U.S. patent application number 16/332627 was filed with the patent office on 2020-05-28 for system and methods for multiplexed analysis of cellular and other immunotherapeutics.
The applicant listed for this patent is IsoPlexis Corporation. Invention is credited to Emily BETTINI, Sean MACKAY, Colin NG.
Application Number | 20200166518 16/332627 |
Document ID | / |
Family ID | 60083408 |
Filed Date | 2020-05-28 |
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United States Patent
Application |
20200166518 |
Kind Code |
A1 |
MACKAY; Sean ; et
al. |
May 28, 2020 |
SYSTEM AND METHODS FOR MULTIPLEXED ANALYSIS OF CELLULAR AND OTHER
IMMUNOTHERAPEUTICS
Abstract
Disclosed are methods of identifying a secretome from a subject
cell within a heterogeneous cell population when the subject cell
contacts a target cell (e.g. a subject immune cell contacts a
target cancer cell) or a stimulatory agent and methods of using the
identified secretome to identify cells that are safe and
efficacious for cellular therapies, including adoptive
Inventors: |
MACKAY; Sean; (New Haven,
CT) ; NG; Colin; (Branford, CT) ; BETTINI;
Emily; (Waterbury, CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
IsoPlexis Corporation |
Branford |
CT |
US |
|
|
Family ID: |
60083408 |
Appl. No.: |
16/332627 |
Filed: |
September 12, 2017 |
PCT Filed: |
September 12, 2017 |
PCT NO: |
PCT/US2017/051223 |
371 Date: |
March 12, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62393612 |
Sep 12, 2016 |
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62431318 |
Dec 7, 2016 |
|
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62480147 |
Mar 31, 2017 |
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62480752 |
Apr 3, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/574 20130101;
G01N 33/5091 20130101; G01N 33/5014 20130101; G01N 2800/7095
20130101; G01N 2800/52 20130101; G01N 33/68 20130101; G01N 33/505
20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68; G01N 33/50 20060101 G01N033/50 |
Claims
1. A method of identifying a secretome from a subject cell within a
heterogeneous cell population comprising: (a) contacting the
subject cell and a target cell or a stimulatory agent in at least
one chamber of a plurality of chambers, wherein the chamber is in
fluid communication with an antibody panel and wherein the antibody
panel is removably attached to the chamber; (b) maintaining the
subject cell and the target cell or the stimulatory agent in the
chamber under conditions sufficient to permit (1) the subject cell
to secrete at least one of a peptide, polypeptide, and protein and
(2) at least one antibody of the antibody panel specific for the at
least one protein to bind the at least one peptide, polypeptide, or
protein, forming at least one of an antibody:secreted peptide,
antibody:secreted polypeptide, or an antibody:secreted protein
complex; (c) removing the antibody panel from the chamber; wherein
the at least one chamber comprises cell media that maintains the
viability of the subject cell from step (a) through (c); and (d)
imaging the at least one peptide, polypeptide, or protein, forming
at least one of an antibody:secreted peptide, antibody:secreted
polypeptide, or an antibody:secreted protein complex, thereby
identifying the secretome of the subject cell when the subject cell
contacts the target cell or the stimulatory agent.
2. A method of identifying a secretome from a subject cell within a
heterogeneous cell population comprising: (a) contacting the
subject cell and a target cell or a stimulatory agent under
conditions sufficient to permit stimulation of the subject cell;
(b) introducing the subject cell to at least one chamber of a
plurality of chambers, wherein the chamber is in fluid
communication with an antibody panel and wherein the antibody panel
is removably attached to the chamber; (c) maintaining the subject
cell in the chamber under conditions sufficient to permit (1) the
subject cell to secrete at least one of a peptide, polypeptide, and
protein and (2) at least one antibody of the antibody panel
specific for the at least one protein to bind the at least one
peptide, polypeptide, or protein, forming at least one of an
antibody:secreted peptide, antibody:secreted polypeptide, or an
antibody:secreted protein complex; wherein the at least one chamber
comprises cell media that maintains the viability of the subject
cell from step (b) through (c); and (d) removing the antibody panel
from the chamber; and (e) imaging the at least one peptide,
polypeptide, or protein, forming at least one of an
antibody:secreted peptide, antibody:secreted polypeptide, or an
antibody:secreted protein complex, thereby identifying the
secretome of the subject cell following contact with the target
cell or the stimulatory agent.
3. The method of claim 2, further comprising the step of disrupting
contact between the subject cell and the target cell or the
stimulatory agent.
4. The method of claim 2, wherein the subject cell and the target
cell are comprised by a composition and wherein the subject cell
and the target cell or the stimulatory agent are in fluid
communication.
5. The method of claim 4, further comprising the step of depleting
the target cell or the stimulatory agent from the composition.
6. The method of claim 1, wherein the heterogeneous cell population
is a functionally heterogeneous cell population.
7. The method of claim 1, wherein the functionally heterogeneous
cell population comprises at least two cells that produce a
secretome in response to a stimulus, wherein the first cell
produces a first secretome, wherein the second cell produces a
second secretome, and wherein the first secretome and the second
secretome are not identical.
8. The method of claim 1, wherein the functionally heterogeneous
cell population comprises one or more immune cells.
9. The method of claim 8, wherein the one or more immune cells
comprise a T-lymphocyte, a B-lymphocyte, a natural killer (NK)
cell, a macrophage, a neutrophil, a mast cell, an eosinophil, or a
basophil.
10. (canceled)
11. The method of claim 9, wherein the T-lymphocyte expresses a
non-naturally occurring antigen receptor.
12. The method of claim 7, wherein the T-lymphocyte expresses a
Chimeric Antigen Receptor (CAR).
13. (canceled)
14. The method of claim 1, wherein the subject cell is an immune
cell.
15. The method of claim 14, wherein the immune cell is a
T-lymphocyte, a B-lymphocyte, a natural killer (NK) cell, a
macrophage, a neutrophil, a mast cell, an eosinophil, or a
basophil.
16. (canceled)
17. The method of claim 15, wherein the T-lymphocyte expresses a
non-naturally occurring antigen receptor.
18. The method of claim 17, wherein the T-lymphocyte expresses a
Chimeric Antigen Receptor (CAR).
19. The method of claim 1, wherein the functionally heterogeneous
cell population comprises one or more neuronal cells.
20. (canceled)
21. The method of claim 1, wherein the subject cell is a neuronal
cell.
22. (canceled)
23. The method of claim 1, wherein the functionally heterogeneous
cell population comprises one or more endocrine cells.
24. (canceled)
25. The method of claim 1, wherein the subject cell is an endocrine
cell.
26. (canceled)
27. The method of claim 1, wherein the functionally heterogeneous
cell population comprises one or more exocrine cells.
28.-29. (canceled).
30. The method of claim 1, wherein the subject cell is an exocrine
cell.
31.-32. (canceled)
33. The method of claim 1, wherein the step of contacting the
subject cell and the target cell or the stimulatory agent in a
chamber comprises direct contact of the subject cell and the target
cell.
34. The method of claim 1, wherein the step of contacting the
subject cell and the target cell or the stimulatory agent in a
chamber comprises indirect contact of the subject cell and the
target cell.
35. The method of claim 34, wherein the indirect contact comprises
fluid communication between the subject cell and the target cell or
the stimulatory agent.
36. The method of claim 34, wherein the indirect contact comprises
communication between the subject cell and the target cell or the
stimulatory agent through a natural or artificial extracellular
matrix.
37. The method of claim 34, wherein the indirect contact comprises
communication between the subject cell and the target cell or the
stimulatory agent through an intermediate cell.
38. The method of claim 1, wherein the target cell is a cancer
cell.
39. The method of claim 38, wherein the cancer cell is a primary
cancer cell or a cultured cancer cell.
40. The method of claim 39, wherein the primary cancer cell is
metastatic.
41. The method of claim 1, wherein the target cell is a
B-lymphocyte.
42. The method of claim 1, wherein the target cell is a bacteria,
yeast, or microbe.
43. The method of claim 1, wherein the target cell is an infected
cell.
44. (canceled)
45. The method of claim 1, wherein the target cell is a host
cell.
46. The method of claim 47, wherein the host cell comprises any
cell isolated or derived from the same individual as the subject
cell.
47. The method of claim 45, wherein the host cell perpetuates or is
a target of an autoimmune response.
48. (canceled)
49. The method of claim 45, wherein the host cell is a functional
cell and wherein the host cell does not stimulate an autoimmune
response.
50. The method of claim 1, wherein the stimulatory agent comprises
a stimulatory antibody.
51.-54. (canceled)
55. The method of claim 50, wherein the stimulatory antibody
specifically binds an epitope of a T cell regulator protein.
56. The method of claim 55, wherein the T cell regulator protein
comprises programmed cell death protein 1 (PD-1).
57. (canceled)
58. The method of claim 1, wherein the stimulatory agent comprises
a stimulatory ligand.
59. The method of claim 58, wherein the stimulatory ligand
comprises programmed death ligand 1 (PD-L1).
60. The method of claim 1, wherein each antibody of the antibody
panel is attached to a surface that is removably attached to the
chamber.
61. The method of claim 60, wherein each antibody of the antibody
panel is attached to the surface to form a repeating pattern and
wherein each chamber of the plurality of chambers comprises a
repeat of the pattern.
62. The method of claim 1, wherein the conditions sufficient to
permit (1) the subject cell to secrete at least one of a peptide,
polypeptide, and protein and (2) at least one antibody of the
antibody panel specific for the at least one protein to bind the at
least one peptide, polypeptide, or protein, forming at least one
and an antibody:secreted peptide, antibody:secreted polypeptide, or
an antibody:secreted protein complex, comprise 5% CO.sub.2 and
37.degree. C. for a period of at least 2 hours.
63. The method of claim 62, wherein the period is at least 4 hours,
at least 8 hours, at least 12 hours, at least 16 hours, or at least
24 hours.
64. (canceled)
65. The method of claim 1, wherein the at least one chamber
comprises a cell media that maintains the viability of the subject
cell from step (a) through (c).
66. The method of claim 1, wherein the secretome comprises one or
more distinct peptide(s), polypeptide(s), or protein(s) that
indicate diminished or decreasing cell function or cell
viability.
67. The method of claim 1, wherein the secretome comprises one or
more distinct peptide(s), polypeptide(s), or protein(s) that
indicate(s) augmented or increasing inflammation.
68. The method of claim 1, wherein the secretome comprises one or
more distinct peptide(s), polypeptide(s), or protein(s) that
indicate(s) increased cell activity or cellular stimulation.
69. The method of claim 1, further comprising determining a
Polyfunctional Strength Index (PSI).
70. The method of claim 69, wherein the Polyfunctional Strength
Index is the product of a percentage of polyfunctional subject
cells within the heterogeneous cell population and an average
signal intensity of two or more cytokines.
71. The method of claim 70, wherein the polyfunctional subject
cells, at a single cell level, secrete at least two cytokines.
72. The method of claim 71, wherein the at least two cytokines
produced by each of the polyfunctional subject cells and the two or
more cytokines of the average signal intensity comprise the same
cytokines.
73. The method of claim 71, wherein the at least two cytokines
produced by each of the polyfunctional subject cells and the two or
more cytokines of the average signal intensity consist of the same
cytokines.
74. The method of claim 69, wherein an increase in the PSI
indicates an increase in the potency of the polyfunctional subject
cells.
75.-136. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Patent Application
No. 62/393,612, filed Sep. 12, 2016, U.S. Patent Application No.
62/431,318, filed Dec. 7, 2016, U.S. Patent Application No.
62/480,147, filed Mar. 31, 2017, and U.S. Patent Application No.
62/480,752, filed Apr. 3, 2017, the contents of each of which are
herein incorporated by reference in their entirety.
FIELD OF THE DISCLOSURE
[0002] The disclosure is directed to molecular biology, and more,
specifically, to methods of identifying the contents of the
secretome of a subject cell and the use of the secretome for
determining the safety and efficacy of the subject cell as a
cell-based therapy.
BACKGROUND
[0003] There have been long-felt but unmet needs in the art for a
system and methods of identifying cells suitable for use in a
cellular therapy as well as methods of evaluating the safety and
efficacy of these cells ex vivo or in vitro in a highly multiplexed
reaction that can simultaneously analyze thousands of single cells
from a heterogeneous cell population. The disclosure provides a
system and methods to solve these long-felt but unmet needs.
SUMMARY
[0004] The systems of the disclosure provide a highly multiplexed
method of evaluating the secretome of individual cells in a
functionally heterogeneous population following contact with a
target cell or a stimulatory agent. Analysis of the composition of
the secretome of the subject cell may be used to determine the
subject cell's identity, viability, safety and efficacy when used
in a cell-based therapy. Cellular therapies may include autologous
or allogeneic cells. Cellular therapies may include modified cells,
including, but not limited to T-cells that express at least one
artificial or chimeric antigen receptor.
[0005] The disclosure provides a method of identifying a secretome
from a subject cell within a heterogeneous cell population
comprising: (a) contacting the subject cell and a target cell or a
stimulatory agent in at least one chamber of a plurality of
chambers, wherein the chamber is in fluid communication with an
antibody panel and wherein the antibody panel is removably attached
to the chamber; (b) maintaining the subject cell and the target
cell or the stimulatory agent in the chamber under conditions
sufficient to permit (1) the subject cell to secrete at least one
of a peptide, polypeptide, and protein and (2) at least one
antibody of the antibody panel specific for the at least one
protein to bind the at least one peptide, polypeptide, or protein,
forming at least one of an antibody:secreted peptide,
antibody:secreted polypeptide, or an antibody:secreted protein
complex; (c) removing the antibody panel from the chamber; and (d)
imaging the at least one peptide, polypeptide, or protein, forming
at least one of an antibody:secreted peptide, antibody:secreted
polypeptide, or an antibody:secreted protein complex, thereby
identifying the secretome of the subject cell when the subject cell
contacts the target cell or the stimulatory agent.
[0006] The disclosure provides a method of identifying a secretome
from a subject cell within a heterogeneous cell population
comprising: (a) contacting the subject cell and a target cell or a
stimulatory agent under conditions sufficient to permit stimulation
of the subject cell; (b) introducing the subject cell to at least
one chamber of a plurality of chambers, wherein the chamber is in
fluid communication with an antibody panel and wherein the antibody
panel is removably attached to the chamber; (c) maintaining the
subject cell in the chamber under conditions sufficient to permit
(1) the subject cell to secrete at least one of a peptide,
polypeptide, and protein and (2) at least one antibody of the
antibody panel specific for the at 1least one protein to bind the
at least one peptide, polypeptide, or protein, forming at least one
of an antibody:secreted peptide, antibody:secreted polypeptide, or
an antibody:secreted protein complex; (d) removing the antibody
panel from the chamber; and (e) imaging the at least one peptide,
polypeptide, or protein, forming at least one of an
antibody:secreted peptide, antibody:secreted polypeptide, or an
antibody:secreted protein complex, thereby identifying the
secretome of the subject cell following contact with the target
cell or the stimulatory agent. In certain embodiments, this method
further comprises the step of disrupting contact between the
subject cell and the target cell or the stimulatory agent. In
certain embodiments of this method, the subject cell and the target
cell or the stimulatory agent are comprised by a composition and
wherein the subject cell and the target cell or the stimulatory
agent are in fluid communication. In certain embodiments, including
those wherein the subject cell and the target cell or the
stimulatory agent are comprised by a composition and wherein the
subject cell and the target cell or the stimulatory agent are in
fluid communication, the method further comprises the step of
depleting the target cell or the stimulatory agent from the
composition.
[0007] In certain embodiments of the systems and methods of the
disclosure, the heterogeneous cell population is a functionally
heterogeneous cell population. In certain embodiments, the
functionally heterogeneous cell population may comprise at least
two cells that produce a secretome in response to a stimulus,
wherein the first cell produces a first secretome, wherein the
second cell produces a second secretome, and wherein the first
secretome and the second secretome are not identical.
[0008] Secretomes of the disclosure may comprise one or more
peptides, polypeptides, proteins, small molecules, and ions. In
certain embodiments, the secretome comprises one or more peptides,
polypeptides or proteins. In certain embodiments, the secretome
comprises one or more small molecules. In certain embodiments, the
secretome comprises one or more ions. When the secretome comprises
a small molecule or an ion, detectable labels may be used in
addition or in place of antibodies to identify, quantify, or
otherwise analyze the small molecule and ions of the secretome.
[0009] Secretomes of the disclosure may be released from a subject
cell actively or passively. For example, secretomes of the
disclosure may be released from a subject cell via a vesicle, an
intercellular gap junction and/or a transmembrane channel or
pump.
[0010] In certain embodiments of the systems and methods of the
disclosure, the functionally heterogeneous cell population
comprises one or more immune cells. In certain embodiments, the one
or more immune cells comprise a T-lymphocyte, a B-lymphocyte, a
natural killer (NK) cell, a macrophage, a neutrophil, a mast cell,
an eosinophil, or a basophil. In certain embodiments, the
T-lymphocyte comprises a naive T-lymphocyte, an activated
T-lymphocyte, an effector T-lymphocyte, a helper T-lymphocyte, a
cytotoxic T-lymphocyte, a gamma-delta T-lymphocyte, a regulatory
T-lymphocyte, a memory T-lymphocyte, or a memory stem T-lymphocyte.
In certain embodiments, the T-lymphocyte expresses a non-naturally
occurring antigen receptor. In certain embodiments, the
T-lymphocyte expresses a Chimeric Antigen Receptor (CAR).
[0011] In certain embodiments of the systems and methods of the
disclosure, the functionally heterogeneous cell population
comprises one or more immune cells. In certain embodiments, the one
or more immune cells comprise a T-lymphocyte, a B-lymphocyte, a
natural killer (NK) cell, a macrophage, a neutrophil, a mast cell,
an eosinophil, or a basophil. In certain embodiments, the
B-lymphocyte comprises a plasmablast, a plasma cell, a memory
B-lymphocyte, a regulatory B cell, a follicular B cell, or a
marginal zone B cell.
[0012] In certain embodiments of the systems and methods of the
disclosure, the subject cell is an immune cell. In certain
embodiments, the immune cell comprises a T-lymphocyte, a
B-lymphocyte, a natural killer (NK) cell, a macrophage, a
neutrophil, a mast cell, an eosinophil, or a basophil. In certain
embodiments, the T-lymphocyte comprises a naive T-lymphocyte, an
activated T-lymphocyte, an effector T-lymphocyte, a helper
T-lymphocyte, a cytotoxic T-lymphocyte, a gamma-delta T-lymphocyte,
a regulatory T-lymphocyte, a memory T-lymphocyte, or a memory stem
T-lymphocyte. In certain embodiments, the T-lymphocyte expresses a
non-naturally occurring antigen receptor. In certain embodiments,
the T-lymphocyte expresses a Chimeric Antigen Receptor (CAR).
