U.S. patent application number 16/343869 was filed with the patent office on 2020-10-01 for methods and systems for t cell expansion.
The applicant listed for this patent is Georgia Tech Research Corporation. Invention is credited to Nate Dwarshuis, Ranjna Madan-lala, Kyung-Ho Roh, Krishnendu Roy, Hannah Kathryn Wilson.
Application Number | 20200306299 16/343869 |
Document ID | / |
Family ID | 1000005087234 |
Filed Date | 2020-10-01 |
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United States Patent
Application |
20200306299 |
Kind Code |
A9 |
Roy; Krishnendu ; et
al. |
October 1, 2020 |
Methods and Systems for T Cell Expansion
Abstract
The present disclosure provides a system for mimicking the
secondary lymphoid organs where suspension cells (e.g., T cells)
are expanded; methods of expanding, activating, and transfecting
the suspension cells in the synthetic microenvironment, and
suspension cells produced by such systems and methods.
Inventors: |
Roy; Krishnendu; (Atlanta,
GA) ; Dwarshuis; Nate; (Atlanta, GA) ;
Madan-lala; Ranjna; (Atlanta, GA) ; Roh;
Kyung-Ho; (Atlanta, GA) ; Wilson; Hannah Kathryn;
(Atlanta, GA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Georgia Tech Research Corporation |
Atlanta |
GA |
US |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20190343879 A1 |
November 14, 2019 |
|
|
Family ID: |
1000005087234 |
Appl. No.: |
16/343869 |
Filed: |
October 20, 2017 |
PCT Filed: |
October 20, 2017 |
PCT NO: |
PCT/US17/57687 PCKC 00 |
371 Date: |
April 22, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62410877 |
Oct 21, 2016 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/2818 20130101;
C07K 16/2809 20130101; A61K 35/17 20130101; A61K 38/20 20130101;
C12M 21/18 20130101; C12N 15/86 20130101; C12M 27/14 20130101 |
International
Class: |
A61K 35/17 20060101
A61K035/17; A61K 38/20 20060101 A61K038/20; C07K 16/28 20060101
C07K016/28; C12N 15/86 20060101 C12N015/86; C12M 1/40 20060101
C12M001/40; C12M 3/06 20060101 C12M003/06 |
Goverment Interests
GOVERNMENT SPONSORSHIP
[0002] This invention was made with government support under Grant
Nos. 12567G5 and 1547638 awarded by the National Science
Foundation. The government has certain rights in the invention.
Claims
1. A system for expanding, activating, and/or transfecting
suspension cells comprising: a three-dimensional functionalized
porous microcarrier; and suspension cells.
2. (canceled)
3. The system of claim 1, wherein the suspension cells are T
cells.
4.-7. (canceled)
8. The system of claim 1, wherein the functionalized porous
microcarrier comprises at least one of antibodies, aptamers, and
phage-display identified peptide ligands.
9. The system of claim 1, wherein the functionalized porous
microcarrier comprises antibodies that are specific for the
suspension cells.
10. The system of claim 3, wherein the functionalized porous
microcarrier comprises antibodies that are specific for T
cells.
11. The system of claim 1, wherein the functionalized porous
microcarrier comprises one or more of anti-CD2, anti-CD3 and
anti-CD28 antibodies.
12.-13. (canceled)
14. The system of claim 1 further comprising an open bioreactor
comprising a static culture vessel with a gas-permeable bottom.
15. The system of claim 1 further comprising a closed bioreactor
selected from the group consisting of a stirred-type bioreactor, a
bag bioreactor, a perfusion bioreactor, and combinations
thereof.
16. A system for expanding, activating, and/or transfecting T cells
comprising: a three-dimensional functionalized macroporous
microcarrier; T cells; and at least one of: a culture medium; a
cytokine; a viral vector; and a growth factor.
17. The system of claim 16 comprising at least the cytokine
selected from the group consisting of IL2, IL7, IL15, and
combinations thereof.
18. The system of claim 16, wherein the T cells are selected from
the group consisting of recombinant T cells, gene modified T cells,
chimeric antigen receptor (CAR) T cells, unmodified T cells,
CCR7+CD62+ central memory T cells, and combinations thereof.
19. The system of claim 16 comprising at least the viral vector
that is configured for use in gene therapy.
20. The system of claim 16 comprising at least the viral vector
comprising a CAR transgene, the system further comprising an
additional gene selected from the group consisting of a therapeutic
gene, surface marker gene, reporter gene, suicide genes, chemokine
receptor gene, cytokine-expressing gene, immune-checkpoint receptor
gene, and combinations thereof.
21. (canceled)
22. A method comprising: obtaining a blood sample from a patient;
isolating suspension cells from the blood sample; introducing at
least a portion of the suspension cells to a bioreactor comprising
a three-dimensional functionalized porous microcarrier; activating
at least a portion of the suspension cells introduced into the
bioreactor; and expanding at least a portion of the activated
suspension cells.
23.-28. (canceled)
29. The method of claim 22, wherein the porous microcarrier
comprises one or more of proteins, carbohydrates, lipids and
nucleic acids.
30.-31. (canceled)
32. The method of claim 22, wherein the functionalized porous
microcarrier comprises at least one of antibodies, aptamers, and
phage-display identified peptide ligands.
33.-34. (canceled)
35. The method of claim 22, wherein the functionalized porous
microcarrier comprises one or more of anti-CD2, anti-CD3 and
anti-CD28 antibodies.
36.-47. (canceled)
48. (canceled)
49. A suspension cell obtained by the method of claim 22.
50.-56. (canceled)
57. A pharmaceutical composition comprising: at least one
suspension cell of claim 49; at least one carrier; and at least one
additional therapeutic agent; wherein the composition is formulated
for intravenous administration.
58.-63. (canceled)
64. The method of of claim 22 further comprising: transfecting at
least a portion of the expanded suspension cells; preparing at
least a portion of the transfected suspension cells for transfusion
into the patient; and transfusing at least a portion of the
prepared suspension cells into the patient; wherein the porous
microcarrier is configured for activation, expansion, and/or
transfection of the suspension cells.
65.-68. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 62/410,877, filed on 21 Oct. 2016, the disclosure
of which is herein incorporated by reference in its entirety.
BACKGROUND OF THE DISCLOSURE
1. Field
[0003] Embodiments of the present disclosure relate generally to
methods and systems for suspension cell (for example and not
limitation, T cells) expansion, and more specifically to synthetic
microenvironments capable of mimicking T cell niches within
secondary lymphoid organs (such as for example and not limitation,
lymph nodes). For example and not limitation, these synthetic
microenvironments comprise functionalized macroporous
three-dimensional (3D) microcarriers that contain antibodies to
enhance T cell expansion and activation, as well as optionally
enable transfection. T cells obtainable by these systems and
methods include gamma-delta T cells as well as alpha-beta T cells,
including for example and not limitation, recombinant T cells, gene
modified T cells, chimeric antigen receptor (CAR) T cells,
unmodified T cells, and CCR7.sup.+CD62.sup.+ central memory T
cells.
2. Background
[0004] Immunotherapy using adoptive T cell transfer (ACT) is a
highly promising approach in treating cancers, infectious and
autoimmune diseases, as well as for transplantation associated
problems. In particular, anti-tumor ACT, using tumor-isolated or
genetically-engineered T cells (e.g., Chimeric Antigen Receptor
(CAR) T cells), has shown great potential in clinical trials of
various cancers (1-3).
[0005] Physiological T cell expansion occurs primarily in secondary
lymphoid organs (e.g., lymph nodes (LNs), spleen, gut-associated
lymphoid tissue etc.). Upon interaction with antigen presenting
cells (APC, such as for example and not limitation, dendritic
cells) displaying foreign antigen epitopes, T cells engage with
APCs through several surface molecules including T cell receptors
(TCRs), CD3, CD28, etc. T cells expand as a result of this direct
signaling aided by a set of locally secreted cytokines by
themselves and APCs. The high cell density of the T cell zone in
LNs ensures that cytokines are presented at high local
concentrations and efficient autocrine and paracrine signaling
takes place. Current T cell expansion methods using suspension
cultures, is therefore non-physiological and requires very high
dosage of cytokines and do not recapitulate the cell-cell
communication required for efficient T cell expansion. Even though
T cells do form loose aggregates in these suspension cultures,
synthetic 3D niches that mimic a more LN like high-density culture
environment with efficient cell-cell communication and relevant
extracellular-matrix, could significantly improve T cell expansion
and quality.
[0006] Currently, the most common approach to T cell expansion is
the use of soluble anti-CD3 or anti-CD3/anti-CD28 dynabeads with
suspension cultures. Although surface-immobilized antibodies (Abs),
such as on microbeads, signal more robustly and mimic the
APC/T-cell interactions better, a key issue with bead-based
expansion is that newly generated cells (progenies) have minimal
interaction with anti-CD3/anti-CD28 beads and thus do not expand
further.
[0007] In adults, each dose of CAR-T cell therapy requires
approximately 10.sup.8-10.sup.9 cells to be injected (4-6).
Considering the additional number of cells that are required for
safety testing and quality control (e.g., testing of sterility,
endotoxin level, cell purity, particulate impurity, stability,
potency, etc.), the total number of cells for each batch of
production must exceed .about.10.sup.9. Unfortunately, the number
of autologous T cells that can be harvested from cancer patients,
especially those with advanced cancer and those undergoing
radiation and chemotherapy could be very limited, which further
decreases significantly in the process of genetic-modification (CAR
transduction with lentivirus). Current expansion protocols using
suspension cultures with large amounts of IL-2, IL-7 or IL-15 can
only achieve 10-100-fold expansion in 1-2 weeks of processing, thus
generally resulting in just over a single dose of T cells from each
batch with no option of multiple dosing or storage. If multiple
doses are to be administered from a single bioprocess, even more
cells would be necessary (especially given inefficient freeze-thaw
process). Furthermore, the transduction/expansion process generally
results in a heterogeneous population of T cells with multiple
phenotypes (effector cells, memory cells, exhausted cells, high or
low cytokine-secreting cells etc.) and which of these phenotypes
are most suited for maximal in vivo anti-tumor efficacy, is still
largely unknown. Although for B cell malignancies, memory-type T
cells (CD62L.sup.+CCR7.sup.+) have been shown to have the highest
correlative potency, primarily due to their ability to survive in
vivo and better home into the tumor site (lymph nodes); that has
not been established for other cancers (7). Thus, new bioprocess
engineering methods to efficiently expand CAR-T populations, in
terms of numbers, cell quality and potency, is critically needed to
enable broad clinical use of this promising therapy.
