. Cryopreservation methods are known to those
skilled in the art. In some alternatives of the method of making
genetically modified T-cells, which have a chimeric antigen
receptor, the method can further comprise cryopreserving the
genetically modified T-cells.
[0222] In some alternatives, a population of genetically modified
T-cells is provided, wherein the population of genetically modified
T-cells, such as T-cells that are derived from thymocytes or
T-cells that are derived from engineered precursors, desirably iPS
cells, comprises a plurality of affinity selected CD8+ and/or CD4+
expressing T-cells, in the absence of, enriched over, or isolated
from CD8- and/or CD4- T-cells, wherein said plurality of affinity
selected CD8+ and/or CD4+ T-cells have stimulated CD2, CD3, CD4
and/or CD28 receptors, wherein said plurality of affinity selected
CD8+ and/or CD4+ T-cells further comprise a gene encoding a
chimeric antigen receptor and a cell surface selectable marker and,
wherein said plurality of affinity selected CD8+ and/or CD4+
expressing T-cells have been re-stimulated with at least one
cytokine, such as for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, or 20 days or any time that is
within a range of times defined by any two of the aforementioned
time points. In some alternatives, the selected CD8+ and/or CD4+
T-cells are generated by methods provided herein. In some
alternatives, the at least one cytokine comprises GM-CSF, IL-7,
IL-12, IL-15, IL-18, IL-2, and/or IL-21. In some alternatives, the
at least one cytokine comprises IL/7, IL-15 and/or IL-21. In some
alternatives, the at least one cytokine comprises IL-2, IL-15
and/or IL-21. In some alternatives of the population of genetically
modified T-cells, the plurality of affinity selected CD8+ and/or
CD4+ T-cells further comprise at least one receptor that promotes,
induces, enhances or contribute to engraftment fitness. In some
alternatives, the at least one receptor that promotes, enhances,
induces, or contributes to engraftment fitness is CD45 RA, CD45 RO,
CCR7, CD25, CD127, CD57, CD137, CD27, CD28 and/or CD62L. In some
alternatives, the at least one receptor that promotes, enhances,
contributes to, or induces engraftment fitness is CD27, CD28 and/or
CD62L. In some alternatives, the plurality of affinity selected
CD8+ and/or CD4+ expressing T-cells further comprises a vector
having a first sequence encoding a leader sequence, a second
sequence encoding a ligand binding domain, a third sequence
encoding a signaling domain and a fourth sequence encoding a
selectable marker. In some alternatives, the vector further
comprises a sequence encoding a spacer. In some alternatives, the
spacer comprises an IgG4 hinge. In some alternatives, the vector is
a viral vector. In some alternatives, the viral vector is derived
from simian virus 40, adenoviruses, adeno-associated virus (AAV),
lentivirus, or retroviruses. In some alternatives, the viral vector
is a recombinant adenovirus, adeno-associated virus, lentivirus or
retrovirus vector. In some alternatives, the viral vector is a
lentivirus vector. In some alternatives, the cell surface
selectable marker codes for a truncated epidermal growth factor
receptor (EGFRt). In some alternatives, the ligand binding domain
comprises an antibody, or a binding portion thereof. In some
alternatives, the ligand binding domain comprises a single chain
variable fragment (scFv), or a binding portion thereof. In some
alternatives, the ligand binding domain comprises FMC63, or a
binding portion thereof. In some alternatives, the ligand binding
domain is specific for CD19. In some alternatives, the population
comprises isolated or enriched CD8+ expressing T-cells in the
absence of, enriched over, substantially depleted of CD4+ T-cells.
In some alternatives, the population comprises isolated or enriched
CD4+ T-cells in the absence of, enriched over, or substantially
depleted of CD8+ T-cells.
[0223] In some alternatives, the adoptive cellular immunotherapy
compositions are useful in the treatment of a disease or cancer. In
some alternatives, a composition or product combination for human
therapy is provided, wherein the composition or product combination
comprises a pharmaceutical excipient and at least one population of
genetically modified T-cells according to any one or more of the
alternative T-cells or population of genetically modified T-cells
prepared or obtained as described herein. In some alternatives of
the composition or product combination for human therapy, the
composition or product combination comprises a population of
genetically modified T-cells, such as T-cells that are derived from
thymocytes or T-cells that are derived from engineered precursors,
desirably iPS cells, wherein the population comprises isolated CD8+
T-cells in the absence of enriched over, substantially depleted of
CD4+ T-cells. In some alternatives of the composition or product
combination for human therapy, the composition or product
combination comprises a population of genetically modified T-cells,
such as T-cells that are derived from thymocytes or T-cells that
are derived from engineered precursors, desirably iPS cells,
wherein the population comprises isolated CD4+ T-cells in the
absence of, enriched over, or substantially depleted of CD8+
T-cells. In some alternatives of the composition or product
combination for human therapy, the composition or product
combination comprises a combination of isolated CD8+ T-cells in the
absence of, enriched over, or substantially depleted of CD4+
T-cells and isolated CD4+ T-cells absence of, enriched over, or
substantially depleted of CD8+ T-cells in a 1:1 ratio. In some
alternatives, the population of genetically modified T-cells are
mixed and administered or co-administered as separate formulations
to a subject in need thereof in a CD4+ T-cell to CD8+ T-cell ratio
that is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or a
ratio within a range defined by any two of these ratios. In some
alternatives, the population of genetically modified T-cells are
mixed and administered or co-administered as separate formulations
to a subject in need thereof in a CD8+ T-cell to CD4+ T-cell ratio
that is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or a
ratio within a range defined by any two of these ratios.
[0224] "Pharmaceutical excipient," or pharmaceutical vehicle as
described herein can refer to a carrier or inert medium used as a
solvent in which the medicinally active agent or T-cells for
treatment is formulated and or administered. Vehicles can include
polymeric micelles, liposomes, lipoprotein-based carriers,
nano-particle carriers, dendrimers, and/or other vehicles for
T-cells that are known to one skilled in the art. An ideal vehicle
or excipient can be non-toxic, biocompatible, non-immunogenic,
biodegradable, and can avoid recognition by the host's defense
mechanisms.
[0225] In some alternatives, a composition or product combination
for human therapy is provided, wherein the composition or product
combination comprises a pharmaceutical excipient and at least one
population of genetically modified T-cells of any of the
alternatives described herein. In some alternatives, the excipients
are pharmaceutical vehicles. In some alternatives, the
pharmaceutical vehicles include pharmaceutical compositions.
[0226] Acute lymphoblastic leukemia (ALL) or acute lymphoid
leukemia is a cancer of the white blood cells, characterized by the
overproduction of cancerous, immature white blood cells, also known
as lymphoblasts. In patients with ALL, lymphoblasts are
overproduced in the bone marrow and continuously multiply, causing
damage and death by inhibiting the production of normal cells, such
as, for example, red and white blood cells and platelets, in the
bone marrow and by spreading to other organs. ALL is most common in
childhood with a peak incidence at 2-5 years of age, and another
peak at an older age.
[0227] The symptoms of ALL are indicative of a reduced production
of functional blood cells, because the leukemia wastes the
resources of the bone marrow, which are normally used to produce
new, functioning blood cells. Symptoms can include fever, increased
risk of infection, increased tendency to bleed, anemia,
tachycardia, fatigue and headache. Between 50 to 70% of children
and 40-50% of adults who achieve complete remission after initial
therapy but then suffer a relapse can be able to go into a second
complete remission. Treatment for relapse after a first remission
can be standard chemotherapy or experimental drugs, or more
aggressive treatments such as stem cell transplants. Treatment for
relapse after a first remission can be standard chemotherapy or
experimental drugs, or more aggressive treatments such as stem cell
transplants. However, for others such as acute myeloid leukemia and
ALL the reduced mortality of the autogenous relative to allogeneic
hematopoietic stem cell transplantation (HSCT) can be outweighed by
an increased likelihood of cancer relapse and related mortality,
and therefore the allogeneic treatment can be preferred for those
conditions. As such methods are needed to improve the cells for
treatment of leukemia, myeloid leukemia and ALL.
[0228] In some alternatives, a method of treating, inhibiting, or
ameliorating a disease, such as a cancer, such as ALL, in a subject
in need thereof is provided, wherein the method comprises
administering to the subject at least one composition or product
combination of any one or more of the alternatives described
herein, wherein the composition or product combination comprises
genetically modified T-cells, which have a chimeric antigen
receptor, wherein the T-cells are transduced, cytokine-stimulated
CD8+ T-cells and/or CD4+ T-cells. In some alternatives of the
method, the method comprises administering a composition or product
combination, wherein the composition or product combination
comprises a population of genetically modified T-cells comprising
isolated CD8+ T-cells absence of, enriched over, or substantially
depleted of CD4+ T-cells. In some alternatives of the method, the
method comprises administering a composition or product
combination, wherein the composition or product combination
comprises a population of genetically modified T-cells comprising
isolated CD4+ T-cells absence of, enriched over, or substantially
depleted of CD8+ T-cells. In some alternatives, the method further
comprises administering a composition or product combination,
wherein the composition or product combination comprises a
population of genetically modified T-cells comprising isolated CD4+
T-cells absence of, enriched over, or substantially depleted of
CD8+ T-cells. In some alternatives of the method, the method
further comprises administering a composition or product
combination, wherein the composition or product combination
comprises a population of genetically modified T-cells comprising
isolated CD8+ T-cells absence of, enriched over, or substantially
depleted of CD4+ T-cells. In some alternatives of the method, the
method further comprises administering the composition or product
combination, wherein the composition or product combination
comprises isolated CD8+ T-cells absence of, enriched over, or
substantially depleted of CD4+ T-cells and isolated CD4+ T-cells
absence of, enriched over, or substantially depleted of CD8+
T-cells in a 1:1 ratio. In some alternatives, the population of
genetically modified T-cells are mixed and administered or
co-administered as separate formulations to a subject in need
thereof in a CD4+ T-cell to CD8+ T-cell ratio that is 1:1, 1:2,
1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or a ratio within a range
defined by any two of these ratios. In some alternatives, the
population of genetically modified T-cells are mixed and
administered or co-administered as separate formulations to a
subject in need thereof in a CD8+ T-cell to CD4+ T-cell ratio that
is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or a ratio
within a range defined by any two of these ratios. In some
alternatives, the subject is identified or selected to receive an
anti-cancer therapy. In some alternatives, the method further
comprises measuring or evaluating an inhibition of a disease. In
some alternatives, the method further comprises providing said
subject an additional anti-cancer therapy before, during, or after
administration of the composition or product combination of any one
or more of the alternatives described herein. In some alternatives
of the method, the composition or product combination of any one or
more of the alternatives described herein, are administered to said
subject by adoptive cell transfer. In some alternatives, the
composition or product combination of any one or more of the
alternatives described herein, are administered to said subject
after said subject has received another form of anti-cancer
therapy. In some alternatives, the composition or product
combination of any one or more of the alternatives described
herein, are administered to said subject after said subject has
received another form of anti-cancer therapy. In some alternatives,
the subject is suffering from leukemia. In some alternatives, the
subject has recurrent and/or refractory CD19+ childhood acute
lymphoblastic leukemia (ALL). In some alternatives of the method,
the subject has recurrent and/or chemotherapy refractory CD19+
acute lymphoblastic leukemia (ALL). In some alternatives of the
method, the subject is suffering from an autoimmune disease. In
some alternatives of the method, the subject is suffering from a
post-HSCT relapse.
Additional Alternatives
Vectors, Cells and Methods of Transducing Cells
[0229] The compositions described herein provide for CD4+ and/or
CD8+ expressing T lymphocytes, such as T-cells that are derived
from thymocytes or T-cells that are derived from engineered
precursors, desirably iPS cells. T lymphocytes can be collected in
accordance with known techniques and enriched or depleted by known
techniques, such as affinity binding to antibodies such as flow
cytometry and/or immunomagnetic selection. After enrichment and/or
depletion steps, in vitro expansion of the desired T lymphocytes
can be carried out in accordance with known techniques (including
but not limited to those described in U.S. Pat. No. 6,040,177 to
Riddell et al., hereby expressly incorporated by reference in its
entirety). In some alternatives, the T-cells are autologous T-cells
i.e., obtained from the patient to which the cells are delivered.
In some alternatives, the T cells are precursor T cells. In some
alternatives, the precursor T cells are a hematopoietic stem cells
(HSC).
[0230] For example, the desired T-cell population or subpopulation
can be expanded by adding an initial T lymphocyte population to a
culture medium in vitro, and then adding to the culture medium
feeder cells, such as non-dividing peripheral blood mononuclear
cells (PBMC), (e.g., such that the resulting population of cells
contains at least 5, 10, 20, or 40 or more PBMC feeder cells for
each T lymphocyte in the initial population to be expanded); and
incubating the culture (e.g. for a time sufficient to expand the
numbers of T-cells). The non-dividing feeder cells can comprise
gamma-irradiated PBMC feeder cells. In some alternatives, the PBMC
are irradiated with gamma rays at 3000, 3200, 3300, 3400, 3500, or
3600 rads to prevent cell division, or within a range of rads
defined by any two of the aforementioned rads. The order of
addition of the T-cells and feeder cells to the culture media can
be reversed if desired. The culture can be incubated under
conditions that are suitable for the growth of T lymphocytes. For
the growth of human T lymphocytes, for example, the temperature
will generally be at least 25 degrees Celsius, preferably at least
30 degrees, more preferably 37 degrees, or any other temperature
within a range defined by any two of these temperatures.
[0231] The T lymphocytes expanded include CD8+ cytotoxic T
lymphocytes (CTL) and CD4+ helper T lymphocytes that can be
specific for an antigen present on a human tumor or a pathogen and
express a chimeric antigen receptor.
[0232] In another alternative, the expansion method or propagation
can further comprise adding non-dividing EBV-transformed
lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated
with gamma rays in the range of 6000, 7000, 8000, 9000, or 10,000
rads, or within a range of rads defined by any two of the
aforementioned rads. The LCL feeder cells can be provided in any
suitable amount, such as a ratio of LCL feeder cells to initial T
lymphocytes of at least 10:1.
