[0002] This invention was made with government support under Grant No.
CA174121 and No. HL110574 awarded by the National Institutes of Health.
The government has certain rights in the invention.
Claims
1. A method for preparing cells for treating a patient with cancer,
comprising the steps: a) purifying T cells from a sample comprising
leukocytes and platelets, wherein the leukocytes comprise T cells and
wherein the T cells are purified by: i) performing a size based
separation using a microfluidic device to produce an enriched product in
which, compared to the sample, the percentage cells that are platelets
has been reduced; and ii) in addition to the size based separation,
performing an affinity based separation; b) after the purification of
step a), activating and expanding the T cells to produce a composition in
which the percentage of T cells that are central memory T cells has
increased compared to the percentage of T cells that are central memory T
cells in the sample; c) genetically engineering activated T cells to
comprise therapeutic benefit in the treatment of said patient's cancer.
2. The method of claim 1, wherein, in step c), the activated T cells are
genetically engineered to comprise modified cell surface receptors of
therapeutic benefit in the treatment of said patient's cancer.
3. The method of claim 2, wherein, the modified cell surface receptors of
therapeutic benefit are chimeric antigen receptors (CARs).
4. The method of claim 1, wherein the sample is obtained by apheresis
5. The method of claim 1, wherein the sample is obtained by
leukapheresis.
6. The method of claim 1, wherein the platelets in the enriched product
of paragraph a)ii) are depleted by at least 80% compared to the sample
and/or there are no more than 5 platelets per leukocyte in the enriched
product.
7. The method of claim 1, wherein, the genetically engineered T cells are
collected by transferring them into a pharmaceutical composition for
administration to a patient.
8. The method of claim 7, wherein cells are not frozen before being
collected.
9. A method of treating a patient for cancer comprising administering to
said patient genetically engineered T cells prepared by a method
comprising the steps of: a) purifying T cells from a sample comprising
leukocytes and platelets, wherein the leukocytes comprise T cells and
wherein the T cells are purified by: i) performing a size based
separation using a microfluidic device to produce an enriched product in
which, compared to the sample, the percentage cells that are platelets
has been reduced; and ii) in addition to the size based separation,
performing an affinity based separation; b) after the purification of
step a), activating and expanding the T cells to produce a composition in
which the percentage of T cells that are central memory T cells has
increased compared to the percentage of T cells that are central memory T
cells in the sample; c) genetically engineering activated T cells to
comprise therapeutic benefit in the treatment of said patient's cancer.
10. The method of claim 9 wherein, in step c), the activated T cells are
genetically engineered to comprise modified cell surface receptors of
therapeutic benefit in the treatment of said patient's cancer.
11. The method of claim 10, wherein, the modified cell surface receptors
of therapeutic benefit are chimeric antigen receptors (CARs).
12. The method of claim 9, wherein the sample is obtained by apheresis
13. The method of claim 9, wherein the sample is obtained by
leukapheresis.
14. The method of claim 9, wherein the platelets in the enriched product
of paragraph a)ii) are depleted by at least 80% compared to the sample
and/or there are no more than 5 platelets per leukocyte in the enriched
product.
15. The method of claim 9, wherein, the genetically engineered T cells
are collected by transferring them into a pharmaceutical composition for
administration to a patient.
16. A method for preparing a therapeutic composition comprising
genetically engineered T cells, comprising: a) purifying T cells from
sample obtained from a patient, wherein the sample comprises leukocytes
and platelets; the leukocytes comprise T cells and the T cells are
purified by: i) performing a size based separation using a microfluidic
device configured to separate cells by deterministic lateral displacement
to produce an enriched product in which, compared to the sample, the
percentage of cells that are platelets has been reduced; ii) in addition
to the size based separation, performing an affinity based separation by
binding T cells to a carrier that binds to T cells with specificity, and
then separating the carrier-bound T cells from cells not bound to
carrier; b) after the purification of step a), activating and expanding
the T cells to produce a composition in which the percentage of T cells
that are central memory T cells has increased compared to the percentage
of T cells that are central memory T cells in the sample; c) genetically
engineering the activated T cells.
17. The method of claim 16, wherein the sample is an apheresis sample.
18. The method of claim 16, wherein the sample is a leukapheresis sample.
19. The method of claim 16, wherein, after the cells are prepared, they
are administered to said patient.
20. The method of claim 16, wherein the platelets in the enriched product
of paragraph a)i) are depleted by at least 80% compared to the sample
and/or there are no more than 5 platelets per leukocyte in the enriched
product.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No. 16/662,033,
filed on Oct. 24, 2019, which is a continuation of U.S. Ser. No.
16/108,365, filed on Aug. 22, 2018, which issued as U.S. Pat. No.
10,844,353 on Nov. 24, 2020 and which claims the benefit of U.S.
Provisional Patent Application No. 62/553,723, filed on Sep. 1, 2017; the
benefit of U.S. Provisional Patent Application No. 62/567,553, filed on
Oct. 3, 2017; the benefit of Provisional Patent Application No.
62/635,304, filed on Feb. 26, 2018; and the benefit of Provisional Patent
Application No. 62/656,939, filed on Apr. 12, 2018; and, in addition, the
is a continuation-in-part of PCT/US2017/057876, filed on Oct. 23, 2017.
These prior applications are all incorporated by reference herein in
their entireties.
FIELD OF THE INVENTION
[0003] The present invention is directed primarily to methods of preparing
cells and compositions for therapeutic uses. The methods employ
microfluidic devices that separate cells based on size.
BACKGROUND OF THE INVENTION
[0004] Cell therapy, and especially CAR-T cell therapy, has demonstrated
extraordinary efficacy in treating B-cell diseases such as B-acute
lymphoid leukemia (B-ALL) and B-Cell Lymphomas. As a result, the demand
for autologous therapies has increased dramatically and development
efforts have broadened to focus on cancers characterized by solid tumors,
such as glioblastomas (Vonderheide, et al., Immunol. Rev. 257:7-13
(2014); Fousek, et al., Clin. Cancer Res. 21:3384-3392 (2015); Wang, et
al., Mol. Ther. Oncolytics 3:16015 (2016); Sadelain, et al., Nature
545:423-431 (2017)). Targeted gene editing with CRISPR/Cas-9 in focused
populations of autologous cells, such as stem cells, may further fuel
demand (Johnson, et al., Cancer Cell Res. 27:38-58 (2017)).
[0005] The preparation of cells for personalized therapy is usually a
labor-intensive process that relies on procedures adapted from blood
banking or protein bioprocessing procedures which are poorly suited for
therapeutic applications. Cell losses associated with processing steps
are typically substantial (Hokland, et al., Scand. J. Immunol. 11:353-356
(1980); Stroncek, et al., J. Transl. Med. 12:241 (2014)), in part because
of processes that use preparations that achieve cell specific separations
(Powell, et al., Cytotherapy 11:923-935 (2009); TerumoBCT. ELUTRA Cell
Separation System. Manufacturer recommendations for the Enrichment of
Lymphocytes from Apheresis Residues) but do so at the expense of cell
viability and yield (Chiche-Lapierre, Cytotherapy 18(6):547 (2016)).
Thus, there is a need for more efficient processes.
SUMMARY OF THE INVENTION
[0006] The present invention is directed, inter alia, to methods of
collecting and rapidly processing cells, particularly cells that have
therapeutic uses. Many of the methods rely on Deterministic Lateral
Displacement (DLD), a process that involves flowing a sample through a
microfluidic device containing a specifically designed array of
microposts that are tilted at a small angle from the direction of fluid
flow (Davis, et al., Proc. Natl. Acad. Sci. USA 103:14779-14784 (2006);
Inglis, et al., Lab Chip 6:655-658 (2006); Chen, et al.,
Biomicrofluidics. 9(5):054105 (2015)). Cells larger than the target size
of the micropost array may be gently deflected ("bumped") by the
microposts into a stream of clean buffer, effectively separating them
from smaller, non-deflected cells and particles, while simultaneously
washing the cells in a process that is non-injurious. Advantageous
characteristics of DLD with respect to cell processing are described in
Table 1:
TABLE-US-00001
TABLE 1
Intrinsic Properties of DLD and Their Implications for Cell Processing
DLD
Feature Enablement Implications
Uniform Fractionate complex Uniform and gentle de-bulking of platelet and
feature and mixtures based on size with RBC from blood products without
gap size ability to discriminate centrifugation up to 99.99% efficiency
particles to within ~0.5 .mu.m. Eliminates open solutions such as Ficoll,
and
avoids need for harsh hypertonic solutions
(Elutriation).
Ability to mix different Dc Use of sequential cut-offs to manage highly
within the same device heterogeneous fractionations
Cell Washing & Buffer Cell Washing >99.9% removal in single pass
Exchange Potential to improve and remove cell culture
while maintaining closed system ensuring
viable cells.
Concentration Concentration of cells in culture to make
downstream processing seamless.
Minimize reagent expense without requiring
open centrifugation or transfer losses.
Closeable Simple, sterilizable Ideal for single use, especially patient
specific
fluid path therapeutic device.
Low Dead <50 .mu.l Dead volume per 14 Excellent cell recovery
Volume lane chip
Requires only Hands free operation Potential to automate complex cell
handling
positive and liquid addition exchange processes within
pressure a closed system
[0007] Methods for Engineering Target Cells
[0008] In its first aspect, the invention is directed to a method of
genetically engineering a population of target cells. This is done by
isolating the target cells from a crude fluid composition by performing
Deterministic Lateral Displacement (DLD) on a microfluidic device. The
device is characterized by the presence of at least one channel which
extends from a sample inlet to one or more fluid outlets, and which is
bounded by a first wall and a second wall opposite from the first wall.
An array of obstacles is arranged in rows in the channel, with each
subsequent row of obstacles being shifted laterally with respect to a
previous row. The obstacles are disposed in a manner such that, when the
crude fluid composition is applied to an inlet of the device and passed
through the channel, target cells flow to one or more collection outlets
where an enriched product is collected, and contaminant cells or
particles flow to one or more waste outlets that are separate from the
collection outlets. Once the target cells have been purified using the
device, they are transfected or transduced with nucleic acids designed to
impart upon the cells a desired phenotype, e.g., to express a chimeric
molecule (preferably a protein that makes the cells of therapeutic
value). The population of cells may then be expanded by culturing in
vitro. When cultured and expanded, the yield of recombinantly engineered
target cells exhibiting the desired phenotype is preferably at least 10%
greater than identical cells not subjected to DLD (and particularly cells
that have been exposed to Ficoll centrifugation but not DLD), and more
preferably at least 20, 30, 40, or 50% greater.
[0009] In a preferred embodiment, the crude fluid composition is blood or,
more preferably, a preparation of leukocytes that has been obtained by
performing apheresis or leukapheresis on the blood of a patient.
Preferred target cells include T cells, B-cells, NK-cells, monocytes and
progenitor cells, with T cells (especially natural killer T cells) being
the most preferred. Apart from leukocytes, other types of cells, e.g.,
dendritic cells or stem cells, may also serve as target cells.
[0010] In general, crude fluid compositions containing target cells will
be processed without freezing (at least up until the time that they are
genetically engineered), and at the site of collection. The crude fluid
composition will preferably be the blood of a patient, and more
preferably be a composition containing leukocytes obtained as the result
of performing apheresis or leukapheresis on such blood. However, the term
"crude fluid composition" also includes bodily fluids such as lymph or
synovial fluid as well as fluid compositions prepared from bone marrow or
other tissues. The crude fluid composition may also be derived from
tumors or other abnormal tissue.
[0011] Although it is not essential that target cells be bound to a
carrier before being genetically engineered, it is preferred that, either
before or after DLD is first performed (preferably before) they be bound
to one or more carriers. The exact means by which this occurs is not
critical to the invention but binding should be done "in a way that
promotes DLD separation." This term, as used in the present context,
means that the method must ultimately result in binding that exhibits
specificity for a particular target cell type, that provides for an
increase in size of the complex relative to the unbound cell of at least
2 .mu.m (and alternatively at least 20, 50, 100, 200, 500 or 1000% when
expressed as a percentage) and, in cases where therapeutic or other uses
require free target cells, that allow the target cell to be released from
complexes by chemical or enzymatic cleavage, chemical dissolution,
digestion, due to competition with other binders, by physical shearing,
e.g., using a pipette to create shear stress, or by other means.
[0012] In a preferred embodiment, the carriers have on their surface an
affinity agent (e.g., an antibody, activator, hapten, aptamer, nucleic
acid sequence, or other compound) that allows the carriers to bind
directly to the target cells with specificity. Alternatively, there may
be an intermediary protein, cell, or other agent that binds to both the
target cell and carrier with specificity. For example, antibodies may be
used that recognize surface antigens on target cells and that also bind
with specificity to carriers (e.g., due to that presence of a second
antibody on the carrier surface, avidin/biotin binding or some other
similar interaction). In addition, target cells may sometimes interact
with specificity with other cells to form a complex and in so doing, the
other cells may serve as a biological carrier, i.e., they may increase
the effective size of the target cell and thereby facilitate its
separation from uncomplexed cells. For example, human T cells may
interact with sheep erythrocytes or autologous human erythrocytes to form
a rosette of cells that can then be purified as a complex. Alternatively,
other carriers may bind with specificity to cells in such a rosette to
further promote a size based separation.
[0013] As used in this context, the word "specificity" means that at least
100 (and preferably at least 1000) target cells will be bound by carrier
in the crude fluid composition relative to each non-target cell bound. In
cases where the carrier binds after DLD, the binding may occur either
before the target cells are genetically engineered or after.
[0014] Binding of the carriers may help to stabilize cells, activate them
(e.g., to divide) or help to facilitate the isolation of one type of cell
from another. As suggested above, the binding of carriers to cells can
take place at various times in the method, including during the time that
cells are being obtained. In order to improve separation, carriers may be
chosen such that the binding of a single carrier to a cell results in a
carrier-cell complex that is substantially larger than the size of the
cell alone. Alternatively carriers may be used that are smaller that the
target cell. In this case, it is preferred that several carriers bind
with specificity to a cell, thereby forming a complex having one cell and
multiple carriers. During DLD, complexed target cells may separate from
uncomplexed cells having a similar size and provide a purification that
would otherwise not occur.
[0015] In order to achieve such separation, the diameter of the complex
should preferably be at least 20% larger than the uncomplexed target
cells and more preferably at least 50% larger, at least twice as large or
at least ten times as large. As stated above this increase in size may be
either due to the binding of a single large carrier to target cells or
due to the binding of several smaller carriers. This may be accomplished
using: a) only carriers with a diameter at least as large (or in other
embodiments, at least twice as large or at least ten times as large) as
that of the target cells; b) only carriers with a diameter no more than
50% (or in other embodiments, no more than 25% or 15%) as large as that
of the target cells; or c) mixtures of large and small carriers with
these size characteristics (e.g., there may be one group of carriers with
a diameter at least as large (or at least twice or ten times as large) as
the target cells and a second group of carriers with a diameter no more
than 50% (or no more than 25% or 15%) as large as that of the target
cells. Typically, a carrier will have a diameter of 1-1000 .mu.m (and
often in the range of 5-600 or 5-400 .mu.m). Ideally, the complexes will
be separated from other cells or contaminants by DLD on a microfluidic
device having an array of obstacles with a critical size lower than the
size of the complexes but higher than the size of uncomplexed non-target
cells or contaminants.
[0016] In addition carriers may act in a way that "complements DLD
separation" rather than directly promoting separation by this technique.
For example, a carrier (e.g., as Janus or Strawberry-like particles) may
comprise two or more discrete chemical properties that support and confer
actionable differential non-size related secondary properties, such as
chemical, electrochemical, or magnetic properties, on the cells that they
bind with and these properties may be used in downstream processes. Thus,
the particles may be used to facilitate magnetic separation,
electroporation, or gene transfer. They may also confer advantageous
changes in cellular properties relating to, for example, metabolism or
reproduction.
[0017] In a particularly important embodiment, the binding of carriers may
be used as a means of separating a specific leukocyte, especially T
cells, including natural killer T cells, from other leukocytes, e.g.,
granulocytes and monocytes, and/or from other cells. This may be done,
for example, in a two step process in which DLD is performed on target
cells that are not bound to a carrier using an array of obstacles with a
critical size smaller than the cells and also performed on complexes
comprising target cells and carriers using an array of obstacles with a
critical size smaller than the complexes but larger than the uncomplexed
cells. The DLD steps can be performed in either order, i.e., DLD may be
performed on the complexes before or after being performed on uncomplexed
target cells.
[0018] No more than four hours (and preferably no more than three, two or
one hour(s)) should elapse from the time that the obtaining of crude
fluid composition is completed until the target cells are first bound to
carriers. In addition, no more that five hours (and preferably no more
than four, three or two hours) should elapse from the time that the
obtaining of crude fluid composition is completed until the first time
that target cells are transfected or transduced.
[0019] In a particularly preferred embodiment, the target cells in the
methods described above are T cells (especially natural killer T cells
and memory T cells) and these are engineered to express chimeric antigen
receptors on their surface. The procedures for making these CAR T cells
are described more specifically below.
[0020] Methods for Making CAR T Cells
[0021] The invention includes a method of producing CAR T cells by
obtaining a crude fluid composition comprising T cells (especially
natural killer T cells and memory T cells) and performing DLD on the
composition using a microfluidic device. Generally, the crude fluid
composition comprising T cells will be an apheresis or leukapheresis
product derived from the blood of a patient and containing leukocytes.
[0022] The microfluidic device must have at least one channel extending
from a sample inlet to one or more fluid outlets, wherein the channel is
bounded by a first wall and a second wall opposite from the first wall.
An array of obstacles is arranged in rows in the channel, each subsequent
row of obstacles being shifted laterally with respect to a previous row.
These obstacles are disposed in a manner such that, when the crude fluid
composition comprising T cells is applied to an inlet of the device and
fluidically passed through the channel, the T cells flow to one or more
collection outlets where an enriched product is collected and other cells
(e.g., red blood cells, and platelets) or other particles of a different
(generally smaller) size than the T cells flow to one or more waste
outlets that are separate from the collection outlets. Once obtained, the
T cells are genetically engineered to produce chimeric antigen receptors
(CARs) on their surface using procedures well established in the art.
These receptors should generally bind antigens that are on the surface of
a cell associated with a disease or abnormal condition. For example, the
receptors may bind antigens that are unique to, or overexpressed on, the
surface of cancer cells. In this regard, CD19 may sometimes be such an
antigen.
[0023] The genetic engineering of CAR-expressing T cells will generally
comprise transfecting or transducing T cells with nucleic acids and, once
produced, the CAR T cells may be expanded in number by growing the cells
in vitro. Activators or other factors may be added during this process to
promote growth, with IL-2 and IL-15 being among the agents that may be
used. The yield of T cells expressing chimeric receptors on their surface
after DLD, recombinant engineering and expansion, should, in some
embodiments be at least 10% greater than T cells prepared in the same
manner but not subjected to DLD and preferably at least 20, 30, 40 or 50%
greater. Similarly, in some embodiments, the yield of T cells expressing
the chimeric receptors on their surface should be at least 10% greater
than T cells isolated by Ficoll centrifugation and not subjected to DLD
and preferably at least 20, 30, 40 or 50% greater.
[0024] Chimeric receptors will typically have a) an extracellular region
with an antigen binding domain; b) a transmembrane region and c) an
intracellular region. The cells may also be recombinantly engineered with
sequences that provide the cells with a molecular switch that, when
triggered, reduce CAR T cell number or activity. In a preferred
embodiment, the antigen binding domain is a single chain variable
fragment (scFv) from the antigen binding regions of both heavy and light
chains of a monoclonal antibody. There is also preferably a hinge region
of 2-20 amino acids connecting the extracellular region and the
transmembrane region. The transmembrane region may have CD3 zeta, CD4,
CD8, or CD28 protein sequences and the intracellular region should have a
signaling domain, typically derived from CD3-zeta, CD137 or a CD28. Other
signaling sequences may also be included that serve to regulate or
stimulate activity.
[0025] After obtaining the crude fluid composition comprising T cells, or
during the time that they are being collected, the T cells may, for the
reasons discussed above, be bound to one or more carriers in a way that
promotes DLD separation. This will preferably take place before
performing DLD. However, it may also occur after performing DLD and
either before or after cells are transfected or transduced for the first
time. In a preferred embodiment, the carriers should comprise on their
surface an affinity agent (e.g., an antibody, activator, hapten or
aptamer) that binds with specificity to T cells, preferably natural
killer T cells. The term "specificity" as used in this context means that
the carriers bind preferentially to the desired T cells as compared to
any other cells in the composition. For example, the carriers may bind to
100 or 1000 CD8+ T cells for each instance in which it binds a different
type of cell.
[0026] Carriers may, in some embodiments, have a spherical shape and be
made of either biological or synthetic material, including collagen,
polysaccharides including polystyrene, acrylamide, alginate and magnetic
material. In addition, carriers may act in a way that complements DLD
separation.
[0027] In order to aid in achieving a separation, the diameter of the
complex formed between T cells and carriers should preferably be at least
20% larger than the uncomplexed T cells and preferably at least 50%
larger, at least twice as large or at least ten times as large. This
increase in size may be either due to the binding of a single large
carrier to the cells or due to the binding of several smaller carriers.
Binding may involve using: a) only carriers with a diameter at least as
large (or in other embodiments, at least twice as large or at least ten
times as large) as that of the T cells; b) only carriers with a diameter
no more than 50% (or in other embodiments, no more than 25% or 15%) as
large as that of the T cells; or c) mixtures of large and small carriers
with these size characteristics (e.g., there may be one group of carriers
with a diameter at least as large (or at least twice or ten times as
large) as the T cells and a second group of carriers with a diameter no
more than 50% (or no more than 25% or 15%) as large as that of the T
cells. Typically a carrier will have a diameter of 1-1000 .mu.m (and
often in the range of 5-600 or 5-400 .mu.m). Ideally, the complexes will
be separated from uncomplexed cells or contaminants by DLD on a
microfluidic device having an array of obstacles with a critical size
lower than the size of the complexes but higher than the size of
uncomplexed non-target cells or contaminants.
[0028] As discussed above in connection with target cells, the
purification of T cells may involve a two step process. For example, DLD
may be performed on T cells that are not bound to carriers using an array
of obstacles with a critical size smaller than the T cells. A composition
containing the separated T cells together with other cells or particles
may then be recovered and bound to one or more carriers in a way that
promotes DLD separation and in which T cells are bound with specificity.
The complexes thereby formed may then be separated on an array of
obstacles with a critical size smaller than the complexes but larger than
uncomplexed cells. In principle, the DLD steps could be performed in
either order, i.e., it might be performed on the complexes first or on
the uncomplexed T cells first.
[0029] Preferably, no more than four hours (and, more preferably, no more
than three, two or one hour(s)) should elapse from the time that the
obtaining of the crude fluid composition comprising T cells is completed
(e.g., from the time that apheresis or leukapheresis is completed) until
the T cells are bound to a carrier. In addition, no more than five hours
(and preferably no more than four hours, three or two hours) should
elapse from the time that the obtaining of T cells is completed until the
first time that T cells are transfected or transduced. Ideally, all steps
in producing the CAR T cells are performed at the same facility where the
crude fluid composition comprising T cells is obtained and all steps are
completed in no more than four (and preferably no more than three) hours
and without the cells being frozen.
[0030] Treating Cancer, Autoimmune Disease or Infectious Disease Using CAR
T Cells
[0031] In another aspect, the invention is directed to a method of
treating a patient for cancer, an autoimmune disease or an infectious
disease by administering CAR T cells engineered to express chimeric
antigen receptors recognizing cancer cell antigens, or antigens on cells
responsible for, or contributing to, autoimmune or infectious disease.
The CAR T cells may be made using the methods discussed in the section
above, i.e., by obtaining a crude fluid composition comprising T cells
(preferably a leukocyte-containing apheresis or leukapheresis product
derived from the patient) and then performing DLD on the composition
using a microfluidic device. The CAR T cells (preferably natural killer T
cells, and memory T cells) recovered in this manner are then expanded by
growing the cells in vitro. Finally, the cells are administered to a
patient, which should generally be the same patient that gave the blood
from which the T cells were isolated.
[0032] Preferably, the yield of T cells expressing chimeric receptors on
their surface after DLD, recombinant engineering and expansion is at
least 10% greater than T cells prepared in the same manner but not
subjected to DLD and more preferably at least 20, 30, 40 or 50% greater.
For example, the yield of T cells expressing the chimeric receptors on
their surface may be at least 10% greater than T cells isolated by Ficoll
centrifugation and not subjected to DLD and preferably at least 20, 30,
40 or 50% greater.
[0033] Chimeric receptors will typically have at least: a) an
extracellular region with an antigen binding domain; b) a transmembrane
region and c) an intracellular region. The cells may also be
recombinantly engineered with sequences that provide the cells with a
molecular switch that, when triggered, reduce CAR T cell number or
activity. In a preferred embodiment, the antigen binding domain is a
single chain variable fragment (scFv) from the antigen binding regions of
both heavy and light chains of a monoclonal antibody. There is also
preferably a hinge region of 2-20 amino acids connecting the
extracellular region and the transmembrane region. The transmembrane
region itself may have CD3 zeta, CD4, CD8, or CD28 protein sequences and
the intracellular region will have a signaling domain, typically derived
from CD3-zeta and/or a CD28 intracellular domain. Other signaling
sequences may also be included that serve to regulate or stimulate
activity.