[0008] Thus, despite its potential and recent success, current
approaches for CAR-based ACT are severely constrained by (a) the
limited availability of autologous T cells from cancer patients;
(b) difficulty in robustly and reproducibly expanding these cells
to enough numbers for multiple administrations; and (c) lack of
methods that selectively expand the most potent sub-population of T
cells for specific applications (for example but not limitation,
memory T cells and/or T cells with superior transport properties).
As a result, new cell-manufacturing concepts that would allow large
scale production of therapeutic T cells, such as for example and
not limitation, therapeutic CAR-T cells, without losing their
potency and safety, are needed.
[0009] What is needed, therefore, is a synthetic microenvironment
capable of mimicking T cell niches within secondary lymphoid organs
such as for example and not limitation, lymph nodes, the anatomical
location where natural T cell activation and expansion take place
in the body. The systems should take advantage of 3D
microcarriers--which are widely used for adherent cells in industry
practice but not for suspension cells--that can be functionalized
with antibodies to promote suspension cell (e.g., T cell)
activation and expansion. The systems should provide suspension
cells with improved potency and efficacy and allow for specific
highly potent sub populations to expand selectively. It is to such
systems and methods of producing suspension cells, such as for
example and not limitation, T cells, that embodiments of the
present disclosure are directed.
BRIEF SUMMARY OF THE DISCLOSURE
[0010] As specified in the Background Section, there is a great
need in the art to identify technologies for improved methods and
systems for large-scale production of suspension cells, such as for
example and not limitation, T cells, and use this understanding to
develop novel bioreactor systems and methods. The present
disclosure satisfies this and other needs. Embodiments of the
present disclosure relate generally to synthetic microenvironment
capable of mimicking T cell niches within secondary lymphoid organs
(such as for example and not limitation, lymph nodes) and more
specifically to macroporous 3D microcarriers that can be
functionalized with antibodies to promote suspension cell (e.g., T
cell) activation and expansion to mimic the environment found in
lymph nodes. The system should provide suspension cells with
improved potency and efficacy. It is to such systems and methods of
producing suspension cells, such as for example and not limitation,
T cells, that embodiments of the present disclosure are directed. T
cells produced by the systems and methods described herein include,
but are not limited to, recombinant T cells, gene modified T cells,
chimeric antigen receptor (CAR) T cells, unmodified T cells, and
CCR7.sup.+CD62.sup.+ central memory T cells.
[0011] The present disclosure provides a system for mimicking the
secondary lymphoid organs where suspension cells (e.g., T cells)
are expanded; methods of expanding, activating, and transfecting
the suspension cells in the synthetic microenvironment, and
suspension cells produced by such systems and methods.
[0012] In one aspect, the disclosure provides a system for
expanding, activating, and/or transfecting suspension cells
comprising: a porous microcarrier; and the suspension cells.
[0013] In some embodiments, the suspension cells are isolated from
a patient's blood or organs, including both normal and/or diseased
tissues.
[0014] In some embodiments, the suspension cells are T cells.
[0015] In other embodiments, the porous microcarrier is three
dimensional. In some embodiments, the porous microcarrier comprises
proteins, carbohydrates, lipids or nucleic acids.
[0016] In some embodiments, the porous microcarrier comprises
gelatin or other extracellular matrix components.
[0017] In yet other embodiments, the porous microcarrier is
functionalized. In some embodiments, the functionalized porous
microcarrier comprises at least one of antibodies, aptamers, and
phage-display identified peptide ligands.
[0018] In some embodiments, the antibodies comprise antibodies that
are specific for the suspension cells. In other embodiments, the
antibodies comprise antibodies that are specific for T cells. In
some embodiments, the antibodies comprise anti-CD2, anti-CD3 and/or
anti-CD28 antibodies.
[0019] In an embodiment, the system in any of the preceding
embodiments further comprises a bioreactor. In some embodiments,
the bioreactor comprises a closed bioreactor and an open
bioreactor. In some embodiments, the open bioreactor comprises a
static culture vessel with a gas-permeable bottom. In other
embodiments, the closed bioreactor comprises a stirred-type
bioreactor, a bag bioreactor, and a perfusion bioreactor.
[0020] In another embodiment, the system in any of the preceding
embodiments further comprises at least one of culture medium, at
least one cytokine, and/or at least one viral vector, and
optionally at least one growth factor.
[0021] In some embodiments, the at least one cytokine comprises IL2
and optionally at least one of IL7 or IL15. In other embodiments,
the at least one viral vector is configured for use in gene therapy
(such as for example and not limitation, a lentiviral vector, a
retroviral vector, an adenoviral vector, and an adeno-associated
viral vector).
[0022] In any of the embodiments described herein, the suspension
cells can comprise recombinant T cells, gene modified T cells,
chimeric antigen receptor (CAR) T cells, unmodified T cells, and/or
CCR7.sup.+CD62.sup.+ central memory T cells.
[0023] In some embodiments, the at least one viral vector comprises
a CAR transgene and optionally at least one additional gene,
wherein the at least one additional gene comprises therapeutic
genes, surface marker genes, reporter genes, suicide genes,
chemokine receptor genes, cytokine-expressing genes, and/or
immune-checkpoint receptor genes.
[0024] In any of the preceding embodiments, the porous microcarrier
is macroporous. In some embodiments, the porous microcarrier is
degradable, such as for example and not limitation,
biodegradable.
[0025] In a related aspect, the disclosure provides a method of
expanding, activating, and/or transfecting suspension cells, the
method comprising: obtaining a blood sample from a patient;
isolating suspension cells from the blood sample; introducing the
suspension cells to a bioreactor comprising a porous microcarrier;
activating the suspension cells; expanding the suspension cells;
optionally transfecting the suspension cells; preparing the
suspension cells for transfusion into the patient; and transfusing
the suspension cells into the patient.
[0026] In some embodiments, the blood or tissue sample is obtained
by leukapharesis.
[0027] In other embodiments, the suspension cells are T cells.
[0028] In some embodiments, the step of isolating suspension cells
from the blood sample further comprises bead separation or magnetic
bead separation.
[0029] In other embodiments, the bioreactor comprises a closed
bioreactor and an open bioreactor. In some embodiments, the closed
bioreactor comprises a stirred-type bioreactor, a bag bioreactor,
and a perfusion bioreactor. In other embodiments, the open
bioreactor comprises a static culture vessel with a gas-permeable
bottom.
[0030] In some embodiments, the porous microcarrier is three
dimensional. In other embodiments, the porous microcarrier
comprises proteins, carbohydrates, lipids or nucleic acids. In
still other embodiments, the porous microcarrier comprises gelatin
or other extracellular matrix components.
[0031] In some embodiments, the porous microcarrier is
functionalized. In other embodiments, the functionalized porous
microcarrier comprises at least one of antibodies, aptamers, and
phage-display identified peptide ligands. In still other
embodiments, the antibodies comprise antibodies that are specific
for the suspension cells. In some embodiments, the antibodies
comprise antibodies that are specific for T cells. In some
embodiments, the antibodies comprise anti-CD2, anti-CD3 and/or
anti-CD28 antibodies.
[0032] In an embodiment of any of the foregoing methods, the method
further comprises at least one of culture medium, at least one
cytokine, and/or at least one viral vector, and optionally at least
one growth factor. In some embodiments, the at least one cytokine
comprises IL2, and optionally at least one of IL7 or IL15.
[0033] In an embodiment of any of the foregoing methods, the
activation step further comprises agitation, optionally under
hypoxic conditions. In some embodiments, the agitation can be
periodic or continuous. In other embodiments, the activation step
can last for at least one day, at least two days, at least three
days, and at least four days, and all ranges of time in
between.
[0034] In an embodiment of any of the foregoing methods, the
expansion step further comprises seed trains, optionally under
hypoxic conditions. For example and not limitation, the expansion
step can include increasing the size of the flask and/or bioreactor
as the culture grows, which can also involve moving the expanding
cells to a new vessel, and culture media exchanges every 2-3 days
(particularly if a static culture). The expansion step can last for
at least one day, at least two days, at least three days, at least
four days, at least five days, at least six days, at least seven
days, at least eight days, at least nine days, at least ten days,
and at least eleven days, and all ranges of time in between.
[0035] In an embodiment of any of the foregoing methods, the
optional transfection step further comprises adding at least one
viral vector to the bioreactor comprising the suspension cells.
[0036] In an embodiment of any of the foregoing methods, the
suspension cells comprise recombinant T cells, gene modified T
cells, chimeric antigen receptor (CAR) T cells, unmodified T cells,
and/or CCR7.sup.+CD62.sup.+ central memory T cells.
[0037] In some embodiments, the at least one viral vector is
configured for use in gene therapy. In other embodiments, the at
least one viral vector comprises a CAR transgene and optionally at
least one additional gene, wherein the at least one additional gene
comprises therapeutic genes, surface marker genes, reporter genes,
suicide genes, chemokine receptor genes, cytokine-expressing genes,
and/or immune-checkpoint receptor genes.
[0038] In an embodiment of any of the foregoing methods, the
preparation step further comprises cell expansion and downstream
bioprocessing. In some embodiments, the cell expansion and
downstream bioprocessing comprises cell separation, purification,
packaging, preservation, storage, shipping and transport, thawing,
formulation, resuspension, and transfusion.
[0039] In an embodiment of any of the foregoing methods, the
transfusion step further comprises injection, intravenous
administration, and implantation (such as for example and not
limitation, implantation of a sustained delivery device).
[0040] In an embodiment of any of the foregoing methods, the porous
microcarrier is macroporous. In any of the preceding embodiments,
the porous microcarrier is macroporous. In some embodiments, the
porous microcarrier is degradable, such as for example and not
limitation, biodegradable.
[0041] In a related aspect, the disclosure provides a T cell
obtained from any of the systems disclosed herein.
[0042] In a related aspect, the disclosure provides a T cell
obtained from any of the methods disclosed herein.
[0043] In one embodiment, the T cell is a memory cell. In another
embodiment, the T cell is CCR7.sup.+CD62L.sup.+. In another
embodiment, the T cell is a central memory T cell. In yet another
embodiment, the T cell is a CD4 T cell or a CD8 T cell. In some
embodiments, the T cell comprises recombinant T cells, gene
modified T cells, chimeric antigen receptor (CAR) T cells,
unmodified T cells, and/or CCR7.sup.+CD62.sup.+ central memory T
cells.