[0013] In certain embodiments of the systems and methods of the
disclosure, the subject cell is an immune cell. In certain
embodiments, the immune cell comprises a T-lymphocyte, a
B-lymphocyte, a natural killer (NK) cell, a macrophage, a
neutrophil, a mast cell, an eosinophil, or a basophil. In certain
embodiments, the B-lymphocyte comprises a plasmablast, a plasma
cell, a memory B-lymphocyte, a regulatory B cell, a follicular B
cell, or a marginal zone B cell.
[0014] In certain embodiments of the systems and methods of the
disclosure, the functionally heterogeneous cell population
comprises one or more neuronal cells. In certain embodiments, the
one or more neuronal cells comprise a neuron, a glial cell, an
astrocyte, a satellite cell, or an enteric glial cell.
[0015] In certain embodiments of the systems and methods of the
disclosure, the subject cell is a neuronal cell. In certain
embodiments, the neuronal cell comprises a neuron, a glial cell, an
astrocyte, a satellite cell, or an enteric glial cell.
[0016] In certain embodiments of the systems and methods of the
disclosure, the functionally heterogeneous cell population
comprises one or more endocrine cells. In certain embodiments, the
one or more endocrine cells are isolated or derived from a pineal
gland, a pituitary gland, a pancreas, an ovary, a testicle, a
thyroid gland, a parathyroid gland, a hypothalamus, or an adrenal
gland.
[0017] In certain embodiments of the systems and methods of the
disclosure, the subject cell is an endocrine cell. In certain
embodiments, the endocrine cell is isolated or derived from a
pineal gland, a pituitary gland, a pancreas, an ovary, a testicle,
a thyroid gland, a parathyroid gland, a hypothalamus, or an adrenal
gland.
[0018] In certain embodiments of the systems and methods of the
disclosure, the functionally heterogeneous cell population
comprises exocrine cells. In certain embodiments, the exocrine cell
is isolated or derived from a salivary gland, a sweat gland or a
component of the gastrointestinal tract. Components of the
gastrointestinal tract may comprise a mouth, a stomach, a small
intestine, and a large intestine.
[0019] In certain embodiments of the systems and methods of the
disclosure, the subject cell is an exocrine cell. In certain
embodiments, the exocrine cell is isolated or derived from a
salivary gland, a sweat gland or a component of the
gastrointestinal tract. Components of the gastrointestinal tract
may comprise a mouth, a stomach, a small intestine, and a large
intestine.
[0020] In certain embodiments of the systems and methods of the
disclosure, the step of contacting the subject cell and the target
cell or the stimulatory agent in a chamber comprises direct contact
of the subject cell and the target cell or the stimulatory
agent.
[0021] In certain embodiments of the systems and methods of the
disclosure, the step of contacting the subject cell and the target
cell or the stimulatory agent in a chamber comprises indirect
contact of the subject cell and the target cell or the stimulatory
agent. In certain embodiments, the indirect contact comprises fluid
communication between the subject cell and the target cell or the
stimulatory agent. In certain embodiments, the indirect contact
comprises communication between the subject cell and the target
cell or the stimulatory agent through a natural or artificial
extracellular matrix. In certain embodiments, the indirect contact
comprises communication between the subject cell and the target
cell or the stimulatory agent through an intermediate cell.
[0022] In certain embodiments of the systems and methods of the
disclosure, the target cell is a cancer cell. In certain
embodiments, the cancer cell is a primary cancer cell or a cultured
cancer cell. In certain embodiments, the primary cancer cell is
metastatic.
[0023] In certain embodiments of the systems and methods of the
disclosure, the target cell is a B-lymphocyte.
[0024] In certain embodiments of the systems and methods of the
disclosure, the target cell is a bacteria, yeast, or microbe.
[0025] In certain embodiments of the systems and methods of the
disclosure, the target cell is an infected cell. Exemplary infected
cells may have contacted or may have been exposed to a virus,
bacteria, yeast, or microbe.
[0026] In certain embodiments of the systems and methods of the
disclosure, the target cell is a host cell. In certain embodiments,
the host cell comprises any cell isolated or derived from the same
individual as the subject cell. In certain embodiments, the host
cell perpetuates an autoimmune response.
[0027] In certain embodiments of the systems and methods of the
disclosure, the target cell is a host cell. In certain embodiments,
the host cell comprises any cell isolated or derived from the same
individual as the subject cell. In certain embodiments, the host
cell is a target of an autoimmune response.
[0028] In certain embodiments of the systems and methods of the
disclosure, the target cell is a host cell. In certain embodiments,
the host cell comprises any cell isolated or derived from the same
individual as the subject cell. In certain embodiments, the host
cell is a functional cell and wherein the host cell does not
stimulate an autoimmune response. The term "functional" cell is
meant to describe a viable cell that does not contribute to a
disease or disorder in the host. Alternatively, or in addition, the
term "functional" cell may describe a cell without any known
mutations that cause a disease or disorder in the host. For
example, a functional cell may be non-cancerous and/or
non-autoimmune.
[0029] In certain embodiments of the systems and methods of the
disclosure, the stimulatory agent comprises a stimulatory antibody.
In certain embodiments, the stimulatory antibody is a monoclonal
antibody. In certain embodiments, the monoclonal antibody is a
fully human antibody. In certain embodiments, the monoclonal
antibody is a humanized antibody, a chimeric antibody, a
recombinant antibody or a modified antibody. In certain
embodiments, the modified antibody comprises one or more sequence
variations when compared to a fully human version of an antibody
having the same epitope specificity, one or more modified or
synthetic amino acids, or a chemical moiety to enhance a
stimulatory function. In certain embodiments, the stimulatory
antibody specifically binds an epitope of a T cell regulator
protein. In certain embodiments, the T cell regulator protein
comprises programmed cell death protein 1 (PD-1). In certain
embodiments, the stimulatory antibody comprises Nivolumab or a
biosimilar thereof.
[0030] In certain embodiments of the systems and methods of the
disclosure, the stimulatory agent comprises a stimulatory ligand.
In certain embodiments, the stimulatory ligand comprises programmed
death ligand 1 (PD-L1).
[0031] In certain embodiments of the systems and methods of the
disclosure, each antibody of the antibody panel is attached to a
surface that is removably attached to the chamber.
[0032] In certain embodiments of the systems and methods of the
disclosure, each antibody of the antibody panel is attached to the
surface to form a repeating pattern and wherein each chamber of the
plurality of chambers comprises a repeat of the pattern. Antibody
panels of the disclosure form patterns in which each repeat
comprises the full panel of antibodies. For examples, if the
antibody panel comprises antibodies "a", "b" and "c", then each
repeat of the pattern also comprises at least one of antibody "a",
"b" and "c". The pattern need only have a size scale such that each
chamber aligns with at least one repeat of the pattern. In
preferred embodiments, the pattern need only have a size scale such
that each chamber aligns with one repeat of the pattern. When
additional detectable labels are added to the panel to identify,
capture or quantify secreted small molecules and/or ions, the
detectable labels also repeat by the same rules set out for the
antibody pattern.
[0033] In certain embodiments of the systems and methods of the
disclosure, the conditions sufficient to permit (1) the subject
cell to secrete at least one of a peptide, polypeptide, and protein
and (2) at least one antibody of the antibody panel specific for
the at least one protein to bind the at least one peptide,
polypeptide, or protein, forming at least one of an
antibody:secreted peptide, antibody:secreted polypeptide, or an
antibody:secreted protein complex, may comprise 5% CO.sub.2 and
37.degree. C. for a period of 2 hours, about 2 hours or at least 2
hours. Alternatively, the period may be 4 hours, about 4 hours or
at least 4 hours; 8 hours, about 8 hours or at least 8 hours; 12
hours, about 12 hours or at least 12 hours; 16 hours, about 16
hours or at least 16 hours; or 24 hours, about 24 hours or at least
24 hours. In certain embodiments, the period is 16 hours, about or
at least 16 hours.
[0034] In certain embodiments of the systems and methods of the
disclosure, at least one chamber comprises a cell media that
maintains the viability of the subject cell from steps (a) through
(c) (e.g., from contacting the subject cell and target cell or the
stimulatory agent through removal of the antibody panel comprising
antibody complexes with one or more of a peptide, polypeptide or
protein secreted from the subject cell).
[0035] In certain embodiments of the systems and methods of the
disclosure, the secretome comprises one or more distinct
peptide(s), polypeptide(s), or protein(s) that indicate diminished
or decreasing cell function or cell viability.
[0036] In certain embodiments of the systems and methods of the
disclosure, the secretome comprises one or more distinct
peptide(s), polypeptide(s), or protein(s) that indicate(s)
augmented or increasing inflammation.
[0037] In certain embodiments of the systems and methods of the
disclosure, the secretome comprises one or more distinct
peptide(s), polypeptide(s), or protein(s) that indicate(s)
increased cell activity or cellular stimulation.
[0038] In certain embodiments of the systems and methods of the
disclosure, the methods further comprise determining a
Polyfunctional Strength Index (PSI). In certain embodiments, the
Polyfunctional Strength Index is the product of a percentage of
polyfunctional subject cells within the heterogeneous cell
population and an average signal intensity of two or more
cytokines. In certain embodiments, the average signal intensity of
two or more cytokines is the average signal intensity of two or
more distinct cytokines (i.e. AB versus AA). In certain
embodiments, the polyfunctional subject cells, at a single cell
level, secrete at least two cytokines. In certain embodiments, the
polyfunctional subject cells, at a single cell level, secrete at
least two distinct cytokines (i.e. AB versus AA). In certain
embodiments, the at least two cytokines produced by each of the
polyfunctional subject cells and the two or more cytokines of the
average signal intensity comprise the same cytokines (e.g. AB and
AB). In certain embodiments, the at least two cytokines produced by
each of the polyfunctional subject cells and the two or more
cytokines of the average signal intensity consist of the same
cytokines. In certain embodiments, an increase in the PSI indicates
an increase in the potency of the polyfunctional subject cells.
[0039] The disclosure provides a use of a secretome produced by a
method of the disclosure for the identification of a T-Lymphocyte
expressing a CAR that specifically binds an antigen presented on a
target cell, a CAR that specifically binds a stimulatory agent, or
a CAR that specifically binds an antigen presented on a target cell
and specifically binds a stimulatory agent.
[0040] The disclosure provides a use of a secretome produced by a
method of the disclosure for the evaluation of the safety of a
cellular therapy, wherein the cellular therapy comprises the
subject cell, the cellular therapy is intended to respond to the
target cell or the stimulatory agent, and wherein the cellular
therapy is considered safe when the secretome lacks one or more
peptide(s), polypeptide(s), or protein(s) that stimulate the immune
system.
[0041] The disclosure provides a use of a secretome produced by a
method of the disclosure for the evaluation of the safety of a
cellular therapy, wherein the cellular therapy comprises the
subject cell, the cellular therapy is intended to respond to the
target cell or the stimulatory agent, and wherein the cellular
therapy is considered safe when the secretome lacks one or more
peptide(s), polypeptide(s), or protein(s) that indicate decreased
cell viability.
[0042] The disclosure provides a use of a secretome produced by a
method of the disclosure for the evaluation of the efficacy of a
cellular therapy, wherein the cellular therapy comprises the
subject cell, the cellular therapy is intended to respond to the
target cell or the stimulatory agent, and wherein the cellular
therapy is considered safe when the secretome contains one or more
peptide(s), polypeptide(s), or protein(s) that indicate a selective
response to the target cell.
[0043] The disclosure provides a use of the secretome produced by a
method of the disclosure for the evaluation of a cellular therapy,
wherein the cellular therapy comprises the subject cell and wherein
the subject cell is a chimeric antigen receptor (CAR)-expressing T
cell; wherein the cellular therapy is intended to respond to the
target cell or the stimulatory agent, wherein the target cell is a
cancer cell that expresses an antigen to which the chimeric antigen
receptor (CAR) specifically binds, and wherein, upon binding the
antigen, the chimeric antigen receptor (CAR) stimulates the T cell;
wherein the cellular therapy is considered efficacious when the
secretome comprises one or more peptide(s), polypeptide(s), or
protein(s) that stimulate the immune system above a first
threshold, wherein the one or more peptide(s), polypeptide(s), or
protein(s) comprise one or more cytokines, and wherein the one or
more cytokines are selected from the group consisting of effector,
stimulatory or chemoattractive cytokines; and wherein the cellular
therapy is considered safe when the secretome comprises one or more
peptide(s), polypeptide(s), or protein(s) that mediate a
deleterious process, wherein the one or more peptide(s),
polypeptide(s), or protein(s) comprise one or more cytokines, and
wherein the one or more cytokines are selected from the group
consisting of regulatory and inflammatory cytokines. In certain
embodiments of this use, the effector cytokines are selected from
the group consisting of Granzyme B, IFN-.gamma., M1P-1.alpha.,
Performin, TNF-.alpha., and TNF-.beta.. In certain embodiments of
this use, the use of claim 59, wherein the stimulatory cytokines
are selected from the group consisting of GM-CSF, IL-12, IL-15,
IL-2, IL-21, IL-5, IL-7, IL-8 and IL-9. In certain embodiments of
this use, the chemoattractive cytokines are selected from the group
consisting of CCL-11, IP-10, MIP-1.beta. and RANTES. In certain
embodiments of this use, the regulatory cytokines are selected from
the group consisting of IL-10, IL-13, IL-22, IL-4, TGF-.beta.1,
sCD137 and sCD40L. In certain embodiments of this use, the
inflammatory cytokines are selected from the group consisting of
IL-17A, IL-17F, IL-1.beta., IL-6, MCP-1 and MCP-4. In certain
embodiments of this use, the deleterious process comprises
inflammation. In certain embodiments of this use, the deleterious
process comprises an autoimmune response. In certain embodiments of
this use, the deleterious process comprises a non-selective
response to the target cell. In certain embodiments of this use,
the subject cell is isolated or derived from an adoptive cell
therapy for use in the treatment of a cancer.
[0044] The disclosure provides a use of the secretome produced by a
method of the disclosure for the evaluation of a cellular therapy,
wherein the cellular therapy comprises the subject cell and wherein
the subject cell is a chimeric antigen receptor (CAR)-expressing T
cell; wherein the cellular therapy is intended to respond to the
target cell or the stimulatory agent, wherein the target cell is an
autoimmune cell that expresses an antigen to which the chimeric
antigen receptor (CAR) specifically binds, and wherein, upon
binding the antigen, the chimeric antigen receptor (CAR) stimulates
the T cell; wherein the cellular therapy is considered efficacious
when the secretome comprises one or more peptide(s),
polypeptide(s), or protein(s) that stimulate the immune system
above a first threshold, wherein the one or more peptide(s),
polypeptide(s), or protein(s) comprise one or more cytokines, and
wherein the one or more cytokines are selected from the group
consisting of effector, stimulatory or chemoattractive cytokines;
and wherein the cellular therapy is considered safe when the
secretome comprises one or more peptide(s), polypeptide(s), or
protein(s) that mediate a deleterious process, wherein the one or
more peptide(s), polypeptide(s), or protein(s) comprise one or more
cytokines, and wherein the one or more cytokines are selected from
the group consisting of regulatory and inflammatory cytokines. In
certain embodiments of this use, the effector cytokines are
selected from the group consisting of Granzyme B, IFN-.gamma.,
M1P-1.alpha., Performin, TNF-.alpha., and TNF-.beta.. In certain
embodiments of this use, the use of claim 59, wherein the
stimulatory cytokines are selected from the group consisting of
GM-CSF, IL-12, IL-15, IL-2, IL-21, IL-5, IL-7, IL-8 and IL-9. In
certain embodiments of this use, the chemoattractive cytokines are
selected from the group consisting of CCL-11, IP-10, MIP-1.beta.
and RANTES. In certain embodiments of this use, the regulatory
cytokines are selected from the group consisting of IL-10, IL-13,
IL-22, IL-4, TGF-.beta.1, sCD137 and sCD40L. In certain embodiments
of this use, the inflammatory cytokines are selected from the group
consisting of IL-17A, IL-17F, IL-1.beta., IL-6, MCP-1 and MCP-4. In
certain embodiments of this use, the deleterious process comprises
inflammation. In certain embodiments of this use, the deleterious
process comprises an autoimmune response. In certain embodiments of
this use, the deleterious process comprises a non-selective
response to the target cell. In certain embodiments of this use,
the autoimmune cell is an immune cell that initiates, effectuates,
or enhances an autoimmune response. In certain embodiments of this
use, the subject cell is isolated or derived from an adoptive cell
therapy for use in the treatment of an autoimmune condition.
[0045] The disclosure provides a use of the secretome produced by a
method of the disclosure for the evaluation of a cellular therapy,
wherein the cellular therapy comprises the subject cell and wherein
the subject cell is a chimeric antigen receptor (CAR)-expressing T
cell; wherein the cellular therapy is intended to respond to the
target cell or the stimulatory agent, wherein the target cell is an
infected cell that expresses an antigen to which the chimeric
antigen receptor (CAR) specifically binds, and wherein, upon
binding the antigen, the chimeric antigen receptor (CAR) stimulates
the T cell; wherein the cellular therapy is considered efficacious
when the secretome comprises one or more peptide(s),
polypeptide(s), or protein(s) that stimulate the immune system
above a first threshold, wherein the one or more peptide(s),
polypeptide(s), or protein(s) comprise one or more cytokines, and
wherein the one or more cytokines are selected from the group
consisting of effector, stimulatory or chemoattractive cytokines;
and wherein the cellular therapy is considered safe when the
secretome comprises one or more peptide(s), polypeptide(s), or
protein(s) that mediate a deleterious process, wherein the one or
more peptide(s), polypeptide(s), or protein(s) comprise one or more
cytokines, and wherein the one or more cytokines are selected from
the group consisting of regulatory and inflammatory cytokines. In
certain embodiments of this use, the effector cytokines are
selected from the group consisting of Granzyme B, IFN-.gamma.,
M1P-1.alpha., Performin, TNF-.alpha., and TNF-.beta.. In certain
embodiments of this use, the use of claim 59, wherein the
stimulatory cytokines are selected from the group consisting of
GM-CSF, IL-12, IL-15, IL-2, IL-21, IL-5, IL-7, IL-8 and IL-9. In
certain embodiments of this use, the chemoattractive cytokines are
selected from the group consisting of CCL-11, IP-10, MIP-1.beta.
and RANTES. In certain embodiments of this use, the regulatory
cytokines are selected from the group consisting of IL-10, IL-13,
IL-22, IL-4, TGF-.beta.1, sCD137 and sCD40L. In certain embodiments
of this use, the inflammatory cytokines are selected from the group
consisting of IL-17A, IL-17F, IL-1.beta., IL-6, MCP-1 and MCP-4. In
certain embodiments of this use, the deleterious process comprises
inflammation. In certain embodiments of this use, the deleterious
process comprises an autoimmune response. In certain embodiments of
this use, the deleterious process comprises a non-selective
response to the target cell. In certain embodiments of this use,
the autoimmune cell is an immune cell that initiates, effectuates,
or enhances an autoimmune response. In certain embodiments of this
use, the subject cell is isolated or derived from an adoptive cell
therapy for use in the treatment of an infection or an
infectious/contagious condition.