[0034] After obtaining the crude fluid composition or during the time the
crude fluid composition is being collected, T cells present in the
composition may be bound to one or more carriers in a way that promotes
or complements DLD separation. This will preferably take place before
performing DLD. However, it may also occur after performing DLD and
either before or after the cells are genetically engineered. Preferably
the binding will promote DLD separation and the carriers will comprise on
their surface an antibody, activator or other agent that binds with
specificity to T cells, especially natural killer T cells. The term
"specificity" as used in this context means that the carrier will be
bound preferentially to the desired T cells as compared to any other
cells in the composition. For example, the carrier may bind to 100 or
1000 CD8+ T cells for every carrier that binds to other types of cells.
[0035] The diameter of the complex formed between T cells and carrier
should preferably be at least 20% larger than the uncomplexed T cells and
more preferably at least 50% larger, at least twice as large or at least
ten times as large. This increase in size may be either due to the
binding of a single large carrier to the cells or due to the binding of
several smaller carriers. Binding may involve using: a) only carriers
with a diameter at least as large (or in other embodiments, at least
twice as large or at least ten times as large) as that of the T cells; b)
only carriers with a diameter no more than 50% (or in other embodiments,
no more than 25% or 15%) as large as that of the T cells; or c) mixtures
of large and small carriers with these size characteristics (e.g., there
may be one group of carriers with a diameter at least as large (or at
least twice or ten times as large) as the T cells and a second group of
carriers with a diameter no more than 50% (or no more than 25% or 15%) as
large as that of the T cells. Typically, a carrier will have a diameter
of 1-1000 .mu.m (and often in the range of 5-600 or 5-400 .mu.m).
Ideally, the complexes will be separated from uncomplexed cells or
contaminants by DLD on a microfluidic device having an array of obstacles
with a critical size lower than the size of the complexes but higher than
the size of uncomplexed non-target cells or contaminants.
[0036] The purification of T cells may involve a two step process. For
example, DLD may be performed on T cells that are not bound to carriers
using an array of obstacles with a critical size smaller than the T
cells. A composition containing the separated T cells together with other
cells or particles may then be recovered and bound to one or more
carriers in a way that promotes DLD separation and in which T cells are
bound with specificity. The complexes thereby formed may then be
separated on an array of obstacles with a critical size smaller than the
complexes but larger than uncomplexed cells. In principle, the DLD steps
could be performed in either order, i.e., it might be performed on the
complexes first or on the uncomplexed T cells first.
[0037] Preferably, no more than four hours (and more preferably no more
than three, two or one hour(s)) should elapse from the time that the
obtaining of T cells is completed (e.g., until apheresis or leukapheresis
is completed) until the T cells are bound to a carrier. In addition, no
more than five hours (and preferably no more than four, three or two
hours) should elapse from the time that the obtaining of T cells is
completed until the first time that T cells are transfected or
transduced. Ideally, all steps in producing the CAR T cells are performed
at the same facility where the crude fluid composition comprising T cells
is obtained and all steps are completed in no more than four (and
preferably no more than three) hours.
[0038] CAR T cells made in this way may be used in treating patients for
leukemia, e.g., acute lymphoblastic leukemia using procedures well
established in the art of clinical medicine and, in these cases, the CAR
may recognize CD19 or CD20 as a tumor antigen. The method may also be
used for solid tumors, in which case antigens recognized may include
CD22; RORI; mesothelin; CD33/IL3Ra; c-Met; PSMA; Glycolipid F77;
EGFRvIII; GD-2; NY-ESO-1; MAGE A3; and combinations thereof. With respect
to autoimmune diseases, CAR T cells may be used to treat rheumatoid
arthritis, lupus, multiple sclerosis, ankylosing spondylitis, type 1
diabetes or vasculitis.
[0039] In some embodiments, the target cells produced by the methods
described above will be available for administration to a patient earlier
than if the cells were generated using methods not including a DLD. These
cells may be administered 1 or more days earlier, and preferably 2, 3, 4,
5 or more days earlier. The cells may be administered within 8-10 days
from the time that obtaining of the crude fluid composition is completed.
[0040] Collection and Processing of Cells
[0041] The current invention is also directed to protocols for collecting
and processing cells from a patient which are designed to process cells
quickly, and which can generally be performed at sites where the cells
are collected. The protocols may be used as a part of the methods for
preparing target cells and CAR T cells described above. Aspects of some
of these protocols are illustrated in FIGS. 13 and 14 and may be
contrasted with the protocol shown in FIG. 12. In the particular
procedures illustrated, a composition obtained by apheresis of whole
blood is obtained and T cells in the composition are then selected. The
term "selected" in this context means that the T cells are bound by
agents that recognize the T cells with specificity (as defined above).
DLD is then used to isolate the selected T cells and transfer these cells
into a chosen fluid medium.
[0042] More generally, the invention concerns a method of collecting
target cells by: a) obtaining a crude fluid composition comprising the
target cells from a patient; and b) performing Deterministic Lateral
Displacement (DLD) on the crude fluid composition to obtain a composition
enriched in target cells wherein either before, or after DLD, the target
cells are bound to a carrier in a way that promotes DLD separation. For
example, a carrier may be used that has on its surface an affinity agent
(e.g., an antibody, activator, hapten or aptamer) that binds with
specificity (as defined above) to the target cells.
[0043] Carrier may, if desired, be bound to target cells during the time
that the cells are being collected from the patient and no more than five
hours (and preferably no more than four, three, two or one hour(s))
should elapse from the time that the obtaining of the crude fluid
composition comprising target cells is completed until the target cells
are bound to the carrier.
[0044] The diameter of the complex formed between target cells and one or
more carriers should preferably be at least 20% larger than the
uncomplexed cells and preferably at least 50% larger, at least twice as
large or at least ten times as large. This increase in size may be either
due to the binding of a single large carrier to the target cells or due
to the binding of several smaller carriers. Binding may involve using: i)
only carriers with a diameter at least as large (or in other embodiments,
at least twice as large or at least ten times as large) as that of the
target cells; ii) only carriers with a diameter no more than 50% (or in
other embodiments, no more than 25% or 15%) as large as that of the
target cells; or iii) mixtures of large and small carriers with these
size characteristics (e.g., there may be one group of carriers with a
diameter at least as large (or at least twice or ten times as large) as
the target cells and a second group of carriers with a diameter no more
than 50% (or no more than 25% or 15%) as large as that of the target
cells. Typically a carrier will have a diameter of 1-1000 .mu.m (and
often in the range of 5-600 or 5-400 .mu.m). Ideally the complexes would
be separated from other cells or contaminants by DLD on a microfluidic
device having an array of obstacles with a critical size lower than the
size of the complexes but higher than the size of uncomplexed cells or
contaminants.
[0045] In a preferred embodiment, the crude fluid composition comprising
target cells is obtained by performing apheresis or leukapheresis on
blood from the patient. This composition may include one or more
additives that act as anticoagulants or that prevent the activation of
platelets. Examples of such additives include ticlopidine, inosine,
protocatechuic acid, acetylsalicylic acid, and tirofiban alone or in
combination.
[0046] The microfluidic devices must have at least one channel extending
from a sample inlet to one or more fluid outlets, wherein the channel is
bounded by a first wall and a second wall opposite from the first wall.
There must also be an array of obstacles arranged in rows in the channel,
with each subsequent row of obstacles being shifted laterally with
respect to a previous row such that, when said crude fluid composition
comprising target cells is applied to an inlet of the device and
fluidically passed through the channel, target cells flow to one or more
collection outlets where an enriched product is collected and contaminant
cells, or particles that are in the crude fluid composition and that are
of a different size than the target cells flow to one more waste outlets
that are separate from the collection outlets.
[0047] In a particularly preferred embodiment, target cells are T cells
selected from the group consisting of: Natural Killer T cells; Central
Memory T cells; Helper T cells and Regulatory T cells, with Natural
Killer T cells being the most preferred. In alternative preferred
embodiments, the target cells are stem cells, B cells, macrophages,
monocytes, dendritic cells, or progenitor cells.
[0048] In addition to steps a) and b), the method of the invention may
include: c) genetically engineering cells by transducing them using a
viral vector. Alternatively, the cells may be transfected electrically,
chemically or by means of nanoparticles and/or expanded cells in number;
and/or d) treating the same patient from which the target cells were
obtained with the target cells collected. In addition, the collected
cells may be cultured and/or cryopreserved. In cases where the target
cells are T cells, culturing should generally be carried out in the
presence of an activator, preferably an activator that is bound to a
carrier. Among the factors that may be included in T cell cultures are
IL-2 and IL-15.
[0049] In some embodiments, the target cells produced by the methods
described above will be available for administration to a patient earlier
than if the cells were generated using methods not including DLD. These
cells may be administered 1 or more days earlier, and preferably 2, 3, 4,
5 or more days earlier. The cells may be administered within 8-10 days
from the time that obtaining of the crude fluid composition is completed.
[0050] In addition to the methods discussed above, the invention includes
the target cells produced by the methods and treatment methods in which
the target cells are administered to a patient.
[0051] Altering the Characteristics of Leukocytes Using DLD
[0052] Reducing the level of platelets in leukocyte preparations has
advantages both with respect to the making of CAR T cells and in
preparing leukocytes for other therapeutic uses. In this regard, the
present invention is based, in part, on the concept that DLD reduces the
total number of platelets in apheresis samples more effectively than
commonly used Ficoll separations (see FIGS. 19-21), especially when a
buffer is used that does not promote platelet aggregation (see FIGS.
22-23). When used in combination with separation based on magnetic beads
that specifically bind to T cells, DLD results in a preparation of cells
that can be expanded more rapidly than when such magnetic beads are used
either alone or in conjunction with Ficoll centrifugation (see FIG. 24).
This effect may be partly due to a reduction in platelet number and
partly due to factors that are independent of the number of platelets
present (see FIG. 24). In addition, the results obtained using the
DLD/magnetic bead procedure are more consistent (see FIG. 25) and the
expanded T cells from this procedure have a higher percentage of T cells
with a central memory phenotype when compared to populations prepared
using a Ficoll/magnetic bead approach (see FIG. 26). The higher initial
cell recovery from DLD combined with: a) a more rapid expansion of T
cells and b) a higher percentage of central memory cells, means that
therapeutically effective levels of T cells can be made available for
patients more rapidly.
[0053] In one aspect, the invention is directed to a method for decreasing
the ratio of platelets to leukocytes in an apheresis sample by performing
deterministic lateral displacement on the sample in the absence of
centrifugation or elutriation to obtain a product in which the ratio of
platelets to leukocytes is at least 20% (and preferably 50% or 70%) lower
than the ratio obtained when the same procedure is performed using
centrifugation (including gradient centrifugation or counterflow
centrifugation) or elutriation instead of DLD. Preferably, this procedure
includes no separation steps performed on the apheresis sample prior to
DLD and DLD is carried out in a buffer that does not comprise
intercalators or other means that alter the size of platelets and that
does not promote platelet aggregation. Agents that should be avoided
include dextran and other highly charge polymers. In addition to lowering
the ratio of platelets to leukocytes, the total number of platelets in
the DLD derived product should be at least 70% lower than in the
apheresis sample and preferably, at least 90% lower.
[0054] In another aspect, the invention is directed to a method for
purifying T cells from an apheresis sample by performing DLD on the
sample, followed by an affinity separation step and expansion of the T
cells by culturing in the presence of activator. This process should
result in a number of T cells that is at least twice as high as the
number produced by the same procedure performed using Ficoll
centrifugation instead of DLD. A preferred affinity method comprises the
use of magnetic beads that bind specifically to T cells by an antibody
that recognizes at least CD3, and might include CD3 together with CD28 or
other costimulatory molecules. The number of T cells obtained after 14
days in culture should be at least two times higher (and preferably at
least four or six time higher) than the number produced by the same
procedure performed using Ficoll centrifugation instead of DLD. The
percentage of memory T cells in the product produced by this method
should be at least 10% (and preferably at least 20%) higher than the
percentage produced using the same procedure but with Ficoll
centrifugation instead of DLD.
[0055] The method is especially well suited to the production of T cells
for CAR T cell therapy. The time needed to produce a sufficient number of
cells to treat a patient is reduced by at least 5% (and preferably at
least 10% or at least 20%) using DLD instead of Ficoll centrifugation and
the CAR T cells can be prepared without the need for freezing. In a
preferred method, cells are collected from a patient, processed by DLD
and, optionally, an affinity method at the same site. Genetic
transformation may also take place at the site and, preferably, no more
than one hour elapses from the time that apheresis is completed until DLD
is begun.
[0056] The invention also encompasses a method for decreasing the ratio of
platelets to leukocytes in an apheresis sample by performing
deterministic lateral displacement (DLD) on the sample. DLD is carried
out in the absence of centrifugation or elutriation, to obtain a product
in which the total number of platelets is at least 70% (and preferably at
least 90%) lower than in the apheresis sample. DLD should preferably be
carried out in a buffer that does not comprise intercalators and that
does not promote platelet aggregation. Preferably, the buffer does not
comprise dextran or other highly charge polymers.
[0057] The invention is not limited to leukocytes but also includes other
therapeutically valuable cells, especially cells that may be present in
apheresis preparations, including circulating stem cells. The benefits of
DLD, including benefits due to the removal unwanted platelets, should
apply to a wide variety of processes.
[0058] Methods of Using DLD for Large Volumes of Leukapheresis Material
[0059] One advantage of DLD is that it can be used to process small
quantities of material with little increase in volume as well as
relatively large quantities of material. The procedure may be used on
leukapheresis products that have a small volume due to the concentration
of leukocytes by centrifugation as well as in processing a large volume
of material.
[0060] Thus, in another aspect, the invention is directed to a system for
purifying cells from large volume leukapheresis processes in which at
least one microfluidic device is used that separates materials by DLD.
The objective is to obtain leukocytes that may be used therapeutically or
that secrete agents that may be used therapeutically. Of particular
importance, the invention includes binding specific types of leukocytes
to one or more carriers in a way that promotes and, optionally, also
complements DLD separation and then performing DLD on the complex. In
this way, specific types of leukocytes may be separated from cells that
are about the same size and that, in the absence of complex formation,
could not be resolved by DLD. In this regard, a two step procedure as
discussed above may sometimes be advantageous in which a one DLD
procedure separates unbound leukocytes from smaller material and a
another DLD procedure separates a carrier-leukocyte complex from
uncomplexed cells. Essentially the same technique can be used in other
contexts as well, e.g., on cultured cells, provided that cell specific
carriers are available. In all instances, the cells may be recombinantly
genetically engineered to alter the expression of one or more of their
genes.
[0061] For leukapheresis material, the microfluidic devices must have at
least one channel extending from a sample inlet to both a "collection
outlet" for recovering white blood cells (WBCs) or specific
leukocyte-carrier complexes and a "waste outlet" through which material
of a different size (generally smaller) than WBCs or uncomplexed
leukocytes flow. The channel is bounded by a first wall and a second wall
opposite from the first wall and includes an array of obstacles arranged
in rows, with each successive row being shifted laterally with respect to
a previous row. The obstacles are disposed in a manner such that, when
leukapheresis material is applied to an inlet of the device and
fluidically passed through the channel, cells or cell complexes are
deflected to the collection outlet (or outlets) where an enriched product
is collected and material of a different (generally smaller) size flows
to one or more separate waste outlets.
[0062] In order to facilitate the rapid processing of large volumes of
starting material, the obstacles in microfluidic devices may be designed
in the shape of diamonds or triangles and each device may have 6-40
channels. In addition, the microfluidic devices may be part of a system
comprising 2-20 microfluidic devices (see FIG. 7). Individual devices may
be operated at flow rates of 14 ml/hr but flow rates of at least 25 ml/hr
(preferably at least 40, 60, 80 or 100 ml per hour) are preferable and
allow large sample volumes (at least 200 ml and preferably 400-600 ml) to
be processed within an hour.
[0063] Separation of Viable Cells
[0064] In another aspect, the invention is directed to methods of
separating a viable cell from a nonviable cell comprising: (a) obtaining
a sample comprising the viable cell and the nonviable cell, where the
viable cell can have a first predetermined size and the nonviable cell
can have a second predetermined size; and where the first predetermined
size can be greater than or equal to a critical size, and the second
predetermined size can be less than the critical size; (b) applying the
sample to a device, where the device can comprise an array of obstacles
arranged in rows, where the rows can be shifted laterally with respect to
one another, where the rows can be configured to deflect a particle
greater than or equal to the critical size in a first direction and a
particle less than the critical size in a second direction; and (c)
flowing the sample through the device, where the viable cell can be
deflected by the obstacles in the first direction, and the non-viable
cell can be deflected in the second direction, thereby separating the
viable cell from the nonviable cell. The critical size can be about
1.1-fold greater than the second predetermined size and in some
embodiments, the viable cell can be an actively dividing cell. In some
embodiments, the device can comprise at least three zones with
progressively smaller obstacles and gaps.
[0065] Separation of Adherent Cells
[0066] The invention also includes a method of obtaining adherent target
cells, preferably cells of therapeutic value, e.g., adherent stem cells,
by: a) obtaining a crude fluid composition comprising the adherent target
cells from a patient; and b) performing Deterministic Lateral
Displacement (DLD) to obtain a composition enriched in the adherent
target cells. During this process, the adherent target cells may be bound
to one or more carriers in a way that promotes or complements DLD
separation. For example carriers may have on their surface an affinity
agent (e.g., an antibody, activator, hapten or aptamer) that binds with
specificity (as defined above) to the adherent target cells and may be
transfected or transduced with nucleic acids designed to impart on the
cells a desired phenotype, e.g., to express a chimeric molecule
(preferably a protein that makes the cells of greater therapeutic value).
[0067] Carriers may be added at the time that the crude fluid composition
is being collected or, alternatively after collection is completed but
before DLD is performed for the first time. In a second alternative, DLD
may be performed for a first time before carrier is added. For example,
if the adherent cell has a size less than the critical size, the crude
fluid composition may be applied to the device before the carrier is
added, the adherent cell may be recovered, the cells may then be attached
to one or more carriers to form a complex that is larger than the
critical size of a device, a second DLD step may then be performed and
the carrier adherent cell complexes may be collected.
[0068] Preferably, no more than three hours (and more preferably no more
than two hours, or one hour) elapse from the time that the obtaining of
the crude fluid composition from the patient is completed until the
adherent cell is bound to a carrier for the first time. In another
preferred embodiment, no more than four hours (and preferably no more
than three or two hours) elapse from the time that the obtaining of the
crude fluid composition from the patient is complete until the first time
that the adherent cell or a carrier adherent cell complex is collected
from the device for the first time.
[0069] The methodology described above may be used to separate adherent
target cells, e.g., adherent stem cells, from a plurality of other cells.
The method involves: a) contacting a crude fluid composition comprising
the adherent target cells and the plurality of other cells, wherein the
adherent target cells are at least partially associated with one or more
carriers in a way that promotes DLD separation and form carrier
associated adherent target cell complexes, wherein the complexes comprise
an increased size relative to the plurality of other cells, and wherein
the size of the carrier associated adherent cell complexes is preferably
at least 50% greater than a critical size, and other, uncomplexed cells
comprise a size less than the critical size; b) applying the crude fluid
composition containing the carrier associated adherent cell complexes to
a device, wherein the device comprises an array of obstacles arranged in
rows, wherein the rows are shifted laterally with respect to one another,
wherein the rows are configured to deflect cells or complexes greater
than or equal to the critical size in a first direction and cells or
complexes less than the critical size in a second direction; c) flowing
the crude fluid composition comprising the carrier associated adherent
target cell complexes through the device, wherein the complexes are
deflected by the obstacles in the first direction, and uncomplexed cells
are deflected in the second direction, thereby separating the carrier
associated adherent cell complexes from the other uncomplexed cells; d)
collecting a fluid composition comprising the separated carrier
associated adherent target cell complexes.
[0070] The diameter of the complex formed between adherent target cells
and one or more carriers should preferably be at least 20% larger than
the uncomplexed cells and preferably at least 50% larger, at least twice
as large or at least ten times as large. This increase in size may be
either due to the binding of a single large carrier to the adherent
target cells or due to the binding of several smaller carriers. Binding
may involve using: a) only carriers with a diameter at least as large (or
in other embodiments, at least twice as large or at least ten times as
large) as that of the adherent target cells; b) only carriers with a
diameter no more than 50% (or in other embodiments, no more than 25% or
15%) as large as that of the adherent target cells; or c) mixtures of
large and small carriers with these size characteristics (e.g., there may
be one group of carriers with a diameter at least as large (or at least
twice or ten times as large) as the adherent target cells and a second
group of carriers with a diameter no more than 50% (or no more than 25%
or 15%) as large as that of the adherent target cells. Typically a
carrier will have a diameter of 1-1000 .mu.m (and often in the range of
5-600 or 5-400 .mu.m).
[0071] The carriers may be made of any of the materials that are known in
the art for the culturing of adherent cells including polypropylene,
polystyrene, glass, gelatin, collagen, polysaccharides, plastic,
acrylamide and alginate. They may be uncoated or coated with materials
that promote adhesion and growth (e.g., serum, collagen, proteins or
polymers) and may have agents (e.g., antibodies, antibody fragments,
substrates, activators or other materials) attached to their surfaces. In
some embodiments, the diluent can be growth media, the steps can be
performed sequentially and, after step (d), buffer exchange can be
performed.
[0072] Examples of specific adherent cells that may be isolated in the
methods described above include: an MRC-5 cell; a HeLa cell; a Vero cell;
an NIH 3T3 cell; an L929 cell; a Sf21 cell; a Sf9 cell; an A549 cell; an
A9 cell; an AtT-20 cell; a BALB/3T3 cell; a BHK-21 cell; a BHL-100 cell;
a BT cell; a Caco-2 cell; a Chang cell; a Clone 9 cell; a Clone M-3 cell;
a COS-1 cell; a COS-3 cell; a COS-7 cell; a CRFK cell; a CV-1 cell; a
D-17 cell; a Daudi cell; a GH1 cell; a GH3 cell; an HaK cell; an HCT-15
cell; an HL-60 cell; an HT-1080 cell; a HEK cell, HT-29 cell; an HUVEC
cell; an I-10 cell; an IM-9 cell; a JEG-2 cell; a Jensen cell; a Jurkat
cell; a K-562 cell; a KB cell; a KG-1 cell; an L2 cell; an LLC-WRC 256
cell; a McCoy cell; a MCF7 cell; a WI-38 cell; a WISH cell; an XC cell; a
Y-1 cell; a CHO cell; a Raw 264.7 cell; a HEP G2 cell; a BAE-1 cell; an
SH-SY5Y cell, and any derivative thereof.
[0073] Separation of Cells Bound to an Activator
[0074] The invention also includes methods of purifying cells capable of
activation using the procedures described above. In a preferred
embodiment, the invention is directed to a method of separating an
activated cell from a plurality of other cells by: a) contacting a crude
fluid composition comprising a cell capable of activation and the
plurality of other cells with one or more carriers, in a way that
promotes DLD separation, wherein one or more of the carriers comprise a
cell activator, wherein one or more carriers are at least partially
associated with the cell capable of activation by the cell activator upon
or after contact to generate a carrier associated cell, wherein the
association of the cell activator with the cell capable of activation at
least partially activates the cell capable of activation, wherein the
carrier associated cell complex comprises an increased size relative to
other cells, and wherein a size of the carrier associated cell complex is
greater than or equal to a critical size, and the cells in the plurality
of other cells comprise a size less than the critical size; b) applying
the crude fluid composition to a device, wherein the device comprises an
array of obstacles arranged in rows; wherein the rows are shifted
laterally with respect to one another, wherein the rows are configured to
deflect a particle greater than or equal to the critical size in a first
direction and a particle less than the critical size in a second
direction; c) flowing the sample through the device, wherein the carrier
associated cell complex is deflected by the obstacles in the first
direction, and the cells in the plurality of other cells are deflected in
the second direction, thereby separating the activated cell from the
other cells of the plurality. The fluid composition comprising the
separated carrier associated cell complex may then be collected. During
this process the cells may optionally be transfected or transduced with
nucleic acids designed to impart on the cells a desired phenotype, e.g.,
to express a chimeric molecule (preferably a protein that makes the cells
of greater therapeutic value).
[0075] The cell capable of activation may be selected from the group
consisting of: a T cell, a B cell, a macrophage, a dendritic cell, a
granulocyte, an innate lymphoid cell, a megakaryocyte, a natural killer
cell, a thrombocyte, a synoviocyte, a beta cell, a liver cell, a
pancreatic cell; a DE3 lysogenized cell, a yeast cell, a plant cell, and
a stem cell.