[0044] In a related aspect, the disclosure provides a composition
comprising at least one T cell as described herein. In some
embodiments, the composition further comprises at least one
carrier. In other embodiments, the composition is formulated for
intravenous administration.
[0045] In a related aspect, the disclosure provides a
pharmaceutical composition comprising at least one T cell as
described herein. In some embodiments, the pharmaceutical
composition further comprises at least one carrier. In other
embodiments, the composition is formulated for intravenous
administration. In some embodiments, the pharmaceutical composition
further comprises at least one additional therapeutic agent.
[0046] In a related aspect, the disclosure provides the use of a
composition (including a pharmaceutical composition) as described
herein to treat a disease or condition in a patient in need
thereof. In some embodiments, the disease or condition comprises
genetic diseases, cancers, infections, autoimmune diseases, and/or
transplant complications. In some embodiments, the use further
comprises a second therapeutic method or agent, such as for example
and not limitation, a cancer drug, an immunotherapeutic, an
immunosuppressant, an autoimmune therapeutic agent, and/or a
therapeutic for treating infections.
[0047] In a related aspect, the disclosure provides method of
treating a disease or condition in a patient in need thereof,
comprising administering a composition (including a pharmaceutical
composition) as described herein to said patient. In some
embodiments, the disease or condition comprises genetic diseases,
cancers, infections, autoimmune diseases, and/or transplant
complications. In some embodiments, the treatment further comprises
a second therapeutic method or agent, such as for example and not
limitation, a cancer drug, an immunotherapeutic, an
immunosuppressant, an autoimmune therapeutic agent, and/or a
therapeutic for treating infections.
[0048] In an embodiment of any of the foregoing systems, the porous
microcarrier is configured for activation, expansion, and/or
transfection of the suspension cells.
[0049] In an embodiment of any of the foregoing methods, the porous
microcarrier is configured for activation, expansion, and/or
transfection of the suspension cells.
[0050] These and other objects, features and advantages of the
present disclosure will become more apparent upon reading the
following specification in conjunction with the accompanying
description, claims and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0051] The accompanying Figures, which are incorporated in and
constitute a part of this specification, illustrate several aspects
described below.
[0052] FIGS. 1A-1B. Optimization of streptavidin coating of
microcarriers. FIG. 1A) Streptavidin surface density as a function
of sulfo-NHS-biotin added per mass of microcarrier for Cultispher S
and Cultispher G microcarriers. FIG. 1B) Binding site density as a
function of sulfo-NHS-biotin amount added per mass of Cultispher S
(CuS) microcarriers.
[0053] FIGS. 2A-2B. Verification of open biotin binding sites.
Zeiss Lightsheet microscope imaging of (FIG. 2A) uncoated CuS
microcarriers and (FIG. 2B) CuS microcarriers optimally coated with
streptavidin and saturated with FITC-biotin.
[0054] FIG. 3. Antibody coating density as function of CD3/CD28
percentage. Microcarriers coated with streptavidin were conjugated
using mAb cocktails that contained varying amounts of biotinylated
isotype control (calculated as percentage CD3/CD28 mAb mass with
CD3/CD28 ratio as 1:1).
[0055] FIG. 4. Comparison of T cell expansion and MACS bead
expansion. Primary human T cells were expanded over 2 weeks in well
plates in various culture conditions. (FIG. 4, Top) Day 14
brightfield images of T cells expanded at the indicated
cell:carrier ratio with the absolute number of carriers held
constant. (FIG. 4, Bottom) Day 14 fold change of microcarrier
cultures at indicated ratios compared with cells grown in
MACSibeads culture (conventional magnetic beads).
[0056] FIG. 5 depicts undisturbed microcarrier cultures forming 3D
cell:carrier clusters.
[0057] FIGS. 6A-6B. Assessment of memory subpopulations in
microcarrier-expanded T cell cultures. T cells were expanded on
microcarriers or MACSibeads cultures for 14 days and assessed via
flow and transmigration assay. FIG. 6A) Expanded T cells assessed
for CCR7 and CD62L populations using flow cytometry. FIG. 6B) The
same cells assessed in (FIG. 6A) were characterized for functional
migratory potential.
[0058] FIGS. 7A-7B. Lentiviral transduction of microcarrier T cell
cultures. T cells were activated with either plate-bound antibodies
or microcarriers and transduced with lentivirus expressing an
anti-CD19 chimeric antigen receptor. FIG. 7A) Flow cytometry plots
of CAR-expressing T cells assessed on day 9 of culture. FIG. 7B)
Transduced T cells were assessed for functionality by measuring
degranulation in response to tumor cells.
[0059] FIGS. 8A-8B. Microcarrier T cell cultures across 3 donors.
FIG. 8A) Fold change and FIG. 8B) viability assessed at day 14 of
culture at varying cell:carrier ratios.
DETAILED DESCRIPTION OF THE DISCLOSURE
[0060] As specified in the Background Section, there is a great
need in the art to identify technologies for improved methods and
systems for large-scale production of suspension cells, such as for
example and not limitation, T cells, and use this understanding to
develop novel bioprocessing systems and methods. The present
disclosure satisfies this and other needs. Embodiments of the
present disclosure relate generally to synthetic microenvironment
capable of mimicking T cell niches within secondary lymphoid organs
(such as for example and not limitation, lymph nodes) and more
specifically to macroporous 3D microcarriers that can be
functionalized with antibodies to promote suspension cell (e.g., T
cell) activation and expansion to mimic the environment found in
lymph nodes. The systems should provide suspension cells with
improved potency and efficacy and allow for specific sub
populations to expand selectively. It is to such systems and
methods of producing suspension cells, such as for example and not
limitation, T cells, that embodiments of the present disclosure are
directed. T cells produced by the systems and methods described
herein include, but are not limited to, recombinant T cells, gene
modified T cells, chimeric antigen receptor (CAR) T cells,
unmodified T cells, and CCR7.sup.+CD62.sup.+ central memory T
cells.
[0061] Current approaches for CAR-based ACT are severely
constrained by (a) the limited availability of autologous T cells
from cancer patients (b) difficulty in robustly and reproducibly
expanding these cells to enough numbers for multiple
administrations and (c) lack of quantifiable biomarkers that are
predictive for functional anti-cancer potency across various
tumors. Current state-of-the-art methods involve culturing
patient-isolated T cells with .alpha.-CD3 antibodies (Ab) or
.alpha.-CD3/.alpha.-CD28 Ab-functionalized beads with high amounts
of interleukins (ILs; such as for example and not limitation, IL2,
IL7 and/or IL15) in single cell suspension. Even industry-based
efforts have adopted this process with only improvements being the
use of (a) closed systems, e.g., bag-based cultures and (b) rocking
platforms (e.g., the Wave bioreactor). The field will benefit
greatly from improved manufacturing processes for reproducible,
rapid, more-efficient expansion of highly-potent T cells, with
reduced cost.
[0062] Thus, new cell-manufacturing and biomarker characterization
concepts, that would allow large scale production of therapeutic
CAR-T cells without losing their potency, are critically needed.
The inventors hypothesized that mimicking the cell-cell and
autocrine/paracrine communication as well as the hypoxic
microenvironment of the lymph node (LN) (where T cell expansion
takes place in the body) along with modern bioreactor technologies
would significantly enhance expansion of CAR-T cells without loss
in potency.
[0063] Herein is demonstrated the use of
.alpha.-CD3/.alpha.-CD28-functionalized microcarriers and cultured
human T cells in LN-mimicking 3D niches where T cells remain at
high density with close cell-cell contact, and allow efficient
paracrine/autocrine signaling. These parameters, absent from
current T-cell manufacturing concepts, are likely critical since T
cells secrete large amounts of ILs locally to promote rapid, large
scale expansion. Thus, the systems and methods described herein
could also reduce culture media and IL requirements, thereby
significantly reducing cost. Although microcarriers are primarily
used for adherent cells, .alpha.-CD3/.alpha.-CD28 functionalization
of microcarriers could allow T cells to be anchored to the
microcarrier 3D structure for improved activation and/or expansion.
Effects of low oxygen tension, various cell-seeding densities and
.alpha.-CD3/.alpha.-CD28 ligand densities on expansion efficacy and
T cell quality were also studied. Methods according to the
disclosure can combine the LN-like niche with stirred tank or
perfusion bioreactors, to affect dynamic culture and flow perfusion
and thus improve expansion efficacy (time and cell numbers),
product quality, scalability and cost effectiveness. Further,
porous microcarriers can mimic 3D LN-like niches. Microcarriers are
widely used for bioreactor cultures of adherent cells (8-10), but
not for non-adherent cells like T cells. The use of porous gelatin
(denatured collagen) carriers with functionalized
anti-CD3/anti-CD28 would allow us to anchor T cells to the
scaffolds, mimic the extracellular matrix (ECM) microenvironment of
LN, and mimic the APC/T-cell signaling events in a controlled
manner. Porous microcarriers also provide a high surface area for
culture and can be used with stirred tank and perfusion bioreactors
to ensure large-scale culture. Stirred tank bioreactors allow for
easy scale-up of cultures and are widely investigated in cell
bioprocessing (11-13). A single bioreactor can replace a large
number of static petri-dishes, can provide a closed-culture system
to reduce handling and contamination during manufacturing,
eventually provide automated monitoring of culture parameters
(oxygen, pH, etc.) and can have better nutrient mixing. Porous
microcarriers can be suspended in stirred tank bioreactors for
rapid scale up.
[0064] Microcarrier cultures were originally developed for
large-scale culture of anchorage-dependent cells. Microcarriers
allow for high density cell culture; typically, about two orders of
magnitude higher cell densities (up to 2.times.10.sup.8 cells/mL,
compared to 2-3 .times.10.sup.6/mL cells without microcarrier)
(18). This enables scaling up of cell manufacturing processes with
smaller footprints with reduced overall consumption of expensive
media, serum and growth factors. Porous microcarriers are
particularly suited for high density culture and significantly
increase the available surface area for cells. In addition, porous
microcarriers are better protected against unwanted mechanical
stress generated in bioreactors (11) However, microcarriers are
typically always used for anchorage dependent cells (8-10). Herein
it is shown that T cells are also ideal candidates for porous
microcarrier-based expansion because: (a) it allows high density
culture similar to that inside LNs, thus providing
autocrine/paracrine IL signaling; (b) allows functionalization of
the carrier surface with anti-CD3/CD28, thus avoiding bead-based
signaling. Specific, non-limiting embodiments described herein use
the Cultispher G microcarriers (Hyclone) due to several reasons: i)
this microcarrier is made of gelatin, which is derived from
collagen, one of the most abundant structural components of the LN
ECM; ii) chemical surface modification is possible by use of
unreacted amine or carboxylic acid groups in gelatin (as shown in
U.S. Pat. No. 8,318,492); iii) upon completion of culture, close to
100% cell harvesting is possible by complete dissolution of gelatin
matrix with enzymatic (e.g. trypsin) digestion; iv) highly
crosslinked cavernous structure with high interior surface area
allows greater number of cells (>2,000) per microcarrier that
are well protected from shear stress; and v) great mechanical
stability that allows for a long term culture.