[0233] In another alternative, the expansion method or propagation
can further comprise adding anti-CD3 and/or anti CD28 antibody to
the culture medium (e.g., at a concentration of at least 0.5
ng/ml). In another alternative, the method of making genetically
modified T-cells, which have a chimeric antigen receptor method can
further comprise adding IL-2, IL-15, and/or IL-21 to the culture
medium (e.g., wherein the concentration of IL-2 is at least 10
units/ml). In another alternative, the method of making genetically
modified T-cells, which have a chimeric antigen receptor method can
further comprise adding IL-7, IL-15, and/or IL21 to the culture
medium (e.g., wherein the concentration of IL-2 is at least 10
units/ml). After isolation of T lymphocytes, both cytotoxic and
helper T lymphocytes can be sorted into naive, memory, and effector
T-cell subpopulations either before or after expansion.
[0234] CD8+ cells can also be obtained by using standard methods.
In some alternatives, CD8+ cells are further sorted into naive,
central memory, and effector memory cells by identifying cell
surface antigens that are associated with each of those types of
CD8+ cells. In some alternatives, memory T-cells are present in
both CD62L+ and CD62L- subsets of CD8+ peripheral blood
lymphocytes. PBMC are sorted into CD62L-CD8+ and CD62L+CD8+
fractions after staining with anti-CD8 and anti-CD62L antibodies.
In some alternatives, the expression of phenotypic markers of
central memory T.sub.CM include CD45RO, CD62L, CCR7, CD28, CD3,
and/or CD127 and/or are negative or low for granzyme B and/or
CD45RA. In some alternatives, central memory T-cells are CD28+,
CD27+, CD45RO+, CD62L+, and/or CD8+ T-cells. In some alternatives,
naive CD8+ T lymphocytes are characterized by the expression of
phenotypic markers of naive T-cells including CD62L, CCR7, CD27,
CD28, CD3, CD127, and/or CD45RA.
[0235] Whether a cell or cell population is positive for or
expresses a particular cell surface marker can be determined by
flow cytometry using a specific antibody that is specific for the
surface marker and an isotype matched control antibody. A cell
population negative for a marker refers to the absence of
significant binding of the cell population with the specific
antibody above the isotype control, indicative of a lack of
expression of said marker; positive refers to uniform binding of
the cell population above the isotype control indicative of the
expression of the marker. In some alternatives, a decrease in
expression of one or markers refers to loss of 1 log 10 in the mean
fluorescence intensity and/or decrease of percentage of cells that
exhibit the marker of at least 20% of the cells, 25% of the cells,
30% of the cells, 35% of the cells, 40% of the cells, 45% of the
cells, 50% of the cells, 55% of the cells, 60% of the cells, 65% of
the cells, 70% of the cells, 75% of the cells, 80% of the cells,
85% of the cells, 90% of the cell, 95% of the cells, or 100% of the
cells or any % within a range of % s defined by any two of these
values when compared to a reference cell population. In some
alternatives, a cell population positive for one or markers refers
to a percentage of cells that exhibit the marker of at least 50% of
the cells, 55% of the cells, 60% of the cells, 65% of the cells,
70% of the cells, 75% of the cells, 80% of the cells, 85% of the
cells, 90% of the cell, 95% of the cells, or 100% of the cells or
any % within a range of % s defined by any two of these values when
compared to a reference cell population.
[0236] CD4+ T helper cells are sorted into naive, central memory,
and effector cells by identifying cell populations that have cell
surface antigens. CD4+ lymphocytes can be obtained by standard
methods. In some alternatives, naive CD4+ T lymphocytes are
CD45RO-, CD45RA+, CD62L+, CD27+, CD28+, and/or CD4+ T-cells. In
some alternatives, central memory CD4+ cells are CD62L+ and/or
CD45RO+. In some alternatives, effector CD4+ cells are CD62L-
and/or CD45RO-. In some alternatives, effector CD4+ cells are
CD28+, CD27+ and/or CD62L+.
[0237] In some alternatives, populations of CD4+ and CD8+ that are
antigen specific can be obtained by stimulating naive or antigen
specific T lymphocytes with antigen. For example, antigen-specific
T-cell lines or clones can be generated to Cytomegalovirus antigens
by isolating T-cells from infected subjects and stimulating the
cells in vitro with the same antigen. Naive T-cells can also be
used. Any number of antigens from tumor cells can be utilized as
targets to elicit T-cell responses. In some alternatives, the
adoptive cellular immunotherapy compositions are useful in the
treatment of a disease or disorder including a solid tumor,
hematologic malignancy, breast cancer or melanoma.
Modification of T Lymphocyte Populations
[0238] In some alternatives it can be desired to introduce
functional genes into the T-cells to be used in immunotherapy in
accordance with the present disclosure. For example, the introduced
gene or genes can improve the efficacy of therapy by promoting the
viability and/or function of transferred T-cells; or they can
provide a genetic marker to permit selection and/or evaluation of
in vivo survival or migration; or they can incorporate functions
that improve the safety of immunotherapy, for example, by making
the cell susceptible to negative selection in vivo as described by
Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell
et al., Human Gene Therapy 3:319-338 (1992); see also the
publications of PCT/US91/08442 and PCT/US94/05601 by Lupton et al.
describing the use of bifunctional selectable fusion genes derived
from fusing a dominant positive selectable marker with a negative
selectable marker, both references hereby expressly incorporated by
reference in their entireties. This can be carried out in
accordance with known techniques (see, e.g., U.S. Pat. No.
6,040,177 to Riddell et al. at columns 14-17, hereby expressly
incorporated by reference in its entirety) or variations thereof
that will be apparent to those skilled in the art based upon the
present disclosure. In some alternatives, the T cells are precursor
T cells. In some alternatives, the precursor T cells are a
hematopoietic stem cells (HSC).
[0239] In some alternatives, T-cells are modified with chimeric
receptors, as described herein. In some alternatives, the T-cells
are obtained from the subject to be treated, in other alternatives,
the lymphocytes are obtained from allogeneic human donors,
preferably healthy human donors. In some alternatives, the T cells
are precursor T cells. In some alternatives, the precursor T cells
are a hematopoietic stem cells (HSC).
[0240] In some alternatives, chimeric receptors comprise a ligand
binding domain that specifically binds to surface molecule on a
cell, a polypeptide spacer region, a transmembrane domain and an
intracellular signaling domain as described herein. In some
alternatives, the ligand binding domain is a single-chain antibody
fragment (scFv) that is derived from the variable heavy (VH) and
variable light (VL) chains of a monoclonal antibody (mAb).
Co-stimulatory signals can also be provided through the chimeric
receptor by fusing the co-stimulatory domain of CD28 and/or 4-1BB
to the CD3.xi. chain. Chimeric receptors are specific for cell
surface molecules independent from HLA, thus overcoming the
limitations of TCR-recognition including HLA-restriction and low
levels of HLA-expression on tumor cells.
[0241] In some alternatives, the same or a different chimeric
receptor can be introduced into each of population of CD4+ and CD8+
T lymphocytes. In some alternatives, the chimeric receptor in each
of these populations has a ligand binding domain that specifically
binds to the same ligand on the cell. The cellular signaling
modules can differ. In some alternatives, the intracellular
signaling domain of the CD8+ cytotoxic T-cells is the same as the
intracellular signaling domain of the CD4+ helper T-cells. In other
alternatives, the intracellular signaling domain of the CD8+
cytotoxic T-cells is different than the intracellular signaling
domain of the CD4+ helper T-cells. In some alternatives, an
anti-CD19 CAR is introduced into one population of lymphocytes and
an EGFR specific CAR is introduced into another population of
lymphocytes.
[0242] In some alternatives each of the CD4 or CD8 T lymphocytes
are sorted in to naive, central memory, effector memory or effector
cells prior to transduction, as described herein. In some
alternatives, each of the CD4 or CD8 T lymphocytes are sorted into
naive, central memory, effector memory, or effector cells after
transduction.
[0243] Various transduction techniques have been developed, which
utilize recombinant infectious virus particles for gene delivery.
This represents a currently preferred approach to the transduction
of T lymphocytes described herein. The viral vectors, which have
been used in this way include virus vectors derived from simian
virus 40, adenoviruses, adeno-associated virus (AAV), lentiviral
vectors, and retroviruses. Thus, gene transfer and expression
methods are numerous but essentially function to introduce and
express genetic material in mammalian cells. Several of the above
techniques have been used to transduce hematopoietic or lymphoid
cells, including calcium phosphate transfection, protoplast fusion,
electroporation, and infection with recombinant adenovirus,
adeno-associated virus and retrovirus vectors. Primary T
lymphocytes have been successfully transduced by electroporation
and by retroviral or lentiviral infection.
[0244] Retroviral and lentiviral vectors provide a highly efficient
method for gene transfer into eukaryotic cells. Moreover,
retroviral or lentiviral integration takes place in a controlled
fashion and results in the stable integration of one or a few
copies of the new genetic information per cell.
[0245] In some alternatives it can be useful to include in the
T-cells a positive marker that enables the selection of cells of
the negative selectable phenotype in vitro. The positive selectable
marker can be a gene that upon being introduced into the host cell
expresses a dominant phenotype permitting positive selection of
cells carrying the gene. Genes of this type are known in the art,
and include, inter alia, hygromycin-B phosphotransferase gene (hph)
which confers resistance to hygromycin B, the amino glycoside
phosphotransferase gene (neo or aph) from Tn5 which codes for
resistance to the antibiotic G418, the dihydrofolate reductase
(DHFR) gene, the adenosine deaminase gene (ADA), and the multi-drug
resistance (MDR) gene. In some alternatives, the positive marker is
an EGFR truncated protein for cell selection.
[0246] A variety of methods can be employed for transducing T
lymphocytes, as is well known in the art. In some alternatives,
transduction is carried out using lentiviral vectors. In some
alternatives, CD4+ and CD8+ cells each can separately be modified
with an expression vector encoding a chimeric receptor to form
defined populations. In some alternatives, these cells are then
further sorted into subpopulations of naive, central memory and
effector cells as described above by sorting for cell surface
antigens unique to each of those cell populations.
[0247] In some alternatives, CD4+ and CD8+ cells that proliferate
in response to antigen or tumor targets are selected. For example,
CD4+ cells that proliferate vigorously when stimulated with antigen
or tumor targets as compared to sham transduced cells, or CD8+
transduced cells are selected. In some alternatives, CD4+ and CD8+
cells are selected that are cytotoxic for antigen bearing cells. In
some alternatives, CD4+ are expected to be weakly cytotoxic as
compared to CD8+ cells.
[0248] In another alternative, transduced lymphocytes, such as CD8+
central memory cells, are selected that provide for cell killing in
vivo using an animal model established for the particular type of
cancer. Such animal models are known to those of skill in the art
and exclude human beings. As described herein, not all chimeric
receptor constructs transduced into lymphocytes confer the ability
to kill tumor cells in vivo despite the ability to become activated
and kill cells in vitro.
[0249] The disclosure contemplates that combinations of CD4+ and
CD8+ T-cells will be utilized in compositions. In one alternative,
combinations of chimeric receptor transduced CD4+ cells can be
combined with chimeric receptor transduced CD8+ cells of the same
ligand specificity or combined with CD8+ T-cells that are specific
for a distinct tumor ligand. In other alternatives, chimeric
receptor transduced CD8+ cells are combined with chimeric receptor
transduced CD4+ cells specific for a different ligand expressed on
the tumor. In yet another alternative, chimeric receptor modified
CD4+ and CD8+ cells are combined. In some alternatives CD8+ and
CD4+ cells can be combined in different ratios for example, a 1:1
ratio of CD8+ and CD4+, a ratio of 10:1 of CD8+ to CD4+, or a ratio
of 100:1 of CD8+ to CD4+ or any other ratio within a range defined
by any two of the aforementioned ratio values. In some
alternatives, the combined population is tested for cell
proliferation in vitro and/or in vivo, and the ratio of cells that
provides for proliferation of cells is selected.
[0250] As described herein, the disclosure contemplates that CD4+
and CD8+ cells can be further separated into subpopulations, such
as naive, central memory, and effector memory cell populations. As
described herein, in some alternatives, naive CD4+ cells are
CD45RO-, CD45RA+, CD62L+, CD28+, CD27+, and/or CD4+ positive
T-cells. In some alternatives, central memory CD4+ cells are CD62L
positive and/or CD45RO positive. In some alternatives, effector
CD4+ cells are CD62L negative and/or CD45RO positive. Each of these
populations can be independently modified with a chimeric receptor.
In some alternatives, central memory CD4+ cells are CD62L+, CD28+
and/or CD27+.
[0251] After transduction and/or selection for chimeric receptor
bearing cells, the cell populations are preferably expanded in
vitro until a sufficient number of cells are obtained to provide
for at least one infusion into a human subject, typically around
10.sup.4 cells/kg to 10.sup.9 cells/kg. In some alternatives, the
transduced cells are cultured in the presence of antigen bearing
cells, anti CD3, anti CD28, IL 2, IL-7, IL 15, and/or IL-21 and/or
combinations thereof. In some alternatives, the T-cells are
stimulated in the presence of an antibody-bound support, such as a
bead or particle. In some alternatives, the T-cells are stimulated
with an antibody-bound support, wherein the antibody-bound support
comprises anti-TCR, anti-CD2, anti-CD3, anti-CD4 and/or anti-CD28
antibodies. In some alternatives, the T-cells are stimulated with
an antibody-bound support, wherein the antibody-bound support
comprises anti-CD3 and/or anti-CD28 antibodies.
[0252] Each of the subpopulations of CD4+ and CD8+ cells can be
combined with one another. In a specific alternative, modified
naive or central memory CD4+ cells are combined with modified
central memory CD8+ T-cells to provide a synergistic cytotoxic
effect on an antigen bearing cells, such as a B-cell comprising
CD19 on the cell surface.
Compositions
[0253] The disclosure provides for an adoptive cellular
immunotherapy composition comprising a genetically modified T
lymphocyte cell preparation as described herein.
[0254] In some alternatives, the T lymphocyte cell preparation
comprises CD4+ T-cells that have a chimeric receptor comprising an
extracellular antibody variable domain specific for a ligand
associated with the disease or disorder, a customizable spacer
region, a transmembrane domain, and an intracellular signaling
domain of a T-cell receptor or other receptors as described herein.
In other alternatives, an adoptive cellular immunotherapy
composition further comprises a chimeric receptor modified
tumor-specific CD8+ cytotoxic T lymphocyte cell preparation that
provides a cellular immune response, wherein the cytotoxic T
lymphocyte cell preparation comprises CD8+ T-cells that have a
chimeric receptor comprising an extracellular single chain antibody
specific for a ligand associated with the disease or disorder, a
customizable spacer region, a transmembrane domain, and an
intracellular signaling domain of a T-cell receptor, as described
herein.