[0076] The cell activator may be selected from the group consisting of: an
antibody or antibody fragment, CD3, CD28, an antigen, a helper T cell, a
receptor, a cytokine, a glycoprotein, and any combination thereof. In
other embodiments, the activator may be a small compound and may be
selected from the group consisting of insulin, IPTG, lactose,
allolactose, a lipid, a glycoside, a terpene, a steroid, an alkaloid, and
any combination thereof.
[0077] In a preferred embodiment, the cell capable of activation is
collected from a patient as part of a crude fluid composition comprising
the cell capable of activation and a plurality of other cells, wherein no
more than four hours (and preferably no more than three hours, two hours
or one hour) elapse from the time that the obtaining of the crude fluid
composition from the patient is completed until the cell capable of
activation is bound to the carrier. It is also preferable that no more
than four hours elapse from the time that the obtaining of the crude
fluid composition from the patient is completed until step c) is
completed. Alternatively, the method may be altered by binding activator
before collection of cells begins.
[0078] Preferably, the diameter of the complex formed between a cell
capable of activation and one or more carriers should be at least 20%
larger than the uncomplexed cells and more preferably at least 50%
larger, at least twice as large or at least ten times as large. This
increase in size may be either due to the binding of a single large
carrier to the cell capable of activation or due to the binding of
several smaller carriers. Binding may involve using: a) only carriers
with a diameter at least as large (or in other embodiments, at least
twice as large or at least ten times as large) as that of the cell
capable of activation; b) only carriers with a diameter no more than 50%
(or in other embodiments, no more than 25% or 15%) as large as that of
the cell capable of activation; or c) mixtures of large and small
carriers with these size characteristics (e.g., there may be one group of
carriers with a diameter at least as large (or at least twice or ten
times as large) as the cell capable of activation and a second group of
carriers with a diameter no more than 50% (or no more than 25% or 15%) as
large as that of the cell capable of activation. Typically a carrier will
have a diameter of 1-1000 .mu.m (and often in the range of 5-600 or 5-400
.mu.m).
[0079] Separating Compounds from Cells
[0080] In another embodiment, the invention includes methods of removing a
compound from a cell comprising: (a) obtaining a fluid composition
comprising the cell and the compound, where the cell has a predetermined
size that is greater than a predetermined size of the compound, and where
the predetermined size of the cell is greater than or equal to a critical
size, and the predetermined size of the compound is less than the
critical size; (b) applying the sample to a device, where the device
comprises an array of obstacles arranged in rows, where the rows are
shifted laterally with respect to one another, where the rows are
configured to deflect a particle greater than or equal to the critical
size in a first direction and a particle less than the critical size in a
second direction; and (c) flowing the sample through the device, during
which the cell is deflected by the obstacles in the first direction, and
the compound can be deflected in the second direction, thereby removing
the compound from the cell. In some embodiments, the method can further
comprise culturing the cell after step (c) or recycling the cells to a
culture from which the fluid composition of step a) was obtained.
[0081] The compound may be a toxic compound and may be selected from the
group consisting of: an antibiotic, an antifungal, a toxic metabolite,
sodium azide, a metal ion, an endotoxin, a plasticizer, a pesticide, and
any combination thereof. In other embodiments, the compound can be a
spent chemical component.
[0082] Continuous Purification of a Secreted Cellular Product
[0083] The invention also includes methods of continuously purifying a
secreted product from a cell comprising: (a) obtaining a fluid
composition comprising the cell (which may be a cell culture
composition), where the cell is suspended in the fluid composition (or
the cell is bound to one or more carriers in a way that promotes DLD
separation and that forms a carrier-cell complex) and where the cell
secretes the secreted product into the fluid composition, where the cell
(or the carrier-cell complex) has a predetermined size that is greater
than a predetermined size of the secreted product, and where the
predetermined size of the cell (or the carrier-cell complex) is greater
than or equal to a critical size, and the predetermined size of the
secreted product is less than the critical size; (b) applying the fluid
composition comprising the cell (or the carrier-cell complex) to a device
for DLD, where the device comprises an array of obstacles arranged in
rows; where the rows are shifted laterally with respect to one another,
where the rows are configured to deflect a particle greater than or equal
to the critical size in a first direction and a particle less than the
critical size in a second direction; (c) flowing the fluid composition
comprising the cell or the carrier-cell complex through the device, where
the cell or carrier-cell complex is deflected by the obstacles in the
first direction, and the secreted product is deflected in the second
direction, thereby separating the secreted product from the cell; (d)
collecting the secreted product, thereby producing a fluid composition of
the secreted product that is purified; (e) collecting a recovered fluid
composition comprising the separated cells or carrier-cell complexes; (f)
re-applying the cells (or the carrier-cell complexes) to the fluid
composition; and repeating steps (a) through (e); thereby continuously
purifying the secreted product from the cell.
[0084] The secreted product can be a protein, an antibody, a biofuel, a
polymer, a small molecule, and any combination thereof and the cell can
be a bacterial cell, an algae cell, a mammalian cell, and a tumor cell.
In one preferred embodiment, the secreted product is a therapeutically
valuable protein, antibody, polymer or small molecule. In addition the
fluid composition of step a) may be obtained from a culture in which
cells are grown on carriers.
[0085] Use of Microfluidic Sizing Devices
[0086] More broadly, the invention is directed to methods of engineering a
population of target cells prepared by any size based microfluidic
separation method. Differences in sorting cells based on size may be the
result of using bump arrays as discussed herein or result from inertial
forces generated by controlling the flow rate during separations or
through the design of the microfluidic devices themselves (see U.S. Pat.
Nos. 9,895,694 and 9,610,582, incorporated herein by reference in their
entirety). There may be only a single separation procedure used or there
may be more than one. For example, target cells may be separated from
smaller particles and cells using one microfluidic procedure and from
larger particles and cells using a second procedure.
[0087] Once target cells are isolated, they are genetically engineered to
have a desired phenotype. This may be accomplished using standard
recombinant methodology for transfecting or transforming cells. For
example, cells may be transfected with a vector to express a recombinant
phenotype. By avoiding centrifugation prior to genetic engineering, there
should be at least a 20% increase in cells with the desired
characteristics.
[0088] Preferred target cells are leukocytes (especially T cells) or stem
cells and the preferred crude fluid composition is blood or an apheresis
preparation obtained from a patient. A central objective is to reduce the
ratio of platelets to target cells in these preparations by at least 50%
and preferably by at least 80% or 90%. The isolation of target cells
should take place under conditions such that a product is obtained in
which the total number of platelets is at least 70% (and preferably at
least 90%) lower than in the starting apheresis preparation.
[0089] During or after genetic engineering, cells are expanded in cell
culture. Using the procedures described above, the number of T cells
obtained after 14 days in culture should be at least two times (and
preferably at least five or ten times) higher than in a procedure in
which cells are isolated using centrifugation. In addition, the
percentage of memory T cells in culture relative to the total number of T
cells should be at least 10% (and preferably 20% or 30%) higher than in a
procedure in which T cells are isolated by centrifugation.
[0090] No more than one hour should elapse from the time that apheresis
collection is completed until the time that DLD is performed and no more
than four hours should elapse from the time the obtaining of the
apheresis sample is completed until the target cells have been isolated
and are genetically engineered.
[0091] In a particularly preferred embodiment, the method described above
is used for the production of CAR T cells. This involves first obtaining
a crude fluid composition containing T cells by apheresis and then
isolating the cells on a microfluidic device using one or more procedures
that separate T cells from platelets based on differences in size. As a
result, a product should be obtained that is an enriched in T cells and
depleted in platelets. In the next step, the isolated T cells are
genetically engineered to express chimeric antigen receptors (CARs) on
their surface. These cells are cultured to expand their number and then
collected. The T cells should not be centrifuged or elutriated at any
step prior to being genetically engineered and, in a preferred
embodiment, reagents used for genetic engineering are separated from
cells by size using a microfluidic device. In an additional preferred
embodiment, T cells are collected by being transferred into a
pharmaceutical composition for administration to a patient.
[0092] T cells should not be frozen before being collected or transferred
into a pharmaceutical composition and preferably at least 90% of
platelets are removed. Prior to, or during, culturing, cells may be
exposed to a T cell activator or a carrier. This may help to stabilize
the cells and may also facilitate size-based microfluidic separation. It
should be noted however, that neither activators nor the carriers
necessarily need to be bound to magnetic beads or particles.
[0093] Compared to a procedure in which cells are isolated or concentrated
by centrifugation, CAR T cells obtained by microfluidic separation should
be available for use by a patient at least one day (and preferably, at
least 3, 5 or 10 days) earlier. Overall, the time necessary to produce a
sufficient number of CAR T cells for treatment should be at least 10%
(and preferably at least 20% or 30%) shorter than when the same method is
carried out using Ficoll centrifugation to isolate cells. This is partly
because, by using a size based microfluidic separation, the number of CAR
T cells obtained after 14 days in culture will typically be at least two
times (and preferably four or eight times) higher than the number in
cultures which use cells obtained by Ficoll centrifugation. In addition,
when cells prepared by the present method are administered to a patient,
they should exhibit at least 10% less senescence than cells isolated from
an apheresis composition by centrifugation.
[0094] The present invention also encompasses treating a patient for a
disease or condition by administering a therapeutically effective amount
of cells prepared by the methods discussed above. This includes any
disease or condition that responds to engineered leukocytes or stem cells
and, at least in the case of CART cells, cancer is among the diseases
that may be treated.
BRIEF DESCRIPTION OF THE DRAWINGS
[0095] FIGS. 1A-1G: FIGS. 1A-1C illustrate different operating modes of
DLD. This includes: i) Separation (FIG. 1A), ii) Buffer Exchange (FIG.
1B) and iii) Concentration (FIG. 1C). In each mode, essentially all
particles above a critical diameter are deflected in the direction of the
array from the point of entry, resulting in size selection, buffer
exchange or concentration as a function of the geometry of the device. In
all cases, particles below the critical diameter pass directly through
the device under laminar flow conditions and subsequently off the device.
FIG. 1D shows a 14 lane DLD design used in separation mode. The full
length of the depicted array and microchannel is 75 mm and the width is
40 mm, each individual lane is 1.8 mm across. FIGS. 1E-1F are enlarged
views of the plastic diamond post array and consolidating collection
ports for the exits. FIG. 1G depicts a photo of a leukapheresis product
being processed using a prototype device at 10 PSI.
[0096] FIGS. 2A-2H: FIG. 2A is a scatter plot showing the range of normal
donor platelet and WBC cell counts used in this study. Mean counts of
WBC: 162.4.times.10.sup.6/mL and Platelets: 2718.times.10.sup.3/.mu.L
respectively (+). The outlier sample (.tangle-solidup.), clogged the 20
.mu.m prefilter and was excluded from the data set. Input sample shown
(FIGS. 2C and 2D). Representative 24-hour old normal donor leukapheresis
input (FIG. 2B) and PBMC product processed by either a 14-lane diamond
post DLD at 10 PSI (FIG. 2E) or Ficoll-Hypaque (FIG. 2F). Representative
DLD product (FIG. 2G) and Ficoll (FIG. 2H) from the same Leukapheresis
donor (#37). Input (FIGS. 2B, 2C, 2D) and product fractions (FIGS. 2E and
2F) were fixed and stained on slides with CD41-FITC (platelets) plus
CD45-Alexa647 (WBC) and counter-stained with DAPI (nuclear DNA).
[0097] FIG. 3: This figure concerns the consistency of cell activation in
DLD vs. Ficoll and Direct Magnet approaches (CD4, CD8 vs CD25 Day 8).
Cell activation and Phenotypic profile shows a shift during expansion
towards classic central memory T cell associated phenotype (Day 8). Cells
were counted and de-beaded as described previously. At each time point
100,000 cells were stained with CD3-BV421, CD45RA-BV605, CD95-FITC,
CD279-PE, CD25-APC, CD4-Alexa 700, and CD8-APC-Cy7, incubated for 30 at
room temperature in the dark and washed with 10 volumes of PBS prior to
centrifugation and fixation in 1.0% Para-formaldehyde in PBS. Samples
were acquired on a BD FACSAria, and analyzed using a CD3 and forward and
side scatter gate using FlowLogic software.
[0098] FIG. 4: FIG. 4 is a graph depicting rapid gain of memory cell
phenotype and consistent activation of samples via DLD compared to Ficoll
& Direct Magnet. Plot of % CD45RA-, CD25+ cells measuring conversion to T
cell activation and conversion via CD45 RO is status shown. Cells were
fed 200 Units IL-2/mL culture at Day 3 and again at day 8 only as the
experiment was designed to address initial ability to expand.
[0099] FIGS. 5A-5C: These figures concern the Fold Expansion of CD3 cells
(.times.10.sup.6) from DLD, Ficoll and Direct Magnet. Aliquots of DLD
product and Ficoll cells were incubated with CD3/CD28 beads following
Thermo-Fisher CTS protocol using a T cell density of 1.times.10.sup.7 T
cells/mL. A ratio of .about.2.5 Beads/T cell and for the Direct Magnet
using .about.5.0 Beads/T cell was used, cells and beads were incubated on
a rotary mixer for 60 min prior to magnetic separation. Either stimulated
or unstimulated (unseparated PMBC) cells were diluted in complete media
(RPMI-1640+10% FBS+antibiotics without IL-2) to 0.5.times.10.sup.6/mL and
were plated in time point specific reactions to avoid any disturbance of
the cultures at intermediate time points. On Day 3, 200 IU of IL-2/mL was
added to the stimulated and separated arm per manufacturer's
recommendation. Cell counts were determined on Day 3, 8, 15 after
de-beading using manufacturers protocol (pipetting) by Coulter count
(Scepter) and verified by bead based absolute counting using flow
cytometry on a BD FACSCalibur using a no-wash approach with a
fluorescence threshold on CD45 and staining with CD3-FITC, CD45-PerCP and
using the DNA stain DRAQ5 to ensure effective discrimination of doublets
and any cells with beads still attached. Correlation between counting
methods was acceptable with a slope of 0.95, R2=0.944. Media was added to
the cultures to maintain cell densities in an acceptable range
(<3.0.times.10.sup.6/mL) on Days 6, and 9. Day 15 data point for the
donor 21 was lost due to contamination. Averages or % CV's shown in
horizontal bars as indicated (FIG. 5A). FIG. 5B shows the percentage of T
central memory cells (day 15) and FIG. 5C shows the number of T central
memory cells (day 15).
[0100] FIG. 6A-6B: FIGS. 6A-6B concern cytometric analysis of T central
memory cells and the number of central memory cells produced. FIG. 6A: T
Central Memory Cells: CD3+ T cells were gated on a singlet gate followed
by a CD3 v Side scatter and central memory phenotyping using 4 parameter
gate of CD45RO, CCR7, CD28 and CD95 to define the central memory
population. The population was back gated to display central memory
cells, which in a color figure are red, as fraction of T cells. Non-red
cells in a color figure represent all non central memory T cells. FIG.
6B: Phenotype Conversion and Key Metrics (Day 15): Key metrics show # of
donors where the number of central memory cells is >50%, with the
average and % CV associated with the central memory expansion.
[0101] FIG. 7: FIG. 7 is a schematic showing how current individual chips
have been designed to be stackable in layers to achieve throughput as
demanded by any particular application using established manufacturing
approaches. Injection molded layers are planned as systems are developed.
[0102] FIGS. 8A-8C: These are supplemental figures showing the
concentration of WBC via DLD. FIG. 8A: DLD Product Derived from Whole
Blood: Whole blood was passed over first DLD to remove erythrocytes. A
second, in line, concentrating DLD, designed to achieve a concentration
factor of 12, was connected to the product output of the separating DLD.
Equal volumes of product and waste were added to tubes with equal numbers
of absolute count beads and analyzed by flow cytometry. The resulting
relative cell:bead ratio for Waste (FIG. 8B) and for Concentrate (FIG.
8C) was calculated compared to the input material to determine fold
concentration. Leukocytes were stained with CD45 PerCP and 1 mM DRAQ5,
which was used as a fluorescence threshold to acquire both the beads and
the leukocytes. 5000 bead events acquired. (all reagents eBioscience).
Designed Concentration Factor: 12.0x; Observed relative concentration:
15.714/1.302=12.07x
[0103] FIG. 9: FIG. 9 is a supplemental figure on the expression of CD25
and CD4 on unstimulated CD3+ T Cells purified by either DLD or Ficoll
methods (Day 8). Cells were prepared as described and analyzed as in FIG.
3. Mean CD4+25+: Ficoll: 20.25%; DLD: 8%.
[0104] FIG. 10: This is a supplemental figure on the allocation of IL-2
expanded central memory T cells by major subsets. In the original
figures: CD8 (Green), CD4 (Blue), CD4+CD8+(Red) Central memory cells were
sequentially gated: CD3+, CD45RO+CCR7+, CD28+CD95+. Relative abundance of
CD4 subset driven by IL2 is evident.
[0105] FIG. 11: FIG. 11 is a supplemental figure depicting estimates of
the number of central memory T cells, post expansion with IL-2, assuming
yields in this study and a typical leukapheresis harvest from a donor
with 50.times.10.sup.6 WBC cells per/mL and containing 50% CD3
lymphocytes in 250 mL.
[0106] FIG. 12: FIG. 12 illustrates a protocol that might, in principle,
be used for producing CAR T cells and administering the cells to a
patient. It has been included to contrast other procedures discussed
herein and does not represent work actually performed.
[0107] FIG. 13: FIG. 13 illustrates a proposed protocol for producing CAR
T cells that differs from the protocol of FIG. 12 in the initial steps of
the procedure. The steps in the center portion of the figure are included
for purposes of comparison. The diagram is intended to illustrate
inventive concepts and does not represent work actually performed.
[0108] FIG. 14: FIG. 14 illustrates a second proposed protocol for
producing CAR T cells that differs from the protocol of FIG. 12 in the
initial steps of the procedure. The steps in the center portion of the
figure are included for purposes of comparison. As with FIGS. 12 and 13,
the diagram is intended to illustrate inventive concepts and does not
represent work actually performed.
[0109] FIG. 15: FIG. 15 shows a schematic of a device for removing
secreted products from spent cells.
[0110] FIG. 16: FIG. 16 shows a schematic of a device for continuous
removal of toxic compounds from actively growing cells.
[0111] FIG. 17: FIG. 17 shows a schematic of a device for continuous
removal of toxic compounds from actively growing cells with the option of
adding carriers between each iteration.
[0112] FIGS. 18A and 18B: FIG. 18 A shows an example of a mirrored array
of obstacles with a downshift. A central channel is between an array of
obstacles on the left and on the right. The central channel can be a
collection channel for particles of at least a critical size (i.e.,
particles of at least a critical size can be deflected by the arrays to
the central channels, whereas particles of less than the critical size
can pass through the channel with the bulk flow). By downshifting rows,
changes in the width of the channel relative to a mirrored array with a
downshift can be achieved. The amount of downshift can vary based on the
size and/or cross-sectional shape of the obstacles. FIG. 18B illustrates
a mirrored array of obstacles with no downshift. An array on the left and
an array on the right can deflect particles of at least a critical size
to the central channel.
[0113] FIGS. 19A and 19B: These figures show the platelets remaining in an
apheresis sample processed using DLD (FIG. 19A) and the platelets
remaining in an apheresis sample processed using Ficoll centrifugation
(FIG. 19B). The platelets are the smaller, brighter cells (some of which
are shown by arrows) and leukocytes are the larger darker cells. It can
be seen that the relative number of platelets is substantially lower in
cells processed by DLD.
[0114] FIG. 20: FIG. 20 graphically shows the percentage of platelets left
in apheresis samples processed by Ficoll centrifugation (white bars) and
apheresis samples processed by DLD (striped bars) using three different
buffers. The X axis is the percentage of platelets remaining. Results for
a 1% BSA/PBS buffer are shown on the far left side of the figure;
percentages for a buffer containing an F127 poloxamer intercalator are in
the center and percentages for an elutriation buffer are on the far
right.
[0115] FIG. 21: FIG. 21 shows the results of FIG. 22 except that the Y
axis represents the number of platelets per leukocyte, i.e., the ratio of
platelets to leukocytes. Bars with triplet stripes represent the ratio
present in the initial apheresis sample, white bars are the ratio after
processing by Ficoll centrifugation and bars with widely spaced single
stripes are the ratio after processing by DLD.
[0116] FIG. 22: FIG. 22 is a bar graph showing the expansion of T cells
that occurs as the result of processing apheresis samples by Ficoll
centrifugation (solid white bars) and DLD (bars with single stripes
adjacent to white bars). Moving from left to right the bars show the
effect of adding 10% (bars with double stripes), 50% (stippled bars) and
100% (bars with dark widely spaced bars adjacent to stippled bars) of the
platelets originally present in the apheresis starting material back to
the cells processed by DLD before expansion. The left side of the figure
depicts cells processed via DLD without EDTA and then stimulated with
CD3/CD28 at day 0, with CD3 positive cells counted on day 3, 7 and 14.
The right side of the figure shows cells processed with 2 mM EDTA and
then stimulated with CD3/CD28 at day 0, with CD3 positive cells counted
on day 3, 7 and 14.
[0117] FIG. 23: FIG. 23 is a comparison of variability in the expansion of
T cells obtained using three methods of processing: separation using
magnetic beads alone (bar on the far left of the figure), separation
using Ficoll centrifugation followed by magnetic beads (center bar), and
separation by DLD followed by magnetic beads (bar on the far right).
[0118] FIG. 24: FIG. 24 shows the percentage of cells present in the
expanded T cell populations of FIG. 23 that are central memory T cells.
[0119] Overview of Workflow Figures: FIGS. 25-32 are all included to
illustrate concepts and do not concern any experiments actually
performed. The figures illustrate the basic workflow typically involved
in obtaining cells from a patient which are then processed and used
therapeutically, typically to treat the same patient from which the cells
were obtained. The figures, for the most part, refer specifically to the
making of CAR T cells but it will be understood that the processes can be
applied generally to the making of leukocytes and other cells for
therapeutic purposes. In the case of many such processes, including those
for CAR T cells, the cells collected from patients are recombinantly
engineered to express genes of therapeutic interest. Any type of such
engineering may be a part of the illustrated processes. The figures
divide steps into those that are performed at a clinic and those that are
performed at a separate site, which for the purposes of illustration has
been termed a "manufacturing site." FIG. 25 shows the workflow that might
be expected for a typical process used, for example, in making CAR T
cells. Transformation of the cells would typically occur at the
manufacturing site after cryopreserved samples are thawed, washed,
debulked and enriched. FIG. 26 shows several places (stippled boxes) in a
prototype CAR T cell process where DLD might be used while maintaining
the same basic workflow. FIGS. 27-32 then present a number of
illustrative examples of how DLD may be used to modify workflow and
improve the protoype process. Primary objectives are to produce cells of
better overall quality, shorten the time needed to obtain a sufficient
number of cells to treat a patient and to automate steps that are
currently more labor intensive.
[0120] FIG. 25: FIG. 25 illustrates the basic workflow generally involved
in current methods for processing cells from a patient for therapeutic
use. As illustrated, the process starts at a clinic with the collection
of cells ("apheresis collection"), proceeds to a manufacturing site where
the cells are expanded and may be engineered, and ends back at the clinic
with the administration of the cells to a patient ("Re-infusion").
[0121] FIG. 26: FIG. 26 shows several steps in the CAR T workflow where
DLD could be used (stippled boxes). In this example, the basic workflow
remains essentially unchanged.
[0122] FIG. 27: FIG. 27 illustrates that DLD may be used immediately after
apheresis to clean up cells prior to freezing (stippled box). The
advantages of using DLD at this point are that it removes platelets from
cells before shipment and thereby eliminates deleterious effects from
platelet activation, clot formation, any storage related degranulation
and overall loss of cells. In this manner, DLD improves the quality of
cell compositions being shipped and replaces one of the hands on labor
steps in the process with an automated counterpart.
[0123] FIG. 28: In this scenario, DLD is used during early steps in which
cells are cleaned up, activated, DLD separated and transfer into a
desired medium, e.g., a growth medium (stippled boxes). Importantly,
freezing and cryopreservation at the clinic and subsequent thawing at the
manufacturing site are avoided which should reduce the time needed to
complete the process, e.g., by about 2-3 days. Although not preferred, a
cryopreservation step could, if desired, still be performed. Other steps
that might be avoided are indicated by crossed through text.
[0124] FIG. 29: In this scenario, DLD is used in conjunction with magnetic
beads that bind to cell surface CD3 on T cells. This scenario has the
same advantage of reducing platelets described above but may be used to
activate cells and begin growth much sooner than in the prototype
procedure. Cells may also be shipped at warm temperatures compatible with
growth. As a result, counterpart steps that would have been performed at
the manufacturing site are eliminated and overall processing time is
correspondingly reduced.
[0125] FIG. 30: The most important difference in the scenario shown in
FIG. 30 compared to the scenario in FIG. 29 is that cells are genetically
transformed with virus immediately after exposure to activator (see
stippled boxes). Rather than several days elapsing before activation and
transformation, cells are purified, activated, transformed and growing in
culture medium within 24 hours after apheresis collection is completed.