[0046] The disclosure provides a use of the secretome produced by a
method of the disclosure for the evaluation of a cellular therapy,
wherein the cellular therapy comprises the subject cell and wherein
the subject cell is a chimeric antigen receptor (CAR)-expressing T
cell; wherein the cellular therapy is intended to respond to the
target cell or the stimulatory agent, wherein the target cell is a
cardiovascular cell that expresses an antigen to which the chimeric
antigen receptor (CAR) specifically binds, and wherein, upon
binding the antigen, the chimeric antigen receptor (CAR) stimulates
the T cell; wherein the cellular therapy is considered efficacious
when the secretome comprises one or more peptide(s),
polypeptide(s), or protein(s) that stimulate the immune system
above a first threshold, wherein the one or more peptide(s),
polypeptide(s), or protein(s) comprise one or more cytokines, and
wherein the one or more cytokines are selected from the group
consisting of effector, stimulatory or chemoattractive cytokines;
and wherein the cellular therapy is considered safe when the
secretome comprises one or more peptide(s), polypeptide(s), or
protein(s) that mediate a deleterious process, wherein the one or
more peptide(s), polypeptide(s), or protein(s) comprise one or more
cytokines, and wherein the one or more cytokines are selected from
the group consisting of regulatory and inflammatory cytokines. In
certain embodiments of this use, the effector cytokines are
selected from the group consisting of Granzyme B, IFN-.gamma.,
M1P-1.alpha., Performin, TNF-.alpha., and TNF-.beta.. In certain
embodiments of this use, the use of claim 59, wherein the
stimulatory cytokines are selected from the group consisting of
GM-CSF, IL-12, IL-15, IL-2, IL-21, IL-5, IL-7, IL-8 and IL-9. In
certain embodiments of this use, the chemoattractive cytokines are
selected from the group consisting of CCL-11, IP-10, MIP-1.beta.
and RANTES. In certain embodiments of this use, the regulatory
cytokines are selected from the group consisting of IL-10, IL-13,
IL-22, IL-4, TGF-.beta.1, sCD137 and sCD40L. In certain embodiments
of this use, the inflammatory cytokines are selected from the group
consisting of IL-17A, IL-17F, IL-1.beta., IL-6, MCP-1 and MCP-4. In
certain embodiments of this use, the deleterious process comprises
inflammation. In certain embodiments of this use, the deleterious
process comprises an autoimmune response. In certain embodiments of
this use, the deleterious process comprises a non-selective
response to the target cell. In certain embodiments of this use,
the autoimmune cell is an immune cell that initiates, effectuates,
or enhances an autoimmune response. In certain embodiments of this
use, the cardiovascular cell is a smooth muscle cell, a cardiac
muscle cell, an endothelial cell, or any other cell type integrated
into a capillary, vein, or an artery. In certain embodiments of
this use, the cardiovascular cell is a component of the local or
circulating blood, including a blood platelet. In certain
embodiments of this use, the platelet is a component of a blood
clot or another form of obstruction to healthy blood flow. In
certain embodiments of this use, the cardiovascular cell is a
damaged cell. In certain embodiments of this use, the
cardiovascular cell is an infected cell. In certain embodiments of
this use, the subject cell is isolated or derived from an adoptive
cell therapy for use in the treatment of a cardiovascular condition
or a vascular condition.
[0047] The disclosure provides a method of identifying a subject
cell population as efficacious for use in an adoptive cell therapy,
comprising: detecting at least one component of a secretome of each
subject cell of the subject cell population according to the method
of identifying a secretome from a subject cell of the disclosure;
identifying a subpopulation of polyfunctional cells of the subject
cell population, wherein a polyfunctional cell of subject cells of
the subject cell population secrete two or more signaling
molecules; calculating a percentage of polyfunctionality of the
subject cell population, wherein the percentage of
polyfunctionality is the percentage of polyfunctional cells within
the subject cell population; measuring a signal intensity of a
first signaling molecule of the secretome of each polyfunctional
cell of the subject cell population; measuring a signal intensity
of a second signaling molecule of the secretome of each
polyfunctional cell of the subject cell population; calculating a
Polyfunctional Strength Index (PSI) for each polyfunctional cell of
the subject cell population, wherein the PSI comprises (a) the
product of the percentage of polyfunctionality of the subject cell
population and the signal intensity of the first signaling molecule
and (b) the product of the percentage of polyfunctionality of the
subject cell population and the signal intensity of the second
signaling molecule; identifying the subject cell population as
efficacious for use in an adoptive cell therapy when the PSI
indicates that at least 50% of the subject cells in the subject
cell population are polyfunctional, the signal intensity of the
first signaling molecule indicates that the concentration of the
first signaling molecule within the chamber is between 2 pg/ml and
10,000 pg/ml, inclusive of the endpoints, and the signal intensity
of the second signaling molecule indicates that the concentration
of the second signaling molecule within the chamber is between 2
pg/ml and 10,000 pg/ml, inclusive of the endpoints; and identifying
the subject cell population as not efficacious for use in an
adoptive cell therapy when the PSI indicates that less than 50% of
the subject cells in the subject cell population are
polyfunctional, the signal intensity of the first signaling
molecule indicates that the concentration of the first signaling
molecule within the chamber is less than 2 pg/ml, and the signal
intensity of the second signaling molecule indicates that the
concentration of the second signaling molecule within the chamber
is less than 2 pg/ml.
[0048] In certain embodiments of the methods of identifying a
subject cell population as efficacious for use in an adoptive cell
therapy, the subject cell population comprises a plurality of
immune cells. In certain embodiments, the plurality of immune cells
comprises a T-lymphocyte, a B-lymphocyte, a natural killer (NK)
cell, a macrophage, a neutrophil, a mast cell, an eosinophil, a
basophil or a combination thereof. In certain embodiments, the
T-lymphocyte is a naive T-lymphocyte, an activated T-lymphocyte, an
effector T-lymphocyte, a helper T-lymphocyte, a cytotoxic
T-lymphocyte, a gamma-delta T-lymphocyte, a regulatory
T-lymphocyte, a memory T-lymphocyte, or a memory stem T-lymphocyte.
In certain embodiments, the T-lymphocyte expresses a non-naturally
occurring antigen receptor. In certain embodiments, the
T-lymphocyte expresses a Chimeric Antigen Receptor (CAR).
[0049] In certain embodiments of the methods of identifying a
subject cell population as efficacious for use in an adoptive cell
therapy, the subject cell population comprises a plurality of
neuronal cells. In certain embodiments, the plurality of neuronal
cells comprises a neuron, a glial cell, an astrocyte, a satellite
cell, an enteric glial cell or a combination thereof.
[0050] In certain embodiments of the methods of identifying a
subject cell population as efficacious for use in an adoptive cell
therapy, the subject cell population comprises a plurality of
endocrine cells. In certain embodiments, the plurality of endocrine
cells comprises one or more cells isolated or derived from a pineal
gland, a pituitary gland, a pancreas, an ovary, a testicle, a
thyroid gland, a parathyroid gland, a hypothalamus, or an adrenal
gland.
[0051] In certain embodiments of the methods of identifying a
subject cell population as efficacious for use in an adoptive cell
therapy, the subject cell population comprises a plurality of
exocrine cells. In certain embodiments, the plurality of exocrine
cells comprises one or more cells isolated or derived from a
salivary gland, a sweat gland or a component of the
gastrointestinal tract. In certain embodiments, the component of
the gastrointestinal tract comprises a mouth, a stomach, a small
intestine, and a large intestine.
[0052] In certain embodiments of the methods of identifying a
subject cell population as efficacious for use in an adoptive cell
therapy, the target cell is a cancer cell. In certain embodiments,
the cancer cell is a primary cancer cell or a cultured cancer cell.
In certain embodiments, the primary cancer cell is metastatic.
[0053] In certain embodiments of the methods of identifying a
subject cell population as efficacious for use in an adoptive cell
therapy, the target cell is a B-lymphocyte.
[0054] In certain embodiments of the methods of identifying a
subject cell population as efficacious for use in an adoptive cell
therapy, the target cell is a bacteria, yeast, or microbe.
[0055] In certain embodiments of the methods of identifying a
subject cell population as efficacious for use in an adoptive cell
therapy, the target cell is an infected cell. Exemplary infected
cells may have contacted or may have been exposed to a virus,
bacteria, yeast, or microbe.
[0056] In certain embodiments of the methods of identifying a
subject cell population as efficacious for use in an adoptive cell
therapy, the target cell is a host cell. In certain embodiments,
the host cell comprises any cell isolated or derived from the same
individual as the subject cell. In certain embodiments, the host
cell perpetuates an autoimmune response.
[0057] In certain embodiments of the methods of identifying a
subject cell population as efficacious for use in an adoptive cell
therapy, the target cell is a host cell. In certain embodiments,
the host cell comprises any cell isolated or derived from the same
individual as the subject cell. In certain embodiments, the host
cell is a target of an autoimmune response.
[0058] In certain embodiments of the methods of identifying a
subject cell population as efficacious for use in an adoptive cell
therapy, the target cell is a host cell. In certain embodiments,
the host cell comprises any cell isolated or derived from the same
individual as the subject cell. In certain embodiments, the host
cell is a functional cell and wherein the host cell does not
stimulate an autoimmune response. The term "functional" cell is
meant to describe a viable cell that does not contribute to a
disease or disorder in the host. Alternatively, or in addition, the
term "functional" cell may describe a cell without any known
mutations that cause a disease or disorder in the host. For
example, a functional cell may be non-cancerous and/or
non-autoimmune.
[0059] In certain embodiments of the methods of identifying a
subject cell population as efficacious for use in an adoptive cell
therapy, the first signaling molecule is a peptide, a polypeptide,
or a protein. In certain embodiments, the first signaling molecule
is a cytokine.
[0060] In certain embodiments of the methods of identifying a
subject cell population as efficacious for use in an adoptive cell
therapy, the second signaling molecule is a peptide, a polypeptide,
or a protein. In certain embodiments, the second signaling molecule
is a cytokine.
[0061] In certain embodiments of the methods of identifying a
subject cell population as efficacious for use in an adoptive cell
therapy, the subject cell population comprises a plurality of
T-lymphocytes; wherein the target cell is a cancer cell, an
infected cell or a host cell that perpetuates an autoimmune
response; the first signaling molecule comprises an effector
cytokine, a stimulatory cytokine, or a chemoattractive cytokine and
the second signaling molecule comprises an effector cytokine, a
stimulatory cytokine, or a chemoattractive cytokine. In certain
embodiments, at least one T-lymphocyte of the plurality of
T-lymphocytes expresses a chimeric antigen receptor (CAR). In
certain embodiments, each T-lymphocyte of the plurality of
T-lymphocytes expresses a chimeric antigen receptor (CAR). In
certain embodiments, the effector cytokine is Granzyme B,
IFN-.gamma., M1P-1.alpha., Performin, TNF-.alpha. or TNF-.beta.. In
certain embodiments, the stimulatory cytokine is GM-CSF, IL-12,
IL-15, IL-2, IL-21, IL-5, IL-7, IL-8 and IL-9. In certain
embodiments, the chemoattractive cytokine is CCL-11, IP-10,
MIP-1.beta. or RANTES.
[0062] In certain embodiments of the methods of identifying a
subject cell population as efficacious for use in an adoptive cell
therapy, the method further comprises identifying the subject cell
population as safe for use in an adoptive cell therapy, when the
PSI indicates that at least 50% of the subject cells in the subject
cell population are polyfunctional, the signal intensity of the
first signaling molecule indicates that the concentration of the
first signaling molecule within the chamber is less than 2 pg/ml,
the signal intensity of the second signaling molecule indicates
that the concentration of the second signaling molecule within the
chamber is less than 2 pg/ml, the subject cell population comprises
a plurality of T-lymphocytes, the first signaling molecule
comprises a regulatory cytokine or an inflammatory cytokine and the
second signaling molecule comprises a regulatory cytokine or an
inflammatory cytokine; and identifying the subject cell population
as not safe for use in an adoptive cell therapy when the PSI
indicates that at least 50% of the subject cells in the subject
cell population are polyfunctional, the signal intensity of the
first signaling molecule indicates that the concentration of the
first signaling molecule within the chamber is greater than 2
pg/ml, the signal intensity of the second signaling molecule
indicates that the concentration of the second signaling molecule
within the chamber is greater than 2 pg/ml, the subject cell
population comprises a plurality of T-lymphocytes, the first
signaling molecule comprises a regulatory cytokine or an
inflammatory cytokine and the second signaling molecule comprises a
regulatory cytokine or an inflammatory cytokine. In certain
embodiments, the regulatory cytokine is IL-10, IL-13, IL-22, IL-4,
TGF-.beta.1, sCD137 and sCD40L. In certain embodiments, the
inflammatory cytokine is IL-17A, IL-17F, IL-1.beta., IL-6, MCP-1
and MCP-4. In certain embodiments, at least one T-lymphocyte of the
plurality of T-lymphocytes expresses a chimeric antigen receptor
(CAR). In certain embodiments, each T-lymphocyte of the plurality
of T-lymphocytes expresses a chimeric antigen receptor (CAR).
[0063] In certain embodiments of the methods of identifying a
subject cell population as efficacious for use in an adoptive cell
therapy, the subject cell population comprises at least 100 cells.
In certain embodiments, the subject cell population comprises at
least 500 cells. In certain embodiments, the subject cell
population comprises at least 1000 cells. In certain embodiments,
the subject cell population comprises at least 5000 cells.
[0064] In certain embodiments of the methods of identifying a
subject cell population as efficacious for use in an adoptive cell
therapy, the detecting step comprises detecting at least 2
components of a secretome from each subject cell of the subject
cell population. In certain embodiments, the detecting step
comprises detecting at least 10 components of a secretome from each
subject cell of the subject cell population. In certain
embodiments, the detecting step comprises detecting at least 20
components of a secretome from each subject cell of the subject
cell population. In certain embodiments, the detecting step
comprises detecting at least 30 components of a secretome from each
subject cell of the subject cell population. In certain
embodiments, the detecting step comprises detecting at least 50
components of a secretome from each subject cell of the subject
cell population. In certain embodiments, the detecting step
comprises detecting at least 100 components of a secretome from
each subject cell of the subject cell population.
[0065] In certain embodiments of the methods of identifying a
subject cell population as efficacious for use in an adoptive cell
therapy, the percentage of polyfunctional cells comprises a first
percentage of polyfunctional cells that secrete two or more
signaling molecules, a second percentage of polyfunctional cells
that secrete three or more signaling molecules, a third percentage
of polyfunctional cells that secrete four or more signaling
molecules, a fourth percentage of polyfunctional cells that secrete
five or more signaling molecules, and a subsequent percentage of
polyfunctional cells that secrete increasing numbers of signaling
molecules.
[0066] In certain embodiments of the methods of identifying a
subject cell population as efficacious for use in an adoptive cell
therapy, the measuring of the signal intensity comprises detecting
a fluorescent signal from a complex of an antibody specific for the
first or second signaling molecule and the first or second
signaling molecule, respectively, and normalizing each fluorescent
signal against a reference signal to determine a relative
fluorescent unit (RFU) value. In certain embodiments, the reference
signal is a maximal signal, a minimal signal, or a signal from a
component of the secretome with a constant or known concentration.
In certain embodiments, the reference signal is a component of the
secretome of a subject cell that is most abundant.
[0067] In certain embodiments of the methods of identifying a
subject cell population as efficacious for use in an adoptive cell
therapy, the method further comprises measuring a third or
subsequent signaling molecule of the secretome of each
polyfunctional cell.
[0068] In certain embodiments of the methods of identifying a
subject cell population as efficacious for use in an adoptive cell
therapy, the method further comprises determining a relative
contribution of the first signaling molecule, the second signaling
molecule or the subsequent signaling molecule to a response of the
subpopulation of polyfunctional cells to a target cell or to a
stimulatory agent, wherein the relative contribution is the product
of an average of a percentage of the PSI of each polyfunctional
cell from the first signaling molecule, the second signaling
molecule or the subsequent signaling molecule from each
polyfunctional cell and a total PSI for the subpopulation of
polyfunctional cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0069] FIG. 1 is a photograph showing a system of the disclosure
for high throughput analysis of Two-Cell Interactions. Natural
Killer (NK) cell and leukemia cell (K562) populations loaded into a
plurality of chambers (e.g. in this example, taking the form of
micro-troughs) are mixed allowing for a high throughput analysis of
individual two-cell interactions within one chamber (e.g. in this
example, a micro-trough).
[0070] FIG. 2 is a series of graphs showing single cell secretion
levels following two cell interactions with a Natural Killer (NK)
cell as the subject cell and a leukemia cell (K562) as the target
cell in a system of the disclosure. The percent (%) of cell
subpopulation secreting is provided as a function of the type of
cytokine secreted by the NK cell in each pairing. The secretomes of
each NK cell were identified according to a method of the
disclosure. The arrows indicate statistically significant
differences between the levels of cytokine(s) identified in the
secretome of single NK cells in each condition of the two-cell
interaction method.
[0071] FIG. 3 is a series of graphs comparing cellular
polyfunctionality following two cell interactions with a Natural
Killer (NK) cell as the subject cell and a leukemia cell (K562) as
the target cell in a system of the disclosure. The data demonstrate
an increased percentage of poly-functional NK cells (secreting
2+cytokines) following an interaction with the leukemia cell
(right-hand graph).
[0072] FIG. 4 is a Process Analytical Tools (PAT)-Principal
Component Analysis (PCA) plot showing cellular polyfunctionality
following two cell interactions with a Natural Killer (NK) cell as
the subject cell and a leukemia cell (K562) as the target cell in a
system of the disclosure. Functional groups are color-coded by
population in which there is a dominant function.
Leukemia-activated NK cells lead to increased effector functional
cell subpopulations and reduced inflammatory cell
subpopulations.
[0073] FIG. 5 is a photograph depicting the isolation and
manipulation of human immune cell subsets in a system of the
disclosure. NK/K562 interactions isolated at the single cell level
are combined with their corresponding secretome (also referred to
as a secretion profile), enabling a comprehensive analysis of a
Target/ Effector relationship.
[0074] FIG. 6 is a pair of graphs depicting a cytokine and
cytotoxicity profile of CD8-positive T cells. The addition of the
bispecific antibody substantially increases both cytokine effector
function and cytotoxicity to the target Raji cells (right-hand
graph). The table above the graphs summarizes the data presented in
the graphs below.
[0075] FIG. 7A-C is a series of graphs and a key identifying the
components of the secretomes of CAR-expressing T-Cells following
stimulation by CD19 Beads. A. Shows the upregulation in cytokine
secretion by CAR T-cells stimulated with CD19 versus control beads.