[0065] To provide activation signals to CAR-T cells in 3-D, the
surface of the porous microcarriers was modified with
anti-CD3/anti-CD28 Abs in varying densities. In some non-limiting
embodiments, sulfo-NHS-biotin (Life Technologies) was conjugated to
the amine groups of gelatin followed by incubation with
streptavidin to generate a fully streptavidin modified
microcarrier. Streptavidin was thus a biolinker for further
modification with biotinylated anti-CD3 and anti-CD28 Abs. By
controlling the relative concentrations of biotinylated Abs, it was
possible to systematically vary the surface density of Abs. In
other embodiments, amine- or carboxylate-reactive reagents (e.g.,
carbodiimide (EDC) or N-hydroxysulfosuccinimide (sulfo-NHS), Life
Technologies) were used to functionalize the gelatin-based
microcarriers with, for example and not limitation, Protein A or G,
which bind in high affinities to Fc region of Abs with different
number of binding sites per molecule (5 and 2, respectively). It
was possible to vary the overall density of anti-CD3/anti-CD28 Abs
on the microcarrier surface by changing incubation concentrations,
and also systemically vary the local valency by changing using
streptavidin, Protein A or Protein G.
[0066] Various bioreactors have been employed for scale-up cell
manufacturing to enhance homogeneity of the system (including
elements such as nutrients, cytokine/growth-factors, oxygen, cell
density, etc.), improve sterility (closed system design), and
augment biological functions (shear, flow) (19). Spinner flasks or
stirred tank bioreactors (11-13) are the most-explored platform
that supports expansion of a variety of cell types including 3D
cellular organizations (20). These bioreactors create a homogeneous
physicochemical environment (19). However, the stirrer (or spinner)
can apply irregular amount of mechanical shear forces on suspended
cells and cell aggregates. Perfusion bioreactors, on the other
hand, minimizes such high shear, and can deliver fresh nutrients
and cytokines continuously while imposing fluid mechanical forces
on cells in a more controlled manner (21-24). In cultures with
microcarriers, the high cell density requires frequent media
exchanges, in which case the application of perfusion has
significant benefit. Perfusion rates can be controlled to mimic the
interstitial fluid flow regime (15), such as for example and not
limitation, flow in the LN niches. In some embodiments of the
present disclosure, stirred-tank type ambr.TM. micro-bioreactor
systems (TAP Biosystems) that allows bioprocess optimization at
microscale (10-15 ml) are used, mimicking the core characteristics
of classical bioreactors, but with reduced use of media and growth
factors. Other embodiments can use the CartiGen perfusion
bioreactor system (model C9-x, Instron), optionally without
employing the compression feature due to the unknown effects of the
associated mechanical stimuli. In either embodiment, fresh media
can be perfused using a common flow loop in a varying flow rate,
closely mimicking the physiological interstitial flow rate, ranging
from 0.1-2.0 .mu.m/s 25.
[0067] Another major challenge for CAR-T cell manufacturing is that
extensive expansion results in decreased potency, which is related
to the differentiation status of T cells before and after the
expansion. The systems and methods of the present disclosure result
in improved potency relative to current methods of expansion.
Definitions
[0068] To facilitate an understanding of the principles and
features of the various embodiments of the disclosure, various
illustrative embodiments are explained below. Although exemplary
embodiments of the disclosure are explained in detail, it is to be
understood that other embodiments are contemplated. Accordingly, it
is not intended that the disclosure is limited in its scope to the
details of construction and arrangement of components set forth in
the following description or examples. The disclosure is capable of
other embodiments and of being practiced or carried out in various
ways. Also, in describing the exemplary embodiments, specific
terminology will be resorted to for the sake of clarity.
[0069] It must also be noted that, as used in the specification and
the appended claims, the singular forms "a," "an" and "the" include
plural references unless the context clearly dictates otherwise.
For example, reference to a component is intended also to include
composition of a plurality of components. References to a
composition containing "a" constituent is intended to include other
constituents in addition to the one named. In other words, the
terms "a," "an," and "the" do not denote a limitation of quantity,
but rather denote the presence of "at least one" of the referenced
item.
[0070] As used herein, the term "and/or" may mean "and," it may
mean "or," it may mean "exclusive-or," it may mean "one," it may
mean "some, but not all," it may mean "neither," and/or it may mean
"both." The term "or" is intended to mean an inclusive "or."
[0071] Also, in describing the exemplary embodiments, terminology
will be resorted to for the sake of clarity. It is intended that
each term contemplates its broadest meaning as understood by those
skilled in the art and includes all technical equivalents which
operate in a similar manner to accomplish a similar purpose. It is
to be understood that embodiments of the disclosed technology may
be practiced without these specific details. In other instances,
well-known methods, structures, and techniques have not been shown
in detail in order not to obscure an understanding of this
description. References to "one embodiment," "an embodiment,"
"example embodiment," "some embodiments," "certain embodiments,"
"various embodiments," etc., indicate that the embodiment(s) of the
disclosed technology so described may include a particular feature,
structure, or characteristic, but not every embodiment necessarily
includes the particular feature, structure, or characteristic.
Further, repeated use of the phrase "in one embodiment" does not
necessarily refer to the same embodiment, although it may.
[0072] Ranges may be expressed herein as from "about" or
"approximately" or "substantially" one particular value and/or to
"about" or "approximately" or "substantially" another particular
value. When such a range is expressed, other exemplary embodiments
include from the one particular value and/or to the other
particular value. Further, the term "about" means within an
acceptable error range for the particular value as determined by
one of ordinary skill in the art, which will depend in part on how
the value is measured or determined, i.e., the limitations of the
measurement system. For example, "about" can mean within an
acceptable standard deviation, per the practice in the art.
Alternatively, "about" can mean a range of up to .+-.20%,
preferably up to .+-.10%, more preferably up to .+-.5%, and more
preferably still up to .+-.1% of a given value. Alternatively,
particularly with respect to biological systems or processes, the
term can mean within an order of magnitude, preferably within
2-fold, of a value. Where particular values are described in the
application and claims, unless otherwise stated, the term "about"
is implicit and in this context means within an acceptable error
range for the particular value.
[0073] By "comprising" or "containing" or "including" is meant that
at least the named compound, element, particle, or method step is
present in the composition or article or method, but does not
exclude the presence of other compounds, materials, particles,
method steps, even if the other such compounds, material,
particles, method steps have the same function as what is
named.
[0074] Throughout this description, various components may be
identified having specific values or parameters, however, these
items are provided as exemplary embodiments. Indeed, the exemplary
embodiments do not limit the various aspects and concepts of the
present disclosure as many comparable parameters, sizes, ranges,
and/or values may be implemented. The terms "first," "second," and
the like, "primary," "secondary," and the like, do not denote any
order, quantity, or importance, but rather are used to distinguish
one element from another.
[0075] It is noted that terms like "specifically," "preferably,"
"typically," "generally," and "often" are not utilized herein to
limit the scope of the claimed disclosure or to imply that certain
features are critical, essential, or even important to the
structure or function of the claimed disclosure. Rather, these
terms are merely intended to highlight alternative or additional
features that may or may not be utilized in a particular embodiment
of the present disclosure. It is also noted that terms like
"substantially" and "about" are utilized herein to represent the
inherent degree of uncertainty that may be attributed to any
quantitative comparison, value, measurement, or other
representation.
[0076] The dimensions and values disclosed herein are not to be
understood as being strictly limited to the exact numerical values
recited. Instead, unless otherwise specified, each such dimension
is intended to mean both the recited value and a functionally
equivalent range surrounding that value. For example, a dimension
disclosed as "50 mm" is intended to mean "about 50 mm."
[0077] It is also to be understood that the mention of one or more
method steps does not preclude the presence of additional method
steps or intervening method steps between those steps expressly
identified. Similarly, it is also to be understood that the mention
of one or more components in a composition does not preclude the
presence of additional components than those expressly
identified.
[0078] The materials described hereinafter as making up the various
elements of the present disclosure are intended to be illustrative
and not restrictive. Many suitable materials that would perform the
same or a similar function as the materials described herein are
intended to be embraced within the scope of the disclosure. Such
other materials not described herein can include, but are not
limited to, materials that are developed after the time of the
development of the disclosure, for example. Any dimensions listed
in the various drawings are for illustrative purposes only and are
not intended to be limiting. Other dimensions and proportions are
contemplated and intended to be included within the scope of the
disclosure.
[0079] As used herein, the term "subject" or "patient" refers to
mammals and includes, without limitation, human and veterinary
animals. In a preferred embodiment, the subject is human.
[0080] In accordance with the present disclosure there may be
employed conventional molecular biology, microbiology, and
recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. See, e.g.,
Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory
Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (herein "Sambrook et al., 1989"); DNA
Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed.
1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic
Acid Hybridization (B. D. Hames & S. J. Higgins eds.(1985);
Transcription and Translation (B. D. Hames & S. J. Higgins,
eds. (1984); Animal Cell Culture (R. I. Freshney, ed. (1986);
Immobilized Cells and Enzymes (IRL Press, (1986); B. Perbal, A
Practical Guide To Molecular Cloning (1984); F. M. Ausubel et al.
(eds.), Current Protocols in Molecular Biology, John Wiley &
Sons, Inc. (1994); among others.
SYNTHETIC MICROENVIRONMENTS OF THE DISCLOSURE
[0081] The present disclosure provides a system for mimicking the
secondary lymphoid organs where suspension cells (e.g., T cells)
are expanded; methods of expanding, activating, and transfecting
the suspension cells in the synthetic microenvironment, and
suspension cells produced by such systems and methods.