Preferably the time from the completion of apheresis until exposure to
vector should be no more than 12 hours, and more preferably, no more than
six or three hours. Most preferably, exposure would occur within two
hours after apheresis is complete and, in all cases, before cells are
frozen. The figure shows many processing steps at the manufacturing site
that may be eliminated and it is expected that the time needed to obtain
sufficient cells for treating a patient would be reduced by at least 3 or
4 days.
[0126] FIG. 31: The scenario shown in FIG. 31 is similar to that in FIG.
30 except that separation by an affinity procedure (exemplified by
magnetic beads that recognize CD3 containing cells) is added in the early
steps to help in the isolation of T cells. DLD is also used to purify
and/or concentrate cells.
[0127] FIG. 32: In the scenario shown in FIG. 32, T cells collected by
apheresis are purified, engineered, expanded and concentrated for
reinfusion without ever undergoing a step in which they are frozen. All
steps can, if desired, be performed at the site where cells are collected
without a need for shipping.
DEFINITIONS
[0128] Apheresis: As used herein this term refers to a procedure in which
blood from a patient or donor is separated into its components, e.g.,
plasma, white blood cells and red blood cells. More specific terms are
"plateletpheresis" (referring to the separation of platelets) and
"leukapheresis" (referring to the separation of leukocytes). In this
context, the term "separation" refers to the obtaining of a product that
is enriched in a particular component compared to whole blood and does
not mean that absolute purity has been attained.
[0129] CAR T cells: The term "CAR" is an acronym for "chimeric antigen
receptor." A "CAR T cell" is therefore a T cell that has been genetically
engineered to express a chimeric receptor.
[0130] CAR T cell therapy: This term refers to any procedure in which a
disease is treated with CAR T cells. Diseases that may be treated include
hematological and solid tumor cancers, autoimmune diseases and infectious
diseases.
[0131] Carrier: As used herein, the term "carrier" refers an agent, e.g.,
a bead, or particle, made of either biological or synthetic material that
is added to a preparation for the purpose of binding directly or
indirectly (i.e., through one or more intermediate cells, particles or
compounds) to some or all of the compounds or cells present. Carriers may
be made from a variety of different materials, including DEAE-dextran,
glass, polystyrene plastic, acrylamide, collagen, and alginate and will
typically have a size of 1-1000 .mu.m. They may be coated or uncoated and
have surfaces that are modified to include affinity agents (e.g.,
antibodies, activators, haptens, aptamers, particles or other compounds)
that recognize antigens or other molecules on the surface of cells. The
carriers may also be magnetized and this may provide an additional means
of purification to complement DLD and they may comprise particles (e.g.,
Janus or Strawberry-like particles) that confer upon cells or cell
complexes non-size related secondary properties. For example the
particles may result in chemical, electrochemical, or magnetic properties
that can be used in downstream processes, such as magnetic separation,
electroporation, gene transfer, and/or specific analytical chemistry
processes. Particles may also cause metabolic changes in cells, activate
cells or promote cell division.
[0132] Carriers that bind "in a way that promotes DLD separation": This
term, refers to carriers and methods of binding carriers that affect the
way that, depending on context, a cell, protein or particle behaves
during DLD. Specifically, "binding in a way that promotes DLD separation"
means that: a) the binding must exhibit specificity for a particular
target cell type, protein or particle; and b) must result in a complex
that provides for an increase in size of the complex relative to the
unbound cell, protein or particle. In the case of binding to a target
cell, there must be an increase of at least 2 .mu.m (and alternatively at
least 20, 50, 100, 200, 500 or 1000% when expressed as a percentage). In
cases where therapeutic or other uses require that target cells, proteins
or other particles be released from complexes to fulfill their intended
use, then the term "in a way that promotes DLD separation" also requires
that the complexes permit such release, for example by chemical or
enzymatic cleavage, chemical dissolution, digestion, due to competition
with other binders, or by physical shearing (e.g., using a pipette to
create shear stress) and the freed target cells, proteins or other
particles must maintain activity; e.g., therapeutic cells after release
from a complex must still maintain the biological activities that make
them therapeutically useful.
[0133] Carriers may also bind "in a way that complements DLD separation":
This term refers to carriers and methods of binding carriers that change
the chemical, electrochemical, or magnetic properties of cells or cell
complexes or that change one or more biological activities of cells,
regardless of whether they increase size sufficiently to promote DLD
separation. Carriers that complement DLD separation also do not
necessarily bind with specificity to target cells, i.e., they may have to
be combined with some other agent that makes them specific or they may
simply be added to a cell preparation and be allowed to bind
non-specifically. The terms "in a way that complements DLD separation"
and "in a way that promotes DLD separation" are not exclusive of one
another. Binding may both complement DLD separation and also promote DLD
separation. For example a polysaccharide carrier may have an activator on
its surface that increases the rate of cell growth and the binding of one
or more of these carriers may also promote DLD separation. Alternatively
binding may just promote DLD separation or just complement DLD
separation.
[0134] Target cells: As used herein "target cells" are the cells that
various procedures described herein require or are designed to purify,
collect, engineer etc. What the specific cells are will depend on the
context in which the term is used. For example, if the objective of a
procedure is to isolate a particular kind of stem cell, that cell would
be the target cell of the procedure.
[0135] Isolate, purify: Unless otherwise indicated, these terms, as used
herein, are synonymous and refer to the enrichment of a desired product
relative to unwanted material. The terms do not necessarily mean that the
product is completely isolated or completely pure. For example, if a
starting sample had a target cell that constituted 2% of the cells in a
sample, and a procedure was performed that resulted in a composition in
which the target cell was 60% of the cells present, the procedure would
have succeeded in isolating or purifying the target cell.
[0136] Bump Array: The terms "bump array" and "obstacle array" are used
synonymously herein and describe an ordered array of obstacles that are
disposed in a flow channel through which a cell or particle-bearing fluid
can be passed.
[0137] Deterministic Lateral Displacement: As used herein, the term
"Deterministic Lateral Displacement" or "DLD" refers to a process in
which particles are deflected on a path through an array,
deterministically, based on their size in relation to some of the array
parameters. This process can be used to separate cells, which is
generally the context in which it is discussed herein. However, it is
important to recognize that DLD can also be used to concentrate cells and
for buffer exchange. Processes are generally described herein in terms of
continuous flow (DC conditions; i.e., bulk fluid flow in only a single
direction). However, DLD can also work under oscillatory flow (AC
conditions; i.e., bulk fluid flow alternating between two directions).
[0138] Critical size: The "critical size" or "predetermined size" of
particles passing through an obstacle array describes the size limit of
particles that are able to follow the laminar flow of fluid. Particles
larger than the critical size can be `bumped` from the flow path of the
fluid while particles having sizes lower than the critical size (or
predetermined size) will not necessarily be so displaced. When a profile
of fluid flow through a gap is symmetrical about the plane that bisects
the gap in the direction of bulk fluid flow, the critical size can be
identical for both sides of the gap; however when the profile is
asymmetrical, the critical sizes of the two sides of the gap can differ.
[0139] Fluid flow: The terms "fluid flow" and "bulk fluid flow" as used
herein in connection with DLD refer to the macroscopic movement of fluid
in a general direction across an obstacle array. These terms do not take
into account the temporary displacements of fluid streams for fluid to
move around an obstacle in order for the fluid to continue to move in the
general direction.
[0140] Tilt angle c: In a bump array device, the tilt angle is the angle
between the direction of bulk fluid flow and the direction defined by
alignment of rows of sequential (in the direction of bulk fluid flow)
obstacles in the array.
[0141] Array Direction: In a bump array device, the "array direction" is a
direction defined by the alignment of rows of sequential obstacles in the
array. A particle is "bumped" in a bump array if, upon passing through a
gap and encountering a downstream obstacle, the particle's overall
trajectory follows the array direction of the bump array (i.e., travels
at the tilt angle relative to bulk fluid flow). A particle is not bumped
if its overall trajectory follows the direction of bulk fluid flow under
those circumstances.
DETAILED DESCRIPTION OF THE INVENTION
[0142] The present invention is primarily concerned with the use of DLD in
preparing cells that are of therapeutic value. The text below provides
guidance regarding methods disclosed herein and information that may aid
in the making and use of devices involved in carrying out those methods.
I. Designing Microfluidic Plates
[0143] Cells, particularly cells in compositions prepared by apheresis or
leukapheresis, may be isolated by performing DLD using microfluidic
devices that contain a channel through which fluid flows from an inlet at
one end of the device to outlets at the opposite end. Basic principles of
size based microfluidic separations and the design of obstacle arrays for
separating cells have been provided elsewhere (see, US 2014/0342375; US
2016/0139012; U.S. Pat. Nos. 7,318,902 and 7,150,812, which are hereby
incorporated herein in their entirety) and are also summarized in the
sections below.
[0144] During DLD, a fluid sample containing cells is introduced into a
device at an inlet and is carried along with fluid flowing through the
device to outlets. As cells in the sample traverse the device, they
encounter posts or other obstacles that have been positioned in rows and
that form gaps or pores through which the cells must pass. Each
successive row of obstacles is displaced relative to the preceding row so
as to form an array direction that differs from the direction of fluid
flow in the flow channel. The "tilt angle" defined by these two
directions, together with the width of gaps between obstacles, the shape
of obstacles, and the orientation of obstacles forming gaps are primary
factors in determining a "critical size" for an array. Cells having a
size greater than the critical size travel in the array direction, rather
than in the direction of bulk fluid flow and particles having a size less
than the critical size travel in the direction of bulk fluid flow. In
devices used for leukapheresis-derived compositions, array
characteristics may be chosen that result in white blood cells being
diverted in the array direction whereas red blood cells and platelets
continue in the direction of bulk fluid flow. In order to separate a
chosen type of leukocyte from others having a similar size, a carrier may
then be used that binds to that cell with in a way that promotes DLD
separation and which thereby results in a complex that is larger than
uncomplexed leukocytes. It may then be possible to carry out a separation
on a device having a critical size smaller than the complexes but bigger
than the uncomplexed cells.
[0145] The obstacles used in devices may take the shape of columns or be
triangular, square, rectangular, diamond shaped, trapezoidal, hexagonal
or teardrop shaped. In addition, adjacent obstacles may have a geometry
such that the portions of the obstacles defining the gap are either
symmetrical or asymmetrical about the axis of the gap that extends in the
direction of bulk fluid flow.
II. Making and Operating Microfluidic Devices
[0146] General procedures for making and using microfluidic devices that
are capable of separating cells on the basis of size are well known in
the art. Such devices include those described in U.S. Pat. Nos.
5,837,115; 7,150,812; 6,685,841; 7,318,902; 7,472,794; and 7,735,652; all
of which are hereby incorporated by reference in their entirety. Other
references that provide guidance that may be helpful in the making and
use of devices for the present invention include: U.S. Pat. Nos.
5,427,663; 7,276,170; 6,913,697; 7,988,840; 8,021,614; 8,282,799;
8,304,230; 8,579,117; US 2006/0134599; US 2007/0160503; US 20050282293;
US 2006/0121624; US 2005/0266433; US 2007/0026381; US 2007/0026414; US
2007/0026417; US 2007/0026415; US 2007/0026413; US 2007/0099207; US
2007/0196820; US 2007/0059680; US 2007/0059718; US 2007/005916; US
2007/0059774; US 2007/0059781; US 2007/0059719; US 2006/0223178; US
2008/0124721; US 2008/0090239; US 2008/0113358; and WO2012094642 all of
which are also incorporated by reference herein in their entirety. Of the
various references describing the making and use of devices, U.S. Pat.
No. 7,150,812 provides particularly good guidance and U.S. Pat. No.
7,735,652 is of particular interest with respect to microfluidic devices
for separations performed on samples with cells found in blood (in this
regard, see also US 2007/0160503).
[0147] A device can be made using any of the materials from which micro-
and nano-scale fluid handling devices are typically fabricated, including
silicon, glasses, plastics, and hybrid materials. A diverse range of
thermoplastic materials suitable for microfluidic fabrication is
available, offering a wide selection of mechanical and chemical
properties that can be leveraged and further tailored for specific
applications.
[0148] Techniques for making devices include Replica molding,
Softlithography with PDMS, Thermoset polyester, Embossing, Injection
Molding, Laser Ablation and combinations thereof. Further details can be
found in "Disposable microfluidic devices: fabrication, function and
application" by Fiorini, et al. (BioTechniques 38:429-446 (March 2005)),
which is hereby incorporated by reference herein in its entirety. The
book "Lab on a Chip Technology" edited by Keith E. Herold and Avraham
Rasooly, Caister Academic Press Norfolk UK (2009) is another resource for
methods of fabrication, and is hereby incorporated by reference herein in
its entirety.
[0149] High-throughput embossing methods such as reel-to-reel processing
of thermoplastics is an attractive method for industrial microfluidic
chip production. The use of single chip hot embossing can be a
cost-effective technique for realizing high-quality microfluidic devices
during the prototyping stage. Methods for the replication of microscale
features in two thermoplastics, polymethylmethacrylate (PMMA) and/or
polycarbonate (PC), are described in "Microfluidic device fabrication by
thermoplastic hot-embossing" by Yang, et al. (Methods Mol. Biol. 949:
115-23 (2013)), which is hereby incorporated by reference herein in its
entirety
[0150] The flow channel can be constructed using two or more pieces which,
when assembled, form a closed cavity (preferably one having orifices for
adding or withdrawing fluids) having the obstacles disposed within it.
The obstacles can be fabricated on one or more pieces that are assembled
to form the flow channel, or they can be fabricated in the form of an
insert that is sandwiched between two or more pieces that define the
boundaries of the flow channel.
[0151] The obstacles may be solid bodies that extend across the flow
channel, in some cases from one face of the flow channel to an opposite
face of the flow channel. Where an obstacle is integral with (or an
extension of) one of the faces of the flow channel at one end of the
obstacle, the other end of the obstacle can be sealed to or pressed
against the opposite face of the flow channel. A small space (preferably
too small to accommodate any particles of interest for an intended use)
is tolerable between one end of an obstacle and a face of the flow
channel, provided the space does not adversely affect the structural
stability of the obstacle or the relevant flow properties of the device.
[0152] The number of obstacles present should be sufficient to realize the
particle-separating properties of the arrays. The obstacles can generally
be organized into rows and columns (Note: Use of the term "rows and
columns" does not mean or imply that the rows and columns are
perpendicular to one another). Obstacles that are generally aligned in a
direction transverse to fluid flow in the flow channel can be referred to
as obstacles in a column. Obstacles adjacent to one another in a column
may define a gap through which fluid flows.
[0153] Obstacles in adjacent columns can be offset from one another by a
degree characterized by a tilt angle, designated E (epsilon). Thus, for
several columns adjacent to one another (i.e., several columns of
obstacles that are passed consecutively by fluid flow in a single
direction generally transverse to the columns), corresponding obstacles
in the columns can be offset from one another such that the corresponding
obstacles form a row of obstacles that extends at the angle .epsilon.
relative to the direction of fluid flow past the columns. The tilt angle
can be selected and the columns can be spaced apart from each other such
that 1/.epsilon. (when expressed in radians) is an integer, and the
columns of obstacles repeat periodically. The obstacles in a single
column can also be offset from one another by the same or a different
tilt angle. By way of example, the rows and columns can be arranged at an
angle of 90 degrees with respect to one another, with both the rows and
the columns tilted, relative to the direction of bulk fluid flow through
the flow channel, at the same angle of E.
[0154] Surfaces can be coated to modify their properties and polymeric
materials employed to fabricate devices, can be modified in many ways. In
some cases, functional groups such as amines or carboxylic acids that are
either in the native polymer or added by means of wet chemistry or plasma
treatment are used to crosslink proteins or other molecules. DNA can be
attached to COC and PMMA substrates using surface amine groups.
Surfactants such as Pluronic.RTM. can be used to make surfaces
hydrophilic and protein repellant by adding Pluronic.RTM. to PDMS
formulations. In some cases, a layer of PMMA is spin coated on a device,
e.g., microfluidic chip and PMMA is "doped" with hydroxypropyl cellulose
to vary its contact angle.
[0155] To reduce non-specific adsorption of cells or compounds, e.g.,
released by lysed cells or found in biological samples, onto the channel
walls, one or more walls may be chemically modified to be non-adherent or
repulsive. The walls may be coated with a thin film coating (e.g., a
monolayer) of commercial non-stick reagents, such as those used to form
hydrogels. Additional examples of chemical species that may be used to
modify the channel walls include oligoethylene glycols, fluorinated
polymers, organosilanes, thiols, poly-ethylene glycol, hyaluronic acid,
bovine serum albumin, poly-vinyl alcohol, mucin, poly-HEMA, methacrylated
PEG, and agarose. Charged polymers may also be employed to repel
oppositely charged species. The type of chemical species used for
repulsion and the method of attachment to the channel walls can depend on
the nature of the species being repelled and the nature of the walls and
the species being attached. Such surface modification techniques are well
known in the art. The walls may be functionalized before or after the
device is assembled.
III. CAR T Cells
[0156] Methods for making and using CAR T cells are well known in the art.
Procedures have been described in, for example, U.S. Pat. Nos. 9,629,877;
9,328,156; 8,906,682; US 2017/0224789; US 2017/0166866; US 2017/0137515;
US 2016/0361360; US 2016/0081314; US 2015/0299317; and US 2015/0024482;
each of which is incorporated by reference herein in its entirety.
IV. Separation Processes that Use DLD
[0157] The DLD devices described herein can be used to purify cells,
cellular fragments, cell adducts, or nucleic acids. As discussed herein,
these devices can also be used to separate a cell population of interest
from a plurality of other cells. Separation and purification of blood
components using devices can be found, for example, in US Publication No.
US2016/0139012, the teaching of which is incorporated by reference herein
in its entirety. A brief discussion of a few illustrative separations is
provided below.
[0158] A. Viable Cells
[0159] In one embodiment devices are used in procedures designed to
separate a viable cell from a nonviable cell. The term "viable cell"
refers to a cell that is capable of growth, is actively dividing, is
capable of reproduction, or the like. In instances where a viable cell
has a size that is greater than a nonviable cell, DLD devices can be
designed to comprise a critical size that is greater than a predetermined
size of the nonviable cell and less than a predetermined size of the
viable cell. The critical size may be as little as 1.1 fold greater than
(or less than) the predetermined size of the nonviable cell but
generally, larger degrees (or smaller) are preferred, e.g., about 1.2
fold-2 fold, and preferably 3-10 fold.
[0160] B. Adherent Cells
[0161] In another embodiment, DLD devices can be used to in procedures to
separate adherent cells. The term "adherent cell" as used herein refers
to a cell capable of adhering to a surface. Adherent cells include
immortalized cells used in cell culturing and can be derived from
mammalian hosts. In some instances, the adherent cell may be trypsinized
prior to purification. Examples of adherent cells include MRC-5 cells;
HeLa cells; Vero cells; NIH 3T3 cells; L929 cells; Sf21 cells; Sf9 cells;
A549 cells; A9 cells; AtT-20 cells; BALB/3T3 cells; BHK-21 cells; BHL-100
cells; BT cells; Caco-2 cells; Chang cells; Clone 9 cells; Clone M-3
cells; COS-1 cells; COS-3 cells; COS-7 cells; CRFK cells; CV-1 cells;
D-17 cells; Daudi cells; GH1 cells; GH3 cells; HaK cells; HCT-15 cells;
HL-60 cells; HT-1080 cells; HT-29 cells; HUVEC cells; I-10 cells; IM-9
cells; JEG-2 cells; Jensen cells; Jurkat cells; K-562 cells; KB cells;
KG-1 cells; L2 cells; LLC-WRC 256 cells; McCoy cells; MCF7 cells; WI-38
cells; WISH cells; XC cells; Y-1 cells; CHO cells; Raw 264.7; BHK-21
cells; HEK 293 cells to include 293A, 293T and the like; HEP G2 cells;
BAE-1 cells; SH-SY5Y cells; and any derivative thereof to include
engineered and recombinant strains.
[0162] In some embodiments, procedures may involve separating cells from a
diluent such as growth media, which may provide for the efficient
maintenance of a culture of the adherent cells. For example, a culture of
adherent cells in a growth medium can be exchanged into a transfection
media comprising transfection reagents, into a second growth medium
designed to elicit change within the adherent cell such as
differentiation of a stem cell, or into sequential wash buffers designed
to remove compounds from the culture.
[0163] In a particularly preferred procedure, adherent cells are purified
through association with one or more carriers that bind in a way that
promotes DLD separation. The carriers may be of the type described herein
and binding may stabilize and/or activate the cells. A carrier will
typically be in the rage of 1-1000 .mu.m but may sometimes also be
outside of this range.
[0164] The association between a carrier and a cell should produce a
complex of increased size relative to other material not associated with
the carrier. Depending of the particular size of the cells and carriers
and the number of cells and carriers present, a complex may be anywhere
from a few percent larger than the uncomplexed cell to many times the
size of the uncomplexed cell. In order to facilitate separations, an
increase of at least 20% is desirable with higher percentages (50; 100;
1000 or more) being preferred.
[0165] C. Activated Cells
[0166] The DLD devices can also be used in procedures for separating an
activated cell or a cell capable of activation, from a plurality of other
cells. The cells undergoing activation may be grown on a large scale but,
in a preferred embodiment, the cells are derived from a single patient
and DLD is performed within at least few hours after collection. The
terms "activated cell" or "cell capable of activation" refers to a cell
that has been, or can be activated, respectively, through association,
incubation, or contact with a cell activator. Examples of cells capable
of activation can include cells that play a role in the immune or
inflammatory response such as: T cells, B cells; regulatory T cells,
macrophages, dendritic cells, granulocytes, innate lymphoid cells,
megakaryocytes, natural killer cells, thrombocytes, synoviocytes, and the
like; cells that play a role in metabolism, such as beta cells, liver
cells, and pancreatic cells; and recombinant cells capable of inducible
protein expression such as DE3 lysogenized E. coli cells, yeast cells,
plant cells, etc.
[0167] Typically, one or more carriers will have the activator on their
surface. Examples of cell activators include proteins, antibodies,
cytokines, CD3, CD28, antigens against a specific protein, helper T
cells, receptors, and glycoproteins; hormones such as insulin, glucagon
and the like; IPTG, lactose, allolactose, lipids, glycosides, terpenes,
steroids, and alkaloids. The activatable cell should be at least
partially associated with carriers through interaction between the
activatable cell and cell activator on the surface of the carriers. The
complexes formed may be just few percent larger than the uncomplexed cell
or many times the size of the uncomplexed cell. In order to facilitate
separations, an increase of at least 20% is desirable with higher
percentages (40, 50 100 1000 or more) being preferred.
[0168] D. Separating Cells from Toxic Material
[0169] DLD can also be used in purifications designed to remove compounds
that may be toxic to a cell or to keep the cells free from contamination
by a toxic compound. Examples include an antibiotic, a cryopreservative,
an antifungal, a toxic metabolite, sodium azide, a metal ion, a metal ion
chelator, an endotoxin, a plasticizer, a pesticide, and any combination
thereof. The device can be used to remove toxic compounds from cells to
ensure consistent production of material from the cells. In some
instances, the cell can be a log phase cell. The term "log phase cell"
refers to an actively dividing cell at a stage of growth characterized by
exponential logarithmic growth. In log phase, a cell population can
double at a constant rate such that plotting the natural logarithm of
cell number against time produces a straight line.
[0170] The ability to separate toxic material may be important for a wide
variety of cells including: bacterial strains such as BL21, Tuner,
Origami, Origami B, Rosetta, C41, C43, DH5.alpha., DH10.beta., or
XL1Blue; yeast strains such as those of genera Saccharomyces, Pichia,
Kluyveromyces, Hansenula and Yarrowia; algae; and mammalian cell
cultures, including cultures of MRC-5 cells; HeLa cells; Vero cells; NIH
3T3 cells; L929 cells; Sf21 cells; Sf9 cells; A549 cells; A9 cells;
AtT-20 cells; BALB/3T3 cells; BHK-21 cells; BHL-100 cells; BT cells;
Caco-2 cells; Chang cells; Clone 9 cells; Clone M-3 cells; COS-1 cells;
COS-3 cells; COS-7 cells; CRFK cells; CV-1 cells; D-17 cells; Daudi
cells; GH1 cells; GH3 cells; HaK cells; HCT-15 cells; HL-60 cells;
HT-1080 cells; HT-29 cells; HUVEC cells; I-10 cells; IM-9 cells; JEG-2
cells; Jensen cells; Jurkat cells; K-562 cells; KB cells; KG-1 cells; L2
cells; LLC-WRC 256 cells; McCoy cells; MCF7 cells; WI-38 cells; WISH
cells; XC cells; Y-1 cells; CHO cells; Raw 264.7; BHK-21 cells; HEK 293
cells to include 293A, 293T and the like; HEP G2 cells; BAE-1 cells;
SH-SY5Y cells; stem cells and any derivative thereof to include
engineered and recombinant strains.
[0171] E. Purification of Material Secreted from Cells
[0172] The DLD devices may also be used in the purification of material
secreted from a cell. Examples of such secreted materials includes
proteins, peptides, enzymes, antibodies, fuel, biofuels such as those
derived from algae, polymers, small molecules such as simple organic
molecules, complex organic molecules, drugs and pro-drugs, carbohydrates
and any combination thereof. Secreted products can include
therapeutically useful proteins such as insulin, Imatinib, T cells, T
cell receptors, Fc fusion proteins, anticoagulants, blood factors, bone
morphogenetic proteins, engineered protein scaffolds, enzymes, growth
factors, hormones, interferons, interleukins, and thrombolytics.