In the up-regulation case, certain cytokines associated with
potency (TNF.alpha., Granzyme, etc.) as well as certain cytokines
associated with toxicity (IL-17A) are detailed. B. Key for the bar
graphs in A. Each bar color represents the inclusion of a cytokine
in a specific functional group. Effector cytokines include, but are
not limited to, Granzyme B, Interferon-gamma (IFN-.gamma.),
MIP-1.alpha., Perforin, TNF-.alpha., and TNF-.beta.. C. Stimulatory
cytokines include, but are not limited to, GM-CSF, Interleukin-12
(IL-12), Interleukin-15 (IL-15), Interleukin-2 (IL-2),
Interleukin-21 (IL-21), Interleukin-5 (IL-5), Interleukin-7 (IL-7),
Interleukin-8 (IL-8), and Interleukin-9 (IL-9). Chemoattractive
cytokines include, but are not limited to, CCL-11, IP-10,
MIP-1.beta., and RANTES. Regulatory cytokines include, but are not
limited to, Interleukin-10 (IL-10), Interleukin-13 (IL-13),
Interleukin-22 (IL-22), Interleukin-4 (IL-4), TGF-.beta.1, sCD137
and sCD40L. Inflammatory cytokines include, but are not limited to,
Interleukin-17A (IL-17A), Interleukin-17F (IL-17F), Interleukin-1b
(IL-1b), Interleukin-6 (IL-6), MCP-1, and MCP-4. Polyfunctionality
of single CAR T-cells after stimulation by control or CD19 beads.
Polyfunctionality is correlated with potency or success of immune
mediated therapies.
[0076] FIG. 8A-G is a series of graphs and a key identifying the
components of the secretomes of CAR-expressing T-Cells following
stimulation by target cells. A. Cytokine secretion levels by
CAR-expressing T-cells after stimulation with K562-CD19 target
cells, where CD19 is a proxy for the leukemia cells or B-cell
malignancies that are the cell therapy target. B. Dampened cytokine
secretion by CAR-expressing T-cells after stimulation with
K562-NGFR control cells (negative control cells because they do not
include the CD19 target). C. Cytokine secretion levels in chambers
(taking the form of micro-troughs for this example) containing a
single CAR-expressing T-cell and a single K562-CD19 target cell. D.
Dampened cytokine secretion in chambers containing a single
CAR-expressing T-cell and a single K562-NGFR control cell. E. Key
for the bar graphs in A-D. Each bar color represents the inclusion
of a cytokine in a specific functional group. Effector cytokines
include, but are not limited to, Granzyme B, Interferon-gamma
(IFN-.gamma.), MIP-1.alpha., Perforin, TNF-.alpha. and TNF-.beta..
C. Stimulatory cytokines include, but are not limited to, GM-CSF,
Interleukin-12 (IL-12), Interleukin-15 (IL-15), Interleukin-2
(IL-2), Interleukin-21 (IL-21), Interleukin-5 (IL-5), Interleukin-7
(IL-7), Interleukin-8 (IL-8), and Interleukin-9 (IL-9).
Chemoattractive cytokines include, but are not limited to, CCL-11,
IP-10, MIP-1.beta., and RANTES. Regulatory cytokines include, but
are not limited to, Interleukin-10 (IL-10), Interleukin-13 (IL-13),
Interleukin-22 (IL-22), Interleukin-4 (IL-4), TGF-.beta.1, sCD137
and sCD40L. Inflammatory cytokines include, but are not limited to,
Interleukin-17A (IL-17A), Interleukin-17F (IL-17F), Interleukin-1b
(IL-1b), Interleukin-6 (IL-6), MCP-1, and MCP-4. F.
Polyfunctionality of single CAR-expressing T cells in response to
K562-CD19 or control stimulation. G. An increase in
polyfunctionality when the K562-CD19 target cell is present in the
chamber (taking the form of a micro-trough for this example) with
the CAR-expressing T-cell.
[0077] FIG. 9 is a series of photographs depicting systems of the
disclosure and the use of fluorescent tracking of cytotoxicity.
K562 cells, labeled with the membrane dye CFSE, are mixed with
non-labeled NK cells and loaded into chambers (taking the form of
micro-troughs for this example) with an excess of SYTOX Orange. The
chambers (taking the form of micro-troughs for this example) are
imaged immediately after loading (TO) and then again after a 16
hour incubation (T1) and assessed for cell death via
fluorescence.
[0078] FIG. 10A-C is a series of signal plots of control and
stimulated CAR (chimeric antigen receptor expressing) cells. Panel
A. PSI (poly-functional strength intensity) Overview and Individual
Cytokine contributions from control CAR cells. Panel B. PSI
Overview and Individual Cytokine contributions from stimulated CAR
cells (stimulated cells were contacted to CD19-conjugated beads).
Panel C. Individual signal plots of both control and stimulated CAR
cells.
[0079] FIG. 11A-F is a series of graphs depicting signals detected
from CAR cells stimulated by target cells. PSI Overview from CAR
cells stimulated with K562-NFGR control cells (A) or K562-CD19 (B)
target cells. Individual Cytokine contributions from CAR cells
stimulated with K562-NGFR (C) and K562-CD19 (D). The final plots
show signal cell secretion points from CAR cells stimulated with
K562-NGFR (E) or K562-CD19 (F).
[0080] FIG. 12A-M is a series of graphs comparing results of
Memorial Sloan-Kettering Cancer Center (MSKCC) assays of cytokine
secretion to exemplary detection sensitivities of the compositions,
systems and methods of the disclosure (referred to as "IsoPlexis"
in the figure). In the study by MSKCC (Brenthens et al. Blood. 2011
Nov. 3; 118(18): 4817-4828), safety and persistence of adoptively
transferred autologous CD19-targeted T cells in patients with
relapsed or chemotherapy refractory B-cell leukemias, MSKCC
stimulated EOP 19-28z transduced T cells with 3T3 fibroblasts
expressing CD19 for 48 hours. MSKCC then analyzed the cytokine
secretion levels from the transduced cells on a Luminex IS100 to
determine pg/mL amounts of analyte. The compositions, systems and
methods of the disclosure can gather the same type of data as the
MSKCC assay with equal or greater sensitivity as the MSKCC assay.
The above side-by-side graphs show the sensitivity, per analyte, on
the compositions, systems and methods of the disclosure versus the
amounts measured from the MSKCC Luminex assay. The measurements
from the MSKCC assay clearly fall within the measurable range of
the compositions, systems and methods of the disclosure (a
concentration of cytokines between about 2 pg/ml and about 10,000
pg/ml, inclusive of the endpoints, per cytokine in a highly
multiplexed reaction). Therefore the same data could have been
gathered using the compositions, systems and methods of the
disclosure with the additional benefit of being able to transform
this data to represent the Polyfunctional Strength Index (PSI) of
individual cell populations. PSI measurements for each cytokine of
a plurality of cytokines for each cell in a large-scale experiment
(simultaneously measuring thousands of individual cells within a
population), enables a prediction of statistically powerful
biomarker and cell subsets that drive patient responses.
[0081] FIG. 13A-B is a pair of tables comparing results of National
Cancer Institute cytokine assays of secretions from CARs to
exemplary detection sensitivities of the compositions, systems and
methods of the disclosure (referred to as "IsoPlexis" in the
figure). In the study by NCI (Kochenderfer et al. Blood, 22 Mar.
2012, 119(12): 2709-2720), B-cell depletion and remissions of
malignancy along with cytokine-associated toxicity in a clinical
trial of anti-CD19 chimeric-antigen-receptor-transduced T cells,
the NCI cultured CD19 directed CAR cells with either CD19-positive
K562 cells or the control NGFR-positive (CD19-negative) overnight
and ran the cytokine secretions next day on a standard ELISA assay.
Cytokine secretion levels for IFN-gamma, TNF, and IL-2 are listed
in the top table. In the bottom table, are listed the limits of
detection on the compositions, systems, and methods of the
disclosure, with either a signal-to-noise ratio (SNR) of greater
than 2 or greater than 5. The compositions, systems, and methods of
the disclosure are able to measure cytokine secretions, with great
confidence, in the same range as those measured by standard ELISA
in this NCI study with the additional benefit of being able to
transform this data to represent the Polyfunctional Strength Index
(PSI) of individual cell populations. PSI measurements for each
cytokine of a plurality of cytokines for each cell in a large-scale
experiment (simultaneously measuring thousands of individual cells
within a population), enables a prediction of statistically
powerful biomarker and cell subsets that drive patient
responses.
[0082] FIG. 14A-C is a series of graphs comparing results of
National Cancer Institute cytokine assays of secretions from CARs
to exemplary detection sensitivities of the compositions, systems
and methods of the disclosure (referred to as "IsoPlexis" in the
figure). In the study by NCI (Zhao et al. The Journal of
Immunology, 2007, 179: 5845-5854), high-affinity TCRs generated by
phage display provide CD4+ cells with the ability to recognize and
kill tumor cell lines, the NCI measured the cytokine secretions
from TCR-transfected T cells. CD8+ and CD4+ T cells were purified
and transfected with the TCRs. The cells were then pulsed with
NY-ESO-1, MART-1 or gp100 peptides, and the resulting secretions of
IL-2 and IFN-gamma were measured using commercially available ELISA
kits at a range of between 200 pg/mL and 30,000 pg/mL, inclusive of
the endpoints. The compositions, systems and methods of the
disclosure are able to measure cytokine secretions, with great
confidence, in the same range as those measured by standard ELISA
in this NCI study with the additional benefit of being able to
transform this data to represent the Polyfunctional Strength Index
(PSI) of individual cell populations. PSI measurements for each
cytokine of a plurality of cytokines for each cell in a large-scale
experiment (simultaneously measuring thousands of individual cells
within a population), enables a prediction of statistically
powerful biomarker and cell subsets that drive patient
responses.
[0083] FIG. 15 is a series of graphs depicting a method of
measuring single-cell Polyfunctional Strength Index (PSI). This
metric quantifies the overall activity of a sample. The PSI is
equal to the percentage of polyfunctional cells (secreting two or
more cytokines) in a sample multiplied by the average signal
intensity of the secreted cytokines. In order to determine which
cytokines are driving the polyfunctional response, each individual
cytokine's contribution to PSI can also be calculated. This is
equivalent to the fraction of the total PSI coming from a specific
cytokine, and is found by calculating the percentage of each cell's
signal corresponding to that cytokine, averaging it across all
cells, and multiplying this percentage by the total PSI.
DETAILED DESCRIPTION
[0084] The disclosure provides systems and methods capable of
measuring key effector proteins at the single cell level. For
example, in certain embodiments, the systems and methods of the
disclosure can simultaneously measure 42 key effector proteins at
the single cell level. The systems and methods of the disclosure
may be used, for example, directly in pipeline drug development and
CAR-T assessment by large-scale developers of cell-based
therapeutics. The multiplexed parameters measured by the systems
and methods of the disclosure cover, for example, the complete
range of relevant immune effector functions including stimulatory,
proinflammatory, regulatory (negative), chemoattractive, pro-growth
and cytolytic (effective).
[0085] The systems and methods of the disclosure require smaller
amounts of cell input (approximately 1000 cells) compared to the
existing single-cell instruments (e.g., flow cytometer) for the
analysis of low quantity patient samples, which minimally 100,000
cells per sample for analysis (and typically require millions of
cells per sample).
[0086] The systems and method of the disclosure provide an
analytical approach capable of evaluating single cell secretion
profiles in a highly multiplex manner. In certain embodiments, this
analysis involves crosstalk of a single immune cell to a diseased
target cell (i.e. direct or indirect contact of a subject cell and
a target cell) while avoiding or minimizing paracrine effects from
the total population (because each pair of subject cell and target
cell may be isolated into a chamber separated from the remaining
plurality of chambers). This approach enables the study and
detection of rare subject to target cell interactions that may be
otherwise masked by other cells within the population or
sample.
[0087] The systems and methods of the disclosure are exemplified in
a nonlimiting manner through demonstrating the ability of a system
of the disclosure to evaluate individual cell-to-cell interactions
for the Target/Effector relationship of immune cells, including CAR
cells, with various target cells or stimulatory agents according to
a method of the disclosure. Highly multiplexed paired immune and
cancer cell assays provide mechanisms for assessing a
polyfunctional cytokine profile at the single cell level, in
response to a cancer cell and its specific antigens. Such assays
may further be used to evaluate the immune response quality in
relation to the magnitude of an immune cell response to a cancer
cell. Even more, such assays may be used to correlate the immune
response quality and/or the magnitude of an immune cell response to
a cancer cell with the ability of the immune cell to kill the
cancer cell. Accordingly, the systems and method of the disclosure
provide novel tools for addressing the critically relevant needs of
identifying and evaluating cellular therapies for safety and
efficacy in the field of cancer immunology, among many other
fields.
[0088] The systems and methods of the disclosure provide the
ability to analyze cells in a highly multiplexed fashion, down to
the single-cell level and are capable of determining the
polyfunctional response to a specific diseased cell. While an
exemplary method includes monitoring killing of a target tumor
cell, by an immune cell, within a chamber, for purposes of
correlating with a highly multiplexed immune response, the systems
and methods of the disclosure include many other applications.
[0089] As shown in the Figures of the disclosure, and in
particular, FIGS. 7, 8, 10, and 11, polyfunctionality is a measure
of efficacy and potency of cells intended for cellular therapy. Of
particular value is the polyfunctional strength index (PSI). The
polyfunctional strength index (PSI) is a metric that factors in the
polyfunctionality of cells in a sample, and the signal intensity of
the cytokines secreted by each cell. It is found by multiplying the
percentage of polyfunctional cells of a sample (single cells
secreting two or more cytokines), by the average signal intensity
of these cytokines. This PSI is shown on the left of FIGS. 10A and
10B, as well as FIGS. 11A and 11B. For example, FIG. 10B
demonstrates that the polyfunctional strength is roughly 2x higher
in the CD19 stimulated sample, relative to the control PSI shown in
FIG. 10A. FIG. 11B demonstrates that the stimulated cells have a
polyfunctional strength roughly 5.times. higher than the control
cells. For more detail regarding PSI, see Example 7.
Methods of Analyzing Immune Cells
[0090] Secreted proteins and in particular cytokines, are key
mediators of intercellular communication within the immune system.
Homeostatic immune response requires tightly regulated cytokine
synthesis and secretion. Many analytical technologies have been
developed to analyze protein secretion during the immune response,
but the methods used are generally restricted to measuring the
average secretions for an entire cellular population. Such
analyses, while helpful in understanding disease pathogenesis and
the immune process, are insufficient to characterize cytokine
activity for individual subsets of cells within a population.
Recent investigations using single-cell analysis have shown that
immune cells display highly heterogeneous cytokine profile even in
cells with similar phenotypes further demonstrating a significant
limitation of focusing only on cellular response at the bulk
population level. These heterogeneous subsets of cells within the
population may dictate a complex signaling interplay between cells
that represent important checks and balances for disease
immunotherapy evaluation. This is particularly notable when a
cellular population's response can be determined by the cell-cell
interactions in a rare subset of cells. As result, understanding
these interactions is crucial to developing more effective
therapeutic treatments in the future.
[0091] Challenges to defining consistent and high functional
quality "drug" in cell-based cancer immunotherapy: Despite the
demonstrated benefit of emerging CAR-T cancer immunotherapeutics,
two major concerns remain: one, manufacturing the cell therapies
consistently, and two, managing the immuno-toxicity, such as
cytokine release syndrome, that could be potentially life
threatening. In cell-based therapies such as Chimeric Antigen
Receptor T cell (CAR-T) therapy, in which the living cells are the
"drug", cellular manufacturing is still relatively new, and each
patient batch generated may differ substantially even if a
standardized operation procedure (SOP) exists to ensure consistency
in manufacturing. Giving clinicians and biotech companies an
effective cellular function monitoring tool could change clinical
paradigms by allowing them to remove or modify the inconsistent or
unsafe cell therapies prior to injection, significantly reducing
risk to the patient, and improving odds of therapeutic success.
[0092] Defining the "quality" of a cytokine mediated anti-tumor T
cell: To evaluate engineered T cells for an immunotherapy or to
evaluate endogenous T cells reactivated to battle cancer or
infection, a T cell's functional status is largely determined by a
spectrum of secreted effector function proteins (e.g., cytokines).
In a protective immune response, the `quality` of an immune cell
correlates to the extent of polyfunctionality (the ability of a T
cell to co-secrete multiple effector proteins).While these
anti-tumor cytolytic, chemoattractive cytokines produce an
effective response, these poly-functional cell subsets must not
also secrete immuno-toxic inflammatory or regulatory cytokines (up
to 15) prevalent in NK or CAR-T cells. To detect consistent
performance of this "quality" effective and safe response of CAR
single-cell subsets, a need exists for a new technology to conduct
highly multiplexed measurement of immune effector proteins in
single T cells. And while single-cell flow cytometry technologies
fix cells to detect cytokines, a more predictive platform would
detect true secretions.
[0093] Current methods of cell function evaluation: Single
parameter ELISpot assay remains the state-of-the-art for T cell
activation assay, but does not measure polyfunctionality. Averaging
bead based multiplexing platform (Luminex based) measure many
cytokines, yet not at the required single-cell level.
Fluorescence-based multicolor flow cytometry is a powerful single
cell analysis tool and has been used to detect cytokines via
retaining and staining proteins within the cytoplasm by blocking
vesicle transport. The number of proteins that can be
simultaneously measured is limited by fluorescence spectral overlap
with ICS (intracellular cytokine staining). Time-of-flight mass
spectrometer-coupled flow cytometry (CyTOF) has been used recently
by CAR-T companies, though not as regularly in trials. Similar to
fluorescence flow cytometry, it does not measure true secretion and
so far the number of cytokines co-measured in single cells by CyTOF
is 11 due in part to the high background of ICS. Other single cell
technologies being developed in research laboratories (e.g.,
microengraving) provides advantages in sensitivity and assay speed
but still limited in the multiplexing capability (typically
<5).
[0094] Risks involved in adoptive cell therapies and other immune
mediated therapies: Cytokine release syndrome (CRS) is a
non-antigen specific, life-threatening toxicity that results from
the over-activation of the immune system due to immune therapies,
such as CAR T-cell therapy. Although CAR T-cells are potent
on-target killers, they activate the immune system far above
naturally occurring levels and due to the nature of their design,
have a large degree of "on target, off tumor" toxicity. Based on
the level of mortalities in recent clinical trials, it has become
apparent that the cytokine profiles of individual CAR-T cells must
be known before introduction to the patient. The systems and
methods of the disclosure determine an abundance of up to 42
cytokines, per single cell, falling into the following groups:
effector, stimulatory, inflammatory, and regulatory. This
information allows the user to identify any potentially toxic
subsets of cells (pro-inflammatory or regulatory) within a
population that would have been missed by conventional means,
providing a safer and more effective means of monitoring
immunotherapies prior to patient introduction.