[0255] In some alternatives, an adoptive cellular immunotherapy
composition comprises a chimeric receptor modified tumor-specific
CD8+ cytotoxic T lymphocyte cell preparation that provides a
cellular immune response, wherein the cytotoxic T lymphocyte cell
preparation comprises CD8+ T-cells that have a chimeric receptor
comprising an extracellular single chain antibody specific for a
ligand associated with the disease or disorder, a customizable
spacer region, a transmembrane domain, and an intracellular
signaling domain of a T-cell receptor, in combination with an
antigen-reactive chimeric receptor modified naive CD4+ T helper
cell derived from CD45RO- CD62L+ and/or CD4+ T-cells, and a
pharmaceutically acceptable carrier.
[0256] In other alternatives, an adoptive cellular immunotherapy
composition comprises an antigen specific CD8+ cytotoxic T
lymphocyte cell preparation that provides a cellular immune
response derived from the patient combined with an antigen-reactive
chimeric receptor modified naive CD4+ T helper cell that augments
the CD8+ immune response, wherein the helper T lymphocyte cell
preparation comprises CD4+ T-cells that have a chimeric receptor
comprising an extracellular antibody variable domain specific for
the antigen associated with the disease or disorder, a customizable
spacer region, a transmembrane domain, and an intracellular
signaling domain of a T-cell receptor.
[0257] In a further alternative, an adoptive cellular immunotherapy
composition comprises an antigen-reactive chimeric receptor
modified naive CD4+ T helper cell that augments the CD8+ immune
response, wherein the helper T lymphocyte cell preparation
comprises CD4+ T-cells that have a chimeric receptor comprising an
extracellular antibody variable domain specific for a ligand
associated with a disease or disorder, a customizable spacer
region, a transmembrane domain, and an intracellular signaling
domain of a T-cell receptor.
[0258] In some alternatives, the CD4+ T helper lymphocyte cell is
selected from the group consisting of naive CD4+ T-cells, central
memory CD4+ T-cells, effector memory CD4+ T-cells, and bulk CD4+
T-cells. In some alternatives, CD4+ helper lymphocyte cell is a
naive CD4+ T-cell, wherein the naive CD4+ T-cell comprises a
CD45RO-, CD45RA+, CD62L+CD28+, CD27+, and/or CD4+. In some
alternatives, the CD8+ T cytotoxic lymphocyte cell is selected from
the group consisting of naive CD8+ T-cells, central memory CD8+
T-cells, effector memory CD8+ T-cells and bulk CD8+ T-cells. In
some alternatives, the CD8+ cytotoxic T lymphocyte cell is a
central memory T-cell wherein the central memory T-cell comprises a
CD45RO+, CD62L+, CD27+, CD28+ and/or CD8+. In yet other
alternatives, the CD8+ cytotoxic T lymphocyte cell is a central
memory T-cell and the CD4+ helper T lymphocyte cell is a naive or
central memory CD4+ T-cell.
[0259] The disclosure provides methods of making adoptive
immunotherapy compositions and uses or methods of using these
compositions for performing cellular immunotherapy in a subject
having a disease or disorder. Proliferation and persistence of the
chimeric receptor modified T-cells can be determined by using an
animal model of the disease or disorder and administering the cells
and determining persistence and/or proliferative capacity of the
transferred cells. In other alternatives, proliferation and
activation can be tested in vitro by going through multiple cycles
of activation with antigen bearing cells.
[0260] In some alternatives, a method of manufacturing the
compositions comprises obtaining a modified naive CD4+ T helper
cell, wherein the modified helper T lymphocyte cell preparation
comprises CD4+ T-cells that have a chimeric receptor comprising a
ligand binding domain specific for a tumor cell surface molecule, a
customized spacer domain, a transmembrane domain, and an
intracellular signaling domain as described herein.
[0261] In another alternative, a method further comprises obtaining
a modified CD8+ cytotoxic T cell, wherein the modified cytotoxic T
lymphocyte cell preparation comprises CD8+ cells that have a
chimeric receptor comprising a ligand binding domain specific for a
tumor cell surface molecule, a customized spacer domain, a
transmembrane domain, and an intracellular signaling domain as
described herein.
[0262] In another alternative, a method comprises obtaining a
modified CD8+ cytotoxic T-cell, wherein the modified cytotoxic T
lymphocyte cell preparation comprises CD8+ T-cells that have a
chimeric receptor comprising a ligand binding domain specific for a
tumor cell surface molecule, a customized spacer domain, a
transmembrane domain, and an intracellular signaling domain, as
described herein, and further comprising combining the modified
CD8+ cytotoxic T-cells with a CD4+ helper cell lymphocyte cell
preparation.
[0263] The preparation of the CD4+ and CD8+ cells that are modified
with a chimeric receptor has been described above, as well as, in
the examples. Antigen specific T lymphocytes can be obtained from a
patient having the disease or disorder or can be prepared by in
vitro stimulation of T lymphocytes in the presence of antigen.
Subpopulations of CD4+ and CD8+ T lymphocytes that are not selected
for antigen specificity can also be isolated as described herein
and combined in the methods of manufacturing. In some alternatives,
the combination of cell populations can be evaluated for uniformity
of cell surface makers, the ability to proliferate through at least
two generations, to have a uniform cell differentiation status.
Quality control can be performed by co-culturing a cell line
expressing the target ligand with chimeric receptor modified
T-cells to determine if the chimeric receptor modified T-cells
recognize the cell line using cytotoxicity, proliferation, or
cytokine production assays that are known in the field. Cell
differentiation status and cell surface markers on the chimeric
receptor modified T-cells can be determined by flow cytometry. In
some alternatives, the markers and cell differentiation status on
the CD8+ cells include CD3, CD8, CD62L, CD28, CD27, CD69, CD25,
PD-1, CTLA-4, CD45RO, and/or CD45RA. In some alternatives, the
markers and the cell differentiation status on the CD4+ cells
include CD3, CD4, CD62L, CD28, CD27, CD69, CD25, PD-1, CTLA-4
CD45RO, and/or CD45RA. In some alternatives, the markers include
CD45 RA, CD45 RO, CCR7, CD25, CD127, CD57, CD137, CD27, CD28 and/or
CD62L. In some alternatives, the markers include CD27, CD28 and/or
CD62L.
[0264] Some alternatives relate to approaches for treating,
inhibiting, or ameliorating a disease, such as cancer, such as ALL
in a subject in need thereof, including methods of inhibiting or
delaying progression and/or metastasis of a cancer, methods of
inhibiting or reducing the presence of a tumor or cancer cell,
and/or a methods of inhibiting or reducing a target population of
CD19 expressing cells in a patient in need thereof. Such methods
involve administering to a subject or a patient in need thereof a
genetically modified cytotoxic T lymphocyte cell preparation that
provides a cellular immune response, wherein the cytotoxic T
lymphocyte cell preparation comprises CD8+ T-cells that have a
chimeric receptor comprising a polynucleotide coding for a ligand
binding domain, wherein the ligand is a tumor specific antigen, or
any other molecule expressed on a target-cell population (e.g.
CD19) that is suitable to mediate recognition and elimination by a
lymphocyte; a polynucleotide coding for a polypeptide spacer
wherein the polypeptide spacer is of a customized length, wherein
the spacer provides for enhanced T-cell proliferation and/or
cytokine production as compared to a reference chimeric receptor; a
polynucleotide coding for a transmembrane domain; and a
polynucleotide coding for one or more intracellular signaling
domains.
[0265] The disclosure also provides methods of performing cellular
immunotherapy in a subject having a disease or disorder such as
cancer, such as ALL comprising: administering a composition of
lymphocytes expressing a chimeric receptor as described herein. In
other alternatives, a method comprises administering to the subject
a genetically modified cytotoxic T lymphocyte cell preparation that
provides a cellular immune response, wherein the cytotoxic T
lymphocyte cell preparation comprises CD8+ T-cells that have a
chimeric receptor comprising a ligand binding domain specific for a
cell surface molecule, a customized spacer domain, a transmembrane
domain, and an intracellular signaling domain as described herein,
and a genetically modified helper T lymphocyte cell preparation
that elicits direct recognition and augments the genetically
modified cytotoxic T lymphocyte cell preparations ability to
mediate a cellular immune response, wherein the helper T lymphocyte
cell preparation comprises CD4+ T-cells that have a chimeric
receptor comprising a ligand binding domain specific for a cell
surface molecule, a customized spacer domain, a transmembrane
domain, and an intracellular signaling domain, as described
herein.
[0266] While not limiting the scope of the disclosure, it is
believed by selecting the chimeric receptor modified T-cell
population that can persist and proliferate in vivo prior to
administration can result in the ability to use a lower dose of
T-cells and provide more uniform therapeutic activity. In some
alternatives, the dose of T-cells can be reduced at least 10%, 20%,
or 30% or greater. Reduction in the dose of T-cells can be
beneficial to reduce the risk or tumor lysis syndrome and cytokine
storm.
[0267] In another alternative, a method of performing cellular
immunotherapy in subject having a disease or disorder comprises:
administering to the subject a genetically modified helper T
lymphocyte cell preparation, wherein the modified helper T
lymphocyte cell preparation comprises CD4+ T-cells that have a
chimeric receptor comprising a ligand binding domain specific for a
tumor cell surface molecule, a customized spacer domain, a
transmembrane domain, and an intracellular signaling domain as
described herein. In some alternatives, the method further
comprises administering to the subject a genetically modified
cytotoxic T lymphocyte cell preparation, wherein the modified
cytotoxic T lymphocyte cell preparation comprises CD4+ cells that
have a chimeric receptor comprising a ligand binding domain
specific for a tumor cell surface molecule, a customized spacer
domain, a transmembrane domain, and an intracellular signaling
domain as described herein.
[0268] Another alternative describes a method of performing
cellular immunotherapy in a subject having a disease or disorder
comprising: analyzing a biological sample of the subject for the
presence of a target molecule (e.g. CD19) associated with the
disease or disorder and administering the adoptive immunotherapy
compositions described herein, wherein the chimeric receptor
specifically binds to the target molecule (CD19).
[0269] In some alternatives, the CD4+ T helper lymphocyte cell is
selected prior to introduction of the chimeric receptor from the
group consisting of naive CD4+ T-cells, central memory CD4+
T-cells, effector memory CD4+ T-cells and bulk CD4+ T-cells. In a
specific alternative, CD4+ helper lymphocyte cell is a naive CD4+
T-cell, wherein the naive CD4+ T-cell comprises a CD45RO-, CD45RA+,
CD28+, CD27+, CD62L+ and/or CD4+. In yet other alternatives, the
CD8+ T cytotoxic lymphocyte cell is selected prior to introduction
of the chimeric receptor from the group consisting of naive CD8+
T-cells, central memory CD8+ T-cells, effector memory CD8+ T-cells
and bulk CD8+ T-cells. In a specific alternative, the CD8+
cytotoxic T lymphocyte cell is a central memory T-cell wherein the
central memory T-cell comprises a CD45RO+, CD62L+, CD28+, CD28+,
and/or CD8+. In a specific alternative, the CD8+ cytotoxic T
lymphocyte cell is a central memory T-cell and the CD4+ helper T
lymphocyte cell is a naive CD4+ T-cell.
[0270] In some alternatives, the CD8+ T-cell and the CD4+ T-cell
are both genetically modified with a chimeric receptor comprising
an antibody heavy chain domain that specifically binds a cell
surface molecule. In other alternatives, the intracellular
signaling domain of the CD8 cytotoxic T-cells is the same as the
intracellular signaling domain of the CD4 helper T-cells. In yet
other alternatives, the intracellular signaling domain of the CD8
cytotoxic T-cells is different than the intracellular signaling
domain of the CD4 helper T-cells.
[0271] The subjects that can be administered the alternatives
described herein include humans, other primates, such as monkeys
and apes, companion animals, such as dogs, cats, and horses, and
domestic animals such as pigs, goats, sheep, and cattle. The
subjects can be male or female and can be any suitable age,
including infant, juvenile, adolescent, adult, and geriatric
subjects. The methods are useful in the treatment of, for example,
CD19 bearing cancer or cells.
[0272] Cells prepared as described above can be utilized in methods
and compositions for adoptive immunotherapy in accordance with
known techniques, or variations thereof that will be apparent to
those skilled in the art based on the instant disclosure.
[0273] In some alternatives, the cells are formulated by first
harvesting them from their culture medium, and then washing and
concentrating the cells in a medium and container system suitable
for administration (a "pharmaceutically acceptable" carrier) in a
treatment-effective amount. Suitable infusion medium can be any
isotonic medium formulation, typically normal saline, Normosol R
(Abbott) or Plasma-Lyte A (Baxter), but also 5% dextrose in water
or Ringer's lactate can be utilized. The infusion medium can be
supplemented with fetal calf serum. Cells are then treated with
cytokines to produce a transduced, cytokine-stimulated population
of CD8+ T-cells and/or CD4+ T-cells with an increased or improved
engraftment fitness as compared to non-cytokine stimulated
populations of CD8+ T-cells and/or CD4+ T-cells. In order to
generate cells with the desired improvement in engraftment fitness,
the cytokines are desirably added in vitro during the propagation
of the T-cells. As such, separated CD4+ and CD8+ T-cells are
cytokine stimulated by the addition of cytokines during the
propagation of the genetically modified T-cells. In some
alternatives, the CD4+ T cells are stimulated with at least one
cytokine, wherein the at least one cytokine comprises IL/7, IL-15
and/or IL-21. In some alternatives, the CD8+ T cells are stimulated
with at least one cytokine, wherein the at least one cytokine
comprises IL-2, IL-15 and/or IL-21.
[0274] A treatment or inhibitory effective amount of cells in the
composition is at least 2 cell subsets (for example, 1 CD8+ central
memory T-cell subset and 1 CD4+ helper T-cell subset) or is more
typically greater than 10.sup.2 cells, and up to 10.sup.6, up to
and including 10.sup.8 or 10.sup.9 cells and can be more than
10.sup.10 cells or any other value in a range defined by any of
these two values. The number of cells will depend upon the ultimate
use for which the composition is intended as will the type of cells
included therein. For example, if cells that are specific for a
particular antigen are desired, then the population will contain
greater than 70%, generally greater than 80%, 85% and/or 90-95% of
such cells. For uses provided herein, the cells are generally in a
volume of a liter or less, can be 500 mls or less, even 250 mls or
100 mls or less, or any other value within a range defined by any
two of these values. Hence the density of the desired cells is
typically greater than 10.sup.4 cells/ml and generally is greater
than 10.sup.7 cells/ml, generally 10.sup.8 cells/ml or greater. The
clinically relevant number of immune cells can be apportioned into
multiple infusions that cumulatively equal or exceed 10.sup.6,
10.sup.7, 10.sup.8, 10.sup.9, 10.sup.10 or 10.sup.11 cells or any
other amount within a range defined by any of these two values.