[0173] FIG. 15 is a schematic depicting the use of DLD in the purification
of secreted products. In some instances, the cells may be in an aqueous
suspension of buffer, growth medium, or the like, such that the cell
secretes product into the suspension. Examples of such secreted products
include proteins, peptides, enzymes, antibodies, fuel, biofuels such as
those derived from algae, polymers, small molecules such as simple
organic molecules, complex organic molecules, drugs and pro-drugs,
carbohydrates and any combination thereof. Secreted products can include
therapeutically useful proteins such as insulin, Imatinib, T cells, T
cell receptors, Fc fusion proteins, anticoagulants, blood factors, bone
morphogenetic proteins, engineered protein scaffolds, enzymes, growth
factors, hormones, interferons, interleukins, and thrombolytics.
[0174] Purification might be carried out, for example, in situations where
cells have a predetermined size that is greater than a predetermined size
of the secreted compound, where the predetermined size of the cell is
greater than or equal to a critical size, and the predetermined size of
the secreted compound is less than the critical size. In such a
configuration, when applied to a DLD device, the cells can be deflected
in a first direction while the secreted compound can be deflected in a
second direction, thereby separating the secreted compound from the cell.
Also, a secreted protein may be captured by a large carrier that binds in
a way that promotes DLD separation. DLD may then be performed and the
carrier-protein complex may then be treated to further purify, or
release, the protein.
[0175] Such processes can be carried out in an iterative fashion such that
a population of separated particles can be continuously looped back into
a device for further separation. In this regard, FIGS. 16 and 17 are
schematics of an iterative process in which separated cells are looped
back into the DLD device after separation. In some instances, the cells
may be looped from a first device into a second, different device with
obstacles comprising different critical sizes. Such a system can allow
systematic separation of a plurality of size ranges by manipulating the
range of critical sizes. In other instances, cells may be looped back to
the same device used previously to separate the isolated particles. This
system can be advantageous for continuous purification of actively
dividing cells or compounds being actively expressed. For example, such a
method could be combined with the method of purifying the secreted
product to both collect the secreted product from one flow stream and the
cell producing the secreted product from another flow stream. Because the
cells can continuously produce the secreted product, the purified cells
can be reapplied to the device to continuously collect the secreted
product from the cells.
[0176] F. Purity and Yields
[0177] The purity, yields and viability of cells produced by the DLD
methods discussed herein will vary based on a number of factors including
the nature of the starting material, the exact procedure employed and the
characteristics of the DLD device. Preferably, purifications, yields and
viabilities of at least 60% should be obtained with, higher percentages,
at least 70, 80 or 90% being more preferred. In a preferred embodiment,
methods may be used to isolate leukocytes from whole blood, apheresis
products or leukapheresis products with at least 70% purity, yield and
viability with higher percentages (at least 80%, 85%, or 90%) being
preferred.
V. Technological Background
[0178] Without being held to any particular theory, a general discussion
of some technical aspects of microfluidics may help in understanding
factors that affect separations carried out in this field. A variety of
microfabricated sieving matrices have been disclosed for separating
particles (Chou, et. al., Proc. Natl. Acad. Sci. 96:13762 (1999); Han, et
al., Science 288:1026 (2000); Huang, et al., Nat. Biotechnol. 20:1048
(2002); Turner et al., Phys. Rev. Lett. 88(12):128103 (2002); Huang, et
al., Phys. Rev. Lett. 89:178301 (2002); U.S. Pat. Nos. 5,427,663;
7,150,812; 6,881,317). Bump array (also known as "obstacle array")
devices have been described, and their basic operation is explained, for
example in U.S. Pat. No. 7,150,812, which is incorporated herein by
reference in its entirety. A bump array operates essentially by
segregating particles passing through an array (generally, a
periodically-ordered array) of obstacles, with segregation occurring
between particles that follow an "array direction" that is offset from
the direction of bulk fluid flow or from the direction of an applied
field (U.S. Pat. No. 7,150,812).
[0179] A. Bump Arrays
[0180] In some arrays, the geometry of adjacent obstacles is such that the
portions of the obstacles defining the gap are symmetrical about the axis
of the gap that extends in the direction of bulk fluid flow. The velocity
or volumetric profile of fluid flow through such gaps is approximately
parabolic across the gap, with fluid velocity and flux being zero at the
surface of each obstacle defining the gap (assuming no-slip flow
conditions) and reaching a maximum value at the center point of the gap.
The profile being parabolic, a fluid layer of a given width adjacent to
one of the obstacles defining the gap contains an equal proportion of
fluid flux as a fluid layer of the same width adjacent to the other
obstacle that defines the gap, meaning that the critical size of
particles that are `bumped` during passage through the gap is equal
regardless of which obstacle the particle travels near.
[0181] In some cases, particle size-segregating performance of an obstacle
array can be improved by shaping and disposing the obstacles such that
the portions of adjacent obstacles that deflect fluid flow into a gap
between obstacles are not symmetrical about the axis of the gap that
extends in the direction of bulk fluid flow. Such lack of flow symmetry
into the gap can lead to a non-symmetrical fluid flow profile within the
gap. Concentration of fluid flow toward one side of a gap (i.e., a
consequence of the non-symmetrical fluid flow profile through the gap)
can reduce the critical size of particles that are induced to travel in
the array direction, rather than in the direction of bulk fluid flow.
This is because the non-symmetry of the flow profile causes differences
between the width of the flow layer adjacent to one obstacle that
contains a selected proportion of fluid flux through the gap and the
width of the flow layer that contains the same proportion of fluid flux
and that is adjacent to the other obstacle that defines the gap. The
different widths of the fluid layers adjacent to obstacles define a gap
that exhibits two different critical particle sizes. A particle
traversing the gap can be bumped (i.e., travel in the array direction,
rather than the bulk fluid flow direction) if it exceeds the critical
size of the fluid layer in which it is carried. Thus, it is possible for
a particle traversing a gap having a non-symmetrical flow profile to be
bumped if the particle travels in the fluid layer adjacent to one
obstacle, but to be not-bumped if it travels in the fluid layer adjacent
to the other obstacle defining the gap.
[0182] In another aspect, decreasing the roundness of edges of obstacles
that define gaps can improve the particle size-segregating performance of
an obstacle array. By way of example, arrays of obstacles having a
triangular cross-section with sharp vertices can exhibit a lower critical
particle size than do arrays of identically-sized and -spaced triangular
obstacles having rounded vertices.
[0183] Thus, by sharpening the edges of obstacles defining gaps in an
obstacle array, the critical size of particles deflected in the array
direction under the influence of bulk fluid flow can be decreased without
necessarily reducing the size of the obstacles. Conversely, obstacles
having sharper edges can be spaced farther apart than, but still yield
particle segregation properties equivalent to, identically-sized
obstacles having less sharp edges.
[0184] B. Fractionation Range
[0185] Objects separated by size on microfluidic include cells,
biomolecules, inorganic beads, and other objects. Typical sizes
fractionated range from 100 nanometers to 50 micrometers. However, larger
and smaller particles may also sometimes be fractionated.
[0186] C. Volumes
[0187] Depending on design, a device or combination of devices might be
used to process between about 10 .mu.l to at least 500 .mu.l of sample,
between about 500 .mu.l and about 40 mL of sample, between about 500
.mu.l and about 20 mL of sample, between about 20 mL of sample and about
200 mL of sample, between about 40 mL of sample and about 200 mL of
sample, or at least 200 mL of sample.
[0188] D. Channels
[0189] A device can comprise one or multiple channels with one or more
inlets and one or more outlets. Inlets may be used for sample or crude
(i.e., unpurified) fluid compositions, for buffers or to introduce
reagents. Outlets may be used for collecting product or may be used as an
outlet for waste. Channels may be about 0.5 to 100 mm in width and about
2-200 mm long but different widths and lengths are also possible. Depth
may be 1-1000 .mu.m and there may be anywhere from 1 to 100 channels or
more present. Volumes may vary over a very wide range from a few .mu.l to
many ml and devices may have a plurality of zones (stages, or sections)
with different configurations of obstacles.
[0190] E. Gap Size (Edge-to-Edge Distance Between Posts or Obstacles)
[0191] Gap size in an array of obstacles (edge-to-edge distance between
posts or obstacles) can vary from about a few (e.g., 1-500) micrometers
or be more than a millimeter. Obstacles may, in some embodiments have a
diameter of 1-3000 micrometers and may have a variety of shapes (round,
triangular, teardrop shaped, diamond shaped, square, rectangular etc.). A
first row of posts can be located close to (e.g. within 5 .mu.m) the
inlet or be more than 1 mm away.
[0192] F. Stackable chips
[0193] A device can include a plurality of stackable chips. A device can
comprise about 1-50 chips. In some instances, a device may have a
plurality of chips placed in series or in parallel or both.
VI. Inventive Concepts
[0194] The numbered paragraphs below present inventive concepts that are
part of the present application. These concepts are expressed in the form
of example paragraphs E1-E273. [0195] E1. A method of engineering a
population of target cells, comprising: [0196] a) isolating the target
cells from a crude fluid composition wherein the isolation procedure
comprises performing Deterministic Lateral Displacement (DLD) on a
microfluidic device, wherein said device comprises: [0197] i) at least
one channel extending from a sample inlet to one or more fluid outlets,
wherein the channel is bounded by a first wall and a second wall opposite
from the first wall; [0198] ii) an array of obstacles arranged in rows in
the channel, each subsequent row of obstacles being shifted laterally
with respect to a previous row, and wherein said obstacles are disposed
in a manner such that, when said crude fluid composition is applied to an
inlet of the device and fluidically passed through the channel, target
cells flow to one or more collection outlets where an enriched product is
collected and contaminant cells or particles that are of a different size
than the target cells flow to one or more waste outlets that are separate
from the collection outlets; [0199] b) genetically engineering the
target cells obtained from the collection outlet(s) to have a desired
phenotype. [0200] E2. The method of E1, wherein said genetic
engineering comprises transfecting or transducing the target cells and
the genetically engineered target cells are expanded by culturing them in
vitro. [0201] E3. The method of E2, wherein the yield of target cells
exhibiting the desired phenotype is at least 10% greater than identical
cells isolated by Ficoll centrifugation and not subjected to DLD. [0202]
E4. The method of E1, wherein the crude fluid composition is blood or a
composition that has been obtained by performing apheresis or
leukapheresis on blood. [0203] E5. The method of any one of E1-4, wherein
the target cells are leukocytes. [0204] E6. The method of any one of
E1-4, wherein the target cells are B-cells, T cells, NK-cells, monocytes
or progenitor cells. [0205] E7. The method of any one of E1-4, wherein
the target cell a dendritic cell. [0206] E8. The method of any one of
E1-7, wherein said crude fluid composition is obtained from a patient.
[0207] E9. The method of E8, wherein, target cells in the crude fluid
composition are not bound to a carrier before being transduced or
transfected. [0208] E10. The method of E8, wherein target cells are bound
to one or more carriers in a way that promotes or complements DLD
separation before performing DLD. [0209] E11. The method of E9, wherein
target cells are bound to one or more carriers in a way that promotes or
complements DLD separation after performing DLD and either before or
after transducing or transfecting them. [0210] E12. The method of E10 or
E11, wherein said one or more carriers comprise on their surface an
affinity agent that binds specifically to said target cells. [0211] E13.
The method of E12, wherein said agent is an antibody, an activator, a
hapten or an aptamer. [0212] E14. The method of any one of E10-13,
wherein the diameter of said carriers is at least as large as that of the
target cells. [0213] E15. The method of any one of E10-13, wherein the
diameters of all of said carriers are no more than 50% as large as that
of the target cells. [0214] E16. The method of any one of E10-13, wherein
the diameters of all of said carriers are at least two times larger than
that of the target cells. [0215] E17. The method of any one of E10-13,
wherein the diameters of all of said carriers are no more than 25% as
large as that of the target cells. [0216] E18. The method of any one of
E10-13, wherein one group of carriers has a diameter at least as large as
the target cells and a second group of carriers has a diameter no more
than 50% as large as that of the target cells. [0217] E19. The method of
any one of E10-13, wherein one group of carriers has a diameter at least
twice as large as the target cells and a second group of carriers has a
diameter no more than 25% as large as the target cells. [0218] E20. The
method of any one of E10-19, wherein said carriers are made of collagen
or a polysaccharide. [0219] E21. The method of any one of E10-20, wherein
said carriers are made of gelatin or alginate. [0220] E22. The method of
any one of E10-21, wherein the crude fluid composition is obtained from a
patient and no more than four hours elapse from the time that the
obtaining of the crude fluid composition is complete until the target
cells are first bound to a carrier. [0221] E23. The method of any one of
E10-21, wherein the crude fluid composition is an apheresis or
leukapheresis product derived from the blood of a patient and no more
than four hours elapse from the time that apheresis or leukapheresis is
completed until the target cells are first bound to a carrier. [0222]
E24. The method of any one of E1-23, wherein the crude fluid composition
is obtained from a patient and no more than five hours elapse from the
time that the obtaining of the crude fluid composition is complete until
the first time that target cells are transfected or transduced. [0223]
E25. The method of any one of E1-23, wherein the crude fluid composition
is an apheresis or leukapheresis product derived from the blood of a
patient and no more than five hours elapse from the time that apheresis
or leukapheresis is completed until the first time that target cells are
transfected or transduced. [0224] E26. The method of either E24 or E25,
wherein no more than four hours elapse until the first time that target
cells are transfected or transduced. [0225] E27. A method of producing
Chimeric Antigen Receptor (CAR) T cells, comprising: [0226] a) obtaining
a crude fluid composition comprising T cells; [0227] b) performing
Deterministic Lateral Displacement (DLD) on the crude fluid composition
using a microfluidic device comprising: [0228] i) at least one channel
extending from a sample inlet to one or more fluid outlets, wherein the
channel is bounded by a first wall and a second wall opposite from the
first wall; [0229] ii) an array of obstacles arranged in rows in the
channel, each subsequent row of obstacles being shifted laterally with
respect to a previous row, and wherein said obstacles are disposed in a
manner such that, when the crude fluid composition is applied to an inlet
of the device and fluidically passed through the channel, T cells in the
composition flow to one or more collection outlets where an enriched
product is collected, and cells, or particles that are in the crude fluid
composition and that are of a different size than the T cells, flow to
one or more waste outlets that are separate from the collection outlets;
[0230] c) genetically engineering the T cells in the enriched product
obtained in step b) to produce the chimeric antigen receptors (CARs) on
their surface. [0231] E28. The method of E27, wherein said crude fluid
composition is an apheresis product or leukapheresis product obtained
from blood from a patient and wherein, when the crude fluid composition
is applied to an inlet of the device and fluidically passed through the
channel, T cells in the composition flow to one or more collection
outlets where an enriched product is collected, and red blood, platelets
or other particles that are in the crude fluid composition and that are
of a different size, flow to one or more waste outlets that are separate
from the collection outlets. [0232] E29. The method of either E27 or E28,
wherein said genetic engineering comprises transfecting or transducing
the target cells and the genetically engineered target cells are expanded
further by growing the cells in vitro. [0233] E30. The method of any one
of E27-29, wherein the yield of T cells expressing the chimeric receptors
on their surface is at least 10% greater than T cells isolated from the
crude fluid composition by Ficoll centrifugation and not subjected to
DLD. [0234] E31. The method of E30, wherein the yield of T cells
expressing the chimeric receptors on their surface is at least 20%
greater than T cells isolated from the crude fluid composition by Ficoll
centrifugation and not subjected to DLD. [0235] E32. The method of E30,
wherein the yield of T cells expressing the chimeric receptors on their
surface is at least 50% greater than T cells isolated from the crude
fluid composition by Ficoll centrifugation and not subjected to DLD.
[0236] E33. The method of any one of E27-32, wherein said CAR comprises
a) an extracellular region comprising antigen binding domain; b) a
transmembrane region; c) an intracellular region and wherein said CAR T
cells optionally comprise one or more recombinant sequences that provide
the cells with a molecular switch that, when triggered, reduce CAR T cell
number or activity. [0237] E34. The method of E33, wherein said antigen
binding domain is a single chain variable fragment (scFv), from antigen
binding regions of both heavy and light chains of a monoclonal antibody.
[0238] E35. The method of E33 or E34 wherein said CAR comprises a hinge
region of 2-20 amino acids connecting the extracellular region and the
transmembrane region. [0239] E36. The method of E35, wherein said
transmembrane region comprises CD8 or CD28 protein sequences. [0240] E37.
The method of any one of E33-36, wherein said intracellular region
comprises a signaling domain derived from CD3-zeta, CD137 or a CD28
intracellular domain. [0241] E38. The method of any one of E27-37,
wherein said crude fluid composition comprising T cells is obtained from
a patient with cancer, an autoimmune disease or an infectious disease.
[0242] E39. The method of E38 wherein, after obtaining the crude fluid
composition comprising T cells, the T cells in the fluid composition are
bound to one or more carriers in a way that promotes DLD separation.
[0243] E40. The method of E39, wherein T cells are bound to one or more
carriers in a way that promotes DLD separation before performing DLD.
[0244] E41. The method of E39, wherein T cells are bound to one or more
carriers in a way that promotes DLD separation after performing DLD and
either before or after they are genetically engineered. [0245] E42. The
method of E39-41, wherein said one or more carriers comprise on their
surface an antibody or activator that binds specifically to said T cells.
[0246] E43. The method of any one of E39-42, wherein the diameters of all
of said carriers are at least as large as that of the T cells. [0247]
E44. The method of any one of E39-42, wherein the diameters of all of
said carriers are no more than 50% as large as that of the T cells.
[0248] E45. The method of any one of E39-42, wherein the diameters of all
of said carriers are at least two times larger than that of the T cells.
[0249] E46. The method of any one of E39-42, wherein the diameters of all
of said carriers are no more than 25% as large as that of the T cells.
[0250] E47. The method of any one of E39-42, wherein one group of
carriers has a diameter at least as large as the T cells and a second
group of carriers has a diameter no more than 50% as large as that of the
T cells. [0251] E48. The method of any one of E39-42, wherein one group
of carriers has a diameter at least twice as large as the T cells and a
second group of carriers has a diameter no more than 25% as large as the
T cells. [0252] E49. The method of any one of E39-48, wherein said
carriers are made of collagen or a polysaccharide. [0253] E50. The method
of any one of E39-49, wherein said carriers are made of gelatin or
alginate. [0254] E51. The method of any one of E39-50, wherein no more
than four hours elapse from the time that obtaining of the crude fluid
composition comprising T cells is completed until the T cells are bound
to a carrier. [0255] E52. The method of any one of E39-50, wherein the
crude fluid composition is an apheresis or leukapheresis product derived
from the blood of a patient and no more than four hours elapse from the
time that apheresis or leukapheresis is completed until the target cells
are bound to a carrier. [0256] E53. The method of any one of E27-50,
wherein no more than five hours elapse from the time that obtaining of
the crude fluid composition comprising T cells is completed until the
first time that T cells are transfected or transduced. [0257] E54. The
method of any one of E27-50, wherein the crude fluid composition is an
apheresis or leukapheresis product derived from the blood of a patient
and no more than five hours elapse from the time that apheresis or
leukapheresis is completed until the first time that T cells are
transfected or transduced. [0258] E55. The method of either E53 or E54,
wherein no more than four hours elapse until the first time that T cells
are transfected or transduced. [0259] E56. The method of any one of
E27-55 where all steps in producing the CAR T cells are performed at the
same facility where the a crude fluid composition comprising T cells is
obtained and all steps are completed in a total of no more than four
hours. [0260] E57. CART cells made by the method of any one of E27-55.
[0261] E58. A method of treating a patient for cancer, an autoimmune
disease, or an infectious disease, comprising administering to said
patient CAR T cells engineered to express chimeric antigen receptors that
recognize antigens on cancer cells, autoimmune cells or infectious cells
from said patient, wherein said CAR T cells have been produced by a
process comprising: [0262] a) obtaining a crude fluid composition
comprising T cells from a patient; [0263] b) performing Deterministic
Lateral Displacement (DLD) on the crude fluid composition using a
microfluidic device comprising: [0264] i) at least one channel extending
from a sample inlet to one or more fluid outlets, wherein the channel is
bounded by a first wall and a second wall opposite from the first wall;
[0265] ii) an array of obstacles arranged in rows in the channel, each
subsequent row of obstacles being shifted laterally with respect to a
previous row, and wherein said obstacles are disposed in a manner such
that, when the crude fluid composition is applied to an inlet of the
device and fluidically passed through the channel, T cells in the
composition flow to one or more collection outlets where an enriched
product is collected, and cells, or particles that are in the crude fluid
composition and that are of a different size than the T cells, flow to
one more or waste outlets that are separate from the collection outlets;
[0266] c) genetically engineering the T cells obtained in step b) to
express chimeric antigen receptors (CARs) on their surface; [0267] d)
expanding the number of engineered T cells by growing the cells in vitro;
and [0268] e) administering the engineered T cells to the patient from
which the crude fluid composition was obtained. [0269] E59. The method
of E58, wherein said crude fluid composition is an apheresis product or
leukapheresis product obtained from blood from said patient and wherein,
when the crude fluid composition is applied to an inlet of the device and
fluidically passed through the channel, T cells in the composition flow
to one or more collection outlets where an enriched product is collected,
and red blood cells, platelets or other particles that are in the crude
fluid composition and that are of a different size, flow to one more
waste outlets that are separate from the collection outlets.
[0270] E60. The method of E58 or E59, wherein genetic engineering
comprises transfecting or transducing the target cells. [0271] E61. The
method of E60, wherein the yield of cells expressing the chimeric
receptors on their surface is at least 10% greater than T cells isolated
from the crude fluid composition by Ficoll centrifugation and not
subjected to DLD. [0272] E62. The method of E60, wherein the yield of
target cells expressing the chimeric receptors on their surface is at
least 50% greater than T cells isolated from the crude fluid composition
by Ficoll centrifugation and not subjected to DLD. [0273] E63. The method
of any one of E58-62, wherein said CAR comprises a) an extracellular
region comprising antigen binding domain; b) a transmembrane region; and
c) an intracellular region and wherein said CAR T cells optionally
comprise one or more recombinant sequences that provide the cells with a
molecular switch that, when triggered, reduce CAR T cell number or
activity. [0274] E64. The method of E63, wherein said antigen binding
domain is a single chain variable fragment (scFv), from the antigen
binding regions of both heavy and light chains of a monoclonal antibody.
[0275] E65. The method of E63 or E64 wherein said CAR comprises a hinge
region of 2-20 amino acids connecting the extracellular region and the
transmembrane region. [0276] E66. The method of any one of E63-65,
wherein said transmembrane region comprises CD8 or CD28 protein
sequences. [0277] E67. The method of any one of E63-66, wherein said
intracellular region comprises a signaling domain derived from CD3-zeta,
CD137, a CD28 intracellular domain. [0278] E68. The method of any one of
E58-67, wherein said patient has leukemia. [0279] E69. The method of E68,
wherein said leukemia is acute lymphoblastic leukemia. [0280] E70. The
method of E68 or E69, wherein said CAR recognizes as an antigen CD19 or
CD20. [0281] E71. The method of any one of E58-67, wherein said patient
has a solid tumor. [0282] E72. The method of E71, wherein said CAR
recognizes an antigen selected from the group consisting of: CD22; RORI;
mesothelin; CD33/IL3Ra; c-Met; PSMA; Glycolipid F77; EGFRvIII; GD-2;
NY-ESO-1 TCR; MAGE A3 TCR; and combinations thereof. [0283] E73. The
method of any one of E58-72 wherein, after obtaining the crude fluid
composition comprising T cells, the T cells in the fluid are bound to a
carrier in a way that promotes DLD separation. [0284] E74. The method of
E73, wherein T cells are bound to one or more carriers in a way that
promotes DLD separation before performing DLD. [0285] E75. The method of
E73, wherein T cells are bound to one or more carriers in a way that
promotes DLD separation after performing DLD and either before or after
the T cells are genetically engineered to express chimeric receptors.