Systems and Arrays
[0095] The disclosure provides a system for the multiplexed
detection of a plurality of compounds from single cells comprising
an array comprising a plurality of chambers and a panel of capture
agents. Preferred capture agents include antibodies, however,
capture agents may include any detectable entity that specifically
binds to a component of a secretome of the disclosure. The
detectable entity may comprise a detectable label, for example.
Detectable labels may include, but are not limited to fluorescent
labels.
[0096] Systems of the disclosure comprise a plurality of individual
chambers, preferably in uniform arrangement. In certain
embodiments, at least some of the plurality of individual chambers
have a length of greater than 50 .mu.m and, optionally, may be
configured to contain an isolated single cell in a sub-nanoliter
volume of contents.
[0097] Capture agent panels of the disclosure may comprise a
plurality of immobilized capture agents, each immobilized capture
agent capable of specifically binding to one of the plurality of
components of a secretome of the disclosure. Preferably, the
immobilized capture agents are arranged in uniform capture agent
panels. Preferably, the immobilized capture agents are attached to
a surface in a repeatable pattern, wherein each repeat of the
pattern aligns with a chamber of the plurality of chambers.
[0098] The array and capture agent panels are coupled to form a
plurality of enclosed interfaces, each enclosed interface
comprising a chamber and a capture agent panel such that the
contents of each chamber are accessible to each and every capture
agent of the capture agent panel.
[0099] Chambers of the array may take on any shape and may have any
dimension, however, in certain embodiments of the disclosure, the
array comprises at least 1, 2, 5, 10, 15, 20, 25, 50, 100, 150,
500, 1000, 1500, 2000 or any integer between of chambers. Each
chamber may have a depth/height of between 1 .mu.m and 2000 .mu.m,
a diameter of between 1 .mu.m and 2000 .mu.m, a width of between 1
.mu.m and 2000 .mu.m and/or a length of between 1 .mu.m and 2000
.mu.m. The distance between any two chambers of the array may be
between 1 .mu.m and 2000 .mu.m.
[0100] In certain embodiments, at least one chamber is a high
aspect ratio rectangular well, having dimensions of about 1-2 mm in
length and about 5-50 .mu.m in depth.
[0101] In certain embodiments, each chamber is rectangular with a
length of about 10-2000 .mu.m, a width of about 10-100 .mu.m, and a
depth of about 10-100 .mu.m.
[0102] In certain embodiments, the capture agent panel may comprise
between 3 and 50 different capture agents, thereby allowing for the
detection of between 3 and 50 different components of a secretome.
In certain embodiments, the capture agent panel may comprise
greater than 3 different capture agents, thereby allowing for the
detection of greater than 3 different components of a secretome. In
certain embodiments, the capture agent panel may comprise greater
than 10 different capture agents, thereby allowing for the
detection of greater than 10 different components of a secretome.
In certain embodiments, the capture agent panel may comprise
greater than 42 different capture agents, thereby allowing for the
detection of greater than 42 different components of a
secretome.
[0103] In certain embodiments, the array comprises a chamber
density of about 200 microwells per cm.sup.2 to about 20,000
microchambers per cm.sup.2.
Definitions
[0104] Unless otherwise defined, scientific and technical terms
used in connection with the disclosure shall have the meanings that
are commonly understood by those of ordinary skill in the art.
Further, unless otherwise required by context, singular terms shall
include pluralities and plural terms shall include the singular.
Generally, nomenclatures utilized in connection with, and
techniques of, cell and tissue culture, molecular biology, and
protein and oligo- or polynucleotide chemistry and hybridization
described herein are those well-known and commonly used in the art.
Standard techniques are used for recombinant DNA, oligonucleotide
synthesis, and tissue culture and transformation (e.g.,
electroporation, lipofection). Enzymatic reactions and purification
techniques are performed according to manufacturer's specifications
or as commonly accomplished in the art or as described herein. The
practice of the present invention will employ, unless indicated
specifically to the contrary, conventional methods of virology,
immunology, microbiology, molecular biology and recombinant DNA
techniques within the skill of the art, many of which are described
below for the purpose of illustration. Such techniques are
explained fully in the literature. See, e.g., Sambrook, et al.
Molecular Cloning: A Laboratory Manual (2nd Edition, 1989);
Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA
Cloning: A Practical Approach, vol. I & II (D. Glover, ed.);
Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid
Hybridization (B. Hames & S. Higgins, eds., 1985);
Transcription and Translation (B. Hames & S. Higgins, eds.,
1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A
Practical Guide to Molecular Cloning (1984).
[0105] The following definitions are useful in understanding the
present invention:
[0106] The term "antibody" (Ab) as used herein includes monoclonal
antibodies, polyclonal antibodies, multispecific antibodies (e.g.,
bispecific antibodies), and antibody fragments, as long as they
exhibit the desired biological activity. The term "immunoglobulin"
(Ig) is used interchangeably with "antibody" herein.
[0107] An "isolated antibody" is one that has been separated and/or
recovered from a component of its natural environment. Contaminant
components of its natural environment are materials that would
interfere with diagnostic or therapeutic uses for the antibody, and
may include enzymes, hormones, and other proteinaceous or
nonproteinaceous solutes. In preferred embodiments, the antibody is
purified: (1) to greater than 95% by weight of antibody as
determined by the Lowry method, and most preferably more than 99%
by weight; (2) to a degree sufficient to obtain at least 15
residues of N-terminal or internal amino acid sequence by use of a
spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under
reducing or non-reducing conditions using Coomassie blue or,
preferably, silver stain. Isolated antibody includes the antibody
in situ within recombinant cells since at least one component of
the antibody's natural environment will not be present. Ordinarily,
however, isolated antibody will be prepared by at least one
purification step.
[0108] Capture agents of the disclosure may comprise one or more
monoclonal antibodies. The term "monoclonal antibody" as used
herein refers to an antibody obtained from a population of
substantially homogeneous antibodies, i.e., the individual
antibodies comprising the population are identical except for
possible naturally occurring mutations that may be present in minor
amounts. Monoclonal antibodies are highly specific, being directed
against a single antigenic site. Furthermore, in contrast to
polyclonal antibody preparations that include different antibodies
directed against different determinants (epitopes), each monoclonal
antibody is directed against a single determinant on the antigen.
In addition to their specificity, the monoclonal antibodies are
advantageous in that they may be synthesized uncontaminated by
other antibodies.
[0109] Monoclonal antibodies contemplated herein include "chimeric"
antibodies in which a portion of the heavy and/or light chain is
identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological
activity. Chimeric antibodies of primary interest herein include
antibodies having one or more human antigen binding sequences
(e.g., CDRs) and containing one or more sequences derived from a
non-human antibody, e.g., an FR or C region sequence. In addition,
chimeric antibodies of primary interest herein include those
comprising a human variable domain antigen binding sequence of one
antibody class or subclass and another sequence, e.g., FR or C
region sequence, derived from another antibody class or subclass.
Chimeric antibodies of interest herein also include those
containing variable domain antigen-binding sequences related to
those described herein or derived from a different species, such as
a non-human primate (e.g., Old World Monkey, Ape, etc). Chimeric
antibodies also include primatized and humanized antibodies.
[0110] Capture agents of the disclosure may comprise humanized
antibodies. A "humanized antibody" is generally considered to be a
human antibody that has one or more amino acid residues introduced
into it from a source that is non-human. These non-human amino acid
residues are often referred to as "import" residues, which are
typically taken from an "import" variable domain. Humanization is
traditionally performed by substituting import hypervariable region
sequences for the corresponding sequences of a human antibody.
Accordingly, such "humanized" antibodies are chimeric antibodies
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species.
[0111] A "human antibody" is an antibody containing only sequences
present in an antibody naturally produced by a human. However, as
used herein, human antibodies may comprise residues or
modifications not found in a naturally occurring human antibody,
including those modifications and variant sequences described
herein. These are typically made to further refine or enhance
antibody performance.
[0112] Capture agents of the disclosure may comprise intact
antibodies. An "intact" antibody is one that comprises an
antigen-binding site as well as a CL and at least heavy chain
constant domains, CH 1, CH 2 and CH 3. The constant domains may be
native sequence constant domains (e.g., human native sequence
constant domains) or amino acid sequence variant thereof.
Preferably, the intact antibody has one or more effector
functions.
[0113] Capture agents of the disclosure may comprise an antibody
fragment. An "antibody fragment" comprises a portion of an intact
antibody, preferably the antigen binding or variable region of the
intact antibody. Examples of antibody fragments include Fab, Fab',
F(ab')2, and Fv fragments; diabodies; linear antibodies;
single-chain antibody molecules; and multispecific antibodies
formed from antibody fragments.
[0114] Capture agents of the disclosure may comprise a functional
fragment or an analog of an antibody. The phrase "functional
fragment or analog" of an antibody is a compound having qualitative
biological activity in common with a full-length antibody. For
example, a functional fragment or analog of an anti-IgE antibody is
one that can bind to an IgE immunoglobulin in such a manner so as
to prevent or substantially reduce the ability of such molecule
from having the ability to bind to the high affinity receptor, Fc
RI.
[0115] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, and a residual
"Fc" fragment, a designation reflecting the ability to crystallize
readily. The Fab fragment consists of an entire L chain along with
the variable region domain of the H chain (VH), and the first
constant domain of one heavy chain (CH 1). Each Fab fragment is
monovalent with respect to antigen binding, i.e., it has a single
antigen-binding site. Pepsin treatment of an antibody yields a
single large F(ab')2 fragment that roughly corresponds to two
disulfide linked Fab fragments having divalent antigen-binding
activity and is still capable of cross-linking antigen. Fab'
fragments differ from Fab fragments by having additional few
residues at the carboxy terminus of the CH1 domain including one or
more cysteines from the antibody hinge region. Fab'-SH is the
designation herein for Fab' in which the cysteine residue(s) of the
constant domains bear a free thiol group. F(ab')2 antibody
fragments originally were produced as pairs of Fab' fragments that
have hinge cysteines between them. Other chemical couplings of
antibody fragments are also known.
[0116] The "Fc" fragment comprises the carboxy-terminal portions of
both H chains held together by disulfides. The effector functions
of antibodies are determined by sequences in the Fc region, which
region is also the part recognized by Fc receptors (FcR) found on
certain types of cells.
[0117] "Fv" is the minimum antibody fragment that contains a
complete antigen-recognition and -binding site. This fragment
consists of a dimer of one heavy- and one light-chain variable
region domain in tight, non-covalent association. From the folding
of these two domains emanate six hypervariable loops (three loops
each from the H and L chain) that contribute the amino acid
residues for antigen binding and confer antigen binding specificity
to the antibody. However, even a single variable domain (or half of
an Fv comprising only three CDRs specific for an antigen) has the
ability to recognize and bind antigen, although at a lower affinity
than the entire binding site.
[0118] Capture agents of the disclosure may comprise single-chain
antibodies (also referred to as scFv). "Single-chain Fv" also
abbreviated as "sFv" or "scFv" are antibody fragments that comprise
the VH and VL antibody domains connected into a single polypeptide
chain. Preferably, the sFv polypeptide further comprises a
polypeptide linker between the VH and VL domains that enables the
sFv to form the desired structure for antigen binding. For a review
of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies,
vol. 113, Rosenburg and Moore eds., Springer-Verlag, N.Y., pp.
269-315 (1994); Borrebaeck 1995, infra.
[0119] Capture agents of the disclosure may comprise diabodies. The
term "diabodies" refers to small antibody fragments prepared by
constructing sFv fragments (see preceding paragraph) with short
linkers (about 5-10 residues) between the VH and VL domains such
that inter-chain but not intra-chain pairing of the V domains is
achieved, resulting in a bivalent fragment, i.e., fragment having
two antigen-binding sites. Bispecific diabodies are heterodimers of
two "crossover" sFv fragments in which the VH and VL domains of the
two antibodies are present on different polypeptide chains.
Diabodies are described more fully in, for example, EP 404,097; WO
93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA,
90:6444-6448 (1993).
[0120] Capture agents of the disclosure may comprise bispecific
antibodies. In certain embodiments, antibodies of the present
invention are bispecific or multi-specific. Bispecific antibodies
are antibodies that have binding specificities for at least two
different epitopes. Exemplary bispecific antibodies may bind to two
different epitopes of a single antigen. Other such antibodies may
combine a first antigen binding site with a binding site for a
second antigen.
[0121] Methods for making bispecific antibodies are known in the
art. Traditional production of full length bispecific antibodies is
based on the co-expression of two immunoglobulin heavy chain-light
chain pairs, where the two chains have different specificities.
Because of the random assortment of immunoglobulin heavy and light
chains, these hybridomas (quadromas) produce a potential mixture of
ten different antibody molecules, of which only one has the correct
bispecific structure. Purification of the correct molecule, which
is usually done by affinity chromatography steps, is rather
cumbersome, and the product yields are low.
[0122] According to a different approach, antibody variable domains
with the desired binding specificities (antibody-antigen combining
sites) are fused to immunoglobulin constant domain sequences.
Preferably, the fusion is with an Ig heavy chain constant domain,
comprising at least part of the hinge, C.sub.H2, and C.sub.H3
regions. It is preferred to have the first heavy-chain constant
region (C.sub.H1) containing the site necessary for light chain
bonding, present in at least one of the fusions. DNAs encoding the
immunoglobulin heavy chain fusions and, if desired, the
immunoglobulin light chain, are inserted into separate expression
vectors, and are co-transfected into a suitable host cell. This
provides for greater flexibility in adjusting the mutual
proportions of the three polypeptide fragments in embodiments when
unequal ratios of the three polypeptide chains used in the
construction provide the optimum yield of the desired bispecific
antibody. It is, however, possible to insert the coding sequences
for two or all three polypeptide chains into a single expression
vector when the expression of at least two polypeptide chains in
equal ratios results in high yields or when the ratios have no
significant effect on the yield of the desired chain
combination.
[0123] As used herein, an antibody is said to be "immunospecific,"
"specific for" or to "specifically bind" an antigen if it reacts at
a detectable level with the antigen, preferably with an affinity
constant, K.sub.a, of greater than or equal to about 10.sup.4
M.sup.-1, or greater than or equal to about 10.sup.5 M.sup.-1,
greater than or equal to about 10.sup.6 M.sup.-1, greater than or
equal to about 10.sup.7 M.sup.-1, or greater than or equal to
10.sup.8 M.sup.-1. Affinity of an antibody for its cognate antigen
is also commonly expressed as a dissociation constant KD, and in
certain embodiments, an antibody specifically binds to a component
of a secretome if it binds with a KD of less than or equal to
10.sup.-1 M, less than or equal to about 10.sup.-5 M, less than or
equal to about 10.sup.-6 M, less than or equal to 10.sup.-7 M, or
less than or equal to 10.sup.-8 M. Affinities of antibodies can be
readily determined using conventional techniques, for example,
those described by Scatchard et al. (Ann. N.Y. Acad. Sci. USA
51:660 (1949)).
[0124] Subject and target cells of the disclosure may be isolated,
derived, or prepared from any species, including any mammal. A
"mammal" for purposes of treating n infection, refers to any
mammal, including humans, domestic and farm animals, and zoo,
sports, or pet animals, such as dogs, cats, cattle, horses, sheep,
pigs, goats, rabbits, etc. Preferably, the mammal is human.
[0125] Subject cells of the disclosure may be used in a cellular
therapy for the treatment of a disease or disorder. "Treating" or
"treatment" or "alleviation" refers to both therapeutic treatment
and prophylactic or preventative measures; wherein the object is to
prevent or slow down (lessen) the targeted pathologic condition or
disorder. Those in need of treatment include those already with the
disorder as well as those prone to have the disorder or those in
whom the disorder is to be prevented. A subject or mammal may be
successfully "treated" when, after receiving a cellular therapy
with a subject cell of the disclosure, the patient shows observable
and/or measurable reduction in or absence of one or more of the
following: reduction in one or more of the symptoms associated with
disease or disorder; reduced morbidity and mortality, and
improvement in quality of life issues. The above parameters for
assessing successful treatment and improvement in the disease are
readily measurable by routine procedures familiar to a physician.
Methods of the disclosure may be used to determine the safety
and/or efficacy of a cellular therapy before, during or after
initiation of treatment of the subject.
[0126] Capture agents of the disclosure may be labeled to render
them detectable using one or more means. "Label" as used herein
refers to a detectable compound or composition that is conjugated
directly or indirectly to the capture agent (e.g. an antibody) so
as to generate a "labeled" capture agent (e.g. an antibody). The
label may be detectable by itself (e.g., a fluorescent label) or,
in the case of an enzymatic label, may catalyze chemical alteration
of a substrate compound or composition that is detectable.
[0127] Capture agents of the disclosure may selectively or
specifically identify, capture, and/or quantify one or more small
molecules in a secretome. A "small molecule" is defined herein to
have a molecular weight below about 500 Daltons.
[0128] Capture agents of the disclosure may include nucleic acids
or labeled nucleic acids. The terms "nucleic acid" and
"polynucleotide" are used interchangeably herein to refer to
single- or double-stranded RNA, DNA, or mixed polymers.
Polynucleotides may include genomic sequences, extra-genomic and
plasmid sequences, and smaller engineered gene segments that
express, or may be adapted to express polypeptides.
[0129] An "isolated nucleic acid" is a nucleic acid that is
substantially separated from other genome DNA sequences as well as
proteins or complexes such as ribosomes and polymerases, which
naturally accompany a native sequence. The term embraces a nucleic
acid sequence that has been removed from its naturally occurring
environment, and includes recombinant or cloned DNA isolates and
chemically synthesized analogues or analogues biologically
synthesized by heterologous systems. A substantially pure nucleic
acid includes isolated forms of the nucleic acid. Of course, this
refers to the nucleic acid as originally isolated and does not
exclude genes or sequences later added to the isolated nucleic acid
by the hand of man.
[0130] The term "polypeptide" is used in its conventional meaning,
i.e., as a sequence of amino acids. The polypeptides are not
limited to a specific length of the product. Peptides,
oligopeptides, and proteins are included within the definition of
polypeptide, and such terms may be used interchangeably herein
unless specifically indicated otherwise. This term also does not
refer to or exclude post-expression modifications of the
polypeptide, for example, glycosylations, acetylations,
phosphorylations and the like, as well as other modifications known
in the art, both naturally occurring and non-naturally occurring. A
polypeptide may be an entire protein, or a subsequence thereof.
[0131] An "isolated polypeptide" is one that has been identified
and separated and/or recovered from a component of its natural
environment. In preferred embodiments, the isolated polypeptide
will be purified (1) to greater than 95% by weight of polypeptide
as determined by the Lowry method, and most preferably more than
99% by weight, (2) to a degree sufficient to obtain at least 15
residues of N-terminal or internal amino acid sequence by use of a
spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under
reducing or non-reducing conditions using Coomassie blue or,
preferably, silver stain. Isolated polypeptide includes the
polypeptide in situ within recombinant cells since at least one
component of the polypeptide's natural environment will not be
present. Ordinarily, however, isolated polypeptide will be prepared
by at least one purification step.