[0275] In some alternatives, the lymphocytes of the invention can
be used to confer immunity to individuals. By "immunity" is meant a
lessening of one or more physical symptoms associated with a
response to infection by a pathogen, or to a tumor, to which the
lymphocyte response is directed. The amount of cells administered
is usually in the range present in normal individuals with immunity
to the pathogen. Thus, the cells are usually administered by
infusion, with each infusion in a range of from 2 cells, up to at
least 10.sup.6 to 3.times.10.sup.10 cells, preferably in the range
of at least 10.sup.7 to 10.sup.9 cells. The T-cells can be
administered by a single infusion, or by multiple infusions over a
range of time. However, since different individuals are expected to
vary in responsiveness, the type and amount of cells infused, as
well as the number of infusions and the time range over which
multiple infusions are given are determined by the attending
physician, and can be determined by routine examination. The
generation of sufficient levels of T lymphocytes (including
cytotoxic T lymphocytes and/or helper T lymphocytes) is readily
achievable using the rapid expansion method of the present
invention, as exemplified herein. See, e.g., U.S. Pat. No.
6,040,177 to Riddell et al. at column 17, hereby expressly
incorporated by reference in its entirety.
[0276] In some alternatives, the compositions, as described herein
are administered intravenously, intraperitoneally, intratumorly,
into the bone marrow, into the lymph node, and/or into
cerebrospinal fluid.
[0277] In some alternatives, the compositions, as described herein
are administered with chemotherapeutic agents and/or
immunosuppressants. In an alternative, a patient is first
administered a chemotherapeutic agent that inhibits or destroys
other immune cells followed by the compositions described herein.
In some cases, chemotherapy can be avoided entirely.
Administration to Patients of ALL
[0278] CD4 and CD8 T cell subsets were immunomagnetically isolated
from apheresis products obtained from the research participant.
Following anti-CD3.times.CD28 bead stimulation, T cell lines were
transduced with a SIN lentiviral vector that directs the
co-expression of the FMC63scFv:IgG4hinge: CD28tm:4-1BB:.xi. CAR and
the selection and tracking suicide construct EGFRt. Transduced
cells were propagated using recombinant human cytokines to numbers
suitable for clinical use over 10-20 days during which time they
were subjected to EGFRt immunomagnetic positive selection. Shortly
following lymphodepleting chemotherapy, cryopreserved CD4/EGFRt+
and CD8/EGFRt+ T cell products were thawed and infused at the
bedside such that patients received a 1:1 ratio of EGFRt+ CD4 and
CD8 T cells at the indicated protocol-prescribed dose level
(1=0.5.times.10.sup.6/kg; 2=1.times.10.sup.6/kg;
3=5.times.10.sup.6/kg). In some alternatives, the enriched CD4+
expressing T-cells were contacted, for example propagated, in 5
ng/mL recombinant human IL-7 (rhIL-7) and/or 0.5 ng/mL recombinant
human IL-15 (rhIL-15). In some alternatives, the enriched CD8+
expressing T-cells were contacted, for example propagated, in 50
U/mL recombinant human IL-2 (rhIL-2) and/or 0.5 ng/mL recombinant
human IL-15 (rhIL-15).
[0279] All enrolled subjects (n=16) had cell products that met
dosing specification and had been cleared for infusion. 13 subjects
(6 m-3 yr s/p HSCT) had been treated at varying dose levels (range
of 0.5.times.10.sup.6/kg-5.times.10.sup.6/kg). The infusions were
well tolerated with only 1 AE>grade 2 (grade 3 anaphylaxis
related to the DMSO). 12 out of 13 subjects had responses for an
ORR of 92%. 11 out of 13 subjects achieved MRD negative CRs (85%).
Subjects with higher disease burden had higher peak PB CAR T cell
levels compared to those with MRD negative marrows (62.7% v 19.6%).
Accumulation with expansion of CAR T cells in bone marrow (n=12),
peripheral blood (n=12) and CSF was observed. The average duration
of persistence for the 5 subjects who lost their T cell grafts was
63 days (range of 42-150 d).
[0280] Each of the twelve responding subjects developed some degree
of CRS with fever and hypotension as the hallmark symptoms. Two
subjects, S02 and S09, required immunomodulatory treatment for sCRS
(tocilizumab for two, and addition of dexamethasone in one). Four
of 13 of these patients developed encephalopathy (2 grade 1, 1
grade 3 and 1 grade 4). The grade 4 encephalopathy was a DLT, and
accompanied by seizures and abnormal MRI findings similar to PRES.
MRI findings were normalized by 9 weeks post therapy, but the
subject, at the time of last assessment, continued to have
seizures, 6 months from therapy.
[0281] As shown, infusions of prescribed dose levels of defined
composition CD4:CD8 CD19CAR/EGFRt+ T cells/kg produced encouraging
rates of MRD-negative CRs in pediatric and young adult ALL patients
who have suffered a post-HSCT relapse. As such, it is feasible to
generate donor-derived products from each of the sixteen enrolled
patients. The expected toxicities for the 13 evaluable patients
include CRS with .about.30% ICU admission rate and encephalopathy
with severity ranging from mild to severe. MRD negative responses
have been seen at all dose levels, although early data suggests
enhanced persistence at escalating doses.
[0282] Although one patient developed acute GVHD post T cell
therapy, the preliminary assessment suggested that CAR T cells were
not mediators of this response. Interestingly, prednisone did not
affect persistence of the T cell graft. As such, the cells that
maintained the markers CD28, CD27, and CD62L showed high
persistence in comparison to cells that were not treated with the
defined cytokine mixtures and lack the markers for engraftment
fitness, which are the T-cells that are used for the current method
of treatment.
[0283] Shown in FIG. 18, is a flow diagram depicting the
development history of a process used to manufacture the
genetically modified T-cell comprising a chimeric antigen receptor.
As indicated, the current manufacturing procedure does not have the
Ficoll processing, whereas initial experimental processes included
a Ficoll processing step. The initial variations of cell
concentrations were also tested as shown in the flow diagram of
FIG. 19. Cells were also expanded in the presence of cytokines
(FIG. 20).
[0284] As shown in FIG. 20, donor sample PD0064: PD0063 CD8+
enriched T-cells were used to test starting cell density in a T25
flask on initiation. Both cells (from PD0064, PD0063) were tested
under normal PLAT-02 culture conditions, IL-2 (50 U/ml)+IL-15 (0.5
ng/mL). Cells from donor sample PD0080, were used in a study to
test the expansion of cells on Tx day, by adding media and
cytokines for a final cell concentration of to 0.7.times.10 6
cell/mL ("early expansion"). By "Tx" it is meant throughout to
refer to Day +1, based on the day of stimulation beads being
defined as Day +0. PD0063 CD8+ cells were used as the enriched
starting material The same cytokine conditions were used as with
the PD0064:PD0063 CD8+ enriched cells. The experiment tested two
cell densities both showing greater viability and expansion when
the cells were volumed up early in the presence of the cytokines.
In this context "volume up" refers to the volume of media/cytokine
addition for initial feeding following Tx was greater in the "early
expansion" conditions, leading to decreased cell density at this
point in culture for "early expansion" conditions.
[0285] As shown in FIG. 21, are the growth of cell samples PD104
and PD0116 and the comparison of cell growth under experimental
conditions. PD0104: PD0063 CD8+ and CD4+ enriched cells were used
to repeat previous experiments to test the expansion of cells on Tx
day to 0.7.times.10.sup.6 cell/mL ("early expansion"), with the
following cytokines and respective concentrations being added: IL-2
(50 U/ml)+IL-15 (0.5 ng/mL) for CD8+ T- cells and IL-7 (5
ng/mL)+IL-15 (0.5 ng/mL) for CD4+ T-cells. Growth and viability
were increased dramatically. These curves mimic what was seen in
clinical products as far as timing of growth/bead
removal/selection/cryopreservation.
[0286] The PD0116: PD0046 CD8+ and CD4+ enriched cells were used to
repeat the experiment to test the expansion of cells on Tx day to
0.7.times.10 6 cell/mL ("early expansion"). Normal PLAT-02 culture
conditions, IL-2 (50 U/ml)+IL-15 (0.5 ng/mL) for CD8 cells and IL-7
(5 ng/mL)+IL-15 (0.5 ng/mL) for CD4's. PD116 was initiated at
180.times.10 6 cells in a V-197 bag and magnetic enrichment and
spinoculation were performed in a transfer pack. As shown, from
these conditions, growth and viability were increased dramatically.
These curves mimic what was seen in clinical products as far as
timing of growth, bead removal, selection, and cryopreservation of
T-cells.
[0287] PD0116: PD0046 CD8+ and CD4+ enriched cells were also used
to repeat an experiment to test the expansion of cells on Tx day by
the addition of media and cytokines for a final volume of to
0.7.times.10.sup.6 cell/mL ("early expansion"). Normal PLAT-02
culture conditions in which additions of IL-2 (50 U/ml)+IL-15 (0.5
ng/mL) for CD8 cells and IL-7 (5 ng/mL)+IL-15 (0.5 ng/mL) for CD4's
was performed. PD116 was initiated at 180.times.10.sup.6 cells in a
V-197 bag and magnetic enrichment and spinoculation were performed
in a transfer pack. This was the full scale data used to transition
to an updated manufacturing process. As shown in the FIG. 21,
growth expansion was increased for CD8+ cells in comparison to the
CD4+ cells. The cytokines used for the CD4.sub.-- and the CD8+
cells and the variations of the combinations of the cytokines used
are depicted in a flow chart as shown in FIG. 22.
[0288] Various cytokine conditions were also tested on the CD8+ and
the CD4+ cells from donors. As shown in FIG. 23 panels A-F, samples
from PD0047 and PD0040 were tested with IL2/IL15, IL7/IL15,
IL2/IL7/IL15, IL2 pulse with IL7/IL15, IL2 only, IL2 pulse with
IL7/IL21 and IL2/IL15. This included incubation with IL-2 alone
(pulse) for 5 days, followed by the addition of IL-7/IL-21, which
then were present throughout the remainder of the culture,
incubation with IL-2 alone (pulse) for 5 days, followed by the
addition of IL-7/IL-21 and IL2/IL15, which then were present
throughout the remainder of the culture. PD0040: PD0038 CD8+
enriched T-cells were used to test various cytokine cocktails.
Concentrations for each condition were: IL-2 (50 U/mL), IL-7 (5
ng/mL), IL-15 (0.5 ng/mL). Condition #4 was an IL-2 pulse for 5
days followed by IL-7/IL-15 throughout the remainder of the
culture. Fold growth and TNC were extrapolated from small scale
experiment. Currently used IL-2/IL-15 combination showed best
expansion. In this study, the IL-2/IL-15 combination was
demonstrated to result in the best expansion.
[0289] As shown, the PD0047: PD0038 CD8+ enriched cells were used
to test various cytokine cocktails. The same cytokine cocktails
were tested using "low dose" IL-2 at 10 U/mL final. Concentrations
of the individual cytokines used in the various combinations were:
IL-2 (50 U/mL) and (10 U/mL) as stated in the legend, IL-7 (5
ng/mL), IL-15 (0.5 ng/mL), IL-21 (5 ng/mL). Pulse conditions were
an IL-2 pulse for 5 days followed by IL-7/IL-21 throughout the
remainder of the culture. Fold growth and TNC are extrapolated from
a small scale experiment. The IL-2/IL-15 combination showed the
best expansion and was used for following experimentations. The
"normal" dose of IL-2 showed best growth. Panel A shows results
from incubation of PD0047 cells at day 15 (D15) and the comparison
of growth with various cytokines and the percent positive cells
(CD3+, CD62L+, CD3+/CD62L+/CD45RO-, CD28+, CDD45RA+/CD4RO-,
CD45RA+/CD45RO and CD45RA-/CD45RO+), following incubation with the
various cytokine conditions.
[0290] Panel B shows results following incubation of PD0047 cells
at day 21 (D21) and the comparison of growth with various cytokines
and the percent positive cells as indicated above. Panel C shows
PD0040 D21 and the comparison of growth with various cytokines, in
which the total cell number is examined after 22 days of growth in
culture. Panel D shows PD0040 D21 and the comparison of growth with
various cytokines, as indicated the CD3+ cell growth is examined
after 22 day of culture growth. Panel E shows PD0047 and the
comparison of growth with various cytokines, in which the total
cell number is examined after 22 days of growth in culture. Panel F
shows PD0047 and the comparison of growth with various cytokines,
as indicated the CD3+ cell growth is examined after 22 day of
culture growth. As shown for PD0040: PD0038 CD8+ enriched cells,
were used to test various cytokine cocktails. Concentrations for
each condition were: IL-2 (50 U/mL), IL-7 (5 ng/mL), IL-15 (0.5
ng/mL). Condition #4 was an IL-2 pulse for 5 days followed by
IL-7/IL-15 throughout the remainder of the culture. Fold growth and
TNC were extrapolated from small scale experiments. IL-2/IL-15
combination showed the best expansion and was used for future
experiments. Additionally, PD0047: PD0038 CD8+ enriched cells were
used to test various cytokine cocktails. The same cytokine
cocktails were tested using "low dose" IL-2 at 10 U/mL final.
Concentrations for each condition were: IL-2 (50 U/mL) and (10
U/mL) stated in legend, IL-7 (5 ng/mL), IL-15 (0.5 ng/mL), IL-21 (5
ng/mL). Pulse conditions were an IL-2 pulse for 5 days followed by
IL-7/IL-21 throughout the remainder of the culture. Fold growth and
TNC were then extrapolated from small scale experiments. The
IL-2/IL-15 cytokine combination used in the study described above
showed the best expansion. The "normal" dose of IL-2 also showed
the best growth. Flow cytometry for phenotypic growth is available
and indicated in further experiments herein.