[0286] E76. The method of E73-75, wherein said one or more carriers
comprise on their surface an antibody or activator that binds
specifically to said T cells. [0287] E77. The method of any one of
E73-76, wherein the diameters of all of said carriers are at least as
large as that of the T cells. [0288] E78. The method of any one of
E73-76, wherein the diameters of all of said carriers are no more than
50% as large as that of the T cells. [0289] E79. The method of any one of
E73-76, wherein the diameters of all of said carriers are at least two
times larger than that of the T cells. [0290] E80. The method of any one
of E73-76, wherein the diameters of all of said carriers are no more than
25% as large as that of the T cells. [0291] E81. The method of E80,
wherein one group of carriers has a diameter at least as large as the T
cells and a second group of carriers has a diameter no more than 50% as
large as that of the T cells. [0292] E82. The method of any one of
E73-81, wherein one group of carriers has a diameter at least twice as
large as the T cells and a second group of carriers has a diameter no
more than 25% as large as the T cells. [0293] E83. The method of any one
of E73-82, wherein said carriers are made of collagen or a
polysaccharide. [0294] E84. The method of any one of E73-83, wherein said
carriers are made of gelatin or alginate. [0295] E85. The method of any
one of E73-84, wherein no more than four hours elapse from the time that
obtaining of the crude fluid composition comprising T cells is completed
until the T cells are bound to a carrier. [0296] E86. The method of any
one of E73-84, wherein the crude fluid composition is an apheresis or
leukapheresis product derived from the blood of a patient and no more
than four hours elapse from the time that apheresis or leukapheresis is
completed until the target cells are bound to a carrier. [0297] E87. The
method of any one of E73-84, wherein no more than five hours elapse from
the time that obtaining of the crude fluid composition comprising T cells
is completed until the first time that the T cells are transfected or
transduced. [0298] E88. The method of any one of E73-84, wherein the
crude fluid composition is an apheresis or leukapheresis product derived
from the blood of a patient and no more than five hours elapse from the
time that apheresis or leukapheresis is completed until the first time
that T cells are transfected or transduced. [0299] E89. The method of E87
or E88, wherein no more than four hours elapse until the first time that
T cells are transfected or transduced. [0300] E90. The method of any one
of E58-89, wherein T cells are available for administration to a patient
at least 1 day earlier than for cells processed via a method not
including DLD. [0301] E91. The method of any one of E58-89, wherein
target cells are available for administration to a patient at least 3
days earlier than for cells processed via a method not including DLD.
[0302] E92. A method of collecting target cells from a patient
comprising: [0303] a) obtaining from the patient a crude fluid
composition comprising target cells; [0304] b) performing Deterministic
Lateral Displacement (DLD) on the crude fluid composition comprising
target cells using a microfluidic device to obtain a composition enriched
in target cells; [0305] wherein, either before or after DLD, target cells
are bound to one or more carriers in a way that promotes DLD separation
and wherein no more than five hours elapse from the time that the
obtaining of the crude fluid composition comprising target cells from the
patient is completed until the target cells are bound to a carrier.
[0306] E93. The method of E92, wherein said one or more carriers comprise
on their surface an antibody or activator that binds specifically to said
target cells. [0307] E94. The method of either E92 or E93, wherein the
diameters of all of said carriers are at least as large as that of the
target cells. [0308] E95. The method of either E92 or E93, wherein the
diameters of all of said carriers are no more than 50% as large as that
of the target cells. [0309] E96. The method of either E92 or E93, wherein
the diameters of all of said carriers are at least two times larger than
that of the target cells. [0310] E97. The method of either E92 or E93,
wherein the diameters of all of said carriers are no more than 25% as
large as that of the target cells. [0311] E98. The method of either E92
or E93, wherein one group of carriers has a diameter at least as large as
the target cells and a second group of carriers has a diameter no more
than 50% as large as that of the target cells. [0312] E99. The method of
either E92 or E93, wherein one group of carriers has a diameter at least
twice as large as the T cells and a second group of carriers has a
diameter no more than 25% as large as the T cells. [0313] E100. The
method of any one of E92-99, wherein said carriers are made of collagen
or a polysaccharide. [0314] E101. The method of any one of E92-100,
wherein said carriers are made of gelatin or alginate. [0315] E102. The
method of any one of E92-101, wherein no more than four hours elapse from
the time that the obtaining of the crude fluid composition comprising
target cells is completed until the target cells are bound to a carrier.
[0316] E103. The method of any one of E92-101, wherein no more than three
hours elapse from the time that the obtaining of the crude fluid
composition comprising target cells is completed until the target cells
are bound to a carrier. [0317] E104. The method of any one of E92-103,
wherein said crude fluid composition comprising target cells is obtained
by performing apheresis or leukapheresis on blood from the patient.
[0318] E105. The method of any one of E92-104 wherein target cells in the
composition enriched in target cells by DLD are transduced using a viral
vector. [0319] E106. The method of E105 wherein target cells are
transfected electrically, chemically or by means of nanoparticles. [0320]
E107. The method of any one of E92-106, wherein said microfluidic device
comprises: [0321] a) at least one channel extending from a sample inlet
to one or more fluid outlets, wherein the channel is bounded by a first
wall and a second wall opposite from the first wall; [0322] b) an array
of obstacles arranged in rows in the channel, each subsequent row of
obstacles being shifted laterally with respect to a previous row, and
wherein said obstacles are disposed in a manner such that, when said
crude fluid composition comprising target cells is applied to an inlet of
the device and fluidically passed through the channel, target cells flow
to one or more collection outlets where an enriched product is collected
and contaminant cells, or particles that are in the crude fluid
composition and that are of a different size than the target cells flow
to one or more waste outlets that are separate from the collection
outlets. [0323] E108. The method of any one of E92-107, wherein said
target cells are T cells. [0324] E109. The method of E108, wherein said T
cells are selected from the group consisting of: Natural Killer T cells;
Central Memory T cells; Helper T cells and Regulatory T cells. [0325]
E110. The method of any one of E92-107, wherein said target cells are
stem cells. [0326] E111. The method of any one of E92-107, wherein said
target cells are B cells, macrophages, dendritic cells, or granulocytes.
[0327] E112. The method of any one of E92-111, wherein said crude fluid
composition comprising target cells comprises one or more additives that
act as anticoagulants or that prevent the activation of platelets. [0328]
E113. The method of E112, wherein said additives are selected from the
group consisting of ticlopidine, inosine, protocatechuic acid,
acetylsalicylic acid, and tirofiban. [0329] E114. The method of any one
of E92-113, wherein steps a) and b) are both carried out at the site
where the crude fluid composition comprising target cells is obtained
from the patient. [0330] E115. The method of any one of E92-114, wherein
no more than four hours elapse from the time that the obtaining of the
crude fluid composition comprising target cells from the patient is
completed until the target cells are bound to a carrier. [0331] E116. The
method of any one of E92-114, wherein the crude fluid composition is an
apheresis or leukapheresis product derived from the blood of the patient
and no more than four hours elapse from the time that apheresis or
leukapheresis is completed until the target cells are bound to a carrier.
[0332] E117. The method of any one of E92-116, wherein said method
further comprises: [0333] c) genetically engineering and/or expanded
cells in number; and/or [0334] d) treating the same patient from which
the target cells were obtained with the target cells collected. [0335]
E118. The method of E117, wherein, after step d), said target cells are
cryopreserved. [0336] E119. The method of either E117 or E118, wherein
target cells that are cultured in step c) are T cells that are cultured
in the presence of an activator. [0337] E120. The method of E119, wherein
the activator is bound to a carrier. [0338] E121. The method of any one
of E58-89, wherein target cells are available for administration to the
patient at least 1 day earlier than for cells processed via a method not
including DLD. [0339] E122. The method of any one of E58-89, wherein
target cells are available for administration to the patient at least 3
days earlier than for cells processed via a method not including DLD.
[0340] E123. Target cells produced by the method of any one of E92-122.
[0341] E124. A method of treating a patient for a disease or condition
comprising administering to said patient the target cells of E123. [0342]
E125. A method of separating an adherent cell from a plurality of other
cells comprising: [0343] a) contacting a crude fluid composition
comprising the plurality of other cells and the adherent cell with one or
more carriers that bind in a way that promotes DLD separation, wherein
the adherent cell is at least partially associated with carriers upon or
after contact to generate a carrier associated adherent cell complex,
wherein the carrier associated adherent cell complex comprises an
increased size relative to cells in the plurality of other cells, and
wherein the size of the carrier associated adherent cell complex is
greater than or equal to a critical size, and the cells in the plurality
of other cells comprise a size less than the critical size; [0344] b)
applying the crude fluid composition to a device, wherein the device
comprises an array of obstacles arranged in rows, wherein the rows are
shifted laterally with respect to one another, wherein the rows are
configured to deflect a particle greater than or equal to the critical
size in a first direction and a particle less than the critical size in a
second direction; and [0345] c) flowing the sample comprising the carrier
associated adherent cell complex through the device, wherein the carrier
associated adherent cell complex is deflected by the obstacles in the
first direction, and the cells in the plurality of other cells are
deflected in the second direction, thereby separating the carrier
associated adherent cell complex from the other cells of the plurality;
[0346] d) collecting a fluid composition comprising the separated carrier
associated adherent cell complex. [0347] E126. The method of E125,
wherein said adherent cell is collected from a patient as part of a crude
fluid composition comprising said adherent cell and a plurality of other
cells, and wherein no more than three hours elapse from the time that the
obtaining of the crude fluid composition from the patient is completed
until the adherent cell is bound to a carrier for the first time. [0348]
E127. The method of E125, wherein no more than two hours elapse from the
time that the obtaining of the crude fluid composition from the patient
is completed until the adherent cell is bound to the carrier for the
first time. [0349] E128. The method of E125, wherein no more than one
hour elapses from the time that the obtaining of the crude fluid
composition from the patient is completed until the adherent cell is
bound to the carrier for the first time.
[0350] E129. The method of E125, wherein no more than four hours elapse
from the time that the obtaining of the crude fluid composition from the
patient is completed until the adherent cell or the carrier adherent cell
complex is collected from the device for the first time. [0351] E130. The
method of E125, wherein no more than four hours elapse from the time that
the obtaining of the crude fluid composition from the patient is
completed until the adherent cell or the carrier adherent cell complex is
collected from the device for the first time. [0352] E131 The method of
any one of E125-130, wherein said carrier comprises on its surface an
antibody or activator that binds specifically to said adherent cell.
[0353] E132. The method of any one of E125-131, wherein the diameter of
said carrier is at least as large as that of the adherent cell. [0354]
E133. The method of any one of E125-131, wherein the diameters of all of
said carriers are at least twice as large as that of the adherent cell.
[0355] E134. The method of any one of E125-131, wherein the diameters of
all of said carriers are at least ten times as large as that of the
adherent cell. [0356] E135. The method of any one of E125-131, wherein
the diameters of all of said carriers are 10-600 .mu.m. [0357] E136. The
method of any one of E125-135, wherein the adherent cell is selected from
the group consisting of: an MRC-5 cell; a HeLa cell; a Vero cell; an NIH
3T3 cell; an L929 cell; a Sf21 cell; a Sf9 cell; an A549 cell; an A9
cell; an AtT-20 cell; a BALB/3T3 cell; a BHK-21 cell; a BHL-100 cell; a
BT cell; a Caco-2 cell; a Chang cell; a Clone 9 cell; a Clone M-3 cell; a
COS-1 cell; a COS-3 cell; a COS-7 cell; a CRFK cell; a CV-1 cell; a D-17
cell; a Daudi cell; a GH1 cell; a GH3 cell; an HaK cell; an HCT-15 cell;
an HL-60 cell; an HT-1080 cell; a HEK cell, HT-29 cell; an HUVEC cell; an
I-10 cell; an IM-9 cell; a JEG-2 cell; a Jensen cell; a Jurkat cell; a
K-562 cell; a KB cell; a KG-1 cell; an L2 cell; an LLC-WRC 256 cell; a
McCoy cell; a MCF7 cell; a WI-38 cell; a WISH cell; an XC cell; a Y-1
cell; a CHO cell; a Raw 264.7 cell; a HEP G2 cell; a BAE-1 cell; an
SH-SY5Y cell, and any derivative thereof [0358] E137. The method of any
one of E125-135, wherein the adherent cell is a stem cell. [0359] E138. A
method of separating an activated cell from a plurality of other cells
comprising: [0360] a) contacting a crude fluid composition comprising a
cell capable of activation and the plurality of other cells with one or
more carriers, wherein at least one carrier comprises a cell activator,
wherein the cell activator is at least partially associated with the cell
capable of activation by the cell activator upon or after contact to
generate a carrier associated cell complex, wherein the association of
the cell activator with the cell capable of activation by the cell
activator at least partially activates the cell capable of activation,
wherein the carrier associated cell complex comprises an increased size
relative to cells in the plurality of other cells, and wherein a size of
the carrier associated cell complex is greater than or equal to a
critical size, and the cells in the plurality of other cells comprise a
size less than the critical size; [0361] b) applying the sample to a
device, wherein the device comprises an array of obstacles arranged in
rows; wherein the rows are shifted laterally with respect to one another,
wherein the rows are configured to deflect a particle greater than or
equal to the critical size in a first direction and a particle less than
the critical size in a second direction; and [0362] c) flowing the sample
through the device, wherein the carrier associated cell complex is
deflected by the obstacles in the first direction, and the cells in the
plurality of other cells are deflected in the second direction, thereby
separating the activated cell from the other cells of the plurality;
[0363] d) collecting a fluid composition comprising the separated carrier
associated cell complex. [0364] E139. The method of E138, wherein the
cell capable of activation is selected from the group consisting of: a T
cell, a B cell, a regulatory T cell, a macrophage, a dendritic cell, a
granulocyte, an innate lymphoid cell, a megakaryocyte, a natural killer
cell, a thrombocyte, a synoviocyte, a beta cell, a liver cell, a
pancreatic cell; a DE3 lysogenized cell, a yeast cell, a plant cell, and
a stem cell. [0365] E140. The method of E138 or E139, wherein the cell
activator is a protein. [0366] E141. The method of E140, wherein the
protein is an antibody. [0367] E142. The method of E140, wherein the
protein is selected from the group consisting of: CD3, CD28, an antigen,
a helper T cell, a receptor, a cytokine, a glycoprotein, and any
combination thereof. [0368] E143. The method of E138, wherein the cell
activator is selected from the group consisting of insulin, IPTG,
lactose, allolactose, a lipid, a glycoside, a terpene, a steroid, an
alkaloid, and any combination thereof. [0369] E144. The method of any one
of E138-143, wherein said cell capable of activation is collected from a
patient as part of a crude fluid composition comprising said cell capable
of activation and a plurality of other cells, and wherein no more than
four hours elapse from the time that the obtaining of the crude fluid
composition from the patient is completed until the cell capable of
activation is bound to the carrier. [0370] E145. The method of any one of
E138-143, wherein no more than three hours elapse from the time that the
obtaining of the crude fluid composition from the patient is completed
until the cell capable of activation is bound to the carrier. [0371]
E146. The method of E138-143, wherein no more than two hours elapse from
the time that the obtaining of the crude fluid composition from the
patient is completed until the cell capable of activation is bound to the
carrier. [0372] E147. The method of any one of E138-143, wherein no more
than four hours elapse from the time that the obtaining of the crude
fluid composition from the patient is completed until step c) is
completed. [0373] E148. The method of any one of E138-143, wherein no
more than three hours elapse from the time that the obtaining of the
crude fluid composition from the patient is completed until step c) is
completed. [0374] E149. The method of any one of E138-148, wherein the
diameters of all of said carriers are at least as large as the cell
capable of activation. [0375] E150. The method of any one of E138-148,
wherein the diameters of all of said carriers are at least twice as large
as that of the cell capable of activation. [0376] E151. The method of any
one of E138-148, wherein the diameters of all of said carriers are at
least ten times as large as that of the cell capable of activation.
[0377] E152. The method of any one of E138-148, wherein the diameters of
said carriers are 10-600 .mu.m. [0378] E153. A method of continuously
purifying a secreted product from a cell comprising: [0379] a) obtaining
a fluid composition comprising the cell, wherein the cell is suspended in
the fluid composition, wherein the cell secretes the secreted product
into the suspension, wherein the cell has a predetermined size that is
greater than a predetermined size of the secreted product, and wherein
the predetermined size of the cell is greater than or equal to a critical
size, and the predetermined size of the secreted product is less than the
critical size; [0380] b) applying the fluid composition comprising the
cell to a device, wherein the device comprises an array of obstacles
arranged in rows; wherein the rows are shifted laterally with respect to
one another, wherein the rows are configured to deflect a particle
greater than or equal to the critical size in a first direction and a
particle less than the critical size in a second direction; [0381] c)
flowing the sample through the device, wherein the cell is deflected by
the obstacles in the first direction, and the secreted product is
deflected in the second direction, thereby separating the secreted
product from the cell; [0382] d) collecting the secreted product, thereby
producing a sample of the secreted product that is substantially pure;
[0383] e) collecting a recovered fluid composition comprising the
separated cell; and [0384] f) re-applying the recovered fluid composition
comprising the separated cell to the device and repeating steps (a)
through (e); thereby continuously purifying the secreted product from the
cell. [0385] E154. The method of E153, wherein the secreted product is
selected from the group consisting of: a protein, an antibody, a biofuel,
a polymer, a small molecule, and any combination thereof. [0386] E155.
The method of E153, wherein the cell is selected from the group
consisting of: a bacterial cell, an algae cell, a mammalian cell, and a
tumor cell. [0387] E156. A method for decreasing the ratio of platelets
to leukocytes in an apheresis sample, comprising performing deterministic
lateral displacement (DLD) on the sample, in the absence of
centrifugation or elutriation, wherein a product is obtained in which the
ratio of platelets to leukocytes is at least 20% lower than the ratio
obtained with the same procedure performed using centrifugation or
elutriation instead of DLD. [0388] E157. The method of E156, wherein a
product is obtained in which the ratio of platelets to leukocytes is at
least 20% lower than the ratio obtained with the same procedure performed
using density gradient centrifugation or counterflow centrifugation.
[0389] E158. The method of E156, wherein a product is obtained in which
the ratio of platelets to leukocytes is at least 20% lower than the ratio
obtained with the same procedure performed using elutriation. [0390]
E159. The method of E156, wherein a product is obtained in which the
ratio of platelets to leukocytes is at least 50% lower than the ratio
obtained using centrifugation or elutriation instead of DLD. [0391] E160.
The method of any one of E156-159, wherein there are no separation steps
performed on the apheresis sample prior to DLD. [0392] E161. The method
of any one of E156-160, wherein DLD is performed in a buffer that does
not comprise intercalators that alter the size of platelets and that does
not promote platelet aggregation. [0393] E162. The method of any one of
E156-159 wherein DLD is performed in a buffer that does not comprise
dextran or other highly charge polymers. [0394] E163. The method of any
one of E156-162, wherein the total number of platelets in the product is
at least 70% lower than in the apheresis sample. [0395] E164. The method
of any one of E156-163, wherein the total number of platelets in the
product is at least 90% lower than in the apheresis sample. [0396] E165.
A method for purifying T cells from an apheresis sample, comprising
performing DLD on the sample, followed by an affinity separation step and
expansion of the T cells by culturing in the presence of activator,
wherein the number of T cells obtained is at least twice as high as the
number produced by the same procedure performed using Ficoll
centrifugation instead of DLD. [0397] E166. The method of E165, wherein
the affinity separation step comprises the use of magnetic beads or
particles comprising an antibody binding to CD3. [0398] E167. The method
of E165, wherein the number of T cells obtained after 14 days in culture
is at least two times higher than the number produced by the same
procedure performed using Ficoll centrifugation instead of DLD. [0399]
E168. The method of E165, wherein the number of T cells obtained after 14
days in culture is at least four times higher than the number produced by
the same procedure performed using Ficoll centrifugation instead of DLD.
[0400] E169. The method of any one of E156-168 wherein, when cells in the
DLD product are transformed with a vector to express a recombinant
phenotype, the yield of cells exhibiting the desired phenotype is at
least 10% greater than for identical cells isolated by Ficoll
centrifugation and not subjected to DLD. [0401] E170. The method of any
one of E156-169, wherein when cells in the DLD product are transformed
with a vector to express a recombinant phenotype, the yield of T cells
exhibiting the desired phenotype is at least 20% greater than for
identical cells isolated by Ficoll centrifugation and not subjected to
DLD. [0402] E171. The method of any one of E163-165 wherein the
percentage of memory T cells in the DLD product relative to the total
number of T cells is at least 10% higher than the percentage produced
using the same procedure but with Ficoll centrifugation instead of DLD.
[0403] E172. The method of any one of E156-171, wherein the method is
used to produce T cells for CAR T cell therapy and the time needed to
produce a sufficient number of cells to treat a patient is reduced by at
least 20% using DLD instead of Ficoll centrifugation. [0404] E173. The
method of E172, wherein the process for producing CART cells does not
include a step in which cells are frozen. [0405] E174. The method of
either E172 or E173, wherein the processing of T cells is performed at
the same site where apheresis is performed. [0406] E175. The method of
any one of E172-174, wherein T cells are genetically transformed at the
same site where apheresis is performed. [0407] E176. The method of any
one of E156-169, wherein no more than one hour elapses from the time that
the apheresis sample collection is completed until the time that DLD is
performed. [0408] E177. A method for decreasing the ratio of platelets to
leukocytes in an apheresis sample, comprising performing deterministic
lateral displacement (DLD) on the sample, in the absence of
centrifugation or elutriation, wherein a product is obtained in which the
total number of platelets in the product is at least 90% lower than in
the apheresis sample. [0409] E178. The method of E177, wherein DLD is
performed in a buffer that does not comprise intercalators that alter the
size of platelets and that does not promote platelet aggregation. [0410]
E179. The method of E177, wherein DLD is performed in a buffer that does
not comprise dextran or other highly charge polymers. [0411] E180. A
method of producing CART cells, comprising: [0412] a) obtaining a sample
composition from a patient by apheresis, wherein said sample composition
comprises T cells; [0413] b) performing DLD on the sample composition to
reduce the total number of platelets present by at least 70%, wherein DLD
is carried out on a microfluidic device comprising: [0414] i) at least
one channel extending from a sample inlet to one or more fluid outlets,
wherein the channel is bounded by a first wall and a second wall opposite
from the first wall; [0415] ii) an array of obstacles arranged in rows in
the channel, each subsequent row of obstacles being shifted laterally
with respect to a previous row, and wherein said obstacles are disposed
in a manner such that, when the crude fluid composition is applied to an
inlet of the device and fluidically passed through the channel, T cells
in the composition flow to one or more collection outlets where an
enriched product is collected, and wherein platelets and other materials
smaller than T cells flow to one or more waste outlets that are separate
from the collection outlets;
[0416] c) genetically engineering the T cells in the enriched product
obtained in step b) to produce chimeric antigen receptors (CARs) on their
surface; [0417] d) culturing the T cells to expand their number; [0418]
e) transferring the T cells into a pharmaceutical composition for
administration to a patient. [0419] E181. The method of E180, wherein
at least 90% of platelets are removed in step b). [0420] E182. The method
of either E180 or E181, wherein all isolation steps and concentration
steps are carried out using DLD. [0421] E183. The method of any one of
E180-182, wherein prior to, or during culturing, cells are exposed to a T
cell activator. [0422] E184. The method of any one of E180-183, wherein,
in step b) and prior to step c), cells are transferred into a medium in
which a T cell activator is present or added. [0423] E185. The method of
E184, wherein the medium containing activator is processed by DLD to
separate activator from cells and to transfer cells into a medium in
which a vector for recombinantly engineering cells is present or added.