[0132] A "native sequence" polynucleotide is one that has the same
nucleotide sequence as a polynucleotide derived from nature. A
"native sequence" polypeptide is one that has the same amino acid
sequence as a polypeptide (e.g., antibody) derived from nature
(e.g., from any species). Such native sequence polynucleotides and
polypeptides can be isolated from nature or can be produced by
recombinant or synthetic means.
[0133] A polynucleotide "variant," as the term is used herein, is a
polynucleotide that typically differs from a polynucleotide
specifically disclosed herein in one or more substitutions,
deletions, additions and/or insertions. Such variants may be
naturally occurring or may be synthetically generated, for example,
by modifying one or more of the polynucleotide sequences of the
invention and evaluating one or more biological activities of the
encoded polypeptide as described herein and/or using any of a
number of techniques well known in the art.
[0134] A polypeptide "variant," as the term is used herein, is a
polypeptide that typically differs from a polypeptide specifically
disclosed herein in one or more substitutions, deletions, additions
and/or insertions. Such variants may be naturally occurring or may
be synthetically generated, for example, by modifying one or more
of the above polypeptide sequences of the invention and evaluating
one or more biological activities of the polypeptide as described
herein and/or using any of a number of techniques well known in the
art.
[0135] Modifications may be made in the structure of the
polynucleotides and polypeptides of the disclosure and still obtain
a functional molecule that encodes a variant or derivative
polypeptide with desirable characteristics. When it is desired to
alter the amino acid sequence of a polypeptide to create an
equivalent, or even an improved, variant or portion of a
polypeptide of the invention, one skilled in the art will typically
change one or more of the codons of the encoding DNA sequence.
[0136] For example, certain amino acids may be substituted for
other amino acids in a protein structure without appreciable loss
of its ability to bind other polypeptides (e.g., antigens) or
cells. Since it is the binding capacity and nature of a protein
that defines that protein's biological functional activity, certain
amino acid sequence substitutions can be made in a protein
sequence, and, of course, its underlying DNA coding sequence, and
nevertheless obtain a protein with like properties. It is thus
contemplated that various changes may be made in the peptide
sequences of the disclosed compositions, or corresponding DNA
sequences that encode said peptides without appreciable loss of
their biological utility or activity.
[0137] In many instances, a polypeptide variant will contain one or
more conservative substitutions. A "conservative substitution" is
one in which an amino acid is substituted for another amino acid
that has similar properties, such that one skilled in the art of
peptide chemistry would expect the secondary structure and
hydropathic nature of the polypeptide to be substantially
unchanged.
[0138] In making such changes, the hydropathic index of amino acids
may be considered. The importance of the hydropathic amino acid
index in conferring interactive biologic function on a protein is
generally understood in the art (Kyte and Doolittle, 1982). It is
accepted that the relative hydropathic character of the amino acid
contributes to the secondary structure of the resultant protein,
which in turn defines the interaction of the protein with other
molecules, for example, enzymes, substrates, receptors, DNA,
antibodies, antigens, and the like. Each amino acid has been
assigned a hydropathic index on the basis of its hydrophobicity and
charge characteristics (Kyte and Doolittle, 1982). These values
are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);
phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);
alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8);
tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine
(-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5);
asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
[0139] Certain amino acids may be substituted by other amino acids
having a similar hydropathic index or score and still result in a
protein with similar biological activity, i.e. still obtain a
biological functionally equivalent protein. In making such changes,
the substitution of amino acids whose hydropathic indices are
within .+-.2 is preferred, those within .+-.1 are particularly
preferred, and those within .+-.0.5 are even more particularly
preferred. The substitution of like amino acids can be made
effectively on the basis of hydrophilicity. The greatest local
average hydrophilicity of a protein, as governed by the
hydrophilicity of its adjacent amino acids, correlates with a
biological property of the protein.
[0140] The following hydrophilicity values have been assigned to
amino acid residues: arginine (+3.0); lysine (+3.0); aspartate
(+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3); asparagine
(+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline
(-0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0);
methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine
(-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
It is understood that an amino acid can be substituted for another
having a similar hydrophilicity value and still obtain a
biologically equivalent, and in particular, an immunologically
equivalent protein. In such changes, the substitution of amino
acids whose hydrophilicity values are within .+-.2 is preferred,
those within .+-.1 are particularly preferred, and those within
.+-.0.5 are even more particularly preferred.
[0141] As outlined above, amino acid substitutions are generally
therefore based on the relative similarity of the amino acid
side-chain substituents, for example, their hydrophobicity,
hydrophilicity, charge, size, and the like. Exemplary substitutions
that take various of the foregoing characteristics into
consideration are well known to those of skill in the art and
include: arginine and lysine; glutamate and aspartate; serine and
threonine; glutamine and asparagine; and valine, leucine and
isoleucine.
[0142] Amino acid substitutions may further be made on the basis of
similarity in polarity, charge, solubility, hydrophobicity,
hydrophilicity and/or the amphipathic nature of the residues. For
example, negatively charged amino acids include aspartic acid and
glutamic acid; positively charged amino acids include lysine and
arginine; and amino acids with uncharged polar head groups having
similar hydrophilicity values include leucine, isoleucine and
valine; glycine and alanine; asparagine and glutamine; and serine,
threonine, phenylalanine and tyrosine. Other groups of amino acids
that may represent conservative changes include: (1) ala, pro, gly,
glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile,
leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.
A variant may also, or alternatively, contain nonconservative
changes. In a preferred embodiment, variant polypeptides differ
from a native sequence by substitution, deletion or addition of
five amino acids or fewer. Variants may also (or alternatively) be
modified by, for example, the deletion or addition of amino acids
that have minimal influence on the immunogenicity, secondary
structure and hydropathic nature of the polypeptide.
[0143] When comparing polynucleotide and polypeptide sequences, two
sequences are said to be "identical" if the sequence of nucleotides
or amino acids in the two sequences is the same when aligned for
maximum correspondence, as described below. Comparisons between two
sequences are typically performed by comparing the sequences over a
comparison window to identify and compare local regions of sequence
similarity. A "comparison window" as used herein, refers to a
segment of at least about 20 contiguous positions, usually 30 to
about 75, 40 to about 50, in which a sequence may be compared to a
reference sequence of the same number of contiguous positions after
the two sequences are optimally aligned.
[0144] Optimal alignment of sequences for comparison may be
conducted using the Megalign program in the Lasergene suite of
bioinformatics software (DNASTAR, Inc., Madison, Wis.), using
default parameters. This program embodies several alignment schemes
described in the following references: Dayhoff, Mo. (1978) A model
of evolutionary change in proteins--Matrices for detecting distant
relationships. In Dayhoff, Mo. (ed.) Atlas of Protein Sequence and
Structure, National Biomedical Research Foundation, Washington D.C.
Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to
Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol.
183, Academic Press, Inc., San Diego, Calif.; Higgins, D. G. and
Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W. and Muller W.
(1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor 11:105;
Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P. H.
A. and Sokal, R. R. (1973) Numerical Taxonomy--the Principles and
Practice of Numerical Taxonomy, Freeman Press, San Francisco,
Calif.; Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad.,
Sci. USA 80:726-730.
[0145] Alternatively, optimal alignment of sequences for comparison
may be conducted by the local identity algorithm of Smith and
Waterman (1981) Add. APL. Math 2:482, by the identity alignment
algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by
the search for similarity methods of Pearson and Lipman (1988)
Proc. Natl. Acad. Sci. USA 85: 2444, by computerized
implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA,
and TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by
inspection.
[0146] One preferred example of algorithms that are suitable for
determining percent sequence identity and sequence similarity are
the BLAST and BLAST 2.0 algorithms, which are described in Altschul
et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al.
(1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0
can be used, for example with the parameters described herein, to
determine percent sequence identity for the polynucleotides and
polypeptides of the invention. Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information.
[0147] "Homology" refers to the percentage of residues in the
polynucleotide or polypeptide sequence variant that are identical
to the non-variant sequence after aligning the sequences and
introducing gaps, if necessary, to achieve the maximum percent
homology. In particular embodiments, polynucleotide and polypeptide
variants have at least 70%, at least 75%, at least 80%, at least
90%, at least 95%, at least 98%, or at least 99% polynucleotide or
polypeptide homology with a polynucleotide or polypeptide described
herein.
[0148] As used in this specification and the appended claims, the
singular forms "a," "an" and "the" include plural references unless
the content clearly dictates otherwise.
EXAMPLES
[0149] The methods detailed below are capable of analyzing distinct
single subject cell to single target cell interactions on a system
of the disclosure (also referred to below as an array). Cell
populations may be cultured separately and briefly mixed before
being loading onto the array. The array may comprise a plurality of
chambers (which, for these examples, may take the form of
micro-troughs) that are enclosed by surface (e.g. a glass slide or
other cover material) creating isolated microenvironments for high
throughput cellular studies such as paired cell interactions. The
surface enclosing each chamber of the array may have attached
thereto one or more detection agents (including, but not limited to
antibodies). The detection agents (e.g. antibodies in the Examples
provided below) may be attached to the surface in a repeated
pattern such that each chamber comprises one repeat of the pattern
on the portion of the surface enclosing the chamber. In this
circumstance, each repeat of the pattern comprises at least one of
every individual detection agent (e.g. antibody specific for a
particular cytokine). The surface may have attached thereto between
20-1000 detection agents that are each specific for a distinct
component of a subject cell's secretome. The systems of the
disclosure include a plurality of chambers. The plurality of
chambers may include, for example, between 2 and 2000 chambers.
Example 1: Analysis of Natural Killer (NK) Cell and Target Cell
[0150] Cell Culture. Isolated peripheral blood mononuclear cells
(PBMCs), stored in-house at a concentration of 2.5.times.10.sup.7
cells/vial, were thawed and cultured overnight in a T25 cell
culture flask with 5 mL of X-Vivo 15 without Gentamicin or phenol
red (Lonza, 04-744Q) supplemented with 1.times. GlutaMAX
(ThermoFisher, 35050061), 1 mM sodium pyruvate (ThermoFisher,
11360-070), 1.times. MEM Vitamin Solution (Gibco; ThermoFisher,
11120-052), 20 mM HEPES (Gibco; ThermoFisher, 15630-080), 2% Human
AB serum (Valley Biomedical, HP1022HI), 1.times.
Penicillin-Streptomycin-Neomycin (Sigma-Aldrich, P4083), 100 ng/mL
recombinant IL-18 (R&D Systems, B001-5) and 10 ng/mL IL-12
(R&D Systems, 219-IL-005). After an overnight recovery, NK
cells were isolated using the Miltenyi NK Cell Isolation Kit
according to the manufacturer's instructions. NK cells were then
cultured at 37.degree. C., 5% CO.sub.2 in supplemented X-Vivo 15
media at a concentration of 1.times.10.sup.6 cells/mL until use. A
human K562 Cell line (ATCC CCL243) was purchased from the American
Type Culture Collection (ATCC) and cultured in Corning.TM.
cellgro.TM. RPMI 1640 Medium (Mod.) 1.times. with L-Glutamine
(Corning; ThermoFisher MT10040CV) supplemented with 10% FBS
(Sigma-Aldrich, F2442), 1.times. Penicillin-Streptomycin-Neomycin
(Sigma-Aldrich, P4083) and 1.times. GlutaMAX supplement
(ThermoFisher, 35050061).
[0151] Single-Cell Secretome Assay. Before performing the single
cell assay, the system of the disclosure (in this example, a
polydimethylsiloxane (PDMS) microchamber array) was plasma treated
for 2.5 minutes with a Plasma Etch PE-25 plasma cleaner to increase
hydrophilicity. The array was then blocked in 3% BSA/PBS for 30
minutes. Immediately prior to the assay, K562 cells were taken from
culture, resuspended in 1 mL of PBS and stained with CellTrace
Carboxyfluorescein succinimidyl ester (CFSE) (1:1000, ThermoFisher)
for 20 minutes. K562 cells were then washed 3.times. with
supplemented X-Vivo 15 media and resuspended at a concentration of
1.times.10.sup.6 cells/mL. K562 cells and NK cells were then
combined at a 1:1 ratio to generate a cell suspension. This
suspension containing K562 cells and NK cells was centrifuged at
300.times.g for 10 minutes and re-suspended in fresh media at a
concentration of 2.5.times.10.sup.6 cells/mL. Immediately before
the assay, the array was rinsed with media and dried using
compressed air. The array was positioned onto a glass slide and
secured into a custom clamping system. Thirty microliters of the
cell suspension was pipetted, chamber-by-chamber, onto the array. A
surface (a glass side in this example) with the a repeated pattern
of antibodies attached thereto was contacted to the array such that
the repeated pattern of antibodies was facing the array and such
that each repeat of the pattern aligned with a chamber of the
array. The array and the surface (with the antibody pattern)
enclosing the array were clamped tightly together in our custom
clamping system. Cells were trapped in microchambers and imaged
immediately after loading as described below. The microchamber
assembly was placed in a standard 5% CO.sub.2, incubator at
37.degree. C. for 16 hours. Following incubation, the surface with
the antibody pattern was removed in a 1% BSA/PBS bath and rinsed
with 1% BSA/PBS. Subsequently, 300 .mu.L of a biotin-labeled
secondary antibody cocktail was introduced to the surface and
incubated for 45 minutes. This cocktail contains detection
antibodies at a concentration of 0.25 .mu.g/mL each in 1:200
suspension of 1% BSA/PBS. Following this step, the surface was
rinsed with 1% BSA, 300 .mu.L of a 1:100 solution of APC
streptavidin (BioLegend, 405207) was added and the surface was
incubated for 30 minutes. Following this incubation, the surface
was washed with 1% BSA/PBS then washed in coplin jars PBS, 50%
PBS/DI water, DI water, DI water sequentially for 3 minutes in a
centrifuge set at 125 rpm. The surface was dried for 30 seconds in
a Labnet slide spinner (C1303-T). The surface was analyzed using a
GenePix microarray scanner as described below.
[0152] Microchamber Array Imaging. A Zeiss Axio Observer.Z1
fluorescent microscope with an automatic stage was used to acquire
both bright field and fluorescent images of the array. Images of
the entire array were taken with a Hamamatsu Orca-Flash4.0 LT
Digital CMOS camera (C11440-42U) of a 54183 uM.times.24866 uM area
corresponding to either 253 tiles (5.times. image) or 494 tiles
(10.times. image). Using Zen2 Pro software, the tiles were stitched
together with 0% overlap and were exported as TIFFs for analysis by
our proprietary CytoSpeak software.
[0153] Imaging of Antibody-Patterned Surface. GenePix 4400A
scanners (Molecular Devices) were used to obtain scanned
fluorescent images of the antibody-patterned surfaces. Two color
channels, 488 (blue, PMT 350, Power 90) and 635 (red, PMT 600,
Power 90) were used to collect fluorescence signals using GenePix
Pro software (Molecular Devices). The image was then exported as a
TIFF before analysis using CytoSpeak software.
Example 2: Analysis of CD8+T-Cell and Target Cell with Bispecific
Antibodies
[0154] Cell Culture. Isolated PBMCs, stored in-house at a
concentration of 2.5.times.10.sup.7 cells/vial, were thawed and
cultured overnight in a T25 cell culture flask with 5 mL of X-Vivo
15 without Gentamicin or phenol red (Lonza, 04-744Q) supplemented
with 1.times. GlutaMAX (ThermoFisher, 35050061), 1 mM sodium
pyruvate (ThermoFisher, 11360-070), 1.times. MEM Vitamin Solution
(Gibco; ThermoFisher, 11120-052), 20 mM HEPES (Gibco; ThermoFisher,
15630-080), 2% Human AB serum (Valley Biomedical, HP1022HI),
1.times. Penicillin-Streptomycin-Neomycin (Sigma-Aldrich, P4083),
and 4 ng/mL recombinant IL-2 (BioLegend). After an overnight
recovery, CD8+ T-cells were isolated using the Miltenyi CD8+ T-Cell
Isolation Kit according to the manufacturer's instructions. CD8+
T-cells were then cultured at 37.degree. C., 5% CO.sub.2 in
supplemented X-Vivo 15 media at a concentration of 1.times.10.sup.6
cells/mL until use. The human Raji B Cell line (purchased from the
American Type Culture Collection (ATCC)) was cultured in
Corning.TM. cellgro.TM. RPMI 1640 Medium (Mod.) 1.times. with
L-Glutamine (Corning; ThermoFisher MT10040CV) supplemented with 10%
FBS (Sigma-Aldrich, F2442), 1.times.
Penicillin-Streptomycin-Neomycin (Sigma-Aldrich, P4083) and
1.times. GlutaMAX supplement (ThermoFisher, 35050061).
[0155] Single-Cell Secretome Assay. Before performing the single
cell assay, the system of the disclosure (in this example, a
polydimethylsiloxane (PDMS) microchamber array) was plasma treated
for 2.5 minutes with a Plasma Etch PE-25 plasma cleaner to increase
hydrophilicity. The array was then blocked in 3% BSA/PBS for 30
minutes. CD8+ T-cells were resuspended in 1 mL of PBS and stained
with CellTrace Violet (1:1000, ThermoFisher) for 20 minutes. CD8+
T-cells were then washed 3.times. with supplemented X-Vivo 15 media
and resuspended at a concentration of 2.times.10.sup.6 cells/mL.
Raji cells were taken from culture and resuspended in 1 mL of PBS
and stained with Cell Trace CFSE (1:1000, ThermoFisher) for 20
minutes. Raji cells were then washed 3.times. with supplemented
X-Vivo 15 media and resuspended at a concentration of
2.times.10.sup.6 cells/mL. CD8+ T-cells and Raji cells were then
combined at a 1:1 ratio to generate a cell suspension.
Subsequently, 100 ng/mL of a bispecific antibody was added to this
suspension. The cell suspension containing CD8+ T-cells and Raji
cells was centrifuged for 10 minutes at 300.times.g and then
resuspended before loading onto the array. Immediately before the
assay, the array was rinsed with media and dried using compressed
air. The array was then positioned on a glass slide and secured
into a custom clamping system. Thirty microliters of the cell
suspension was pipetted, chamber-by-chamber, onto the array. A
surface (a glass side in this example) with the a repeated pattern
of antibodies attached thereto was contacted to the array such that
the repeated pattern of antibodies was facing the array and such
that each repeat of the pattern aligned with a chamber of the
array. The array and the surface (with the antibody pattern)
enclosing the array were clamped tightly together in our custom
clamping system. Cells were trapped in microchambers and imaged
immediately after loading as described below. The microchamber
assembly was placed in a standard 5% CO.sub.2, incubator at
37.degree. C. for 16 hours. Following incubation, the surface with
the antibody pattern was removed in a 1% BSA/PBS bath and rinsed
with 1% BSA/PBS. Subsequently, 300 .mu.L of a biotin-labeled
secondary antibody cocktail was introduced to the surface and
incubated for 45 minutes. This cocktail contains detection
antibodies at a concentration of 0.25 .mu.g/mL each in 1:200
suspension of 1% BSA/PBS. Following this step, the surface was
rinsed with 1% BSA, 300 .mu.L of a 1:100 solution of APC
streptavidin (BioLegened, 405207) was added and the surface was
incubated for 30 minutes. Following this incubation, the surface
was washed with 1% BSA/PBS then washed in coplin jars PBS, 50%
PBS/DI water, DI water, DI water sequentially for 3 minutes in a
centrifuge set at 125 rpm. The surface was dried for 30 seconds in
a Labnet slide spinner (C1303-T). The surface was analyzed using a
GenePix microarray scanner as described below.