[0291] Shown in FIG. 24, are results from a series of experiments
to test the different combination of cytokines PD0051: CD8+ cells
from two different donors were used to test the "normal" cytokine
conditions using IL-2 (50 U/mL) and IL-15 (0.5 ng/mL) were used.
Pulse conditions were an IL-2 pulse for 5 days followed by IL-7 (5
ng/mL), IL-21 (5 ng/mL), throughout the remainder of the culture.
Fold growth and TNC that are shown, are from a scaled experiment
that started with 30.times.10 6 cells. The currently used
IL-2/IL-15 combination showed the best expansion, but expansion was
also seen from the cultures pulsed with IL-2 and switched to
IL-7/IL-21. Fold expansion was similar in the pulsed conditions to
PD0047.
[0292] For PD0055, the same cytokine conditions were tested as
those assessed for PD0051 but with CD4+ donor cells (same donor
PD0038 as above). Cytokine conditions were IL-7 (5 ng/mL) and IL-15
(0.5 ng/mL) for the experiments. Pulse conditions were an IL-2
pulse for 5 days followed by IL-7 (5 ng/mL), IL-21 (5 ng/mL),
throughout the remainder of the culture. Fold growth and TNC are
from a scaled experiment that started with 30.times.10 6 cells. The
currently used IL-7/IL-15 combination showed the usual expansion,
however better expansion was seen from the cultures pulsed with
IL-2 and switched to IL-7/IL-21. Panel A shows PD0051 at day 15
(D15) and the comparison of growth with various cytokines and the
percent positive cells (EGFRt+, EGFRt+/CD4+, EGFRt+/CD8+, CD3+,
CD3+/CD4+, CD3+/CD4+/CD8+, CD3+/CD8+, CD62L+, CD3+/CD8+, CD62L+,
CD3+/CD28+, CD27+, CD127+, CD45RA+/CD45RO-,
CD45RA+/CD45RO_/CD45RO_/CD45RA-, CD62+/CD28+) in a donor versus
donor cytokine comparison. Panel B shows PD0055 at day 17 (D17) and
the comparison of growth with various cytokines and the percent
positive cells. Panel C shows the PD0051 growth curve and the
comparison of growth with various cytokines, in which the total
cell number is examined after 20 days of growth in culture. Panel D
shows PD0051 and the comparison of growth with various cytokines,
as indicated the CD3+ cell growth is examined after 20 days of
culture growth. Panels C and D show a growth curves (total cells
and CD3+) for PD0051, comparing growth following incubation of the
cells with the indicated various cytokines, through 20 days in
culture. Panel E and F shows growth curves (total cells and CD3+
respectively) for donor PD0055 following incubation with the
indicated various cytokines, at the indicated time point at day 17
of growth in culture.
[0293] Shown in FIG. 25, is a cytokine testing experiment in which
different combinations of cytokines were evaluated for their
influence on cell expansion. For sample PD0059, the same cytokine
testing experiment as shown for PD0051 was performed except that
this experiment involved testing a new CD4+ and CD8+ donor
(PD0057). Testing the "normal" cytokine conditions using IL-7 (5
ng/mL) and IL-15 (0.5 ng/mL) for CD4's and IL-2 (50 U/mL) and IL-15
(0.5 ng/mL) for CD8+ cells were performed. Pulse conditions were an
IL-2 pulse for 5 days followed by IL-7 (5 ng/mL), IL-21 (5 ng/mL),
throughout the remainder of the culture. Fold growth and TNC are
from a scaled experiment that started with 30.times.10.sup.6 cells.
The "normal" cytokine combinations in this study resulted in
expansion; good expansion was seen following pulsing with IL-2 and
switching to IL-7/IL-21.
[0294] For PD0078, a similar cytokine study was performed as in
PD0051, except that the experiment was performed with a different
CD4+ and CD8+ donor (PD0063). The test was performed using "normal"
cytokine conditions using IL-7 (5 ng/mL) and IL-15 (0.5 ng/mL) for
CD4's and IL-2 (50 U/mL) and IL-15 (0.5 ng/mL) for CD8's versus an
IL-2 only condition. All growth through day 14 (D+14) was on a
normal plane, cultures were then EGFRt selected. Recovery of
viability and growth was not seen following selection for the CD8+
cells. As shown in Panel A, are the percent of positive cells after
thawing the cells. Panel B shows the PD0059 PLAT-02 cytokine
comparison. Panel C shows PD0059 PLAT-02 cytokine comparison and
the CD3+ cell growth. Panel D shows the PD0078 growth curves. Panel
D shows the PD0078 growth curves and the CD3+ cell growth.
[0295] Shown in FIG. 26, are two flow charts illustrating an
initial expansion methodology and the updated (or "current")
expansion methodology of cell expansion based on experimentation,
which was then used for future experimentations.
[0296] Shown in FIG. 27, are growth curves for samples from PD0080,
PD0084, and PD0085 and a comparison between all three. These three
experiments were repeated experiments of the "early expansion"
methodology. For these experiments, the standard cytokine
conditions were used: IL-2 (50 U/mL)+IL-15 (0.5 ng/mL) for CD8+
T-cells and IL-7 (5 ng/mL)+IL-15 (0.5 ng/mL) for CD4+ T-cells. For
PD0080, two starting densities were tested in duplicate using
PD0063 CD8+ T-cells. One of each of the cell duplicates received
volumes of up to 0.7.times.10.sup.6 cell/mL following a 3-hour
incubation on transduction day. For PD0084, two additional CD8+
donor T-cells were assessed for effects of early expansion.
30.times.10.sup.6 cell starting conditions were used as a control
for each donor and a 60.times.10.sup.6 cell starting density was
used with early expansion as test conditions. For PD0085, a similar
test to PD0084 was performed except the CD4+ T-cells were used to
test early expansion. Again 30.times.10.sup.6 starting cell density
were used as "normal" control condition and 60.times.10.sup.6 cell
starting density was used as a test condition.
[0297] As shown in FIG. 28, the starting material of PD0038
included selected CD4+ and CD8+ T-cells. The growth curves show TNC
from both V-197 bags of cells that were pooled together. From the
early development, it was intended to pool to 50:50 CD4:CD8 product
prior to cryopreservation. Flow data shows post thaw of the pooled
product. Bead removal and EGFRt enrichment occurred D+14 for CD4+
and D+15 for CD8+ T-cells. CD8+ T-cells were grown in IL-2 (50
U/mL)/IL-15 (0.5 ng/mL) and CD4+ T-cells were grown in IL-7 (5
ng/mL)/IL-15 (0.5 ng/mL). This process was changed to a
cryopreservation of separate products and infusing a 50:50 product
upon thawing.
[0298] Shown in FIG. 29, is the growth curve for PD0046. Growth
curves show TNC from both V-197 bags of cells together. In early
development there was an intent to pool a 50:50 CD4:CD8 product
prior to cryopreservation. Flow data shows post thaw of the pooled
product. Bead removal and EGFRt enrichment then occurred. D+14 for
CD4+ T-cells and D+15 for CD8+ T-cells. CD8+ T-cells were grown in
IL-2 (50 U/mL)/IL-15 (0.5 ng/mL) and CD4+ T-cells were grown in
IL-7 (5 ng/mL)/IL-15 (0.5 ng/mL). However, not much was done
following this product, due to the donor cells thawing poorly.
Cells were used later to test 10% HA thawing and proved to be much
better.
[0299] Shown in FIG. 30, is the growth curve for PD0063. Growth
curves show TNC from both V-197 bags of cells together. Bead
removal and EGFRt enrichment then occurred D+14 for CD4's and D+15
for CD8's are shown. CD8+ cells were grown in IL-2 (50 U/mL)/IL-15
(0.5 ng/mL) and CD4+ cells were grown in IL-7 (5 ng/mL)/IL-15 (0.5
ng/mL). Bead removal and EGFRt enrichment then occurred.
[0300] Shown in FIG. 31, is the expansion of cells from bulk PBMC
cultures when grown in the presence of cytokines. As shown, CD4+
cells ( ) were grown in the presence of IL2 and IL15. CD8+ cells
(.box-solid.) were grown in the presence of IL7 and IL15. The lower
panel, (phenotypic comparison) of FIGS. 31A and 31B, shows
percentage of total cells expressing the indicated markers on their
surfaces following incubation with the different conditions, each
of which resulted in cells expressing markers of engraftment
fitness, e.g., CD62L, CD28, CD27, CD45RA. Growing CD8-enriched
cultures in IL-2/IL-15 and CD4-enriched cultures in IL-7/IL-15
yielded robust expansion of both cultures and strong expression of
memory phenotype.
[0301] Shown in FIG. 32, is the expansion of enriched CD8+ and CD4+
cells in cytokine mixtures. As shown in sample PD0044, enriched
CD8+ T-cells were expanded in the presence of IL2 and IL15 (top
panel). In sample PD0044, enriched CD4+ T-cells were expanded in
the presence of IL7 and IL15 for over 20 days. As shown, the pooled
CD4+ and CD8+ T-cells expressed CD3, CD4, CD8, CD62L, CD28, CD27,
CD45RA and CD45RO. Also tested was sample 14602-S14 (See FIG. 32B).
As shown, growing CD8+ enriched cultures in IL-2/IL-15 and CD4+
enriched cultures in IL-7/IL-15 yielded robust expansion of both
cultures and strong expression of memory phenotype. Importantly,
from the data generated and shown in FIGS. 31 and 32, CD4+ T-cells
and CD8+ T-cells grew optimally with different cytokine
combinations such that CD4+ T-cells performed best in these studies
with IL7 and IL15, while CD8+ T-cells grew best in these studies
with the cytokines IL2 and IL15. Growth with their respective
optimal cytokine combinations led to cells that had a phenotype
following cell expansion in which the cells expressed the surface
markers CD45RA, CD62L, CD28, and CD27, which indicate a high or
improved or enhanced engraftment fitness upon adoptive
transfer.
[0302] Shown in FIG. 33, are samples from 14602-S01 through
14602-S06 using the original PLAT-02 (Phase I and Phase II of
clinical trials) methodology of cell expansion. The cells were
positive for EGFRt prior to cyropreservation. The cells were tested
for viability. As shown in the bottom table, the cells 14602-S03
CD8+ and 14602-S03-CD8+ cells did not express CD3, CD62L, CD27,
CD127, CD45RA/CD45RO- or CD45RA. Shown in FIG. 34, are tests on the
cell product 14602-SO4 and 14602-SO4-02 which have both CD4+ and
CD8+ T-cells. The cells were grown in cultures using the "early
expansion" methodology. The cells were tested positive for EGFRt
expression prior to cryopreservation.
[0303] Shown in FIG. 35, are the cells from samples 14602-S07,
14602-S08, and 14602-S09. The CD4+ and CD8+ cells were expanded
using the early expansion methodology. In the top panel, as shown
in the table for 14602-S07 CD4+, cells and 14602-S07 CD8+ cells,
shows percentage of cells expressing various markers including CD3,
CD62L, CD27, CD27, CD127, and CD45RA+ at various times. In the
bottom panel for sample 14602-S08, CD4+ and CD8+ enriched T-cells
remained viable after 10 days in culture.
[0304] Shown in FIG. 36, are the cells tested for viability from
the batch samples of 14602-S10, 14602-S11, 14602-S12 and 14602-S13.
As shown in the accompanying tables, the cells all expressed EGFRt
prior to cryopreservation. The cells were tested for viability in
cell culture for at least 12 days.
[0305] Shown in FIG. 37, are the cells tested for viability from
the batch samples of 14602-S14, 14602-S15 and 14602-S16. As shown
in the accompanying tables, the cells all expressed EGFRt prior to
cryopreservation. The cells were tested for viability in cell
culture for at least 12 days.
[0306] As shown in FIG. 38, the cells from the sample of 14602,
which CD4+ and CD8+ T-cells were enriched from, were positive for
CD3, CD62L, CD28, CD127, CD45RA+/CD45RO-, CD45RA and CD45RO. These
are specific markers which to indicate that both CD4+ and CD8+
cells had a high level of engraftment fitness following the
treatment with their specific cytokine combinations.
[0307] Shown in FIG. 39, are the results for a mouse after T-cell
administration after tumor inoculation (survival, tumor
progression). As shown, the mouse injected with the cells from the
sample PD0046 indicated that the cells had a high, improved, or
enhanced engraftment fitness due to the survival of the mouse after
80 days of administration using multiple concentrations of cells.
The sample PD00044 did not have a high, improved, or enhanced
engraftment fitness as the percent survival dropped at day 40 (FIG.
39B).
[0308] As shown in FIG. 40 is the average tumor progression in mice
treated with cells from the PD0044 and PD0046 cell batches. As
shown in the graphs, the tumor progression in the mouse decreased
if they were administered the CD8+ and CD4+ T-cells from the PD0046
batch, in which the cells were originally grown in the appropriate
cytokine mixtures for CD4+ and CD8+ T-cells (IL7/IL15 for CD4+
T-cells and IL2/IL15 for CD8+ T-cells). As a control, the vehicle
cells did not halt tumor progression in either study with PD0044
and PD0046. As shown in the bottom panels, tumor progression
increased in mice treated with cells from the PD0044 sample, and in
particular increased substantially when treated with the least
concentration of T-cells. Tumor progression decreased in comparison
to the vehicle control.
[0309] As a further test, three groups of mice were tested in which
each group had a total number of 5 mice per group. For Group A, the
mice were treated with a placebo of phosphate buffered saline
(PBS). Group B utilized cells from "normal" expansion" which had a
1:1 EGFRt+CD4:CD8 transduced with Zrx-014. The cells used were from
group PD0055 #1 (Donor PD0038 CD4 with 5 ng/ml IL7 & 0.5 ng/ml
IL15, S1D14 at 75.9% EGFRt+) and group PD0051 #1 (Donor PD0038 CD8
with 50 U/ml IL2 & 0.5 ng/ml IL15, S1D21 @ 76.8% EGFRt+) Group
C utilized cells that were "pulsed" with cytokine treatments which
comprised 1:1 EGFRt+CD4:CD8 transduced with Zrx-014 PD0055 #2
(Donor PD0038 CD4 with 50 U/ml IL2 pulse 5 days, then 5 ng/ml IL7
& 5 ng/ml IL21, S1D11 at 81.2% EGFRt+) PD0051 #2 (Donor PD0038
CD8 with 50 U/ml IL2 pulse 5 days, then 5 ng/ml IL7 & 5 ng/ml
IL21, S1D21 at 89.5% EGFRt+).