[0082] In one aspect, the disclosure provides a system for
expanding, activating, and/or transfecting suspension cells
comprising: a porous microcarrier; and the suspension cells.
[0083] In some embodiments, the suspension cells are isolated from
a patient's blood or organs, including both normal and/or diseased
tissues.
[0084] In some embodiments, the suspension cells are T cells.
[0085] In other embodiments, the porous microcarrier is three
dimensional. In some embodiments, the porous microcarrier comprises
proteins, carbohydrates, lipids or nucleic acids. In some
embodiments, the porous microcarrier comprises gelatin or other
extracellular matrix components.
[0086] In yet other embodiments, the porous microcarrier is
functionalized. In some embodiments, the functionalized porous
microcarrier comprises at least one of antibodies, aptamers, and
phage-display identified peptide ligands.
[0087] In some embodiments, the antibodies comprise antibodies that
are specific for the suspension cells. In other embodiments, the
antibodies comprise antibodies that are specific for T cells. In
some embodiments, the antibodies comprise anti-CD2, anti-CD3 and/or
anti-CD28 antibodies.
[0088] In an embodiment, the system in any of the preceding
embodiments further comprises a bioreactor. In some embodiments,
the bioreactor comprises a closed bioreactor and an open
bioreactor. In some embodiments, the open bioreactor comprises a
static culture vessel with a gas-permeable bottom. In other
embodiments, the closed bioreactor comprises a stirred-type
bioreactor, a bag bioreactor, and a perfusion bioreactor.
[0089] In another embodiment, the system in any of the preceding
embodiments further comprises at least one of culture medium, at
least one cytokine, and/or at least one viral vector, and
optionally at least one growth factor.
[0090] In some embodiments, the at least one cytokine comprises IL2
and optionally at least one of IL7 or IL15. In other embodiments,
the at least one viral vector is configured for use in gene therapy
(such as for example and not limitation, a lentiviral vector, a
retroviral vector, an adenoviral vector, and an adeno-associated
viral vector).
[0091] In any of the embodiments described herein, the suspension
cells can comprise recombinant T cells, gene modified T cells,
chimeric antigen receptor (CAR) T cells, unmodified T cells, and/or
CCR7.sup.+CD62.sup.+ central memory T cells.
[0092] In some embodiments, the at least one viral vector comprises
a CAR transgene and optionally at least one additional gene,
wherein the at least one additional gene comprises therapeutic
genes, surface marker genes, reporter genes, suicide genes,
chemokine receptor genes, cytokine-expressing genes, and/or
immune-checkpoint receptor genes.
[0093] In any of the preceding embodiments, the porous microcarrier
is macroporous. In some embodiments, the porous microcarrier is
degradable, such as for example and not limitation,
biodegradable.
[0094] In a related aspect, the disclosure provides a method of
expanding, activating, and/or transfecting suspension cells, the
method comprising: obtaining a blood sample from a patient;
isolating suspension cells from the blood sample; introducing the
suspension cells to a bioreactor comprising a porous microcarrier;
activating the suspension cells; expanding the suspension cells;
optionally transfecting the suspension cells; preparing the
suspension cells for transfusion into the patient; and transfusing
the suspension cells into the patient.
[0095] In some embodiments, the blood or tissue sample is obtained
by leukapharesis.
[0096] In other embodiments, the suspension cells are T cells.
[0097] In some embodiments, the step of isolating suspension cells
from the blood sample further comprises bead separation or magnetic
bead separation.
[0098] In other embodiments, the bioreactor comprises a closed
bioreactor and an open bioreactor. In some embodiments, the closed
bioreactor comprises a stirred-type bioreactor, a bag bioreactor,
and a perfusion bioreactor. In other embodiments, the open
bioreactor comprises a static culture vessel with a gas-permeable
bottom.
[0099] In some embodiments, the porous microcarrier is three
dimensional. In other embodiments, the porous microcarrier
comprises proteins, carbohydrates, lipids or nucleic acids. In
still other embodiments, the porous microcarrier comprises gelatin
or other extracellular matrix components.
[0100] In some embodiments, the porous microcarrier is
functionalized. In other embodiments, the functionalized porous
microcarrier comprises at least one of antibodies, aptamers, and
phage-display identified peptide ligands. In still other
embodiments, the antibodies comprise antibodies that are specific
for the suspension cells. In some embodiments, the antibodies
comprise antibodies that are specific for T cells. In some
embodiments, the antibodies comprise anti-CD2, anti-CD3 and/or
anti-CD28 antibodies.
[0101] In an embodiment of any of the foregoing methods, the method
further comprises at least one of culture medium, at least one
cytokine, and/or at least one viral vector, and optionally at least
one growth factor. In some embodiments, the at least one cytokine
comprises IL2, and optionally at least one of IL7 or IL15.
[0102] In an embodiment of any of the foregoing methods, the
activation step further comprises agitation, optionally under
hypoxic conditions. In some embodiments, the agitation can be
periodic or continuous. In other embodiments, the activation step
can last for at least one day, at least two days, at least three
days, and at least four days, and all ranges of time in
between.
[0103] In an embodiment of any of the foregoing methods, the
expansion step further comprises seed trains, optionally under
hypoxic conditions. For example and not limitation, the expansion
step can include increasing the size of the flask and/or bioreactor
as the culture grows, which can also involve moving the expanding
cells to a new vessel, and culture media exchanges every 2-3 days
(particularly if a static culture). The expansion step can last for
at least one day, at least two days, at least three days, at least
four days, at least five days, at least six days, at least seven
days, at least eight days, at least nine days, at least ten days,
and at least eleven days, and all ranges of time in between.
[0104] In an embodiment of any of the foregoing methods, the
optional transfection step further comprises adding at least one
viral vector to the bioreactor comprising the suspension cells.
[0105] In an embodiment of any of the foregoing methods, the
suspension cells comprise recombinant T cells, gene modified T
cells, chimeric antigen receptor (CAR) T cells, unmodified T cells,
and/or CCR7.sup.+CD62.sup.+ central memory T cells.
[0106] In some embodiments, the at least one viral vector is
configured for use in gene therapy. In other embodiments, the at
least one viral vector comprises a CAR transgene and optionally at
least one additional gene, wherein the at least one additional gene
comprises therapeutic genes, surface marker genes, reporter genes,
suicide genes, chemokine receptor genes, cytokine-expressing genes,
and/or immune-checkpoint receptor genes.
[0107] In an embodiment of any of the foregoing methods, the
preparation step further comprises cell expansion and downstream
bioprocessing. In some embodiments, the cell expansion and
downstream bioprocessing comprises cell separation, purification,
packaging, preservation, storage, shipping and transport, thawing,
formulation, resuspension, and transfusion.
[0108] In an embodiment of any of the foregoing methods, the
transfusion step further comprises injection, intravenous
administration, and implantation (such as for example and not
limitation, implantation of a sustained delivery device).
[0109] In an embodiment of any of the foregoing methods, the porous
microcarrier is macroporous. In any of the preceding embodiments,
the porous microcarrier is macroporous. In some embodiments, the
porous microcarrier is degradable, such as for example and not
limitation, biodegradable.
[0110] In a related aspect, the disclosure provides a T cell
obtained from any of the systems disclosed herein.
[0111] In a related aspect, the disclosure provides a T cell
obtained from any of the methods disclosed herein.
[0112] In one embodiment, the T cell is a memory cell. In another
embodiment, the T cell is CCR7.sup.+CD62L.sup.+. In another
embodiment, the T cell is a central memory T cell. In yet another
embodiment, the T cell is a CD4 T cell or a CD8 T cell. In some
embodiments, the T cell comprises recombinant T cells, gene
modified T cells, chimeric antigen receptor (CAR) T cells,
unmodified T cells, and/or CCR7.sup.+CD62.sup.+ central memory T
cells.
[0113] In a related aspect, the disclosure provides a composition
comprising at least one T cell as described herein. In some
embodiments, the composition further comprises at least one
carrier. In other embodiments, the composition is formulated for
intravenous administration.
[0114] In a related aspect, the disclosure provides a
pharmaceutical composition comprising at least one T cell as
described herein. In some embodiments, the pharmaceutical
composition further comprises at least one carrier. In other
embodiments, the composition is formulated for intravenous
administration. In some embodiments, the pharmaceutical composition
further comprises at least one additional therapeutic agent.
[0115] In a related aspect, the disclosure provides the use of a
composition (including a pharmaceutical composition) as described
herein to treat a disease or condition in a patient in need
thereof. In some embodiments, the disease or condition comprises
genetic diseases, cancers, infections, autoimmune diseases, and/or
transplant complications. In some embodiments, the use further
comprises a second therapeutic method or agent, such as for example
and not limitation, a cancer drug, an immunotherapeutic, an
immunosuppressant, an autoimmune therapeutic agent, and/or a
therapeutic for treating infections.
[0116] In a related aspect, the disclosure provides method of
treating a disease or condition in a patient in need thereof,
comprising administering a composition (including a pharmaceutical
composition) as described herein to said patient. In some
embodiments, the disease or condition comprises genetic diseases,
cancers, infections, autoimmune diseases, and/or transplant
complications. In some embodiments, the treatment further comprises
a second therapeutic method or agent, such as for example and not
limitation, a cancer drug, an immunotherapeutic, an
immunosuppressant, an autoimmune therapeutic agent, and/or a
therapeutic for treating infections.
[0117] In an embodiment of any of the foregoing systems, the porous
microcarrier is configured for activation, expansion, and/or
transfection of the suspension cells.
[0118] In an embodiment of any of the foregoing methods, the porous
microcarrier is configured for activation, expansion, and/or
transfection of the suspension cells.
EXAMPLES
[0119] The present disclosure is also described and demonstrated by
way of the following examples. However, the use of these and other
examples anywhere in the specification is illustrative only and in
no way limits the scope and meaning of the disclosure or of any
exemplified term. Likewise, the disclosure is not limited to any
particular preferred embodiments described here. Indeed, many
modifications and variations of the disclosure may be apparent to
those skilled in the art upon reading this specification, and such
variations can be made without departing from the disclosure in
spirit or in scope. The disclosure is therefore to be limited only
by the terms of the appended claims along with the full scope of
equivalents to which those claims are entitled.
Example 1
Development of the Synthetic Microcarrier System
[0120] Current T cell expansion technologies do not fully
recapitulate the secondary lymphoid organs where T cell are
expanded with close cell-cell contact under hypoxic conditions.