[0156] Microchamber Array Imaging. A Zeiss Axio Observer.Z1
fluorescent microscope with an automatic stage was used to acquire
both bright field and fluorescent images of the array. Images of
the entire array were taken with a Hamamatsu Orca-Flash4.0 LT
Digital CMOS camera (C11440-42U) of a 54183 uM.times.24866 uM area
corresponding to either 253 tiles (5.times. image) or 494 tiles
(10.times. image). Using Zen2 Pro software, the tiles were stitched
together with 0% overlap and were exported as TIFFs for analysis by
our proprietary CytoSpeak software.
[0157] Imaging of Antibody-Patterned Surface. GenePix 4400A
scanners (Molecular Devices) were used to obtain scanned
fluorescent images of the antibody-patterned surfaces. Two color
channels, 488 (blue, PMT 350, Power 90) and 635 (red, PMT 600,
Power 90) were used to collect fluorescence signals using GenePix
Pro software (Molecular Devices). The image was then exported as a
TIFF before analysis using CytoSpeak software.
Example 3: Analysis of Interactions between CAR T-Cells and
Stimulation Beads
[0158] CD19 Bead Design. 1.times.10.sup.8 M450 Tosylactivated
Dynabeads (ThermoFisher) (250 .mu.L of the M450 Tosylactivated
Dynabeads) were washed 3.times. with PBS. Recombinant human
CD19-Histidine (His) (Sino) was resuspeneded in dH20 at 1
.mu.g/.mu.L and incubated at room temperature for 30 minutes. Fifty
microliters of CD19-His was then added to the beads and incubated
at room temperature overnight, with rotation. The CD19-coated beads
were then washed 3.times. with PBS. To inactivate the remaining
tosyl groups on the beads, the beads were resuspended in 250 .mu.L
of 0.2M Tris with 0.1% BSA (pH 8.5) and incubated with rotation
overnight at room temperature. The beads were then resupended in
PBS at a concentration of 4.times.10.sup.6 beads/mL and stored at
4.degree. C. until use.
[0159] CAR T-cell Stimulation. CAR-T cells were mixed with
CD19-coated beads in a 1:4 ratio and plated at a concentration of
1.times.10.sup.6 cells/mL in a 96-well plate. The plate was
incubated for 6 hours at 37.degree. C. and 5% CO.sub.2 before
performing the assay.
[0160] Cell Culture. Target cells were cultured in Corning.TM.
cellgro.TM. RPMI 1640 Medium (Mod.) 1.times. with L-Glutamine
(Corning; ThermoFisher MT10040CV) supplemented with 10% FBS
(Sigma-Aldrich, F2442), 1.times. Penicillin-Streptomycin-Neomycin
(Sigma-Aldrich, P4083) and 1.times. GlutaMAX supplement
(ThermoFisher, 35050061). CAR T-cells were thawed and cultured
overnight at a concentration of 1.times.10.sup.6 cells/mL in X-Vivo
15 without Gentamicin or phenol red (Lonza, 04-744Q) supplemented
with 1.times. GlutaMAX (ThermoFisher, 35050061), 1mM sodium
pyruvate (ThermoFisher, 11360-070), 1.times. MEM Vitamin Solution
(Gibco; ThermoFisher, 11120-052), 20 mM HEPES (Gibco; ThermoFisher,
15630-080), 2% Human AB serum (Valley Biomedical, HP1022HI),
1.times. Penicillin-Streptomycin-Neomycin (Sigma-Aldrich, P4083)
and 4 ng/mL recombinant human IL-2 (BioLegend, 589104). Immediately
prior to use, dead cells were removed from culture using a Dead
Cell Removal Kit (Miltenyi, 130-090-101) and LS Magnetic Column
(Miltenyi, 130-042-401) or by using a standard Ficoll-Paque Plus
(GE, 17-1440-02) dead cell removal protocol. If it is desired to
separate the CD8+ from CD4+ cells in culture, CD8 microbeads
(Miltenyi, 130-045-201) may be used for isolation according to the
manufacturer's instructions following dead cell removal.
Single-Cell Secretome Assay. Before performing the single cell
assay, the array was plasma treated for 2.5 minutes with a Plasma
Etch PE-25 plasma cleaner to increase hydrophilicity. The array was
then blocked in 3% BSA/PBS for 30 minutes. Stimulated T-cells were
collected, stained at 1:100 with an anti-human CD4 RPE antibody
(ThermoFisher, MHCD0404) and an anti-human CD8a Alexa Fluor 647
antibody (BioLegend, 300918) and incubated at room temperature for
10 minutes. The stimulated T-cells were centrifuged at 300.times.g
for 10 minutes and re-suspended in fresh media at a concentration
of 2.5.times.10.sup.6 cells/mL. Immediately before the assay, the
array was rinsed with media and dried using compressed air. The
array was then positioned on a glass slide and secured into a
custom clamping system. Thirty microliters of the cell suspension
was pipetted, chamber-by-chamber, onto the array. A surface (a
glass side in this example) with the a repeated pattern of
antibodies attached thereto was contacted to the array such that
the repeated pattern of antibodies was facing the array and such
that each repeat of the pattern aligned with a chamber of the
array. The array and the surface (with the antibody pattern)
enclosing the array were clamped tightly together in our custom
clamping system. Cells were trapped in microchambers and imaged
immediately after loading as described below. The microchamber
assembly was placed in a standard 5% CO.sub.2, incubator at
37.degree. C. for 16 hours. Following incubation, the surface with
the antibody pattern was removed in a 1% BSA/PBS bath and rinsed
with 1% BSA/PBS. Subsequently, 300 .mu.L of a biotin-labeled
secondary antibody cocktail was introduced to the surface and
incubated for 45 minutes. This cocktail contains detection
antibodies at a concentration of 0.25 .mu.g/mL each in 1:200
suspension of 1% BSA/PBS. Following this step, the surface was
rinsed with 1% BSA, 300 .mu.L of a 1:100 solution of APC
streptavidin (BioLegened, 405207) was added and the surface was
incubated for 30 minutes. Following this incubation, the surface
was washed with 1% BSA/PBS then washed in coplin jars PBS, 50%
PBS/DI water, DI water, DI water sequentially for 3 minutes in a
centrifuge set at 125 rpm. The surface was dried for 30 seconds in
a Labnet slide spinner (C1303-T). The surface was analyzed using a
GenePix microarray scanner as described below.
[0161] Microchamber Array Imaging. A Zeiss Axio Observer.Z1
fluorescent microscope with an automatic stage was used to acquire
both bright field and fluorescent images of the array. Images of
the entire array were taken with a Hamamatsu Orca-Flash4.0 LT
Digital CMOS camera (C11440-42U) of a 54183 uM.times.24866 uM area
corresponding to either 253 tiles (5.times. image) or 494 tiles
(10.times. image). Using Zen2 Pro software, the tiles were stitched
together with 0% overlap and were exported as TIFFs for analysis by
our proprietary CytoSpeak software.
[0162] Imaging of Antibody-Patterned Surface. GenePix 4400A
scanners (Molecular Devices) were used to obtain scanned
fluorescent images of the antibody-patterned surfaces. Two color
channels, 488 (blue, PMT 350, Power 90) and 635 (red, PMT 600,
Power 90) were used to collect fluorescence signals using GenePix
Pro software (Molecular Devices). The image was then exported as a
TIFF before analysis using CytoSpeak software.
Example 4: Analysis of Interactions between CAR T-Cells and Target
Cells
[0163] Target Cell Design. Target cells are generated by cloning
full length cDNA (see list of target cDNAs below) into a pcDNA3.1
(+) vector (ThermoFisher, V79020) using the EcoRI and XhoI cut
sites. The target plasmid is transfected into K562 cells (ATCC,
CCL243) in a 24-well plate following the standard Lipofectamine
3000 (ThermoFisher) protocol. Stable pool lines are created by
selecting transformants with 500 .mu.g/mL Geneticin (ThermoFisher)
for 3-4 weeks. Once stable pools are created, clonal lines are
developed by serially diluting cells in a 96 well plate. Wells with
a single cell are cultured and selected with Geneticin for 3-4
weeks, until a stable clonal line is developed. Clonal lines were
frozen at a concentration of 1.times.10.sup.7 cells/vial in
Corning.TM. cellgro.TM. RPMI 1640 Medium (Mod.) 1.times. with
L-Glutamine (Corning; ThermoFisher MT10040CV) supplemented with 10%
FBS (Sigma-Aldrich, F2442), 1.times.
Penicillin-Streptomycin-Neomycin (Sigma-Aldrich, P4083), 1.times.
GlutaMAX supplement (ThermoFisher, 35050061) and 10% DMSO. Target
cDNAs include, but are not limited to, the following: Nerve Growth
Factor Receptor (NGFR; Negative control cDNA), CD19, Epidermal
Growth Factor Receptor (EGFR), Epidermal Growth Factor Receptor
type III mutation (EGFRvIII; also referred to as de2-7 EGFR or
AEGFR), Melanoma-associated antigen 3 (MAGE A3), NY-ESO-1 (also
known as CTAG-1B; an immunogenic cancer antigen), Prostate Stem
Cell Antigen (PSCA), Preferentially Expressed Antigen In Melanoma
(PRAME), human epidermal growth factor receptor 2 (HER2), B-cell
maturation antigen (BCMA; also known as CD296), carcinoembryonic
antigen (CEA), Mucin 1 (MUC-1; also known as episialin, PEM, H23Ag,
EMA, CA15-3, and MCA), Mucin 16 (MUC-16), and Mesothelin.
[0164] Cell Culture. Target cells were cultured in Corning.TM.
cellgro.TM. RPMI 1640 Medium (Mod.) 1.times. with L-Glutamine
(Corning; ThermoFisher MT10040CV) supplemented with 10% FBS
(Sigma-Aldrich, F2442), 1.times. Penicillin-Streptomycin-Neomycin
(Sigma-Aldrich, P4083) and 1.times. GlutaMAX supplement
(ThermoFisher, 35050061). CAR T-cells were thawed and cultured
overnight at a concentration of 1.times.10.sup.6 cells/mL in X-Vivo
15 without Gentamicin or phenol red (Lonza, 04-744Q) supplemented
with 1.times. GlutaMAX (ThermoFisher, 35050061), 1mM sodium
pyruvate (ThermoFisher, 11360-070), 1.times. MEM Vitamin Solution
(Gibco; ThermoFisher, 11120-052), 20 mM HEPES (Gibco; ThermoFisher,
15630-080), 2% Human AB serum (Valley Biomedical, HP1022HI),
1.times. Penicillin-Streptomycin-Neomycin (Sigma-Aldrich, P4083)
and 4 ng/mL recombinant human IL-2 (BioLegend, 589104). Immediately
prior to use, dead cells were removed from culture using a Dead
Cell Removal Kit (Miltenyi, 130-090-101) and LS Magnetic Column
(Miltenyi, 130-042-401) or by using a standard Ficoll-Paque Plus
(GE, 17-1440-02) dead cell removal protocol. If it is desired to
separate the CD8+ from CD4+ cells in culture, CD8 microbeads
(Miltenyi, 130-045-201) may be used for isolation according to the
manufacturer's instructions following dead cell removal.
[0165] CAR T-cell Stimulation. Target cells were stained for 20
minutes with CellTrace CFSE (ThermoFisher, C34554) and rinsed
3.times. with supplemented X-Vivo 15 media prior to use. Target
cells (at a concentration of 1.times.10.sup.6 cells/mL) and CAR
T-cells (at a concentration of 1.times.10.sup.6 cells/mL) were
combined in a 1:1 ratio to generate a cell suspension. Two hundred
microliters of the cell suspension containing the CAR T-cells and
the target cells was plated per well into a 96-well plate and
incubated at 37.degree. C., 5% CO.sub.2 for 1-2 hours.
[0166] Single-Cell Secretome Assay. Before performing the single
cell assay, the array was plasma treated for 2.5 minutes with a
Plasma Etch PE-25 plasma cleaner to increase hydrophilicity. The
array was then blocked in 3% BSA/PBS for 30 minutes. Stimulated
T-cells were collected, stained at 1:100 with an anti-human CD4 RPE
antibody (ThermoFisher, MHCD0404) and an anti-human CD8a Alexa
Fluor 647 antibody (BioLegend, 300918) and incubated at room
temperature for 10 minutes. The stimulated T-cells were centrifuged
at 300.times.g for 10 minutes and re-suspended in fresh media at a
concentration of 2.5.times.10.sup.6 cells/mL. Immediately before
the assay, the array was rinsed with media and dried using
compressed air. The array was then positioned on a glass slide and
secured into a custom clamping system. Thirty microliters of the
cell suspension was pipetted, chamber-by-chamber, onto the array. A
surface (a glass side in this example) with the a repeated pattern
of antibodies attached thereto was contacted to the array such that
the repeated pattern of antibodies was facing the array and such
that each repeat of the pattern aligned with a chamber of the
array. The array and the surface (with the antibody pattern)
enclosing the array were clamped tightly together in our custom
clamping system. Cells were trapped in microchambers and imaged
immediately after loading as described below. The microchamber
assembly was placed in a standard 5% CO.sub.2, incubator at
37.degree. C. for 16 hours. Following incubation, the surface with
the antibody pattern was removed in a 1% BSA/PBS bath and rinsed
with 1% BSA/PBS. Subsequently, 300 .mu.L of a biotin-labeled
secondary antibody cocktail was introduced to the surface and
incubated for 45 minutes. This cocktail contains detection
antibodies at a concentration of 0.25 .mu.g/mL each in 1:200
suspension of 1% BSA/PBS. Following this step, the surface was
rinsed with 1% BSA, 300 .mu.L of a 1:100 solution of APC
streptavidin (BioLegened, 405207) was added and the surface was
incubated for 30 minutes. Following this incubation, the surface
was washed with 1% BSA/PBS then washed in coplin jars PBS, 50%
PBS/DI water, DI water, DI water sequentially for 3 minutes in a
centrifuge set at 125 rpm. The surface was dried for 30 seconds in
a Labnet slide spinner (C1303-T). The surface was analyzed using a
GenePix microarray scanner as described below.
[0167] Microchamber Array Imaging. A Zeiss Axio Observer.Z1
fluorescent microscope with an automatic stage was used to acquire
both bright field and fluorescent images of the array. Images of
the entire array were taken with a Hamamatsu Orca-Flas4.0 LT
Digital CMOS camera (C11440-42U) of a 54183 uM.times.24866 uM area
corresponding to either 253 tiles (5.times. image) or 494 tiles
(10.times. image). Using Zen2 Pro software, the tiles were stitched
together with 0% overlap and were exported as TIFFs for analysis by
our proprietary CytoSpeak software.
[0168] Imaging of Antibody-Patterned Surface. GenePix 4400A
scanners (Molecular Devices) were used to obtain scanned
fluorescent images of the antibody-patterned surfaces. Two color
channels, 488 (blue, PMT 350, Power 90) and 635 (red, PMT 600,
Power 90) were used to collect fluorescence signals using GenePix
Pro software (Molecular Devices). The image was then exported as a
TIFF before analysis using CytoSpeak software.
Example 5: Analysis of Interactions between CAR T-Cells and Target
Cells with Target Cell Depletion
[0169] Target Cell Design. Target cells are generated by cloning
full length cDNA (see list of target cDNAs below) into a pcDNA3.1
(+) vector (ThermoFisher, V79020) using the EcoRI and XhoI cut
sites. The target plasmid is transfected into K562 cells (ATCC,
CCL243) in a 24-well plate following the standard Lipofectamine
3000 (ThermoFisher) protocol. Stable pool lines are created by
selecting transformants with 500 .mu.g/mL Geneticin (ThermoFisher)
for 3-4 weeks. Once stable pools are created, clonal lines are
developed by serially diluting cells in a 96 well plate. Wells with
a single cell are cultured and selected with Geneticin for 3-4
weeks, until a stable clonal line is developed. Clonal lines were
frozen at a concentration of 1.times.10.sup.7 cells/vial in
Corning.TM. cellgro.TM. RPMI 1640 Medium (Mod.) 1.times. with
L-Glutamine (Corning; ThermoFisher MT10040CV) supplemented with 10%
FBS (Sigma-Aldrich, F2442), 1.times.
Penicillin-Streptomycin-Neomycin (Sigma-Aldrich, P4083), 1.times.
GlutaMAX supplement (ThermoFisher, 35050061) and 10% DMSO. Target
cDNAs include, but are not limited to, the following: Nerve Growth
Factor Receptor (NGFR; Negative control cDNA), CD19, Epidermal
Growth Factor Receptor (EGFR), Epidermal Growth Factor Receptor
type III mutation (EGFRvIII; also referred to as de2-7 EGFR or
AEGFR), Melanoma-associated antigen 3 (MAGE A3), NY-ESO-1 (also
known as CTAG-1B; an immunogenic cancer antigen), Prostate Stem
Cell Antigen (PSCA), Preferentially Expressed Antigen In Melanoma
(PRAME), human epidermal growth factor receptor 2 (HER2), B-cell
maturation antigen (BCMA; also known as CD296), carcinoembryonic
antigen (CEA), Mucin 1 (MUC-1; also known as episialin, PEM, H23Ag,
EMA, CA15-3, and MCA), Mucin 16 (MUC-16), and Mesothelin.
[0170] Cell Culture. Target cells were cultured in Corning.TM.
cellgro.TM. RPMI 1640 Medium (Mod.) 1.times. with L-Glutamine
(Corning; ThermoFisher MT10040CV) supplemented with 10% FBS
(Sigma-Aldrich, F2442), 1.times. Penicillin-Streptomycin-Neomycin
(Sigma-Aldrich, P4083) and 1.times. GlutaMAX supplement
(ThermoFisher, 35050061). CAR T-cells were thawed and cultured
overnight at a concentration of 1.times.10.sup.6 cells/mL in X-Vivo
15 without Gentamicin or phenol red (Lonza, 04-744Q) supplemented
with 1.times. GlutaMAX (ThermoFisher, 35050061), 1 mM sodium
pyruvate (ThermoFisher, 11360-070), 1.times. MEM Vitamin Solution
(Gibco; ThermoFisher, 11120-052), 20 mM HEPES (Gibco; ThermoFisher,
15630-080), 2% Human AB serum (Valley Biomedical, HP1022HI),
1.times. Penicillin-Streptomycin-Neomycin (Sigma-Aldrich, P4083)
and 4 ng/mL recombinant human IL-2 (BioLegend, 589104). Immediately
prior to use, dead cells were removed from culture using a Dead
Cell Removal Kit (Miltenyi, 130-090-101) and LS Magnetic Column
(Miltenyi, 130-042-401) or by using a standard Ficoll-Paque Plus
(GE, 17-1440-02) dead cell removal protocol. If it is desired to
separate the CD8+ from CD4+ cells in culture, CD8 microbeads
(Miltenyi, 130-045-201) may be used for isolation according to the
manufacturer's instructions following dead cell removal.