[0310] As shown in FIG. 42, are the FACS analysis on cells post
thaw that were used for injection into mice in JME13-25 (PD0051 and
PD00055 cells). As shown both samples of cells had CD8+ and CD4+
T-cells that expressed EGFRt.
[0311] As shown in FIG. 43, are the three groups of mice from
groups A, B, and C that were treated with placebo PBS (group A),
group B (PD00051 "normal expansion cells") and group C (PD00055,
cells pulsed with cytokine combinations). As shown, mice that were
treated with the placebo in Group A had tumor progression that is
visibly seen at day 20. For the Group B mice treated with PD0051
(normal expansion cells), the mice, it is shown that tumor
progression increases in two mice substantially. In the Group C,
mice, the mice treated with PD00055 show only a small amount of
tumor progression, therefore indicating the cells treated with the
cytokine mixture prior to use in treatment had a higher engraftment
fitness.
[0312] As shown in FIG. 44, is the persistence of the CAR
expressing T-cells in the mice after three days of administration.
As indicated, the CD4+ T-cells expressing the CARs had a higher
persistence compared to the cells of normal expansion as well as
the CD8+ T-cells.
[0313] Following experiments examining tumor progression after
T-cell treatments with CAR expressing T-cells, experiments were
performed to determine if there is an in vivo difference in killing
ability between cells that have been grown in various cytokine
conditions in vitro with repeat antigen encounters (FIG. 45). For
the experiment 3 groups of mice were used in which the groups
utilized 3 mice each. For Group A, the mice were treated with a
placebo (PBS). Group B were treated with cells that were expanded
by the "normal expansion method" ("Normal" 1:1 EGFRt+ CD4:CD8
transduced with Zrx-014 PD0055 #1 (Donor PD0038 CD4 with 5 ng/ml
IL7 & 0.5 ng/ml IL15, S1D14 @ 75.9% EGFRt+) PD0051 #1 (Donor
PD0038 CD8 with 50 U/ml IL2 & 0.5 ng/ml IL15, S1D21 @ 76.8%
EGFRt+) (FIG. 18). Group C were treated with cells pulsed with
cytokines during expansion ("Pulsed" 1:1 EGFRt+CD4:CD8 transduced
with Zrx-014 PD0055 #2 (Donor PD0038 CD4 with 50 U/ml IL2 pulse 5
days, then 5 ng/ml IL7 & 5 ng/ml IL21, S1D11 at 81.2% EGFRt+)
PD0051 #2 (Donor PD0038 CD8 with 50 U/ml IL2 pulse 5 days, then 5
ng/ml IL7 & 5 ng/ml IL21, S1D21 at 89.5% EGFRt+).
[0314] As shown in FIG. 46, group A mice died by day 20 following
injection with PBS. Group B mice treated with cells that were
expanded by the "normal expansion method" had tumor progression in
one mouse by day 120. However, for mice treated with the cells that
were expanded in cytokines, the mice were tumor free by day 120. As
such the data indicate that the cells treated in the cytokine
mixtures specific for CD4+ and CD8+ led to a higher engraftment
rate and increased survival in the mice. Additionally, the cells
were more efficient in preventing tumor progression.
[0315] As shown in FIG. 47, the mice had a 100% survival rate when
treated with PD0051 and PD0055 cells post tumor inoculation.
However, the tumor progression was shown to decrease with the
treatment using PD0055 cells.
[0316] As shown in FIG. 48, the average tumor progression was
examined in mice treated with placebo, T-cells grown under "normal
expansion" methods, and T-cells that were pulsed with cytokine
combinations. For the experiment at day 0, mice were tumor
inoculated and imaged for tumors at day 6. At day 7, T-cells or a
placebo was then administered. Tumor inoculation was then repeated
at day 14 and day 21.
[0317] The mice were examined for tumor progression at day 28, 35,
49, 63, 78, 92, 105 and 120, in which images of the tumors were
taken. As shown in the groups of mice, tumor progression decreased
in the mice treated with the cells that were pulsed with cytokines
thus indicating that the cells pulsed with cytokines had a higher,
improved, or enhanced engraftment fitness than the cells that were
expanded under normal conditions without cytokines.
[0318] As shown in FIG. 49, experiments were set up to determine if
there is an in vivo difference in killing ability between cells
that have been grown under the same conditions as PLAT-01 and
PLAT-02 as well as "in between" protocols. For the experiment 17
groups of 5 mice were used test the cells in the treatment of mice
after tumor inoculation. The groups were given a dose of cells
(1.25.times.10.sup.6, 2.5.times.10.sup.6, 5.times.10.sup.6,
10.times.10.sup.6) per product condition as indicated on the Table
of FIG. 49. The cells were thawed, counted and injected into the
mice on the same day. As shown in FIG. 50, are the amounts of CD4+
and CD8+ cells in the different cell products from the two
expansion methods and their amounts of EGFRt in the cells.
[0319] As shown in FIG. 51, the 17 groups of mice were tumor
inoculated then treated with dose titrations with T-cells that were
expanded under normal expansion methods or pulsed with cytokine
combinations. As seen in the graphs, mice that were dosed with
PLAT-1.00 cells at the higher concentrations had a decrease in
tumor progression. Mice that were dosed with PLAT-2.00 also saw a
decrease in tumor progression after 25 days of administration at a
higher concentration of cells. However, for mice dosed with
PLAT-1.33, the mice saw an increase in tumor progression regardless
of cell concentration indicating a low or reduced engraftment
fitness for these cells. For mice that were given cells from
PLAT-1.67, the mice had a decrease in tumor progression when
treated with the highest concentration of cells in the
administration. However as noted in FIG. 50, the cells of PLAT-1.33
also had a low EGFRt count which could also indicate a decreased
amount of CAR on the cell surface.
[0320] Shown in the FIG. 51, is the initiation comparisons between
the PD104 and the PD116 cell product. For the PD0104 produce,
PD0063 CD8+ and CD4+ enriched cells were used to repeat experiment
to test the expansion of cells on Tx day to 0.7.times.10.sup.6
cell/mL ("early expansion"). For normal PLAT-02 culture conditions,
IL-2 (50 U/ml)+IL-15 (0.5 ng/mL) for CD8+ cells and IL-7 (5
ng/mL)+IL-15 (0.5 ng/mL) for CD4+ T cells were used. Growth and
viability were increased dramatically. These curves mimic what was
seen in clinical products as far as timing of growth/bead
removal/selection/cryo.
[0321] As shown in FIG. 52, mice were treated with dose titrations
of cell products of PLAT-1.00, PLAT-1.33, PLAT-1.67 and PLAT-2.00.
As shown in all graphs, the lowest concentration of cells was least
effective in the treatment of tumor progression. However, the cells
that showed the most engraftment fitness were the PLAT-1.00 and the
PLAT-2.00 at higher concentration.
[0322] As shown in FIG. 53, all the cells were least effective at
concentration 1.25.times.10.sup.6. The cells from PLAT-1.00 showed
to be the most effective at inhibiting tumor progression indicating
that the engraftment fitness of the cells were superior or
improved, or enhanced as compared to the other groups and indicated
that the treatment with cytokines also promoted, improved, or
enhanced engraftment fitness for the cells. As shown at a
concentration of 5.times.10.sup.6, the tumor progression was halted
at 25 days in comparison to the other groups of cells used to treat
the mice.
[0323] As shown in FIG. 54, the mice had a higher rate of survival
if treated with a dose of cells between 2.5.times.10.sup.6 and
10.times.10.sup.6 cells per dose. However, the mice treated with
the cells from the PLAT-1.00 product had a longer survival rate
indicating that the T-cells from this group were more robust and
exhibited a higher, improved, or enhanced engraftment fitness.
[0324] As shown in FIG. 55, the survival of the mice was dependent
on the dose concentration of the cells. For all cells, the survival
of the mice decreased with the lowest dose of T-cell treatments.
However, as previously shown the mice treated with the cells from
the PLAT-1.00 product had a longer survival rate indicating that
the T-cells from this group were more robust and had an improved
engraftment fitness.
Additional Alternatives
[0325] In some alternatives, a method of making genetically
modified T-cells, which have a chimeric antigen receptor is
provided, wherein the method comprises separating, isolating, or
enriching a CD8+ population of T-cells and/or a CD4+ population of
T-cells from a mixed population of T-cells, such as T-cells that
are derived from thymocytes or T-cells that are derived from
engineered precursors, desirably iPS cells, so as to generate an
isolated population of T-cells, stimulating the obtained population
of T-cells so as to generate a stimulated population of CD8+
T-cells and/or CD4+ T-cells, transducing the stimulated population
of CD8+ T-cells and/or CD4+ T-cells with a vector, wherein the
vector encodes a chimeric antigen receptor and a marker sequence,
wherein said marker sequence encodes a cell surface selectable
marker, so as to generate a transduced population of CD8+ T-cells
and/or CD4+ T-cells, contacting the transduced population of CD8+
T-cells and/or CD4+ T-cells with at least one cytokine so as to
generate a transduced, cytokine-stimulated population of CD8+
T-cells and/or CD4+ T-cells, enriching the transduced,
cytokine-stimulated population of CD8+ T-cells and/or CD4+ T-cells
by selection of the marker sequence so as to generate an enriched
population of transduced, cytokine-stimulated CD8+ T-cells and/or
CD4+ T-cells and propagating the enriched population of transduced,
cytokine-stimulated CD8+ T-cells and/or CD4+ T-cells for at least
one day so as to obtain said genetically modified T-cells, which
have a chimeric antigen receptor.
[0326] In some alternatives, the separating of the CD8+ population
of T-cells and/or a CD4+ population of T-cells from a mixed
population of T-cells is performed by affinity selection for
T-cells having an epitope present on CD8 and/or CD4. In some
alternatives, the separating of the CD8+ population of T-cells
and/or a CD4+ population of T-cells from a mixed population of
T-cells is performed by flow cytometry. In some alternatives, the
separating of the CD8+ population of T-cells and/or a CD4+
population of T-cells from a mixed population of T-cells is
performed by immuno-magnetic selection. In some alternatives, the
genetically modified CD8+ T-cells and/or CD4+ T-cells comprise at
least one receptor that promotes engraftment fitness. In some
alternatives, the at least one receptor is CD45 RA, CD45 RO, CCR7,
CD25, CD127, CD57, CD137, CD27, CD28 and/or CD62L. In some
alternatives, the at least one receptor is CD27, CD28 and/or CD62L.
In some alternatives, the stimulating of the isolated population of
T-cells is performed by contacting the CD8+ and/or CD4+ T-cells
with an antibody-bound support, such as a bead or particle. In some
alternatives, the antibody-bound support comprises anti-TCR,
anti-CD2, anti-CD3, anti-CD4 and/or anti-CD28 antibodies. In some
alternatives, the antibody-bound support comprises anti-CD3 and/or
anti-CD28 antibodies. In some alternatives, the vector further
comprises a first sequence encoding a leader sequence, a second
sequence encoding a ligand binding domain, a third sequence
encoding a signaling domain and a fourth sequence encoding a
selectable marker sequence. In some alternatives, the vector
further comprises a sequence encoding a spacer. In some
alternatives, the spacer comprises an IgG4 hinge. In some
alternatives, the vector is a viral vector. In some alternatives,
the viral vector is derived from simian virus 40, adenoviruses,
adeno-associated virus (AAV), lentivirus, or retroviruses. In some
alternatives, the viral vector is a recombinant adenovirus,
adeno-associated virus, lentivirus or retrovirus vector. In some
alternatives, the viral vector is a lentivirus vector. In some
alternatives, the marker sequence encodes a truncated epidermal
growth factor receptor (EGFRt). In some alternatives, the at least
one cytokine comprises GM-CSF, IL-7, IL-12, IL-15, IL-18, IL-2,
and/or IL-21. In some alternatives, the at least one cytokine
comprises IL/7, IL-15 and/or IL-21. In some alternatives, the at
least one cytokine comprises IL-2, IL-15 and/or IL-21. In some
alternatives, the contacting is performed for 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or a period
of time within a range defined by any two of these values. In some
alternatives, the method is performed with isolated CD4+ cells in
the absence of CD8+ cells. In some alternatives, the method is
performed with isolated CD8+ in the absence of or enriched over
CD4+ cells. In some alternatives, the CD4+ expressing T-cells are
propagated for at least 1 day and may be propagated for 20 days,
such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, or 20 days or for a period that is within a range defined
by any two of the aforementioned time periods. In some
alternatives, the CD8+ expressing T-cells are propagated for at
least 1 day and may be propagated for 20 days, such as 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days
or for a period that is within a range defined by any two of the
aforementioned time periods. In some alternatives, the method
further comprises removing the antibody-bound support, such as
beads or particles. In some alternatives, the ligand binding domain
of the chimeric antigen receptor comprises an antibody, or a
binding portion thereof. In some alternatives, the ligand binding
domain of the chimeric antigen receptor comprises a single chain
variable fragment (scFv), or a binding portion thereof. In some
alternatives, the ligand binding domain of the chimeric antigen
receptor comprises FMC63, or a binding portion thereof. In some
alternatives, the ligand binding domain of the chimeric antigen
receptor is specific for CD19. In some alternatives, the method
further comprises cryopreserving the genetically modified CD8+
and/or CD4+ T-cells.
[0327] In some alternatives, a population of genetically modified
T-cells is provided wherein the population of genetically modified
T-cells comprises a plurality of affinity selected CD8+ and/or CD4+
T-cells, in the absence of, enriched over, or isolated from CD8-
and/or CD4- T-cells, wherein said plurality of affinity selected
CD8+ and/or CD4+ T-cells have stimulated CD2, CD3, CD4 and/or CD28
receptors, wherein said plurality of affinity selected CD8+ and/or
CD4+ T-cells further comprise a gene encoding a chimeric antigen
receptor and a cell surface selectable marker and, wherein said
plurality of affinity selected CD8+ and/or CD4+ T-cells have been
re-stimulated with at least one cytokine. In some alternatives, the
at least one cytokine comprises GM-CSF, IL-7, IL-12, IL-18, IL-15,
IL-2, and/or IL-21. In some alternatives, the at least one cytokine
comprises IL/7, IL-15 and/or IL-21. In some alternatives, the at
least one cytokine comprises IL-2, IL-15 and/or IL-21. In some
alternatives, the said plurality of affinity selected CD8+ and/or
CD4+ T-cells further comprise at least one receptor that promotes,
induces, contribute to or enhances engraftment fitness. In some
alternatives, the at least one receptor that promotes, induces,
contribute to or enhances engraftment fitness is CD45 RA, CD45 RO,
CCR7, CD25, CD127, CD57, CD137, CD27, CD28 and/or CD62L. In some
alternatives, the at least one receptor that promotes, induces,
contribute to or enhances engraftment fitness is CD27, CD28 and/or
CD62L. In some alternatives, the plurality of affinity selected
CD8+ and/or CD4+ T-cells further comprises a vector having a first
sequence encoding a leader sequence, a second sequence encoding a
ligand binding domain, a third sequence encoding a signaling domain
and a fourth sequence encoding a selectable marker sequence. In
some alternatives, the vector further comprises a sequence encoding
a spacer. In some alternatives, the spacer comprises an IgG4 hinge.
