Note: Descriptions are shown in the official language in which they were submitted.
WO 2021/252898
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COMPOSITIONS AND METHODS OF MANUFACTURING AUTOLOGO-US T CELL
THERAPIES
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Patent Application Serial
No.
63/038,516, file on June 12, 2020, and to U.S. Provisional Patent Application
Serial No.
63/161,283, file on March 15, 2021, the content of each of which is
incorporated by reference in
its entirety, and to each of which priority is claimed.
BACKGROUND OF THE INVENTION
Various methods are available for preparing cells for therapeutic use. For
example,
methods are available for isolating, processing, and engineering cells,
including T cells and other
immune cells. Methods are available to isolate such cells and to express
genetically engineered
antigen receptors, such as high affinity T cell receptors (TCRs) and chimeric
antigen receptors
(CARs). Methods are available to adoptively transfer such cells into subjects.
Presently, standardized methods for cell therapy manufacturing are still
missing, with the
overall processes being extremely complex, as comprising multiple handling
steps, each one
capable of causing operator errors, compromising the overall reproducibility.
Thus, improved
and standardized methods are needed for the preparation (e.g., isolation,
processing, culturing,
and engineering) of cells for use in cell therapy. In particular, methods are
needed for the
preparation and engineering of cells, e.g., a plurality of isolated cell types
or sub-types, with
improved efficiency, safety, variability, and conservation of resources. The
present disclosure
provides methods, cells, compositions, kits, and systems that meet such needs.
SUMMARY OF THE INVENTION
The present disclosure provides for compositions and methods for manufacturing
autologous cell therapies. In certain embodiments, the presently disclosed
subject matter
provides a method of producing a therapeutic population of T cells comprising:
a) obtaining a
sample comprising T cells from a subject; b) isolating a first population of T
cells; c) activating
the first population of T cells, wherein the activation is performed in a
closed container providing
a gas-permeable surface area; d) transfecting the first population of T cells
to express an
exogenous nucleic acid; e) expanding the first population of T cells to obtain
a second population
of T cells, wherein the expansion is performed in a closed container providing
a gas-permeable
surface area; f) harvesting the second population of T cells; and g)
transferring the harvested
second population of T cells in an infusion bag, whereby the second population
of T cells is the
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therapeutic population of T cells. In certain embodiments, the method is
performed within a
closed system. In certain embodiments, the closed system is a sterile
environment.
In certain embodiments, the expansion is performed by culturing the T cells to
produce a
population of young T cells In certain embodiments, the expansion is performed
by culturing
the first population of T cells in the presence of IL2, IL7, IL15, IL21, or
any combination thereof
In certain embodiments, the expansion is performed by culturing the first
population of T cells in
the presence of IL7 and IL15. In certain embodiments, the population of young
T cells
comprises cells that are CD45RA+, CD62L+, CD28+, CD95-, CCR7+, and CD27+.
In certain embodiments, the population of young T cells comprises cells that
are
CD45RA+, CD62L+, CD28+, CD95+, CD27+, CCR7+. In certain embodiments, the
population
of young T cells comprises cells that are CD45R0+, CD62L+, CD28+, CD95+,
CCR7+, CD27+,
CD127+ In certain embodiments, the expansion is performed by culturing the
first population of
T cells in the presence of fibronectin, insulin, transferrin, or any
combination thereof. In certain
embodiments, the expansion is performed by culturing the first population of T
cells at a glucose
concentration of at least about 3.7 g/L.
In certain embodiments, the activation is performed by using non-magnetic
beads. In
certain embodiments, the first population of T cells comprises CD4 T cells. In
certain
embodiments, the first population of T cells comprises CD8 T
In certain embodiments, the method further comprises cryopreserving the second
population of T cells. In certain embodiments, the cryopreservation is
performed by adding
CS10 media to the infusion bag. In certain embodiments, the CS10 media has a
final
concentration of about 50%. In certain embodiments, the cryopreservation is
performed by
adding human serum albumin to the infusion bag. In certain embodiments, the
human serum
albumin has a final concentration of about 1%. In certain embodiments, the
cryopreservation is
performed by adding a crystalloid solution to the infusion bag. In certain
embodiments, the
crystalloid solution has a final concentration of about 46%.
In certain embodiments, the first population of T cells is concentrated by
centrifugation.
In certain embodiments, the centrifugation occurs between the activating and
the transfecting. In
certain embodiments, the second population of T cells is concentrated by
centrifugation. In
certain embodiments, the centrifugation occurs during the harvesting. In
certain embodiments,
the centrifugation is a counter-flow centrifugation. In certain embodiments,
the transfer of cells
occurs by using a peristaltic pump.
In certain embodiments, the transfecting occurs via viral vector. In certain
embodiments,
transfecting occurs via electroporation. In certain embodiments, the
transfecting comprises
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introducing into a T cell a polynucleotide, comprising: first and second
homology arms
homologous to first and second target nucleic acid sequences and oriented to
facilitate
homologous recombination; a nucleotide sequence encoding a TCR polypeptide
sequence
positioned between the first and second homology arms; and a first nucleotide
sequence encoding
a P2A ribosome skipping element positioned upstream of the nucleotide sequence
encoding the
TCR polypeptide and a second nucleotide sequence encoding a P2A ribosome
skipping element
positioned downstream of the nucleotide sequence encoding the TCR polypeptide,
wherein the
first and second nucleotide sequences encoding the P2A ribosome skipping
elements are codon-
diverged relative to each other.