[0171] CAR T-cell Stimulation. CAR T-cells (at a concentration of
1.times.10.sup.6 cells/mL) and target cells (at a concentration of
1.times.10.sup.6 cells/mL) are mixed in a ratio of 1:1 to generate
a cell suspension. Two hundred microliters of the cell suspension
is plated per well of a 96-well plate. The plate is then incubated
overnight (in this example is approximately 20 hours) in an
incubator at 37.degree. C. with 5% CO.sub.2.
[0172] Target Cell Depletion. After an overnight stimulation, the
cell suspension containing CAR T-cells and target cells is removed
from the wells and centrifuged for 10 minutes at 300.times.g. The
supernatant is then removed and the cells are resuspended in a
conjugated bead solution. The conjugated bead solution contains
M280 Dynabeads conjugated to an antibody against the target
receptor. The beads of the conjugated bead solution are designed to
remove all of the target cells from the solution. This conjugated
bead solution containing the CAR T-cells and the target cells is
incubated, with gentle mixing, at room temperature for 10 minutes.
After the incubation, the solution containing the CAR T-cells and
the target cells is brought up to a total volume of 1 mL with PBS
and transferred to a 5 mL round bottom polypropylene falcon tube
(Corning, 352063). The tube is then placed in an EasySep Magnet
(StemCell Technologies, 18000) for 2 minutes before decanting the
solution of CAR T-cells into a clean tube. The beads are washed
with media and decanted 2.times. before centrifuging the CAR
T-cells and resuspending the target cell depleted CAR-T cells for
use in the assay.
[0173] Single-Cell Secretome Assay. Before performing the single
cell assay, the array was plasma treated for 2.5 minutes with a
Plasma Etch PE-25 plasma cleaner to increase hydrophilicity. The
array was then blocked in 3% BSA/PBS for 30 minutes. Stimulated
T-cells were collected, stained at 1:100 with an anti-human CD4 RPE
antibody (ThermoFisher, MHCD0404) and an anti-human CD8a Alexa
Fluor 647 antibody (BioLegend, 300918) and incubated at room
temperature for 10 minutes. The stimulated T-cells were centrifuged
at 300.times.g for 10 minutes and re-suspended in fresh media at a
concentration of 2.5.times.10.sup.6 cells/mL. Immediately before
the assay, the array was rinsed with media and dried using
compressed air. The array was then positioned on a glass slide and
secured into a custom clamping system. Thirty microliters of the
cell suspension was pipetted, chamber-by-chamber, onto the array. A
surface (a glass side in this example) with the a repeated pattern
of antibodies attached thereto was contacted to the array such that
the repeated pattern of antibodies was facing the array and such
that each repeat of the pattern aligned with a chamber of the
array. The array and the surface (with the antibody pattern)
enclosing the array were clamped tightly together in our custom
clamping system. Cells were trapped in microchambers and imaged
immediately after loading as described below. The microchamber
assembly was placed in a standard 5% CO.sub.2, incubator at
37.degree. C. for 16 hours. Following incubation, the surface with
the antibody pattern was removed in a 1% BSA/PBS bath and rinsed
with 1% BSA/PBS. Subsequently, 300 .mu.L of a biotin-labeled
secondary antibody cocktail was introduced to the surface and
incubated for 45 minutes. This cocktail contains detection
antibodies at a concentration of 0.25 .mu.g/mL each in a 1:200
suspension of 1% BSA/PBS. Following this step, the surface was
rinsed with 1% BSA, 300 .mu.L of a 1:100 solution of APC
streptavidin (BioLegened, 405207) was added and the surface was
incubated for 30 minutes. Following this incubation, the surface
was washed with 1% BSA/PBS then washed in coplin jars PBS, 50%
PBS/DI water, DI water, DI water sequentially for 3 minutes in a
centrifuge set at 125 rpm. The surface was dried for 30 seconds in
a Labnet slide spinner (C1303-T). The surface was analyzed using a
GenePix microarray scanner as described below.
[0174] Microchamber Array Imaging. A Zeiss Axio Observer.Z1
fluorescent microscope with an automatic stage was used to acquire
both bright field and fluorescent images of the array. Images of
the entire array were taken with a Hamamatsu Orca-Flash4.0 LT
Digital CMOS camera (C11440-42U) of a 54183 uM.times.24866 uM area
corresponding to either 253 tiles (5.times. image) or 494 tiles
(10.times. image). Using Zen2 Pro software, the tiles were stitched
together with 0% overlap and were exported as TIFFs for analysis by
our proprietary CytoSpeak software.
[0175] Imaging of Antibody-Patterned Surface. GenePix 4400A
scanners (Molecular Devices) were used to obtain scanned
fluorescent images of the antibody-patterned surfaces. Two color
channels, 488 (blue, PMT 350, Power 90) and 635 (red, PMT 600,
Power 90) were used to collect fluorescence signals using GenePix
Pro software (Molecular Devices). The image was then exported as a
TIFF before analysis using CytoSpeak software.
Example 6: Fluorescent Cytotoxicity Analysis of Target/Effector
Interaction
[0176] This method can be applied to any of the target/effector
(subject) cell types of the disclosure with the addition of the
following step to the Single-Cell Secretome Assay Protocol. If
fluorescent tracking of cytotoxicity is desired, 1 .mu.L/mL of
SYTOX orange (ThermoFisher) may be added to the cell suspension
containing the target cells and effector (subject) cells
immediately prior to loading the cell suspension onto the
array.
Example 7: Assessing Polyfunctionality in CAR-Expressing Anti-Tumor
T-Cells
[0177] To evaluate engineered T cells for an immunotherapy or to
evaluate endogenous T cells reactivated to battle cancer or
infection, a T cell's functional status is largely determined by a
spectrum of secreted effector function proteins (e.g., cytokines).
In a protective immune response, the `quality` of an immune cell
correlates to the extent of polyfunctionality (the ability of a T
cell to co-secrete multiple effector proteins).While these
anti-tumor cytolytic, chemoattractive cytokines produce an
effective response, these poly-functional cell subsets must not
also secrete immuno-toxic inflammatory or regulatory cytokines (up
to 15) prevalent in NK or CAR-T cells.
[0178] The following provides one method for determining whether a
stimulated CAR-expressing anti-tumor T cell responds in a
therapeutically effective and safe manner before administering the
cell or substantially similar cell of a population of
CAR-expressing anti-tumor T-cells to a patient as a
therapeutic.
[0179] Target Cell Design. Target cells are generated by cloning
full length cDNA (see list of target cDNAs below) into a pcDNA3.1
(+) vector (ThermoFisher, V79020) using the EcoRI and XhoI cut
sites. The target plasmid is transfected into K562 cells (ATCC,
CCL243) in a 24-well plate following the standard Lipofectamine
3000 (ThermoFisher) protocol. Stable pool lines are created by
selecting transformants with 500 .mu.g/mL Geneticin (ThermoFisher)
for 3-4 weeks. Once stable pools are created, clonal lines are
developed by serially diluting cells in a 96 well plate. Wells with
a single cell are cultured and selected with Geneticin for 3-4
weeks, until a stable clonal line is developed. Clonal lines were
frozen at a concentration of 1.times.10.sup.7 cells/vial in
Corning.TM. cellgro.TM. RPMI 1640 Medium (Mod.) 1.times. with
L-Glutamine (Corning; ThermoFisher MT10040CV) supplemented with 10%
FBS (Sigma-Aldrich, F2442), 1.times.
Penicillin-Streptomycin-Neomycin (Sigma-Aldrich, P4083), 1.times.
GlutaMAX supplement (ThermoFisher, 35050061) and 10% DMSO. Target
cDNAs include, but are not limited to, the following: Nerve Growth
Factor Receptor (NGFR; Negative control cDNA), CD19, Epidermal
Growth Factor Receptor (EGFR), Epidermal Growth Factor Receptor
type III mutation (EGFRvIII; also referred to as de2-7 EGFR or
AEGFR), Melanoma-associated antigen 3 (MAGE A3), NY-ESO-1 (also
known as CTAG-1B; an immunogenic cancer antigen), Prostate Stem
Cell Antigen (PSCA), Preferentially Expressed Antigen In Melanoma
(PRAME), human epidermal growth factor receptor 2 (HER2), B-cell
maturation antigen (BCMA; also known as CD296), carcinoembryonic
antigen (CEA), Mucin 1 (MUC-1; also known as episialin, PEM, H23Ag,
EMA, CA15-3, and MCA), Mucin 16 (MUC-16), and Mesothelin.
[0180] Cell Culture. Target cells were cultured in Corning.TM.
cellgro.TM. RPMI 1640 Medium (Mod.) 1.times. with L-Glutamine
(Corning; ThermoFisher MT10040CV) supplemented with 10% FBS
(Sigma-Aldrich, F2442), 1.times. Penicillin-Streptomycin-Neomycin
(Sigma-Aldrich, P4083) and 1.times. GlutaMAX supplement
(ThermoFisher, 35050061). CAR T-cells were thawed and cultured
overnight at a concentration of 1.times.10.sup.6 cells/mL in X-Vivo
15 without Gentamicin or phenol red (Lonza, 04-744Q) supplemented
with 1.times. GlutaMAX (ThermoFisher, 35050061), 1 mM sodium
pyruvate (ThermoFisher, 11360-070), 1.times. MEM Vitamin Solution
(Gibco; ThermoFisher, 11120-052), 20 mM HEPES (Gibco; ThermoFisher,
15630-080), 2% Human AB serum (Valley Biomedical, HP1022HI),
1.times. Penicillin-Streptomycin-Neomycin (Sigma-Aldrich, P4083)
and 4 ng/mL recombinant human IL-2 (BioLegend, 589104). Immediately
prior to use, dead cells were removed from culture using a Dead
Cell Removal Kit (Miltenyi, 130-090-101) and LS Magnetic Column
(Miltenyi, 130-042-401) or by using a standard Ficoll-Paque Plus
(GE, 17-1440-02) dead cell removal protocol. If it is desired to
separate the CD8+ from CD4+ cells in culture, CD8 microbeads
(Miltenyi, 130-045-201) may be used for isolation according to the
manufacturer's instructions following dead cell removal.
[0181] CAR T-cell Stimulation. Target cells were stained for 20
minutes with CellTrace CFSE (ThermoFisher, C34554) and rinsed
3.times. with supplemented X-Vivo 15 media prior to use. Target
cells (at a concentration of 1.times.10.sup.6 cells/mL) and CAR
T-cells (at a concentration of 1.times.10.sup.6 cells/mL) were
combined in a 1:1 ratio to generate a cell suspension. Two hundred
microliters of the cell suspension containing the CAR T-cells and
the target cells was plated per well into a 96-well plate and
incubated at 37.degree. C., 5% CO.sub.2 for 1-2 hours.
[0182] Single-Cell Secretome Assay. Before performing the single
cell assay, the array was plasma treated for 2.5 minutes with a
Plasma Etch PE-25 plasma cleaner to increase hydrophilicity. The
array was then blocked in 3% BSA/PBS for 30 minutes. Stimulated
T-cells were collected, stained at 1:100 with an anti-human CD4 RPE
antibody (ThermoFisher, MHCD0404) and an anti-human CD8a Alexa
Fluor 647 antibody (BioLegend, 300918) and incubated at room
temperature for 10 minutes. The stimulated T-cells were centrifuged
at 300.times.g for 10 minutes and re-suspended in fresh media at a
concentration of 2.5.times.10.sup.6 cells/mL. Immediately before
the assay, the array was rinsed with media and dried using
compressed air. The array was then positioned on a glass slide and
secured into a custom clamping system. Thirty microliters of the
cell suspension was pipetted, chamber-by-chamber, onto the array. A
surface (a glass side in this example) with the a repeated pattern
of antibodies attached thereto was contacted to the array such that
the repeated pattern of antibodies was facing the array and such
that each repeat of the pattern aligned with a chamber of the
array. The array and the surface (with the antibody pattern)
enclosing the array were clamped tightly together in our custom
clamping system. Cells were trapped in microchambers and imaged
immediately after loading as described below. The microchamber
assembly was placed in a standard 5% CO.sub.2, incubator at
37.degree. C. for 16 hours. Following incubation, the surface with
the antibody pattern was removed in a 1% BSA/PBS bath and rinsed
with 1% BSA/PBS. Subsequently, 300 .mu.L of a biotin-labeled
secondary antibody cocktail was introduced to the surface and
incubated for 45 minutes. This cocktail contains detection
antibodies at a concentration of 0.25 .mu.g/mL each in 1:200
suspension of 1% BSA/PBS. Following this step, the surface was
rinsed with 1% BSA, 300 .mu.L of a 1:100 solution of APC
streptavidin (BioLegened, 405207) was added and the surface was
incubated for 30 minutes. Following this incubation, the surface
was washed with 1% BSA/PBS then washed in coplin jars PBS, 50%
PBS/DI water, DI water, DI water sequentially for 3 minutes in a
centrifuge set at 125 rpm. The surface was dried for 30 seconds in
a Labnet slide spinner (C1303-T). The surface was analyzed using a
GenePix microarray scanner as described below.
[0183] Microchamber Array Imaging. A Zeiss Axio Observer.Z1
fluorescent microscope with an automatic stage was used to acquire
both bright field and fluorescent images of the array. Images of
the entire array were taken with a Hamamatsu Orca-Flash4.0 LT
Digital CMOS camera (C11440-42U) of a 54183 uM.times.24866 uM area
corresponding to either 253 tiles (5.times. image) or 494 tiles
(10.times. image). Using Zen2 Pro software, the tiles were stitched
together with 0% overlap and were exported as TIFFs for analysis by
our proprietary CytoSpeak software.
[0184] Imaging of Antibody-Patterned Surface. GenePix 4400A
scanners (Molecular Devices) were used to obtain scanned
fluorescent images of the antibody-patterned surfaces. Two color
channels, 488 (blue, PMT 350, Power 90) and 635 (red, PMT 600,
Power 90) were used to collect fluorescence signals using GenePix
Pro software (Molecular Devices). The image was then exported as a
TIFF before analysis using CytoSpeak software.
[0185] Analysis of polyfunctionality: The absolute and relative
contributions of each cytokine in the secrotome of the control and
target-cell stimulated CAR T-cells were measured. FIGS. 10 and 11
demonstrate on a single-cell basis the Polyfunctional Strength
Index (PSI) contributions of each detected cytokine in the
secretome. As shown in FIG. 11, panels E and F, when a population
of cells are detected individually, but analyzed as a population, a
percentage of cells in the given population that express any one
cytokine above a threshold level may be determined. The cytokines
used in this plot may include those cytokines that indicate a risk
of the CAR-T cell inducing a deleterious or unwanted reaction in
vivo and the threshold may be raised and lowered according to what
is considered safe and tolerable. Using this method, any population
of cells intended for administration in vivo as a cell therapy may
be tested in vitro to reveal potential harmful reactions before the
cell population can negatively impact a patient.
[0186] The polyfunctional strength index (PSI) is a metric that
factors in the polyfunctionality of cells in a sample, and the
signal intensity of the cytokines secreted by each cell. It is
found by multiplying the percentage of polyfunctional cells of a
sample (single cells secreting two or more cytokines), by the
average signal intensity of these cytokines. This PSI is shown on
the left of FIGS. 10A and 10B, as well as FIGS. 11A and 11B. For
example, FIG. 10B demonstrates that the polyfunctional strength is
roughly 2.times. higher in the CD19 stimulated sample, relative to
the control PSI shown in FIG. 10A. FIG. 11B demonstrates that the
stimulated cells have a polyfunctional strength roughly 5.times.
higher than the control cells.
[0187] As seen in the bar graphs of FIGS. 10 and 11, the
polyfunctional strength can be further broken down into defined
cytokine groups (see the key shown in FIG. 8E), illustrating the
impact of particular groups of cytokines on the sample's
polyfunctionality. In the left graph of FIG. 10B, for example,
effector cytokines are major drivers of the polyfunctionality, as
they account for about 75% of the total PSI. The corresponding
graph on the right shows the contribution of each individual
cytokine to the overall PSI. In this case, Granzyme B is the major
driver of the sample's polyfunctionality.
[0188] In the right graph of FIG. 11D, we see that there are
several cytokines that drive the polyfunctional strength of the
sample. The PSI is primarily composed of inflammatory cytokines
IL-6 and MCP-1 and effector cytokines Granzyme B and Perforin. Many
other cytokines contribute to a lesser degree to the PSI of the
sample.
[0189] FIGS. 10C and 11C show vertical scatterplots of all the
single-cell secretions from the specified population. Each orange
dot corresponds to a specific cytokine that was secreted at a
particular intensity by a single cell. These are
background-subtracted intensities; all intensities shown here are
above 0, and thus correspond to single-cell secretions. Data
points.ltoreq.0, indicating that a single cell did not secrete a
specific cytokine, are not shown.
[0190] The units of the intensity levels (y-axis) shown in the
graphs are arbitrary, but based on "on-chip" calibration curves
("on chip" may also be referred to as "in situ" with respect to the
compositions of the disclosure), these values were converted to
approximate pg/mL secretion amounts. The percentage label above
each cytokine's vertical scatterplot corresponds to the percentage
of single cells in the sample that secreted that cytokine. The
larger percentage at the top of the graph indicates how many
secretions (as a fraction of all secretions) fell above 1000, to
highlight strong secretions (1000 is an arbitrary threshold that
may be adjusted and/or made specific to each cytokine).
INCORPORATION BY REFERENCE
[0191] Every document cited herein, including any cross referenced
or related patent or application is hereby incorporated herein by
reference in its entirety unless expressly excluded or otherwise
limited. The citation of any document is not an admission that it
is prior art with respect to any invention disclosed or claimed
herein or that it alone, or in any combination with any other
reference or references, teaches, suggests or discloses any such
invention. Further, to the extent that any meaning or definition of
a term in this document conflicts with any meaning or definition of
the same term in a document incorporated by reference, the meaning
or definition assigned to that term in this document shall
govern.
Other Embodiments
[0192] While particular embodiments of the disclosure have been
illustrated and described, various other changes and modifications
can be made without departing from the spirit and scope of the
disclosure. The scope of the appended claims includes all such
changes and modifications that are within the scope of this
disclosure.
* * * * *