[0424] E186. The method of any one of E180-185, wherein cells are not
frozen until they are transferred into a pharmaceutical composition for
administration to a patient. [0425] E187. The method of any one of
E180-185, wherein cells are not frozen at any step. [0426] E188. The
method of any one of E180-187, wherein, compared to a procedure in which
cells are isolated or concentrated by a method other than DLD, the
process is completed at least one day sooner. [0427] E189. The method of
any one of E180-187, wherein, compared to a procedure in which cells are
isolated or concentrated by a method other than DLD, the process is
completed at least three days sooner. [0428] E190. The method of any one
of E180-187, wherein, compared to a procedure in which cells are isolated
or concentrated by a method other than DLD, the process is completed at
least five days sooner. [0429] E191. The method of any one of E180-187,
wherein, compared to a procedure in which cells are isolated or
concentrated by a method other than DLD, the process is completed at
least ten days sooner. [0430] E192. The method of any one of E180-191,
wherein, in step d), the number of T cells obtained after 14 days in
culture is at least two times higher than the number produced by the same
procedure performed using Ficoll centrifugation instead of DLD. [0431]
E193. The method of any one of E180-191, wherein, in step d), the number
of T cells obtained after 14 days in culture is at least four times
higher than the number produced by the same procedure performed using
Ficoll centrifugation instead of DLD. [0432] E194. A method of preparing
CART cells, comprising: [0433] a) collecting cells from a patient by
apheresis; [0434] b) performing DLD on the cells obtained in step a) to
separate leukocytes from other cells and particles, and to transfer the
leukocytes into a medium that supports their growth and that has, or is
supplemented with, a T cell activator; [0435] c) performing DLD to
separate T cells from the medium of step b) and to transfer cells into a
medium where they are recombinantly engineered to express chimeric
antigen receptors (CARs) on their surface; [0436] d) performing DLD to
separate the T cells from reagents and to transfer the cells into a
growth medium; [0437] e) culturing the cells to expand their number
[0438] f) performing DLD to transfer expanded T cells into a medium for
administration to a patient. [0439] E195. The method of E194, wherein
cells are not frozen until they are transferred into a pharmaceutical
composition for administration to a patient. [0440] E196. The method of
E194, wherein cells are not frozen at any step. [0441] E197. The method
of any one of E194-196, wherein, compared to a procedure in which cells
are isolated or concentrated by a method other than DLD, the process is
completed at least three days sooner. [0442] E198. The method of any one
of E194-196, wherein, compared to a procedure in which cells are isolated
or concentrated by a method other than DLD, the process is completed at
least five days sooner. [0443] E199. The method of any one of E194-196,
wherein, compared to a procedure in which cells are isolated or
concentrated by a method other than DLD, the process is completed at
least ten days sooner. [0444] E200. A method of preparing CAR T cells for
the treatment of a patient comprising: [0445] a) collecting cells from a
patient by apheresis; [0446] b) performing DLD on the cells obtained in
step a) to separate leukocytes from other cells and particles, and to
transfer the leukocytes into a medium that supports their growth and that
has, or is supplemented with, a T cell activator; [0447] c) separating T
cells from the medium of step b) into medium that contains a vector for
genetically engineering the T cells to produce chimeric antigen receptors
(CARs) on their surface; [0448] d) performing DLD to separate T cells
from reagents and to transfer the cells into a medium for administration
to a patient. [0449] E201. The method of E200, wherein cells are not
frozen until they are transferred into a pharmaceutical composition for
administration to a patient. [0450] E202. The method of E200, wherein
cells are not frozen at any step. [0451] E203. The method of any one of
E200-202, wherein, compared to a procedure in which cells are isolated or
concentrated by a method other than DLD, the process is completed at
least three days sooner. [0452] E204. The method of any one of E200-202,
wherein, compared to a procedure in which cells are isolated or
concentrated by a method other than DLD, the process is completed at
least five days sooner. [0453] E205. The method of any one of E200-202,
wherein, compared to a procedure in which cells are isolated or
concentrated by a method other than DLD, the process is completed at
least ten days sooner. [0454] E206. The method of any one of E180-205,
wherein in step b) a product is obtained in which the ratio of platelets
to leukocytes is at least 50% lower than the ratio obtained using
centrifugation or elutriation instead of DLD. [0455] E207. The method of
any one of E180-205, wherein the yield of T cells exhibiting the desired
CAR T phenotype is at least 10% greater than identical cells isolated by
Ficoll centrifugation and not subjected to DLD. [0456] E208. The method
of any one of E180-205, wherein the yield of T cells exhibiting the
desired CAR T phenotype is at least 20% greater than identical cells
isolated by Ficoll centrifugation and not subjected to DLD. [0457] E209.
The method of any one of E180-208, wherein the time necessary to produce
a sufficient number of T cells for the treatment of a patient is at least
5% shorter than when the same method is carried out using Ficoll
centrifugation rather than DLD to isolate cells from apheresis starting
material. [0458] E210. The method of any one of E180-209, wherein the
time necessary to produce a sufficient number of T cells for the
treatment of a patient is at least 10% shorter than when the same method
is carried out using Ficoll centrifugation rather than DLD to isolate
cells from apheresis starting material. [0459] E211. The method of any
one of E180-209, wherein, when cells prepared by said method are
administered to a patient, they exhibit at least 10% less senescence than
cells that have been processed from an apheresis composition using
centrifugation or elutriation instead of DLD. [0460] E212. The method of
any one of E180-209, wherein, when cells prepared by said method are
administered to a patient, they exhibit increased efficacy when compared
to cells that have been processed from an apheresis composition using
centrifugation or elutriation instead of DLD. [0461] E213. A method for
treating a patient for a disease or condition comprising administering to
said patient a therapeutically effective amount of cells prepared by the
method of any one of E180-209. [0462] E214. The method of E213, wherein
said disease or condition is cancer. [0463] E215. A method of producing
therapeutically active cells, comprising: [0464] a) obtaining a sample
composition from a patient comprising said cells; [0465] b) performing
DLD on the sample to produce a composition enriched in therapeutically
active cells, wherein DLD is performed on a microfluidic device
comprising: [0466] i) at least one channel extending from a sample inlet
to one or more fluid outlets, wherein the channel is bounded by a first
wall and a second wall opposite from the first wall; [0467] ii) an array
of obstacles arranged in rows in the channel, each subsequent row of
obstacles being shifted laterally with respect to a previous row, and
wherein said obstacles are disposed in a manner such that, when the crude
fluid composition is applied to an inlet of the device and fluidically
passed through the channel, the therapeutically active cells in the
composition flow to one or more collection outlets where an enriched
product is collected, and wherein cells and materials smaller than the
therapeutically active cells flow to one more waste outlets that are
separate from the collection outlets; [0468] c) optionally genetically
engineering the therapeutically active cells in the enriched product
obtained in step b); [0469] d) optionally culturing the therapeutically
active cells to expand their number; [0470] e) transferring the
therapeutically active cells into a pharmaceutical composition for
administration to a patient. [0471] E216. The method of E215, wherein
the therapeutically active cells are stem cells. [0472] E217. The method
of E216, wherein the stem cells are found in the circulation and the
sample composition is prepared by apheresis. [0473] E218. The method of
E217, wherein at least 70% of platelets are removed from the enriched
product of step b). [0474] E219. The method of E217, wherein at least 90%
of platelets are removed from the enriched product of step b). [0475]
E220. The method of any one of E215-219, wherein all isolation steps and
concentration steps are carried out using DLD. [0476] E221. The method of
any one of E215-220, wherein cells are not frozen until they are
transferred into a pharmaceutical composition for administration to a
patient. [0477] E222. The method of any one of E215-220, wherein cells
are not frozen at any step. [0478] E223. The method of any one of
E215-222, wherein, compared to a procedure in which cells are isolated or
concentrated by a method other than DLD, the process is completed at
least one day sooner. [0479] E224. The method of any one of E215-222,
wherein, compared to a procedure in which cells are isolated or
concentrated by a method other than DLD, the process is completed at
least three days sooner. [0480] E225. The method of any one of E215-222,
wherein, compared to a procedure in which cells are isolated or
concentrated by a method other than DLD, the process is completed at
least five days sooner. [0481] E226. The method of any one of E215-222,
wherein the yield of therapeutically active cells obtained from the
sample composition is at least 25% higher than the number obtained by a
procedure in which cells are isolated or concentrated by a method other
than DLD. [0482] E227. The method of any one of E215-222, wherein,
compared to a procedure in which cells are isolated or concentrated by a
method other than DLD, the process is completed at least three days
sooner. [0483] E228. The method of any one of E215-222, wherein, compared
to a procedure in which cells are isolated or concentrated by a method
other than DLD, the process is completed at least five days sooner.
[0484] E229. The method of any one of E215-222, wherein the time
necessary to produce a sufficient number of therapeutically active cells
for the treatment of a patient is at least 5% shorter than when the same
method is carried out using Ficoll centrifugation rather than DLD to
isolate cells from the sample composition. [0485] E230. The method of any
one of E215-222, wherein the time necessary to produce a sufficient
number of therapeutically active cells for the treatment of a patient is
at least 10% shorter than when the same method is carried out using
Ficoll centrifugation rather than DLD to isolate cells from the sample
composition. [0486] E231. The method of any one of E215-230, wherein,
when the therapeutically active cells prepared by said method are
administered to a patient, they exhibit at least 10% less senescence than
cells that have been processed from an apheresis composition using
centrifugation or elutriation instead of DLD. [0487] E232. The method of
any one of E215-230, wherein, when the therapeutically active cells
prepared by said method are administered to a patient, they exhibit
increased efficacy when compared to cells that have been processed from
the sample composition using centrifugation or elutriation instead of
DLD. [0488] E233. A method for treating a patient for a disease or
condition, comprising administering to said patient a therapeutically
effective amount of the therapeutically active cells prepared by the
method of any one of E215-230. [0489] E234. The method of E233, wherein
said the therapeutically active cells are stem cells and the disease or
condition is a genetic disease. [0490] E235. A method of engineering a
population of target cells, comprising: [0491] a) isolating the target
cells from a crude fluid composition wherein isolation is carried out on
a microfluidic device, using one or more procedures which separate target
cells from other cells in the crude fluid composition based on
differences in size and wherein the isolation produces a composition
enriched in target cells; [0492] b) genetically engineering the target
cells obtained from step a) to have a desired phenotype; [0493] wherein
target cells are not centrifuged or elutriated prior to being genetically
engineered. [0494] E236. The method of E235, wherein said target cells
are leukocytes or stem cells. [0495] E237. The method of E235, wherein
said target cells are T cells. [0496] E238. The method of any one of
E235-237, wherein said crude fluid composition is blood or an apheresis
preparation obtained from a patient. [0497] E239. The method of E238,
wherein isolation of target cells takes place under conditions such that
the composition enriched in target cells has a total number of platelets
that is at least 70% lower than in the apheresis preparation. [0498]
E240. The method of E239, wherein the total number of platelets in the
composition enriched in target cells is at least 90% lower than in the
apheresis preparation. [0499] E241. The method of E240, wherein the ratio
of platelets to target cells is at least 50% lower than in the apheresis
preparation. [0500] E242. The method of E238, wherein said target cells
are T cells that, after isolation are expanded in cell culture. [0501]
E243. The method of E242, wherein the number of T cells obtained after 14
days in culture is at least two times higher than in a procedure in which
T cells are isolated by a process including a centrifugation step.
[0502] E244. The method of E242, wherein the number of T cells obtained
after 14 days in culture is at least four times higher than in a
procedure in which T cells are isolated by a process including a
centrifugation step. [0503] E245. The method of E244, wherein the
percentage of memory T cells in culture relative to the total number of T
cells is at least 10% higher than in a procedure in which T cells are
isolated by a process including a centrifugation step. [0504] E246. The
method of E244, wherein the percentage of memory T cells in culture
relative to the total number of T cells is at least 20% higher than in a
procedure in which T cells are isolated by a process including a
centrifugation step. [0505] E247. The method of any one of E235-246,
wherein, when cells in the composition enriched in target cells are
transformed with a vector to express a recombinant phenotype, the yield
of target cells exhibiting the desired phenotype is at least 20% greater
than for identical cells isolated by centrifugation. [0506] E248. The
method of any one of E238-247, wherein no more than one hour elapses from
the time that apheresis sample collection is completed until the time
that separation using the microfluidic device is performed. [0507] E249.
The method of any one of E238-247, wherein no more than four hours elapse
from the time the obtaining of the apheresis sample from the patient is
completed until the isolation of target cells is completed. [0508] E250.
The method of any one of E238-247, wherein no more than four hours elapse
from the time the obtaining of the apheresis sample from the patient is
completed until cells are genetically engineered. [0509] E251. A method
of producing CART cells, comprising: [0510] a) obtaining a crude fluid
composition from a patient by apheresis, wherein said sample composition
comprises T cells; [0511] b) isolating the T cells from the crude fluid
composition, wherein isolation is carried out on a microfluidic device
using one or more procedures which separate T cells from platelets and
other cells in the crude fluid composition based on differences in size
and wherein the isolation produces a composition enriched in T cells and
depleted in platelets; [0512] c) genetically engineering the T cells
obtained from step a) to produce chimeric antigen receptors (CARs) on
their surface, and wherein the T cells are not centrifuged or elutriated
at any step prior to being genetically engineered; [0513] d) culturing
the genetically engineered T cells to expand their number; [0514] e)
collecting the cultured cells produced in step d). [0515] E252. The
method of E251, wherein, in step e), T cells are collected by being
transferred into a pharmaceutical composition for administration to a
patient. [0516] E253. The method of E251 or 252, wherein cells are not
frozen before being collected. [0517] E254. The method of any one of
E251-253, wherein at least 90% of platelets are removed in step b).
[0518] E255. The method of any one of E251-254, wherein prior to, or
during culturing, cells are exposed to a T cell activator or a carrier.
[0519] E256. The method of E255, wherein neither said activator nor said
carrier are bound to a magnetic bead or particle. [0520] E257. The method
of any one of E251-256, wherein, compared to a procedure in which cells
are isolated or concentrated by a method not involving the use of a
microfluidic device, the CAR T cells are available for administration to
a patient at least one day earlier. [0521] E258. The method of any one of
E251-256, wherein, compared to a procedure in which cells are isolated or
concentrated by a method not involving the use of a microfluidic device,
the CAR T cells are available for administration to a patient at least
three days earlier. [0522] E259. The method of any one of E251-258,
wherein, in step d), the number of CAR T cells obtained after 14 days in
culture is at least two times higher than the number produced by cells
obtained using Ficoll centrifugation. [0523] E260. The method of any one
of E251-258, wherein, in step d), the number of CAR T cells obtained
after 14 days in culture is at least four times higher than the number
produced by the same procedure performed using Ficoll centrifugation.
[0524] E261. The method of any one of E251-260, wherein, T cells and CAR
T cells are never frozen before being administered to a patient. [0525]
E262. A method of preparing CAR T cells for the treatment of a patient
comprising: [0526] a) obtaining a crude fluid composition from the
patient by apheresis, wherein said composition comprises T cells; [0527]
b) isolating the T cells from the crude fluid composition, wherein
isolation is carried out on a microfluidic device using one or more
procedures which separate T cells from platelets and other cells in the
crude fluid composition based on differences in size and wherein the
isolation produces a composition enriched in T cells and depleted in
platelets; [0528] c) genetically engineering the T cells obtained from
step a) to produce chimeric antigen receptors (CARs) on their surface,
and wherein the T cells are not centrifuged or elutriated at any step
prior to being genetically engineered; [0529] d) culturing the
genetically engineered T cells to expand their number; [0530] e)
separating T cells on a microfluidic device using one or more procedures
which separate T cells from reagents based on differences in size.
[0531] E263. The method of E262, wherein cells are not frozen until they
are transferred into a pharmaceutical composition for administration to a
patient. [0532] E264. The method of E262, wherein cells are not frozen at
any step. [0533] E265. The method of any one of E262-264, wherein,
compared to a procedure in which cells are isolated or concentrated by a
method that does not use a microfluidic device, the process is completed
at least three days sooner. [0534] E266. The method of any one of
E262-265, wherein, compared to a procedure in which cells are isolated or
concentrated by a method not involving the use of a microfluidic device,
the CAR T cells are available for administration to a patient at least
one day earlier. [0535] E267. The method of any one of E262-265, wherein,
compared to a procedure in which cells are isolated or concentrated by a
method not involving the use of a microfluidic device, the CAR T cells
are available for administration to a patient at least three days
earlier. [0536] E268. The method of any one of E262-267, wherein in step
b) a product composition is obtained in which the ratio of platelets to
leukocytes is at least 50% lower than the ratio obtained using
centrifugation or elutriation. [0537] E269. The method of any one of
E262-268, wherein the time necessary to produce a sufficient number of
CAR T cells for the treatment of a patient is at least 5% shorter than
when the same method is carried out using cells isolated by Ficoll
centrifugation. [0538] E270. The method of any one of E262-269, wherein
the time necessary to produce a sufficient number of CAR T cells for the
treatment of a patient is at least 10% shorter than when the same method
is carried out using Ficoll centrifugation to isolate cells from
apheresis starting material. [0539] E271. The method of any one of
E262-270, wherein, when cells prepared by said method are administered to
a patient, they exhibit at least 10% less senescence than cells that have
been processed from an apheresis composition using centrifugation or
elutriation. [0540] E272. A method for treating a patient for a disease
or condition comprising administering to said patient a therapeutically
effective amount of cells prepared by the method of any one of E235-271.
[0541] E273. The method of E272, wherein said disease or condition is
cancer.
EXAMPLES
[0542] The following examples are intended to illustrate, but not limit
the invention.
Example 1
[0543] This study focuses on apheresis samples, which are integral to
CAR-T-cell manufacture. The inherent variability associated with donor
health, disease status and prior chemotherapy all impact the quality of
the leukapheresis collection, and likely the efficacy of various steps in
the manufacturing protocols (Levine, et al., Mol. Therapy: Meth. Clin.
Dev. 4:92-101 (2017)). To stress test the automated DLD leukocyte
enrichment, residual leukocytes (LRS chamber fractions) were collected
from plateletpheresis donations which generally have near normal
erythrocyte counts, 10-20-fold higher lymphocytes and monocytes and
almost no granulocytes. They also have .about.10-fold higher platelet
counts, as compared to normal peripheral blood.
[0544] 12 donors were processed and yields were compared of major blood
cell types and processivity by DLD versus Ficoll-Hypaque density gradient
centrifugation, a "gold standard." 4 of these donors were also assessed
for "T-cell expansion capacity" over a 15-day period. Each donor sample
was processed by both DLD, and Ficoll, and for the 4 donors studied for
T-cell expansion capacity the sample was processed using direct magnetic
extraction.
Materials and Methods
[0545] Microchip design and fabrication: The DLD array used in this study
consisted of a single-zone, mirrored, diamond post design (see D'Silva,
J., "Throughout Microfluidic Capture of Rare Cells from Large Volumes of
Blood;" A Dissertation Presented to the Faculty of Princeton University
in Candidacy for the Degree of Doctor of Philosophy (2016)). There were
14 parallel arrays per chip resulting in a 14-lane DLD device (FIG. 1D).
The device was designed with a 16 .mu.m gap between posts and a 1/42
tilt, resulting in a critical diameter of .about.4 .mu.m. The plastic DLD
device was generated using a process called soft-embossing. First, a
silicon (Si) master for the plastic DLD microchip was made using standard
photolithographic and deep reactive ion etching techniques (Princeton
University, PRISM). The features on the silicon master were then
transferred to a soft elastomeric mold (Edge Embossing, Medford, Mass.)
by casting and curing the elastomer over the Si features. The elastomer
was peeled off to create a reusable, negative imprint of the silicon
master. A plastic blank sheet was placed between the elastomer molds, and
then using a combination of pressure and temperature, the plastic was
extruded into the features (wells) of the soft-elastomer negative mold,
replicating the positive features and depth of the original silicon
master. The soft tool was then peeled off from the plastic device,
producing a flat piece of plastic surface-embossed to a depth .about.100
.mu.m with a pattern of flow channels and trenches around an array of
microposts (FIG. 1D, inset). Ports were created for fluidic access to the
Input and Output ends of the microchip. After cleaning by sonication, the
device was lidded with a heat-sensitive, hydrophilic adhesive (ARFlow
Adhesives Research, Glen Rock, Pa.). The overall chip was 40.times.75 mm,
and 1 mm thick--smaller than the size of a credit card.
[0546] DLD Microchip operation: The microfluidic device was assembled
inside an optically transparent and pressure resistant manifold with
fluidic connections. Fluids were driven through the DLD microchip using a
constant pneumatic pressure controller (MFCS-EZ, Fluigent, Lowell,
Mass.). Two separate pressure controls were used, one for buffer and one
for sample. The flow path for the buffer line included tubing connecting
a buffer reservoir (60 mL syringe), an in-line degasser (Biotech DEGASi,
Minneapolis, Minn.) and the buffer inlet port of the manifold. The flow
path for the sample included tubing connecting a sample reservoir (20 mL
syringe), a 20 .mu.m PureFlow nylon filter of 25 mm diameter (Clear
Solutions, Inc. San Clemente, Calif.) to retain aggregates larger than
the microchips nominal gap size (16 .mu.m), and the sample inlet port on
the manifold. The outlet ports of the manifold were connected by tubing
to collection reservoirs for the waste and product fractions.
[0547] The microchips, filter and tubing were primed and blocked for 15
min with running buffer before the sample was loaded. The DLD setup was
primed by loading running buffer into the buffer reservoir (60 mL
syringe) and then pressurizing; fluid then passed through the tubing and
into the manifold "Buffer in" port (FIG. 1). Air in the manifold port was
vented via another port on that inlet, and then that port was sealed. The
buffer was then driven through the microchip and out both the product and
waste outlets, evacuating all air in the micropost array. At the same
time, buffer was back flushed up through the "Sample IN" port on the
manifold and through the in-line filter, flushing any air. This priming
step took .about.5 min of hands-on time, and removed all air from the
microchip, manifold and tubing. Following the prime step, buffer
continued to flush the setup for an additional 15 minutes to block all
the interior surfaces; this step was automated and did not require
hands-on time.
[0548] Following the block step, the system was depressurized, and sample
was loaded into the sample container (20 mL syringe). The sample (see
below) was diluted 1-part sample to 4 parts running buffer (0.2.times.)
prior to loading on the DLD. The buffer source was re-pressurized first,
then the sample source, resulting in both buffer and sample entering
their respective ports on the manifold and microchip and flowing through
the microchip in parallel (see separation mode, FIG. 1 Ai). Once the
sample was loaded and at running pressure, the system automatically
processed the entire sample volume. Both product and waste fractions were
collected in pre-weighed sterile conical 50 mL tubes and weighed after
the collection to determine the volumes collected.
[0549] Buffer systems. Three different EDTA free buffer formulations were
tested on the DLD: 0.5% F127 (Pluronic F-127, Sigma Aldrich, St. Louis,
Mo.) in phosphate-buffered saline [Ca.sup.++/Mg.sup.++ free) (Quality
biological, Gaithersburg, Md.), 1% Bovine Serum Albumin (BSA)
(Affymetrix, Santa Clara, Calif.) in phosphate-buffered saline
[Ca.sup.++/Mg.sup.++ free], and an isotonic Elutriation Buffer (EB)
composed of 50% Plasmalyte A (Baxter, Deerfield, Ill.) and 50% of a
mixture containing 1.0% BSA (Affymetrix, Santa Clara, Calif.) 1.0 mM
N-Acetyl-Cysteine, 2% Dextrose and 0.45% NaCl (all from Sigma-Aldrich,
St. Louis, Mo.). The buffers were prepared fresh each day, and were
sterile-filtered through a 0.2 .mu.m filter flask prior to use on the
DLD. All samples in the expansion group were processed using the isotonic
elutriation buffer to best align with current CAR-T-cell manufacturing
approaches, even though better DLD performance has been established with
the addition of poloxamer (Johnson, et al., Cancer Cell Res. 27:38-58
(2017)).
[0550] Biological Samples. Leucoreduction System (LRS) chamber samples
from plateletpheresis donations of normal screened donors using a Trima
system (Terumo, Tokyo, Japan) were obtained from the local blood bank.
Cell counts were done at the time of collection by the blood bank. Counts
were verified in our lab, using a Beckman Coulter AcT2 Diff2 clinical
blood analyzer, and ranged between 76-313.3.times.10.sup.3 WBC/.mu.L and
0.8-4.87.times.10.sup.6 platelets/.mu.L. All samples were kept overnight
at room temperature on an orbital shaker (Biocotek, China), and then
processed the following day (.about.24 hours later) to mimic overnight
shipment. Each donor sample was processed by both DLD, and Ficoll, and
for the 4 donors used for T-cell expansion and immunophenotypic studies
the sample was also processed using direct magnetic extraction.
[0551] Ficoll-Hypaque. Peripheral blood mononuclear cells (PBMCs) were
obtained by diluting the LRS sample to 0.5.times. in RPMI (Sigma-Aldrich.
St Louis, Mo.), layered on top of an equal volume of Ficoll-Hypaque (GE,
Pittsburgh, Pa.) in a 50 mL conical tube, and centrifuged for 35 min with
a free-swinging rotor, and no brake, at 400.times.g. After
centrifugation, the top layer was discarded and the interface PBMC
fraction transferred to a new 50 mL tube and brought up to 20 mL of RPMI.
PBMCs were washed by centrifugation for 10 min at 400.times.g, the
supernatant discarded and the pellet resuspended with 20 mL of RPMI and
washed again at 200.times.g for 10 min. The supernatant was removed and
the pellet resuspended in full media containing RPMI-1640+10% Fetal
Bovine Serum (FBS) (Sigma-Aldrich, St. Louis, Mo.) plus penicillin 100
units/mL and streptomycin 100 .mu.g/mL antibiotics (Thermo-Fisher,
Waltham, Mass.).
[0552] Cell Isolation, Counting, and Immunofluorescence Staining. Prior to
and after isolation using the methods described above, the cell counts of
the resulting products were determined using a blood cell analyzer
(Beckman-Coulter AcT2 Diff2). Once in culture, and after activation, cell
counts were determined using the Scepter.TM. 2.0 hand-held cell counter
(Millipore, Billerica, Mass.) and by absolute counting using flow
cytometry. Cells from the input, product and waste fractions were then
loaded onto poly-lysine-coated slides for 10 min and then fixed for 15
min in 4% p-formaldehyde+0.5% Triton X-100 in PBS, before washing 3 times
in PBS by centrifugation. Slides were incubated with the conjugated
primary antibodies CD41-A647 and CD41-FITC (both from BioLegend San
Diego, Calif.) for 60 min in the dark and washed three times with PBS
before mounting in slow-fade mounting media containing the DNA stain DAPI
(Thermo-Fisher, Waltham, Mass.). Slides were viewed with an Etaluma.TM.
Lumascope 620 fluorescence inverted microscope (Carlsbad, Calif.).
Antibodies (mAb) conjugated to fluorochromes were obtained from BioLegend
(San Diego, Calif.): CD25-PE, CD25-APC, CD95-FITC, CD45RA-BV605,
CD45RO-PECy7, CD197/CCR7 PE, CD279-PE, CD28 PE-Cy5, CD45-PerCP, CD3-FITC,
CD3-BV421, CD4-AF700, CD8-APC-AF780, CD61-FITC, CD41-FITC, CD45-Alexa647.