Herein, functionalized microcarriers were used in combination with
modern bioreactors to create 3D niches where T cells are stimulated
to expand with anti-CD3 and anti-CD28 antibodies while remaining in
close cell-cell contact. The high surface density of these
microcarriers encouraged high cell density and efficient signaling,
while cytokine requirements, media usage, and bioreactor footprint
were reduced.
Development and Optimization of Antibody-Coated Microcarriers
[0121] Anti-CD3 and anti-CD28-coated microcarriers were generated
by functionalizing the surface of Cultispher gelatin microcarriers
by (1) conjugating sulfo-NHS-biotin to amine groups of gelatin, (2)
incubating with streptavidin to create a fully streptavidin-coated
microcarrier, and (3) adding biotinylated anti-CD3 and anti-CD28
antibodies at controlled densities to regulate the antibody surface
density.
[0122] Cultispher-S (CuS) and Cultispher-G (CuG) are two types of
microcarriers commonly used in cell manufacturing and
bioprocessing. They are gelatin-based and thus have many lysine
residues that can be functionalized using N-hydroxysuccinamide
(NHS) chemistry. These carriers were functionalized using
streptavidin-biotin chemistry due to the availability of
biotinylated antibodies, the ease of use for this method, and the
relative stability of this system. Sulfo-NHS-biotin was the chosen
crosslinker for this system due to its solubility in water and its
relatively short spacer arm, which likely maximized the
availability of open streptavidin binding sites that can bind to
antibodies.
[0123] First, the inventors quantified the amount of streptavidin
that could be bound to the surface as a function of biotinylation
(FIG. 1A). Microcarriers were first biotionylated using varying
amounts sulfo-NHS-biotin, and streptavidin was then introduced in
excess to evenly coat the surface. Unreacted streptavidin from the
supernatant was measured using the BCA assay, which was then used
in combination with the estimated surface area of CuS and CuG
microcarriers to determine the streptavidin surface density as a
function of the sulfo-NHS-biotin concentration. It was assumed that
the difference between the amount of streptavidin added to the
suspension and the amount of streptavidin remaining in the
supernatant was equal to the amount of bound streptavidin. To make
each curve in FIG. 1A, all samples were repeated in duplicate and
error bars in the chart represented standard deviation about the
mean. The curve fit was found using a modified Scatchard plot
analysis, which followed standard receptor-ligand kinetics and thus
showed saturation of the binding sites in a hyperbolic fashion. The
molecules per area was determined using the amount of streptavidin
remaining in the supernatant and dividing by the surface area of
the microcarriers (mass of microcarriers was known and the
manufacturer supplied the surface area per carrier mass. The
estimated surface areas for CuS and CuG microcarriers were 19,000
cm.sup.2 and 24,000 cm.sup.2, respectively). Cultisper S has much
higher binding efficiency, therefore this microcarrier was used in
all further experiments.
[0124] Excess biotin linked to the surface may negatively impact
antibody attachment efficiency, as it could theoretically block all
available binding sites on the bound streptavidin molecules. As
streptavidin has four binding sites, each molecule generally has a
few sites occupied by biotins attached to the surface of the
microcarrier and the remainder are available to attach to
biotinylated ligands such as antibodies; FIG. 1B quantified the
remaining open binding sites as a function of biotinylation.
Microcarriers were coated as described herein and the number of
available binding sites was quantified using a fluorimeter with
FITC-biotin as a surrogate ligand. FITC-biotin was added to the
suspension of streptavidin coated microcarriers and the supernatant
was assessed for fluorescence to quantify the amount of unbound
FITC-biotin which was then used to find the amount of bound
FITC-biotin (analogous to (FIG. 1A) above). One mole of FITC-biotin
was assumed to be one mole of available binding sites. The area
used for the calculation of binding sites/um.sup.2 was 19000
cm.sup.2. Together, these show that approximately 5000 nmol/g of
sulfo-NHS-biotin is optimal for maximizing ligand surface density,
as this keeps the streptavidin surface density near saturation (and
thus ensured an even coating throughout the carrier), while also
maintaining a significant number of open binding sites.
[0125] FIG. 2 demonstrated a verification of open biotin binding
sites via Zeiss Lightsheet microscope imaging of (A) uncoated CuS
microcarriers and (B) CuS microcarriers optimally coated with
streptavidin and saturated with FITC-biotin. Laser power was fixed
to 10% in both cases. Since the microcarriers were macroporous and
thus could support cell growth on the interior, it was important to
show that the conjugation strategy as described could evenly and
comprehensively coat the entire surface of the microcarriers,
including within the macropores on the interior surface. FIG. 2
demonstrated this even coating because the interior of the
microcarriers appears comparably bright to the outer surface at a
middle cross section, showing that we have coated the core.
Furthermore, the uncoated sample rules out autofluorescence
(background signal) which one might expect due to the fact that the
carriers are protein-based.
TABLE-US-00001 TABLE 1 Scale up of CuS microcarrier coating
procedure. Anti-CD3 and anti-CD28 antibodies were added in a 1:1
ratio, and the Ab coating procedure was repeated for small, medium,
and large batch sizes to verify process scalability. Parameters in
medium and large batches were scaled proportionally to the small
batch size. Microcarrier dry weight Medium Small (15 mg) (150 mg)
Large (300 mg) Streptavidin/.mu.m.sup.2 7500 8500 8200
Biotin-binding 1200 2700 1400 sites/.mu.m.sup.2
Antibodies/.mu.m.sup.2 2600 3000 2600 Antibody:Streptavidin 0.35
0.35 0.31 Ratio
[0126] As shown in FIG. 3, microcarriers coated with streptavidin
were conjugated using monoclonal (mAb) cocktails that contained
varying amounts of biotinylated isotype control (calculated as
percentage CD3/CD28 mAb mass with CD3/CD28 ratio as 1:1). This
demonstrated that the isotype control mAbs and CD3/CD28 antibodies
likely bind very similarly, thus showing that it is possible to
control the amount of activating antibodies (CD3 and CD28) by
spiking the cocktail used to coat the microcarriers with a
non-binding antibody (isotype control). In summary, it is possible
to fine-tune the activation signals delivered on the microcarriers
to expand T cells, which can make the microcarrier platform more
adaptable.
Variable Signaling Density on Microcarriers
[0127] An interesting parameter to vary is the signal strength
delivered on the surface of each microcarrier; this will require
varying the number of CD3 and CD28 antibodies conjugated to the
carriers. To accomplish this, the inventors spiked the CD3/CD28
biotinylated mAb cocktail with a biotinylated isotype control (by
definition, an antibody with unknown target) prior to adding the
cocktail to the carriers for conjugation. In theory, the isotype
control should displace the available binding sites for the
CD3/CD28 mAbs, effectively reducing the signal strength that can be
delivered per unit surface area. Signal strength was expressed as a
percentage of full saturation. Microcarriers were coated at
different signal strengths according to this method, and the total
mAb density was determined indirectly by measuring the supernatant
with the BCA protein assay. The slight downward slope of these data
indicate that the binding efficiency appears to be consistent, but
the isotype control may have a slightly less binding capacity than
the CD3/CD28 mAbs (possibly due to differences in biotinylation
capacity).
[0128] To test the effectiveness of the varied signal strength, the
inventors seeded primary human T cells on microcarriers with signal
densities ranging from 100% to 0% with 20% intervals. Cells were
assessed on day 3 for qualitative differences in activation
potential (cell clusters, cell enlargement, media color changes).
The media color ranged from yellow (100%) to pinkish red (0%)
indicating a differential metabolic activity between the groups,
which in turn was indicative of differential activation. This
demonstrated that changing the signal density on the microcarrier
surface can have significant influence on activation state, and
therefore likely can contribute to T cell expansion and phenotype.
T cell activation was further confirmed with the appearance of T
cell clusters in the microcarriers.
Cell Seeding onto Antibody-Coated Microcarriers
[0129] The loading procedure for Ab-coated CuS microcarriers was
optimized using Jurkat cells, an immortalized line of human T
lymphocytes. Two seeding methods were investigated, including a
column seeding method and an extended orbital shaking method. The
first procedure was designed to promote cell contact between cells
and microcarriers in a syringe-based column. Briefly, the needle of
a 0.5 mL syringe was removed using a dremel, and the mesh from a 40
um cell strainer was wrapped around the bottom. The syringe/filter
was placed in a FACS tube and loaded with Ab-coated microcarriers,
which were held in place by the cell strainer mesh. Media was then
added such that the liquid level inside and outside the syringe was
the same. Finally, 1 million Jurkat cells were pre-labeled with
CFSE fluorescent dye and added to the column, and the assembly was
centrifuged for 3 cycles of 5 minutes at 300 g followed by 10
minutes rest to promote attachment. Visualization of the cells
within the microcarriers demonstrated that cell loading using this
method was heterogeneous, with many microcarriers lacking cells. It
was hypothesized that the short length of time (.about.30 min) may
not be sufficient to allow for cell attachment to
microcarriers.
[0130] An alternative cell seeding method using overnight orbital
shaking was next investigated. Anti-CD3-coated carriers were loaded
into a 12-well plate at a density of 12,000 microcarriers per well,
to which 1 million Jurkat cells were added (83:1 cell to
microcarrier ratio). The plates were placed in an incubator and
continuously agitated on an orbital shaker at 60 rpm to promote
cell attachment to the microcarriers. After 14 hours of attachment,
cells appeared to preferentially surround the microcarriers. To
directly quantify the degree of attachment, the contents of each
well were filtered through a cell strainer to separate unattached
cells from microcarriers plus attached cells. Recovered
microcarriers were digested with dispase, which dissolved the
carriers and allowed for cell recovery and direct quantification,
while unattached cells were quantified in the flow-through
fraction. CD3-coated microcarriers achieved a greater percent cell
attachment compared to non-coated microcarriers (11.2.+-.2.1% vs.
3.9.+-.0.3%), as well as a concomitant decrease in the percent of
unattached cells in the flow-through (73.2.+-.8.4% vs.
92.5.+-.1.6%), suggesting productive binding of T cells to
anti-CD3-coated microcarriers.
Expansion of Primary Human T Cells Using Ab-Coated
Microcarriers
[0131] Having established preferential binding of Jurkat T cells to
anti-CD3/CD28-coated microcarriers (albeit at low efficiency), it
was next determined whether these functionalized microcarriers
promoted the expansion of primary human T cells. As a first step, T
cell expansion was verified using CD3/CD28-loaded magnetic beads,
which is the current state-of-the-art method for expansion of
patient-isolated T cells. First, T cells were isolated from
cryopreserved human PBMCs using a MACS pan-T cell isolation kit and
seeded with anti-CD3/CD28-loaded MACSiBeads (cell:bead ratio=1:2).