In some alternatives, the vector is a viral vector. In some
alternatives, the viral vector is derived from simian virus 40,
adenoviruses, adeno-associated virus (AAV), lentivirus, or
retroviruses. In some alternatives, the viral vector is a
recombinant adenovirus, adeno-associated virus, lentivirus or
retrovirus vector. In some alternatives, the viral vector is a
lentivirus vector. In some alternatives, the cell surface
selectable marker encodes for a truncated epidermal growth factor
receptor (EGFRt). In some alternatives, the ligand binding domain
comprises an antibody, or a binding portion thereof. In some
alternatives, the ligand binding domain comprises a single chain
variable fragment (scFv), or a binding portion thereof. In some
alternatives, the ligand binding domain comprises FMC63, or a
binding portion thereof. In some alternatives, the ligand binding
domain is specific for CD19. In some alternatives, the population
comprises isolated CD4+ T-cells in the absence of or enriched over
CD8+ T-cells. In some alternatives, the population comprises
isolated CD8+ T-cells in the absence of or enriched over CD4+
T-cells.
[0328] In some alternatives, a composition or product combination
for human therapy is provided, wherein the composition or product
combination comprises a pharmaceutical excipient and at least one
population of genetically modified T-cells. In some alternatives,
the at least one population of genetically modified T-cells
comprises a plurality of affinity selected CD8+ and/or CD4+
T-cells, in the absence of, enriched over, or isolated from CD8-
and/or CD4- T-cells, wherein said plurality of affinity selected
CD8+ and/or CD4+ T-cells have stimulated CD2, CD3, CD4 and/or CD28
receptors, wherein said plurality of affinity selected CD8+ and/or
CD4+ T-cells further comprise a gene encoding a chimeric antigen
receptor and a cell surface selectable marker and, wherein said
plurality of affinity selected CD8+ and/or CD4+ T-cells have been
re-stimulated with at least one cytokine. In some alternatives, the
at least one cytokine comprises GM-CSF, IL-7, IL-12, IL-15, IL-18,
IL-2, and/or IL-21. In some alternatives, the at least one cytokine
comprises IL/7, IL-15 and/or IL-21. In some alternatives, the at
least one cytokine comprises IL-2, IL-15 and/or IL-21. In some
alternatives, the said plurality of affinity selected CD8+ and/or
CD4+ T-cells further comprise at least one receptor that promotes,
induces, contribute to or enhances engraftment fitness. In some
alternatives, the at least one receptor that promotes, induces,
contribute to or enhances engraftment fitness is CD45 RA, CD45 RO,
CCR7, CD25, CD127, CD57, CD137, CD27, CD28 and/or CD62L. In some
alternatives, the at least one receptor that promotes, induces,
contribute to or enhances engraftment fitness is CD27, CD28 and/or
CD62L. In some alternatives, the plurality of affinity selected
CD8+ and/or CD4+ T-cells further comprises a vector having a first
sequence encoding a leader sequence, a second sequence encoding a
ligand binding domain, a third sequence encoding a signaling domain
and a fourth sequence encoding a selectable marker sequence. In
some alternatives, the vector further comprises a sequence encoding
a spacer. In some alternatives, the spacer comprises an IgG4 hinge.
In some alternatives, the vector is a viral vector. In some
alternatives, the viral vector is derived from simian virus 40,
adenoviruses, adeno-associated virus (AAV), lentivirus, or
retroviruses. In some alternatives, the viral vector is a
recombinant adenovirus, adeno-associated virus, lentivirus or
retrovirus vector. In some alternatives, the viral vector is a
lentivirus vector. In some alternatives, the cell surface
selectable marker encodes for a truncated epidermal growth factor
receptor (EGFRt). In some alternatives, the ligand binding domain
comprises an antibody, or a binding portion thereof. In some
alternatives, the ligand binding domain comprises a single chain
variable fragment (scFv), or a binding portion thereof. In some
alternatives, the ligand binding domain comprises FMC63, or a
binding portion thereof. In some alternatives, the ligand binding
domain is specific for CD19. In some alternatives, at least one
population comprises isolated CD4+ T-cells in the absence of or
enriched over CD8+ T-cells. In some alternatives, at least one
population comprises isolated CD8+ T-cells in the absence of or
enriched over CD4+ T-cells. In some alternatives, the composition
or product combination comprises the population of genetically
modified T-cells, wherein the population of genetically modified
T-cells comprises isolated CD8+ T-cells in the absence of or
enriched over CD4+ T-cells. In some alternatives, the composition
or product combination comprises the population of genetically
modified T-cells, wherein the population of genetically modified
T-cells comprises isolated CD4+ T-cells in the absence of or
enriched over CD8+ T-cells. In some alternatives, the population
comprises isolated CD8+ T-cells in the absence of or enriched over
CD4+ T-cells and isolated CD4+ T-cells in the absence of or
enriched over CD8+ T-cells mixed or co-administered in a 1:1
ratio.
[0329] In some alternatives, a method of treating, inhibiting, or
ameliorating a disease in a subject in need thereof is provided,
wherein the method comprises administering to the subject at least
one composition or product combination. In some alternatives, the
at least one composition or product combination comprises a
pharmaceutical excipient and at least one population of genetically
modified T-cells. In some alternatives, the at least one population
of genetically modified T-cells comprises a plurality of affinity
selected CD8+ and/or CD4+ T-cells, in the absence of, enriched over
or isolated from CD8- and/or CD4- T-cells, wherein said plurality
of affinity selected CD8+ and/or CD4+ T-cells have stimulated CD2,
CD3, CD4 and/or CD28 receptors, wherein said plurality of affinity
selected CD8+ and/or CD4+ T-cells further comprise a gene encoding
a chimeric antigen receptor and a cell surface selectable marker
and, wherein said plurality of affinity selected CD8+ and/or CD4+
T-cells have been re-stimulated with at least one cytokine. In some
alternatives, the at least one cytokine comprises GM-CSF, IL-7,
IL-12, IL-15, IL-18, IL-2, and/or IL-21. In some alternatives, the
at least one cytokine comprises IL/7, IL-15 and/or IL-21. In some
alternatives, the at least one cytokine comprises IL-2, IL-15
and/or IL-21. In some alternatives, the said plurality of affinity
selected CD8+ and/or CD4+ T-cells further comprise at least one
receptor that promotes engraftment fitness. In some alternatives,
the at least one receptor that promotes, induces, contribute to or
enhances engraftment fitness is CD45 RA, CD45 RO, CCR7, CD25,
CD127, CD57, CD137, CD27, CD28 and/or CD62L. In some alternatives,
the at least one receptor that promotes, induces, contribute to or
enhances engraftment fitness is CD27, CD28 and/or CD62L. In some
alternatives, the plurality of affinity selected CD8+ and/or CD4+
T-cells further comprises a vector having a first sequence encoding
a leader sequence, a second sequence encoding a ligand binding
domain, a third sequence encoding a signaling domain and a fourth
sequence encoding a selectable marker sequence. In some
alternatives, the vector further comprises a sequence encoding a
spacer. In some alternatives, the spacer comprises an IgG4 hinge.
In some alternatives, the vector is a viral vector. In some
alternatives, the viral vector is derived from simian virus 40,
adenoviruses, adeno-associated virus (AAV), lentivirus, or
retroviruses. In some alternatives, the viral vector is a
recombinant adenovirus, adeno-associated virus, lentivirus or
retrovirus vector. In some alternatives, the viral vector is a
lentivirus vector. In some alternatives, the cell surface
selectable marker encodes for a truncated epidermal growth factor
receptor (EGFRt). In some alternatives, the ligand binding domain
comprises an antibody, or a binding portion thereof. In some
alternatives, the ligand binding domain comprises a single chain
variable fragment (scFv), or a binding portion thereof. In some
alternatives, the ligand binding domain comprises FMC63, or a
binding portion thereof. In some alternatives, the ligand binding
domain is specific for CD19. In some alternatives, at least one
population comprises isolated CD4+ T-cells in the absence of or
enriched over CD8+ T-cells. In some alternatives, at least one
population comprises isolated CD8+ T-cells in the absence of or
enriched over CD4+ T-cells. In some alternatives, the composition
or product combination comprises the population of genetically
modified T-cells, wherein the population of genetically modified
T-cells comprises isolated CD8+ T-cells in the absence of or
enriched over CD4+ T-cells. In some alternatives, the composition
or product combination comprises the population of genetically
modified T-cells, wherein the population of genetically modified
T-cells comprises isolated CD4+ T-cells in the absence of or
enriched over CD8+ T-cells. In some alternatives, the population
comprises isolated CD8+ T-cells in the absence of or enriched over
CD4+ T-cells and isolated CD4+ T-cells in the absence of or
enriched over CD8+ T-cells mixed or co-administered in a 1:1 ratio.
In some alternatives, the method comprises administering the
composition or product combination, wherein the composition or
product combination comprises at least one population, wherein the
at least one population comprises isolated CD4+ T-cells in the
absence of or enriched over CD8+ T-cells. In some alternatives, the
method comprises administering the composition or product
combination, wherein the composition or product combination
comprises at least one population, wherein the at least one
population comprises isolated CD8+ T-cells in the absence of or
enriched over CD4+ T-cells. In some alternatives, the method
comprises administering the composition or product combination,
wherein the composition or product combination comprises at least
one population, wherein the at least one population comprises
isolated CD4+ T-cells in the absence of or enriched over CD8+
T-cells and isolated CD8+ T-cells in the absence of or enriched
over CD4+ T-cells mixed or co-administered in a 1:1 ratio. In some
alternatives, the method further comprises administering the
composition or product combination, wherein the composition or
product combination comprises at least one population, wherein the
at least one population comprises isolated CD8+ T-cells in the
absence of or enriched over CD4+ T-cells. In some alternatives, the
method further comprises administering the composition or product
combination, wherein the composition or product combination
comprises at least one population, wherein the at least one
population comprises isolated CD4+ T-cells in the absence of or
enriched over CD8+ T-cells. In some alternatives, the subject is
identified or selected to receive an anti-cancer therapy. In some
alternatives, the method further comprises measuring or evaluating
an inhibition of a disease. In some alternatives, the method
further comprises providing said subject an additional anti-cancer
therapy before, during, or after administration of the composition
or product combination. In some alternatives, the composition or
product combinations are administered to said subject by adoptive
cell transfer. In some alternatives, the composition or product
combinations are administered to said subject after said subject
has received another form of anti-cancer therapy. In some
alternatives, the composition or product combinations are
administered to said subject after said subject has received
another form of anti-cancer therapy. In some alternatives, the
subject is suffering from leukemia. In some alternatives, the
subject has recurrent and/or chemotherapy refractory CD19+
childhood acute lymphoblastic leukemia (ALL). In some alternatives,
the subject has recurrent and/or chemotherapy refractory CD19+
acute lymphoblastic leukemia (ALL). In some alternatives, the
subject is suffering from an autoimmune disease. In some
alternatives, the subject is suffering from a post-HSCT
relapse.
[0330] With respect to the use of substantially any plural and/or
singular terms herein, those having skill in the art can translate
from the plural to the singular and/or from the singular to the
plural as is appropriate to the context and/or application. The
various singular/plural permutations may be expressly set forth
herein for sake of clarity.
[0331] It will be understood by those within the art that, in
general, terms used herein, and especially in the appended claims
(e.g., bodies of the appended claims) are generally intended as
"open" terms (e.g., the term "including" should be interpreted as
"including but not limited to," the term "having" should be
interpreted as "having at least," the term "includes" should be
interpreted as "includes but is not limited to," etc.). It will be
further understood by those within the art that if a specific
number of an introduced claim recitation is intended, such an
intent will be explicitly recited in the claim, and in the absence
of such recitation no such intent is present. For example, as an
aid to understanding, the following appended claims may contain
usage of the introductory phrases "at least one" and "one or more"
to introduce claim recitations. However, the use of such phrases
should not be construed to imply that the introduction of a claim
recitation by the indefinite articles "a" or "an" limits any
particular claim containing such introduced claim recitation to
alternatives containing only one such recitation, even when the
same claim includes the introductory phrases "one or more" or "at
least one" and indefinite articles such as "a" or "an" (e.g., "a"
and/or "an" should be interpreted to mean "at least one" or "one or
more"); the same holds true for the use of definite articles used
to introduce claim recitations. In addition, even if a specific
number of an introduced claim recitation is explicitly recited,
those skilled in the art will recognize that such recitation should
be interpreted to mean at least the recited number (e.g., the bare
recitation of "two recitations," without other modifiers, means at
least two recitations, or two or more recitations). Furthermore, in
those instances where a convention analogous to "at least one of A,
B, and C, etc." is used, in general such a construction is intended
in the sense one having skill in the art would understand the
convention (e.g., "a system having at least one of A, B, and C"
would include but not be limited to systems that have A alone, B
alone, C alone, A and B together, A and C together, B and C
together, and/or A, B, and C together, etc.). In those instances
where a convention analogous to "at least one of A, B, or C, etc."
is used, in general such a construction is intended in the sense
one having skill in the art would understand the convention (e.g.,
"a system having at least one of A, B, or C" would include but not
be limited to systems that have A alone, B alone, C alone, A and B
together, A and C together, B and C together, and/or A, B, and C
together, etc.). It will be further understood by those within the
art that virtually any disjunctive word and/or phrase presenting
two or more alternative terms, whether in the description, claims,
or drawings, should be understood to contemplate the possibilities
of including one of the terms, either of the terms, or both terms.
For example, the phrase "A or B" will be understood to include the
possibilities of "A" or "B" or "A and B."