Viability of the WBCs obtained by DLD and PBMCs purified by
Ficoll-Hypaque was determined by Trypan blue exclusion.
[0553] Activation and Magnetic Separation. For T-cell stimulations in
expansion group, DLD, Ficoll and LRS product were diluted to
1.times.10.sup.7 T cells/mL then activated with washed and equilibrated
anti-CD3/CD28 conjugated magnetic beads (5.0 .mu.m) (Thermo-Fisher,
Waltham, Mass.) at a ratio of 3.2:1 beads per cell for 60 min, and then
the activated T cells were separated by a magnetic depletion for 5 min.
Unbound cells were removed, and the bead-bound cells were cultured
further in full media (below). In the direct magnet protocol, 0.5 mL of
LRS sample (same donor as was processed via DLD or Ficoll) was incubated
with immunomagnetic CD3/CD28 beads for one hour. The mixture was then
placed against a magnet for 5 minutes to capture the T cells. The
magnetic bead-bound cells (activated cells) were removed and then diluted
to 0.5.times.10.sup.6/mL as above for culture in full media.
[0554] After three days in culture, recombinant human IL-2 (BioLegend, San
Diego, Calif.) was added at 200 IU/mL to wells. Following cell culture
for up to 15 days, beads were removed from cells and cells counted at
each time point. To remove beads, the cells in the well were resuspended
by passing the cells through a 5-mL pipette for 10 times. Next, the cell
suspension was passed throughout a 1 mL pipette 40 times followed by
vigorous pipetting using a 200 .mu.L tip for 1 min. Then the cell
suspension was placed on the side of a magnet for 5 min and the
nonmagnetic fraction was transferred to a fresh tube and counted. The
number of cells in the culture wells was determined using a Scepter
hand-held cell counter and by flow cytometry.
[0555] Cell Culture and Cell Activation. For each of the T-cell
preparations put into cell culture, in addition to the stimulated cells
described above, unstimulated cells (controls) were adjusted to
0.5.times.10.sup.6/mL in complete media (RPMI+10% FBS+antibiotics) and
plated in 6-well plates (Corning, N.Y.) and cultured at 37.degree. C., 5%
CO2 in a humidified incubator. Individual wells, for each condition,
unstimulated, and stimulated with and stimulated without IL2, were
dedicated to each donor at each time point to eliminate any possibility
of disruption in expansion due to sampling and the de-beading activity
required for reliable counts, particularly at Day 3.
[0556] Flow Cytometry. No-wash absolute counting by flow cytometry was
used for CD3+ cell counts at all time points, Initial day 0 counts used
TruCount tubes (BD Biosciences, San Jose, Calif.) to accurately determine
the number of cells recovered and counted. Subsequent days used 25,000
123 beads (Affymetrix, Santa Clara, Calif.) which were indexed against
TruCount tubes as an internal control. 100 .mu.L of a cell suspension was
stained with the CD3 FITC, CD25 PE and CD45 PerCP of conjugated
antibodies for 30 min in the dark in either TruCount tubes or with
addition of 25,000 123 beads (Affymetrix, Santa Clara, Calif.). The cells
were then diluted to 2504 of PBS with a final DRAQ5.TM. DNA dye
(Thermo-Fisher, Waltham, Mass.) concentration of 1.0 mM. Next, the
stained cells were fixed with an additional 250 .mu.L 1.2% p-formaldehyde
in PBS overnight prior to acquisition. For absolute count cytometry, a
minimum of 25,000 events or 2500 bead events were acquired on a BD
FACSCalibur (BD Biosciences, San Jose, Calif.) using a fluorescence
threshold (CD45 PerCP). Phenotypic analysis was also performed at all
time points, using a 7-color activation/anergy panel consisting of CD3,
CD45RA, CD95, CD279, CD25, CD4, and CD8. At day 15 the panel was modified
to create a 9-color panel focused on T central memory cells which added
CD45RO PE-Cy7, CD28 PE-Cy5 and substituted CD197/CCR7 PE for CD279/PD1
PE. For multicolor staining, 100 .mu.l of a cell suspension was stained
as above, and resuspended in 7504 PBS and washed by centrifugation at
400.times.g and then resuspending in 2504 1.2% p-formaldehyde and fixed
overnight prior to acquiring 20,000 events using forward scatter
threshold on a four laser BD FACSAria II. (BD Biosciences, San Jose,
Calif.). All data analysis was performed using Flowlogic Software
(Inivai, Melbourne, Australia).
Results
[0557] DLD Microchip and Ficoll Processing of Apheresis Products
[0558] The DLD and Ficoll separation methods were used to process 12 LRS
samples obtained from 12 separate normal donors. Of those 12 samples
received and processed, 11 samples clustered around a mean of
148.7.times.10.sup.3/.mu.L WBC and 2.52.times.10.sup.6/.mu.L platelet
counts respectively (FIG. 2A, 2B). The 12.sup.th sample, with
313.3.times.10.sup.3/.mu.L WBC and 4.87.times.10.sup.6/.mu.L platelet
counts can be seen in the scatter plot as a red triangle, (FIG. 2A). This
sample was sufficiently aggregated at the time of processing that it
rapidly clogged the 20 .mu.m prefilter and thus did not fully enter the
DLD. Microscopic examination of the input sample showed that this sample
was full of platelet-WBC aggregates ranging in size from 25-50 .mu.m with
multiple aggregates observed as large as 250 .mu.m in diameter (FIG. 2C,
2D). Further, both WBC and platelet counts were greater than 3 standard
deviations above the mean WBC and platelet count. Using the quartile
method, this sample was classified as a mild outlier; using the Grubbs
test for outliers and an alpha level of 0.05, this sample was also
classified as an outlier..sup.20 As a result, this donor was excluded
from the study based on extremely high WBC and platelet counts and being
too badly agglutinated and damaged.
[0559] A representative image of the input material (LRS product diluted
to 0.2.times.) is shown in (FIG. 2A). Typical micrographs of DLD (FIG.
2E) and Ficoll (FIG. 2C) cell products from the same input donor, with
significantly lower background platelet levels (CD41-FITC in green) found
in the DLD compared to Ficoll. Also shown are the respective cell
products, as collected in tubes (FIG. 2 G, H). DLD processing automated
the process of removing the WBCs from the RBCs and platelets, generating
one tube for product and one for waste, while the Ficoll sample still
requires further manual processing to pipet the PMBC layer at the
operationally-defined interface of the plasma layer above and Ficoll
layer below (FIG. 2H); plus, an additional minimum of two centrifugal
washes are required to remove most of the contaminating platelets.
[0560] The recovery of WBC, and RBC and platelet depletions of the 11
samples are summarized in Table 2. Mean cell recoveries of PBMC from DLD
were .about.80%, 17% higher than Ficoll (63%), and, after accounting for
the number of CD3 cells in both the DLD and magnetic samples, the DLD
product was 36% higher than Direct Magnet (44%). Mean platelet depletion
via DLD (83%) was superior to both Ficoll (56.5%) and direct magnet
(77%). Mean erythrocyte depletion in these 24-hour old samples was 97%
for both DLD and Ficoll, and 94% for the direct magnet approach. The
average viability of cells obtained by DLD was 96% compared to Ficoll
which were 97%.
[0561] The average total time taken to process equivalent aliquots of a
single sample in a 50 mL conical tube via the Ficoll technique was timed
at .about.90 minutes, with approximately 30 minutes of skilled hands-on
time required. Timed runs using our single microchip layer breadboard
system processed in much shorter time, 50 minutes and required 25 minutes
of hands on time, with approximately 20 minutes being due solely to
assembly of fluidics components because of the prototypic nature of the
otherwise intervention free device.
[0562] Cell Expansion and Characterization
[0563] Following DLD or Ficoll enrichment, cells were activated using
CD3/CD28 magnetic beads for 60 minutes at a target of 3.2 beads per CD3+
cell, separated and then counted prior to plating. Due to limited access
to a flow cytometer, and concerns regarding potential bead interference
in product cell counts, we estimated the T cell count by counting both
the input and non-magnetic fraction and getting the number of T cells
bound to the magnet by subtraction, using an assumption of a 90%
efficient magnetic separation (based on manufacturer reported
efficiencies). Accurate T-cell counts were determined post-plating into
culture using absolute counts by flow cytometry and by coulter counts x %
CD3 positive cells; these counts established that the original magnetic
CD3+ cell depletion process was only 44% efficient (Table 2). This meant
that original calculations pertaining to a target of 3.2 beads per CD3+
cell were in fact on average 2.3 for both the DLD and Ficoll fractions
(fewer beads per T-cell than targeted), and a 5:1 ratio in the direct
magnet fraction (significantly more beads per T-cell than targeted),
potentially causing the direct magnet fraction to have even higher fold
expansion compared to both the DLD and Ficoll arms.
[0564] Flow cytometric characterization of the cultures was performed at
each time point to assess consistency of cell activation. Changes in CD25
expression of CD3+ cells, as measured on Day 8, for Ficoll, DLD and
direct magnet (FIG. 3). IL-2 Receptor positive (CD25) CD3 cells are shown
in Blue (CD4+ plots) and Red (CD8+ plots). DLD prepared cells show more
consistent phenotypic expression across the 4 donors for CD25, an
indicator of response to CD3/CD28 stimulation, as compared to both Ficoll
and direct magnet preparations. DLD prepared CD3+ cells had an average
73% response to co-stimulation compared to Ficoll at 51% (both stimulated
at 2.3 beads/cell), while the direct magnet fraction, stimulated at a
higher 5:1 ratio, had only a 54% response.
[0565] Unstimulated controls for Ficoll and DLD show a marked difference,
with DLD prepared cells remaining CD25 negative in culture compared to
Ficoll (FIG. 9). Interestingly, Donor 37 in the direct magnet fraction
did not respond by day 8, but did expand at later time points (also shown
in (FIG. 5A)) indicating a potentially delayed response of some samples
to the direct magnetic approach.
[0566] In addition to evaluating CD25, conversion to a memory cell
phenotype was tracked using percentage of CD3+ cells that were CD45RA-
and CD25+. The results shown in FIG. 4 indicate a greater percentage of
the cultured cells, as generated via DLD, were responsive to
co-stimulation compared to cells processed by Ficoll and direct
magnetics. Further, the percent of CD3 cells that were CD25- CD45RA- was
lowest in the DLD fraction at 12% as compared to 33 and 29% for Ficoll
and Direct Magnet respectively, indicating a more complete conversion
towards the CD25+CD45RA- population with the DLD CD3 cells. The standard
deviation of the CD45RA-CD25+ population at day 8 for DLD was 10.1% as
compared to 24.8% for Ficoll and 53.4% for Direct Magnet.
[0567] The fold expansion of the individual cultures was determined at day
3, day 8 and day 15; that data is shown in FIG. 5A. The plot shows the
expansion of each donor sample, across each method. While the direct
magnet approach appears to show higher expansion, the counts are likely
significantly affected by the different bead:cell ratios (and
corresponding differences in plating density). Regardless, the 4 donors
show significant variability in the fold expansion. In addition, the day
15 culture for the direct magnet arm donor #21 became contaminated and
had to be discarded, despite having antibiotics present. It is not
possible to know if the day 8 expansion data for donor #21 were
influenced by the contaminant.
[0568] Comparisons between the Ficoll and DLD are valid and much more
direct: these cells were plated at the same density and stimulated at the
same bead:cell ratio. While the average fold expansion of the DLD cells
is not significantly higher than that of the Ficoll cells, the
consistency of expansion across the set of 4 donors, and at all days
surveyed, is striking. Further the percent of cells in culture that are a
central memory phenotype is on average 74% for the DLD arm, contrasted to
47% and 48% respectively for the Ficoll and Direct Magnet arms.
Multiplying fold expansion in 5A by percent yield (table 1) and percent
memory (FIG. 5B) shows that, despite the sub optimal comparison with
bead:cell ratios, that on average twice as many memory cells were
produced from the DLD arm as compared to either Ficoll or Direct Magnet
arms.
[0569] FIG. 6 shows the phenotypic approach to identifying memory cells
used in this study, which is designed to eliminate any issues with shed
antigens such as CD62L (Mahnke, et al., Eur. J. of Immunol. 43:2797-2809
(2013)). Central memory cells are sequentially gated and then backgated
to show the CD3+ T cells are positive for CD45R0+, CD95+, CD28+ and
CD197/CCR7+ against all other CD3+ cells in the culture. Using an
arbitrary greater than 50% of the culture as being a central memory
phenotype as a conversion metric, the DLD arm showed 100% (4/4) donors
achieving central memory conversion with an average of 74% of cells being
of memory phenotype, with coefficient of variation across donors of 13%.
In contrast, the Ficoll arm showed 50% (2/4) converting with an average
of 47% memory cells, and a 29% variation. The direct magnet arm achieved
33% (1/3) conversion with an average of 48% memory cells and an
associated 79% variation.
TABLE-US-00002
TABLE 2
Comparison of DLD, Ficoll and Direct Magnetic Enrichment
WBC RBC Platelet
Recovery Depletion Depletion
DLD (n = 11)
Average 79.6% 96.9% 83.1%
STDEV 13.4% 1.1% 12.3%
Range 46.5-93.7% 95.5-98.6% 60.5-100.0%
Median 80.1% 97.0% 87.6%
Ficoll (n = 11)
Average 63.5% 97.1% 56.5%
STDEV 16.3% 1.7% 22.8%
Range 22.4-83.7% 94.1-99.9% 67.0-92.1%
Median 65.6% 97.0% 52.3%
Direct Magnet (CD3 positive) (n = 4)
Average 44.0% 94.1% 77.6%
STDEV 5.8% 3.3% 10.4%
Range 36.8-50.7% 90.1-97.6% 25.0-99.1%
Median 65.6% 94.5% 76.0%
Example 2: Platelet Add Back Experiment
[0570] Rationale
[0571] Previously, it has been found that WBC derived from the DLD
isolation and purification are healthy and responsive to activation by
CD3/CD28 antibodies and differentiate towards their Tcm (T central
memory) phenotype (Campos-Gonzalez, et al., SLAS, Jan. 23, 2018,
published online doi.org/10.1177/2472630317751214). Additionally, in the
presence of IL-2 Tcm cells expand and proliferate accordingly and
similarly to cells derived from other methods, like Ficoll.
[0572] A key feature of the DLD cell purification is the efficient removal
of red blood cells and platelets to provide a highly purified white blood
cells (WBC) product. In comparison, Ficoll-derived white blood cells
(PBMC's) show more contaminating red blood cells and platelets depending
on the sample quality. On average the platelet "contamination" in the
Ficoll-derived cells is 44% has a range of about 22% of variability
whereas the DLD cells exhibit only a 17% platelet contamination with
variability of +/-12%.
[0573] Because of the striking differences in the platelet depletion in
DLD-processed Apheresis blood when compared to the Ficoll-separated
Apheresis blood, an investigation was made of whether the addition of
autologous platelets to the DLD-purified white blood cells affects the
proliferation and Tcm production over a period of time.
[0574] Experimental Details
[0575] Two different Leukoreduction System-apheresis ("LRS-apheresis")
samples were collected from a local blood bank either as controls or in
2.0 mM EDTA. All four samples were processed identically by two different
methods in parallel: DLD processing and Ficoll gradient centrifugation.
The original platelet:WBC ratios provided by the blood bank were
annotated and confirmed by coulter counter determinations.
[0576] 3.0 ml of each LRS blood were processed by DLD according to our
previously described protocol by diluting the blood to 0.2.times. with
1.0% BSA/5.0 mM EDTA in PBS. Samples were run with a 1.0% BSA/PBS buffer
under standard pressure and condition using an individual DLD-14 lane
chip for each sample. Product and waste were collected, and the
cellularity was measured using a coulter counter.
[0577] 3.0 ml of each LRS blood product from the two different donors and
conditions (collected in 2.0 mM EDTA or control) were diluted 1:1 with
3.0 ml of Phosphate-buffered saline (minus Calcium and Magnesium) and
layered on top of 6.0 ml of Ficoll-Paque in a 50 ml conical tube. The
peripheral mononuclear cells of PBMC's were obtained by centrifugation
for 35 min at 400.times.g with no brake. The top layer, or plasma-rich
fraction, was removed and transferred to another tube and diluted 1:1
with PBS/1.0% BSA. The PBMC were washed with an excess of PBS by
centrifugation, once at 400.times.g for 10 min and the second time at
200.times.g for 10 min. Both supernatants were transferred to new 50 ml
conical tubes and diluted 1:1 with PBS/1.0% BSA. The diluted plasma-rich
fraction and the two supernatants were centrifuged at 1,200.times.g for
15 min, the supernatants discarded, and the pellets resuspended in
PBS/1.0% BSA, combined and centrifuged once more at 1,200.times.g for 15
min. Supernatant was discarded and the pellet- or platelet
fraction-resuspended in 1.0 ml of PBS/1.0% BSA by gentle pipetting. The
platelets were measured using the coulter counter. The corresponding
platelets were added back to the DLD-derived WBC at the desired ratios
and incubated for 1 h before activation with CD3/CD28 magnetic beads
(Thermo Fisher).
[0578] After activation, the cells were placed in complete RPMI media+10%
FBS+antibiotics and cultured over set times in a humidified incubator at
37.degree. C. and 5% CO2. Cell aliquots were analyzed at days 3, 7, and
14 by multi-color flow cytometry using the combination of antibodies
indicated in the figures. Cell culture aliquots were obtained at the
different days and the cells were de-beaded as previously described
before preparation for flow cytometry. Also, cell proliferation was
measured by using a Scepter manual cell counter. We then compared the
differences between the Ficoll-derived cells with those obtained from the
DLD processing under control conditions and when platelets--at different
ratios--were added back to the DLD-cells. The parameters we used were the
number of cells at the different time points and the number of Tcm cells
according to their phenotype by flow cytometry.
[0579] The scheme below illustrates the overall experimental design
followed during this experiment with steps proceeding from top to bottom.
##STR00001##
[0580] Results and Conclusions
[0581] White blood cells obtained using DLD consistently showed less
platelets than white blood cells obtained using Ficoll (see FIGS. 19-21).
The results also demonstrate a clearly superior expansion of T cells
derived from the DLD as compared to their counterparts from Ficoll (FIG.
22). Furthermore, the addition of platelets back to the DLD-isolated
cells reduced their ability to expand to the same levels as the
platelet-free DLD cells (FIG. 22). These results support the hypothesis
that the more efficient platelet-reduction during DLD processing of blood
products produces white blood cells more responsive to activation by
CD3/CD28 and expansion by IL-2.
REFERENCES
[0582] 1. Vonderheide, R. H., and June, C. H. Engineering T-cells for
Cancer: Our Synthetic Future. Immunol. Rev. 2014, 257, 7-13. [0583] 2.
Fousek, K., and Ahmed, N. The Evolution of T-cell Therapies for Solid
Malignancies. Clin. Cancer Res. 2015, 21, 3384-3392. [0584] 3. Wang, X
and Riviere, I. Clinical Manufacturing of CAR-T-cells: Foundation of a
Promising Therapy. Mol. Ther. Oncolytics 2016, 3, 16015. [0585] 4.
Sadelain, M., Riviere, I. and Riddell, S. Therapeutic T-cell engineering.
Nature, 2017, 545, 423-431. [0586] 5. National Cell Manufacturing
Consortium. Achieving Large-Scale, Cost-Effective, Reproducible
Manufacturing of High Quality Cells. A Technology Roadmap to 2025.
February 2016. [0587] 6. Levine, B. L., Miskin, J., Wonnacott, K., et al.
Global Manufacturing of CAR T-cell Therapy. Mol. Therapy: Meth. Clin.
Dev. 2017, 4, 92-101. [0588] 7. Couzin-Frankel, J. Supply of Promising
T-cell Therapy is Strained. Science 2017, 356, 1112. [0589] 8. Johnson,
L. A., and June, C. H. Driving Gene-engineered T-cell Immunotherapy of
Cancer. Cell Res. 2017, 27, 38-58. [0590] 9. Hokland, P., and Heron, I.
The Isopaque-Ficoll Method Re-evaluated: Selective Loss of Autologous
Rosette-forming Lymphocytes During Isolation of Mononuclear Cells from
Human Peripheral Blood. Scand. J. Immunol. 1980, 11, 353-356. [0591] 10.
Stroncek, D. F., Fellowes, V., Pham, C., et al. Counter-flow Elutriation
of Clinical Peripheral Blood Mononuclear Cell Concentrates for the
Production of Dendritic and T-cell Therapies. J. Transl. Med. 2014, 12,
241. [0592] 11. Powell Jr, D. J., Brennan, A. L., Zheng, Z., et al.
Efficient Clinical-scale Enrichment of Lymphocytes for Use in Adoptive
Immunotherapy Using a Modified Counterflow Centrifugal Elutriation
Program. Cytotherapy 2009, 11, 923-935. [0593] 12. TerumoBCT. ELUTRA Cell
Separation System. Manufacturer recommendations for the Enrichment of
Lymphocytes from Apheresis Residues. [0594] 13. C. E. Chiche-Lapierre, C.
E., Tramalloni, D, Chaput, N. et al. Comparative Analysis of Sepax S-100,
COBE 2991, and Manual DMSO Removal Techniques From Cryopreserved
Hematopoietic Stem Cell Apheresis Product Cytotherapy 2016 18, 6:S47.
[0595] 14. Huang, L, Cox, E, Austin, R. Continuous particle separation
through deterministic lateral displacement, Science 2004, 304:987-990.
[0596] 15. Davis, J. A., Inglis, D. W., et al. Deterministic
Hydrodynamics: Taking Blood Apart. Proc. Natl. Acad. Sci. USA 2006, 103,
14779-14784. [0597] 16. Inglis, D. W., Davis, J. A., Austin, R. H.
Critical particle Size for Fractionation by Deterministic Lateral
Displacement. Lab Chip 2006, 6, 655-658. 17. Chen, Y, D'Silva, J. Austin,
R et al. Microfluidic chemical processing with on-chip washing by
deterministic lateral displacement arrays with separator walls.
Biomicrofluidics. 2015 9(5): 054105. [0598] 18. Shilun, F. Skelley, A.,
Anwer, A. G., et al. Maximizing Particle Concentration in Deterministic
Lateral Displacement Arrays. Biomicrofluidics 2017, 11, 024121. [0599]
19. D'Silva, J. Throughout Microfluidic Capture of Rare Cells from Large
Volumes of Blood. A Dissertation Presented to the Faculty of Princeton
University in Candidacy for the Degree of Doctor of Philosophy. 2016.
[0600] 20. NIST/SEMATECH e-Handbook of Statistical Methods, 2017,
www.itl.nist.gov/div898/handbook/August [0601] 21. Mahnke, Y. D., Brodie,
T. M., Sallusto, F., et al. The Who's Who of T-cell Differentiation:
Human Memory T-cell Subsets. Eur. J. of Immunol. 2013, 43, 2797-2809.
[0602] 22. Civin, C. I., Ward, T., Skelley, A. M., et al. Automated
Leukocyte Processing by Microfluidic Deterministic Lateral Displacement.
Cytometry A. 2016, 89, 1073-1083. [0603] 23. Trickett, A., and Kwan, Y.
L. T-cell Stimulation and Expansion Using Anti-CD3/CD28 Beads. J.
Immunol. Meth. 2003, 275, 251-255. [0604] 24. Marktkamcham, S., Onlamoon,
N., Wang, S., et al. The Effects of Anti-CD3/CD28 Coated Beads and IL-2
on Expanded T-cell for Immunotherapy. Adv. Clin. Exp. Med. 2016, 25,
821-828. [0605] 25. Li, Y., and Kurlander, R. J. Comparison of Anti-CD3
and Anti-CD28-coated Beads with Soluble Anti-CD3 for Expanding Human
T-cells: Differing Impact on CD8 T-cell Phenotype and Responsiveness. J.
Transl. Med. 2010, 8, 104-118. [0606] 26. Agrawal S., Ganguly, S., Hjian,
P., et al. PDGF Upregulates CLEC-2 to Induce T Regulatory Cells.
Oncotarget. 2015, 6, 28621-28632. [0607] 27. Zhu, L., Huang, Z.,
Stalesen, R. et al. Platelets Provoke Distinct Dynamics of Immune
Response by Differentially Regulating CD4.sup.+ T-cell Proliferation. J.
Throm. Haem. 2014, 12, 1156-1165. [0608] 28. Koesdjojo, M., Lee, Z.,
Dosier, C., et al. DLD Microfluidic Purification and Characterization of
Intact and Viable Circulating Tumor Cells in Peripheral Blood. AACR
Annual Meeting 2016, Abstract #3956. [0609] 29. Loutherback, K.
"Microfluidic Devices for High Throughput Cell Sorting and Chemical
Treatment," A Dissertation Presented to the Faculty of Princeton
University 2011.
[0610] All references cited herein are fully incorporated by reference.
Having now fully described the invention, it will be understood by one of
skill in the art that the invention may be performed within a wide and
equivalent range of conditions, parameters and the like, without
affecting the spirit or scope of the invention or any embodiment thereof
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