Cultures were expanded over a period of 28 days per manufacturer's
instructions, which ultimately achieved a 50-fold expansion with a
high exogenous IL-2 concentration of 400 U/mL. When exogenous IL-2
was omitted, only a 7-fold expansion was achieved, which is
consistent with previous findings that IL-2 enhances T cell
expansion7. Further, T cells did not expand in the no-bead negative
control, confirming that CD3/CD28 stimulation is required for T
cell expansion.
[0132] Primary human T cells were expanded using either
anti-CD3/CD28-coated microcarriers or IgG negative control-coated
microcarriers. As a positive control, T cells were also expanded
using anti-CD3/CD28-loaded MACSiBead to compare degree of
expansion. The process of seeding T cells onto microcarriers was
carried out as described (83:1 cell:microcarrier ratio using
overnight attachment on an orbital shaker), after which cultures
were removed from the orbital shaker and allowed to expand for an
additional 6 days with periodic media exchanges. At this 7 day
timepoint, T cells cultured with anti-CD3/CD28-coated microcarriers
expanded and formed aggregates of cells adjacent to microcarriers,
while no such expansion occurred in cultures with IgG
control-coated microcarriers. Anti-CD3/CD28-coated microcarriers
achieved a 2.5-fold expansion over 7 days, which compared favorably
with the 1.4-fold expansion observed with the MACSiBead culture.
Therefore, anti-CD3/CD28 functionalized microcarriers were capable
of stimulating primary human T cell expansion.
[0133] To determine whether T cells were expanding within the
interior macropores of the microcarriers, microcarriers plus
attached cells were separated from any unattached cells using a 40
um strainer, and cells were quantified in both the flow-through
fraction (unattached) or the microcarrier-associated fraction
(attached) as described previously. A majority of the cells were
recovered in the flow-through fraction, with only 200,000 cells (7%
of the total cell number) recovered in the microcarrier-attached
fraction, suggesting low cell attachment to microcarriers.
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
staining of attached cells within isolated microcarriers confirmed
the low level of cell loading and further demonstrated the
heterogeneity of cell loading across microcarriers. Indeed, a few
microcarriers had a dozen or more attached cells, while many more
had no cell attachment at all. Therefore, it is likely that
activation and expansion of T cells is mostly occurring adjacent to
the microcarriers rather than within the macropores.
[0134] FIG. 4 shows a comparison of T cell expansion and MACS bead
expansion. Primary human T cells were expanded over 2 weeks in well
plates in various culture conditions. FIG. 4, top shows Day 14
brightfield images of T cells expanded at the indicated
cell:carrier ratio with the absolute number of carriers held
constant. FIG. 4, bottom shows Day 14 fold change of microcarrier
cultures at indicated ratios compared with cells grown in
MACSibeads culture (conventional magnetic beads). MACSibeads cells
were expanded according to the manufacturer's instructions at 1:2
cells:beads with a cell density of 2.5e6 cells/ml. Fold change was
assessed after day 14 by counting cells using an automated cell
counter. The data show that in the case of microcarrier culture
there is an optimal cell:carrier ratio where the fold change
outperforms conventional magnetic bead culture.
[0135] When left undisturbed over the course of 14 day culture, T
cells and microcarriers will form clusters as shown in FIG. 5. As
shown in FIG. 5, the microcarriers (approximately 10, seen as dark
ovals) were pulled together by T cells (fuzzy grey clusters on the
periphery of the microcarriers). These findings demonstrated that
the microcarriers can facilitate cell:carrier adhesion and high
cell density. High cell density is a key characteristic found in
human lymph nodes that the invented system is configured to
mimic.
Process Optimization of Microcarrier-Based Expansion
[0136] The inventors then sought to determine the effects of IL-2
and cell density on the expansion of T cells on microcarriers. To
this end, the inventors created a 2.times.2 design matrix with high
and low IL-2 and cell density levels. The levels for IL-2 were 50
and 400 U/ml, respectively, and the levels for cell density were
1e4/ml and 2.5e6/ml. Expansion and phenotype were evaluated after
day 14. It was observed that within these conditions, only the
higher density cell cultures expanded (the lower level did not
expand at any measurable level). Maximal expansion can vary as a
function of cell:carrier ratio depending on the donor. Between the
two groups that did expand, IL-2 did not appear to affect expansion
but may have influenced memory phenotype, as assessed using flow
cytometry and markers CD62L, CCR7, and CD45RA, resulting in a
higher population of central memory T cells (Tcm), including CD4
and CD8 central memory T cells.
[0137] The inventors then further characterized the memory T cell
subpopulations expanded using the invented microcarrier system. As
shown in FIG. 6, T cells were expanded on microcarriers or
MACSibeads cultures for 14 days and assessed via flow and
transmigration assay. FIG. 6A) Expanded T cells were assessed for
CCR7 and CD62L populations using flow cytometry. T cells that are
CCR7.sup.+CD62L.sup.+ are memory T cells that home to the lymph
nodes and represent a population of T cells that have enhanced
proliferative and migratory potential compared to effector T cells
(CCR7.sup.-CD82L.sup.-). The qualities are important for cell
immunotherapies because highly proliferative cells can divide more
in response to tumor antigen encounter, thus increasing efficacy of
the therapy. Migratory capacity is also important because migratory
cells can travel to more locations throughout the body (especially
the lymph nodes where tumor antigens may be encountered) and will
last longer, providing long-lived protection. The data show that
compared to MACSibeads cultures, the use of microcarriers in the
invented system led to a higher frequency of CCR7.sup.+CD62L.sup.+
phenotypes. Thus, the invented system comprising microcarriers can
produce higher quality T cells for immunotherapy. FIG. 6B) The same
cells assessed in (FIG. 6A) were characterized for functional
migratory potential. CCR7 is a receptor for the chemokine CCL21;
binding of CCL21 to CCR7 will thus trigger a T cell to migration.
When T cells were incubated in a transwell with CCL21 on the
opposite side of a membrane, it was observed that more T cells
crossed the membrane in the case of microcarrier-expanded T cells
than MACSibeads expanded T cells. This confirmed the observations
from (FIG. 6A) by demonstrating that the increased number of
CCR7.sup.+CD62L.sup.+ T cells correlates with increased
migration.
Lentiviral Transduction of Microcarrier T Cell Cultures
[0138] T cells were activated with either plate-bound antibodies or
microcarriers and transduced with lentivirus expressing an
anti-CD19 chimeric antigen receptor. FIG. 7A shows flow cytometry
plots of CAR-expressing T cells assessed on day 9 of culture. CAR
was labelled using biotinylated Protein L with APC-conjugated
streptavidin as secondary. Plate-bound antibody stimulation is a
conventional, small scale technique for T cell activation. T cells
must be activated to be transduced by a lentivirus. These data
demonstrated that microcarriers, while fundamentally different from
widely used standards such as plate-bound activation, can still
provide a strong enough activation signal for T cells to be
transduced at similar levels. FIG. 7B shows the functionality of
transduced T cells by measuring degranulation in response to tumor
cells. Degranulation was assessed in unstimulated (neg), K562
coculature (CD19.sup.-), K562.sup.- CD19.sup.+ coculture
(CD19.sup.+), and anti-CD3-stimulated (pos) groups and was measured
using flow cytometry and quantifying surface expression of CD107a.
Degranulation of T cells is a hallmark of T cell cytotoxocity,
which is important in assessing their ability to kill tumors. The
model tumor cells in this case were K562 cells, and the target the
T cells were programmed to attack was CD19. K562s are naturally do
not express CD19 (the CD19.sup.- group in the chart) so a
genetically engineered K562 line with CD19 inserted (CD19.sup.+)
was utilized as a target with the ligand that the CAR T cells
should recognize. This was also compared to a non-stimulated
control, which should produce no degranulation, and a fully
stimulated positive control using antibodies to trigger
degranulation. Each group was compared using an ANOVA and Control
Dunnett's test with the control as the "neg" group and alpha of
0.05. In FIG. 7B, the circles to the right represent significance;
non-overlapping circles indicate that groups are very significantly
different. The data demonstrate that the in both plate-bound and
microcarrier-activated groups, T cells showed elevated
degranulation when presented with either CD19.sup.+ target cells or
when stimulated with antibodies. The fact that the T cells did not
show elevated degranulation in the CD19.sup.- group demonstrated
that the CAR T cells did not recognize any other proteins on the
K562 cells, ruling out off-target effects. In summary, these data
show that T cells transduced using microcarriers are functionally
equivalent to a widely used standard.
Microcarrier T Cell Cultures Across Multiple Donors
[0139] As shown herein, microcarriers can induce expansion of T
cells, and this expansion may be superior to conventional
techniques (such as MACS) in terms of expansion capacity. The
inventors next asked if this technique could work across multiple
donors. The invented microcarrier system was tested at varying
cell:carrier ratios (80:1 to 10:1) with constant carrier/well using
T cells from 3 healthy donors (aged 25, 47, and 48) (FIG. 8). All
donors showed similar trends, with lower cell:carrier ratios
showing higher expansion. Furthermore, all groups showed similarly
high viability (with the exception of one). Cell expansion roughly
correlated with age, with the youngest donor (number 3) expanding
the greatest in the majority of groups and the oldest (donor 1)
expanding the least. This could be due to metabolic dysfunction
that accumulates with age. Fold change (FIG. 8A) and viability
(FIG. 8B) were assessed at day 14 of culture at varying
cell:carrier ratios. These results demonstrated that the
functionalized microcarriers showed the same trend across different
donors, thus showing broad applicability. However, the exact
behavior depends on each specific donor, with younger donors
(donors 2 and 3) generally showing better expansion than the older
donor (donor 1). Viability in most cases was also very high across
all donors, which further demonstrates that the invented systems
and methods are robust.
Materials and Methods
Microcarrier Functionalization
[0140] Cultispher S and Cultipher G microcarriers (HyClone) were
suspended in 1.times. PBS at 15 mg/ml and autoclaved at 121.degree.