More Alternatives
[0332] In some alternatives, the separating, enriching, or
isolating of the CD8+ expressing population of T-cells and/or a
CD4+ expressing population of T-cells, such as T-cells that are
derived from thymocytes or T-cells that are derived from engineered
precursors, desirably iPS cells, from a mixed population of T-cells
is performed by affinity selection for T-cells having an epitope
present on CD8 and/or CD4. In some alternatives, the separating,
enriching, or isolating of the CD8+ expressing population of
T-cells and/or a CD4+ expressing population of T-cells from a mixed
population of T-cells is performed by flow cytometry. In some
alternatives, the separating, enriching, or isolating of the CD8+
expressing population of T-cells and/or a CD4+ expressing
population of T-cells from a mixed population of T-cells is
performed by immuno-magnetic selection. In some alternatives, the
genetically modified CD8+ expressing T-cells and/or CD4+ expressing
T-cells comprise at least one receptor that promotes, induces, or
contributes to engraftment fitness. In some alternatives, the at
least one receptor that promotes, induces, or contributes to
engraftment fitness is CD45 RA, CD45 RO, CCR7, CD25, CD127, CD57,
CD137, CD27, CD28 and/or CD62L. In preferred alternatives, the at
least one receptor that promotes, induces, or contributes to
engraftment fitness is CD27, CD28 and/or CD62L. In some
alternatives, the stimulating of the isolated, enriched, or
separated population of T-cells is performed by contacting the CD8+
and/or CD4+ expressing T-cells with an antibody-bound support, such
as a bead or particle. In some of these alternatives, the
antibody-bound support comprises anti-TCR, anti-CD2, anti-CD3,
anti-CD4 and/or anti-CD28 antibodies. In preferred alternatives,
the antibody-bound support comprises anti-CD3 and/or anti-CD28
antibodies.
[0333] In many of the aforementioned alternatives, the vector
further comprises a first sequence encoding a leader sequence, a
second sequence encoding a ligand binding domain, a third sequence
encoding a signaling domain and a fourth sequence encoding a
selectable marker sequence. In some of these alternatives, the
vector further comprises a sequence encoding a spacer, which in
some alternatives may comprise an IgG4 hinge. In many of the
aforementioned alternatives, the vector is a viral vector or a
mini-circle.
[0334] In many of the aforementioned alternatives, the viral vector
is derived from simian virus 40, adenoviruses, adeno-associated
virus (AAV), lentivirus, or retroviruses. In some alternatives, the
viral vector is a recombinant adenovirus, adeno-associated virus,
lentivirus or retrovirus vector. Preferably, the viral vector is a
lentivirus vector. In many of the aforementioned alternatives, the
marker sequence encodes a truncated epidermal growth factor
receptor (EGFRt). In many of the aforementioned alternatives, the
at least one cytokine that is utilized comprises GM-CSF, IL-7,
IL-12, IL-15, IL-18, IL-15, IL-2, and/or IL-21 and said cytokine is
provided exogenously to the T-cells e.g., in addition to any
cytokine that may be produced by the cells or present in media.
[0335] In desirable alternatives, the at least one cytokine
comprises IL-7, IL-15 and/or IL-21. In many of the aforementioned
alternatives, the at least one cytokine comprises IL-2, IL-15
and/or IL-21. In desirable alternatives, the at least one cytokine
comprises IL-21 and another cytokine, and/or comprises IL-7 and at
least one other cytokine, and/or comprises IL-15 and at least one
other cytokine, such as comprising IL-21 and IL-15, comprising
IL-21 and IL-7, or comprising IL-15 and IL-7, or comprising IL-2
and IL-15, or comprising IL-7 and IL-15. In preferred alternatives,
the contacting period is performed for at least 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or a
period of time within a range defined by any two of these time
points. In many of the aforementioned alternatives, the methods are
performed with isolated, separated, or enriched populations of CD4+
expressing T-cells, such as T-cells that are derived from
thymocytes or T-cells that are derived from engineered precursors,
desirably iPS cells, in the absence of or having a reduced amount
CD8+ expressing T-cells, as compared to a native population of
unseparated, non-enriched, or non-isolated population of T-cells.
In many of the aforementioned alternatives, these methods are
performed with isolated, separated, or enriched populations of CD8+
expressing T-cells, such as T-cells that are derived from
thymocytes or T-cells that are derived from engineered precursors,
desirably iPS cells, in the absence of or having a reduced amount
of CD4+ expressing T-cells, as compared to a native population of
unseparated, non-enriched, or non-isolated population of T-cells.
In many of the aforementioned alternatives, the CD4+ expressing
T-cells are propagated for at least 1 day, such as 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or
any time that is within a range of times defined by any two of the
aforementioned time points. In many of the aforementioned
alternatives, the CD8+ expressing T-cells are propagated for at
least 1 day, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, or 20 days or any time that is within a range
of times defined by any two of the aforementioned time points.
[0336] In some alternatives, a method of making genetically
modified T-cells, which have a chimeric antigen receptor is
provided, wherein the method comprises separating or enriching a
CD8+ expressing population of T-cells and/or a CD4+ expressing
population of T-cells, such as T-cells that are derived from
thymocytes or T-cells that are derived from engineered precursors,
wherein the precursors are optionally iPS cells, from a mixed
population of T-cells, so as to generate a separated or enriched
population of T-cells, stimulating the separated or enriched
population of T-cells so as to generate a stimulated population of
CD8+ T-cells and/or CD4+ T-cells, transducing the stimulated
population of CD8+ T-cells and/or CD4+ T-cells with a vector,
wherein the vector encodes a chimeric antigen receptor so as to
generate a transduced population of CD8+ T-cells and/or CD4+
T-cells, contacting the transduced population of CD8+ T-cells
and/or CD4+ T-cells with at least one cytokine, provided
exogenously, for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, or 20 days, or for a period that is within a range
defined by any two of the aforementioned time periods, so as to
generate a transduced, cytokine-stimulated population of CD8+
T-cells and/or CD4+ T-cells, wherein the method thereby obtains
said genetically modified T-cells, which have a chimeric antigen
receptor. In some alternatives, the method further comprises
enriching the transduced, cytokine-stimulated population of CD8+
T-cells and/or CD4+ T-cells by selection of a marker, so as to
generate an enriched population of transduced, cytokine-stimulated
CD8+ T-cells and/or CD4+ T-cells. In some alternatives, the marker
is encoded by the vector and optionally is a cell surface marker.
In some alternatives, the method further comprises further
comprising propagating the enriched population of transduced,
cytokine-stimulated CD8+ T-cells and/or CD4+ T-cells for at least
one day, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, or 20 days or for a period that is within a range
defined by any two of the aforementioned time periods.
[0337] In some alternatives, a method of producing genetically
modified T cells is provided, wherein the method comprises
introducing a vector encoding a recombinant protein into cells of a
CD4+-enriched T cell composition, and incubating cells of the CD4+
enriched T cell composition with a combination of cytokines
comprising IL-7 and IL-15, and/or introducing a vector encoding a
recombinant protein into cells of a CD8+ enriched T cell
composition and incubating cells of the CD8+ enriched T cell
composition with a combination of cytokines comprising IL-2 and
IL-15, wherein the method thereby generates a transduced CD4+
enriched population and/or a transduced CD8+ enriched population.
In some alternatives, the transduced CD4+ enriched population has a
higher level of surface expression of CD62L, CD27, and/or CD28, as
compared to a reference enriched population of transduced CD4+
cells, and/or has an increased indicator of engraftment fitness
and/or expansion, and/or persistence, upon administration to a
subject as compared to the reference enriched population of
transduced CD4+ cells, wherein the reference population is a
CD4+-enriched population prepared by another method, which is
identical to the method, except that the IL-2 alone is used in
place of the combination of cytokines. In some alternatives, the
transduced CD8+ enriched population has a higher level of surface
expression of CD62L, CD27, and/or CD28, as compared to a reference
enriched population of transduced CD8+ cells, and/or has an
increased indicator of engraftment fitness and/or expansion, and/or
persistence, upon administration to a subject as compared to the
reference enriched population of transduced CD8+ cells, wherein the
reference population is a population of CD8+-enriched cells
prepared by another method that is identical to the method, except
that the IL-2 alone is used in place of the combination of
cytokines.
[0338] In some alternatives, a method of producing genetically
modified T cells is provided, wherein the method comprises
introducing a vector encoding a recombinant protein into cells of a
CD4+ enriched T cell composition and incubating cells of the CD4+
enriched T cell composition with a first combination of cytokines
and introducing a vector encoding a recombinant protein into cells
of a CD8+ enriched T cell composition and incubating cells of the
CD8+ enriched T cell composition with a second combination of
cytokines which is distinct from the first combination of
cytokines, wherein the method thereby generates a transduced CD4+
enriched population and a transduced CD8+ enriched population. In
some alternatives, the transduced CD4+ enriched population has a
higher level of surface expression of CD62L, CD27, and/or CD28, as
compared to a reference enriched population of transduced CD4+
cells prepared by another method that is identical to the method,
except that the first and second combination of cytokines are
identical, and/or has an increased indicator of engraftment fitness
and/or expansion, and/or persistence, upon administration to a
subject as compared to the reference enriched population of
transduced CD4+ cells. In some alternatives, the transduced CD8+
enriched population has a higher level of surface expression of
CD62L, CD27, and/or CD28, as compared to a reference enriched
population of transduced CD8+ cells prepared by another method that
is identical to the method, except that the first and second
combination of cytokines are identical, and/or has an increased
indicator of engraftment fitness and/or expansion, and/or
persistence, upon administration to a subject as compared to the
reference enriched population of transduced CD8+ cells. In some
alternatives, the first combination comprises IL-7 and IL-15 and
the second combination comprises IL-2 and IL-15. In some
alternatives, the cytokine combination(s) is added subsequently to
transduction initiation, and optionally on the same day as
transduction initiation. In some alternatives, the concentration of
IL-2 is at or about 50 U/mL, where applicable, the concentration of
IL-15 is at or about 0.5 ng/mL, where applicable, and/or the
concentration of IL-7 is at or about 5 ng/mL, where applicable. In
some alternatives, the addition of the combination of cytokines to
the CD4+-enriched and/or to the CD8+-enriched composition comprises
increasing the volume of the composition in which the cells are
incubated, thereby decreasing cell density. In some alternatives,
the indicator of engraftment fitness comprises a percentage of
cells in the composition or total surface expression levels for the
population of a cell surface marker selected from the group
consisting of CD62L, CD27, and/or CD28. In some alternatives, the
indicator of engraftment fitness comprises persistence in a subject
upon administration. In some alternatives, the cells are derived
from the subject. In some alternatives, transduced CD8+ and/or CD4+
cells persist upon administration to a subject from which the cells
were derived, for at least at or about 30 or 60 days post injection
of the cells into the subject. In some alternatives, the
recombinant protein is a chimeric antigen receptor. In some
alternatives, the method further comprises prior to the
transduction, enriching a T cell composition for cells expressing
CD4, thereby generating the enriched CD4+ enriched T cell
composition so-transduced, and/or, prior to the transduction,
enriching a T cell composition for cells expressing CD8, thereby
generating the enriched CD8+ enriched T cell composition
so-transduced. In some alternatives, the method further comprises
cryopreserving the engineered cells. In some alternatives, the
method further comprises administering the engineered cells to a
subject, and optionally further comprising pooling the transduced
CD4+ enriched and the transduced CD8+ enriched compositions prior
to said administering.
[0339] In some alternatives, a cell or composition is produced by
any of the alternatives of the methods listed in the previous
paragraphs of this section of more alternatives. In some
alternatives, a method of administering the cell produced by any of
the alternatives of the method or the alternatives of the
compositions listed in the previous paragraphs is contemplated. In
some alternatives, the cell or composition is administered to a
subject. In some alternatives, the administration is to a subject
from which the cells were derived.
[0340] In some alternatives, a method of adoptive cell therapy is
provided, wherein the method comprises (a) incubating a
CD4+-enriched T cell composition under stimulating conditions and
in the presence of IL-15 and IL-7, thereby generating an expanded
CD4+ composition, (b) separately incubating a CD8+-enriched T cell
composition under stimulating conditions and in the presence of
IL-15 and IL-2, thereby generating an expanded CD8+ composition,
and (c) administering the expanded CD4+ and expanded CD8+
populations to a subject, optionally simultaneously. In some
alternatives, the CD4+ and CD8+ populations are obtained from the
subject. In some alternatives, the one or more of the recited
cytokines or combinations of cytokines further comprises CD21. In
some alternatives, the CD4+-enriched population or the total number
of T cells therein comprises at least 70, 80, 90, 95, 96, 97, 98,
or 99% CD4+ cells, and/or the C8+-enriched population or the total
number of T cells therein comprises at least 70, 80, 90, 95, 96,
97, 98, or 99% CD8+ cells.
[0341] In addition, where features or aspects of the disclosure are
described in terms of Markush groups, those skilled in the art will
recognize that the disclosure is also thereby described in terms of
any individual member or subgroup of members of the Markush group.
Sequence CWU 1
1
7112PRTArtificial SequenceModified IgG4 hinge region 1Glu Ser Lys
Tyr Gly Pro Pro Cys Pro Pro Cys Pro1 5 10 29PRTArtificial
SequenceModified IgG4 hinge region 2Tyr Gly Pro Pro Cys Pro Pro Cys
Pro1 5 310PRTArtificial SequenceModified IgG4 hinge region 3Lys Tyr
Gly Pro Pro Cys Pro Pro Cys Pro1 5 10 413PRTArtificial
SequenceModified IgG4 hinge region 4Glu Val Val Lys Tyr Gly Pro Pro
Cys Pro Pro Cys Pro1 5 10 528PRTArtificial SequenceCD28
transmembrane domain 5Met Phe Trp Val Leu Val Val Val Gly Gly Val
Leu Ala Cys Tyr Ser1 5 10 15 Leu Leu Val Thr Val Ala Phe Ile Ile
Phe Trp Val 20 25 642PRTArtificial Sequence4-1BB domain 6Lys Arg
Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met1 5 10 15
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20
25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40
7112PRTArtificial SequenceCD3-zeta domain 7Arg Val Lys Phe Ser Arg
Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly1 5 10 15 Gln Asn Gln Leu
Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val
Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50
55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
Arg65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu
Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln
Ala Leu Pro Pro Arg 100 105 110
* * * * *