C. for 15 minutes. All subsequent steps were performed in a sterile
environment. Carriers were biotinylated by adding 0.5 .mu.l 10 mM
sulfo-NHS-biotin (Thermo Fisher) per mg microcarriers to the
suspension and vortexing continuously for 60 minutes at room
temperature. Excess reagent was removed by washing the carriers 3
times with 1.times. PBS by diluting 15-fold. Streptavidin (Jackson
ImmunoResearch) was added to the carrier suspension at 40 .mu.g/ml
and vortexed for 45 minutes. Supernatent samples for streptavidin
quantification were taken after this step, and this was followed by
2 wash steps in PBS by diluting 15-fold. For antibody coating,
biotinylated low endotoxin, azide free (LEAF) anti-CD3, anti-CD28,
or mouse IgG1 isotype controls (Biolegend) were added at predefined
mixtures to make a total antibody concentration of 30 .mu.g/ml and
vortexed for 60 minutes. The anti-CD3 and anti-CD28 ratio was
always kept 1:1. Mouse IgG1 isotype control was added to some
microcarriers to displace the anti-CD3/anti-CD28 antibodies for
purposes of lowering the signal strength presented on the surface.
Antibody-coated microcarriers were washed 2 times with 1.times. PBS
by diluting 15-fold. Prior to cell culture, 1 ml of microcarriers
at 15-20 mg/ml was washed with 9 ml of appropriate media.
Quantification of microcarriers was determined at the end of the
process by sampling the microcarriers into a 96 well plate and
counting manually.
Streptavidin Binding Quantification
[0141] Streptavidin binding was quantified indirectly by measuring
unbound streptavidin in the supernatant immediately after the
streptavidin conjugation and incubation steps. Streptavidin protein
concentration was measured using a BCA assay (Thermo Fisher)
according to the manufacturer's instructions with several
modifications. Briefly, a log 2 standard curve was created starting
at 40 .mu.g/ml streptavidin (Jackson ImmunoResearch). The assay was
performed in a TC-treated 96 well plate. All sample or standard
volumes were 150 ul, and the added BCA reagent was 150 ul. The
reaction was allowed to proceed in a dry incubator at 37.degree. C.
for 45-90 minutes prior to reading at 562 nm on a Biotek Plate
Reader.
Microcarrier Binding Site Quantification
[0142] The number of open streptavidin binding sites was indirectly
quantified by measuring the supernatant following FITC-biotin
incubation. Immediately after washing excess streptavidin,
microcarriers were suspended at 15 mg/ml, and 80 .mu.l of 5 .mu.M
FITC-biotin (Thermo Fisher) was added to the carrier suspension and
vortexed for 20 minutes. The carrier suspension was centrifuged at
4500 g for 1 min to pull carriers to the bottom, and a 200 .mu.l
sample was assayed against a standard curve to quantify the unbound
FITC-biotin.
Lightsheet Imaging of Functionalized Microcarriers
[0143] Binding site uniformity was confirmed qualitatively using a
Zeiss Lightsheet microscope. Microcarriers were coated with
streptavidin as described earlier and all binding sites were
saturated with FITC-biotin. Microcarriers were then resuspended in
0.1% agarose heated to 70.degree. C. and cast into a thin
capillary. Streptavidin coated microcarriers without FITC-biotin
were used as negative control for autofluorescence.
Microcarrier and MACSiBeads Cell Culture
[0144] Primary human T cells were obtained from cryopreserved
peripheral blood mononuclear cells (PBMCs) (Zenbio) after
separation with a negative selection magnetic activated cell
sorting kit (Miltenyi Biotech). MACSibeads cell culture was
performed according to the manufacturer's instructions (Miltenyi
Biotek). Briefly, magnetic MACSibeads were conjugated with the
provided anti-CD3 and anti-CD28 antibodies. Beads were added to the
T cell cultures at a 1:2 bead:cell ratio, and initial cell density
was 2.5e6/ml in 96 well plates with total volume of 300 ul.
Microcarrier cultures were performed by adding 45 mg (approximately
36,000) microcarriers/well in 12 well plates and adding T cells in
cell:bead ratios of 83, 25, or 10 with final media volume of 2
ml.
[0145] Cultures were allowed to expand for 14 days, after which
they were assessed for fold change and phenotype via flow cytometry
and chemotaxis assay. Fold change was quantified using a Countess
automatic cell counter (Thermo Fisher). Media was added after day 3
every 1-2 days based on media color. In all cases, recombinant
human IL2 (Peprotech) was added to media at 400 U/ml. Media in all
cases was either RPMI-1640 (Thermo Fisher)+10% FBS (Hyclone) or
OpTmizer with T cell expansion supplement (Thermo Fisher).
Viral Transduction
[0146] T cells were transduced using RetroNectin (Thermo Fisher)
following the plate-coating protocol provided by the manufacturer.
Briefly, 50 .mu.l of 50 .mu.g/ml working solution of Rectronectin
was added to a non-TC-treated 96 well plate. The plate was sealed
and incubated overnight at 4.degree. C. The plate was then washed
and blocked with 2% BSA solution. After BSA was aspirated, plate
was stored at 4.degree. C. until future use.
[0147] For plate-bound antibody T cell cultures, the plate was
prepared by adding a working solution of anti-CD3 and anti-CD28 low
endotoxin, azide free (LEAF) antibodies (Biolegend) at 1 ug/ml and
2 ug/ml respectively to a non-TC-treated 96 well plate. Plate was
incubated overnight at 4.degree. C. and washed twice with PBS prior
to culture. T cells were added to each well at 2.5e6 cell/ml with
300 .mu.l total media per well (OpTmizer, Thermo Fisher).
Microcarrier cultures were carried out as described.
[0148] Cells were transduced on day 1 of culture using a VSV-G
pseudotyped lentivirus with the .alpha.CD19-41BB-CD3.zeta. (30)
chimeric antigen receptor as the genetic payload (Emory Viral
Vector Core). In retronectin plates, each coated well was filled
with 50 ul media and appropriate amount of virus to achieve an MOI
of 5 or 15 based on starting cell number. Wells were mixed well
using a micropipette to ensure even coating and centrifuged for 2
hours at 32.degree. C. and 2000 g. Media was removed from
retronectin wells, and T cells were transferred to the retronectin
plate to begin transfection. In the case of microcarrier samples,
the microcarriers were resuspended well and transferred to the
retronectin plate along with the cells. Transfection was allowed to
proceed for 24 hours, after which cells were transferred again to a
fresh plate where culture continued until day 14 with normal media
additions. Each experimental group was performed in triplicate.
Flow Cytometry for Memory Cell Populations
[0149] Live cells were stained in flow buffer (PBS containing 0.5%
BSA and 2 mM EDTA) at a density of 100,000 cells per 100 .mu.L.
Antibodies were added at a 1:20 dilution and incubated for 2 hours
at 4 oC protected from light (anti-CD3-APC-H7,
anti-CD4-PerCP-Cy5.5, anti-CD45RA-FITC, anti-CCR7-Alexa 647,
anti-CD62L-PE, BD Biosciences). Cells were subsequently washed in
flow buffer and analyzed using a BD LSRFortessa. The data were
analyzed using FlowJo software.
Chemotaxis
[0150] T cells were resuspended in RPMI medium (Life Technologies)
containing 0.2% BSA (Sigma) at a density of 300,000 cells per 100
.mu.L, and 100 .mu.I of this cell suspension was added to the
apical side of 24-well Transwell filters (Costar, 5 .mu.m pore
size). The chemokine CCL21 (PeproTech) was added to the basolateral
chamber of the Transwells at a concentration of 0, 250, or 1000
ng/mL in RPMI+0.2% BSA. After loading, Transwells were placed at
37.degree. C., and the cells were allowed to migrate for 4 hours.
Cells were then collected from the basolateral chamber and
quantified via CountBright beads (Thermo Fisher) using an Accuri C6
flow cytometer. The data were analyzed using FlowJo software.
Quantification of CAR Expression Using Protein L
[0151] CAR expression was quantified as described previously (38).
Briefly, at least 1e5 cells were transferred to flow tubes. Cells
were washed three times with FACS buffer (PBS with 2% bovine serum
albumin and 5 mM EDTA). 1 .mu.l Protein L (Thermo Fisher) at 1
mg/ml was added to each sample. Following incubation for 45
minutes, Protein L was washed out 3 three times with FACS buffer.
In all samples 500 ng/ml streptavidin-PE conjugate was added and
allowed to bind for 45 minutes. Cells were then washed again three
times with FACS buffer. Cells were analyzed on a BD Accuri and
events were quantified using FlowJo software. The positive gate was
set at the boundary of an untransduced population.
Degranulation Assay
[0152] CD8+ T cell degranulation was assayed as previously
described (6). Briefly, after 14 days of expansion, T cells from
each group were pooled, and 1e5 were added to a V-bottom 96 well
plate in OpTmizer media with T cell expansion supplement. Each well
received a stimulation cocktail of anti-CD49d (eBioscience),
anti-CD28 (Biolegend) and monensin (Thermo Fisher) at
concentrations of 1 .mu.g/ml, 1 .mu.g/ml, and 2 .mu.M respectively.
Positive control wells received an additional stimulation of 5
.mu.g/ml anti-CD3 (Biolegend). Tumor cell wells received either
wild-type K562 cells or CD19-transduced K562 cells (each at
1e5/well). Negative control wells received only media. The plate
was centrifuged at 100 g for 1 minute and incubated for 4 hours at
37.degree. C.
[0153] Data was analyzed via flow cytometry by staining for CD107a
and CD4 and quantifying the CD107a+ fraction of the CD4-
population. T cells were distinguished from K562 cells via FSC/SSC.
All data was quantified using FlowJo Software.
[0154] While several possible embodiments are disclosed above,
embodiments of the present disclosure are not so limited. These
exemplary embodiments are not intended to be exhaustive or to
unnecessarily limit the scope of the disclosure, but instead were
chosen and described in order to explain the principles of the
present disclosure so that others skilled in the art may practice
the disclosure. Indeed, various modifications of the disclosure in
addition to those described herein will become apparent to those
skilled in the art from the foregoing description. Such
modifications are intended to fall within the scope of the appended
claims. The embodiments of the present invention are also not
limited to the particular formulations, process steps, and
materials disclosed herein as such formulations, process steps, and
materials may vary somewhat. Further, the terminology employed
herein is used for the purpose of describing exemplary embodiments
only and the terminology is not intended to be limiting since the
scope of the various embodiments of the present invention will be
limited only by the appended claims and equivalents thereof.
[0155] All patents, applications, publications, test methods,
literature, and other materials cited herein are hereby
incorporated by reference in their entirety as if physically
present in this specification.
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References