In certain embodiments, the first and second homology arms of the
polynucleotide are
each from about 300 bases to about 2,000 bases in length. In certain
embodiments, the
polynucleotide further comprises: a nucleotide sequence encoding for the amino
acid sequence
Gly Ser Gly positioned immediately upstream of the nucleotide sequences
encoding the 2A
ribosome skipping elements; a nucleotide sequence encoding for a Furin
cleavage site upstream
of the second nucleotide sequence encoding a 2A ribosome skipping element; and
a nucleotide
sequence encoding for a human growth hormone signal peptide positioned
upstream of the
nucleotide sequence encoding the TCR. In certain embodiments, the
polynucleotide further
comprises a second nucleotide sequence encoding a TCR polypeptide sequence
between the
second nucleotide sequence encoding a P2A ribosome skipping element and the
second
homology arm. In certain embodiments, the polynucleotide further comprises a
second
nucleotide sequence encoding for a human growth hormone signal peptide
positioned upstream
of the second nucleotide sequence encoding the TCR polypeptide. In certain
embodiments, the
polynucleotide is a circular DNA.
In certain embodiments, the second population of T cells expresses an
exogenous TCR
gene sequence encoding for a TCR that recognizes a tumor antigen. In certain
embodiments, the
tumor antigen is a neoantigen. In certain embodiments, the tumor antigen is a
patient specific
neoantigen. In certain embodiments, the exogenous TCR gene sequence is a
patient specific
TCR gene sequence. In certain embodiments, the transfecting comprises cleavage
of an
endogenous locus by a nuclease. In certain embodiments, the nuclease is a
Clustered Regularly
Interspaced Short Palindromic Repeats (CRISPR) family nuclease or derivative
thereof. In
certain embodiments, the nuclease further comprises an sgRNA.
In certain embodiments, the activating is performed within a period of about 2
days, or
about 3 days In certain embodiments, the expanding is performed within a
period of about 10
days, about 11 days, or about 12 days. In certain embodiments, the method is
performed within a
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period of about 13 days, about 14 days, about 15 days. In certain embodiments,
the second
population of T cells comprises at least 20% Tmsc and Tcm collectively, at
least 25% Tmsc and
Tern collectively, at least 30% Tmsc and Tcm collectively, at least 35% Tmsc
and Tcm
collectively, at least 40% Tmsc and Tern collectively, at least 45% Tmsc and
Tcm collectively, at
least 50% Tmsc and Tern collectively, at least 55% Tmsc and Tern collectively,
at least 60%
Tmsc and Tern collectively or more than 61% Tmsc and Tern collectively. In
certain
embodiments, the therapeutic population of T cells is infused into the
subject.
In certain embodiments, the presently disclosed subject matter provides a
composition
comprising the therapeutic population of T cells produced by any one of the
method disclosed
herein, hi certain embodiments, the composition comprises a pharmaceutical
excipient. In
certain embodiments, the composition comprises a therapeutically effective
amount. In certain
embodiments, the therapeutic population of T cells comprises at least 1 x 106
cells/ml. In certain
embodiments, the therapeutic population of T cells comprises at least 10 x 106
cells/ml. In
certain embodiments, the therapeutic population of T cells comprises at least
100 x 106 cells/ml.
In certain embodiments, the presently disclosed subject matter provides a
method of
treating cancer comprising administering the therapeutic population of T cells
produced by any
one of the methods disclosed herein to a subject in need thereof In certain
embodiments, the
presently disclosed subject matter provides a method of treating cancer
comprising administering
the composition disclosed herein to a subject in need thereof.
In certain embodiments, the presently disclosed subject matter provides a
method
comprising: contacting a cell with an activation reagent; editing the cell to
express an exogenous
nucleic acid; culturing the edited cell in a cell culture medium; harvesting
the edited cell; and
transferring the edited cell in a container; wherein the method is performed
within a closed
system. In certain embodiments, the presently disclosed subject matter
provides a method
comprising: contacting a cell with an activation reagent; editing the cell to
express an exogenous
nucleic acid; culturing the cell in a cell culture medium to obtain a
population of cells; harvesting
the population of cells; and transferring the population of cells in a
container; wherein the
method is performed within a closed system. In certain embodiments, the method
further
comprises a counter-flow centrifugation. In certain embodiments, the method
occurs within a
period of about 13 days, about 14 days, about 15 days.
In certain embodiments, the editing comprises introducing into the cell a
polynucleotide.
In certain embodiments, the polynucleotide comprises: first and second
homology arms
homologous to first and second target nucleic acid sequences and oriented to
facilitate
homologous recombination; a nucleotide sequence encoding a TCR polypeptide
sequence
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positioned between the first and second homology arms; and a first nucleotide
sequence encoding
a P2A ribosome skipping element positioned upstream of the nucleotide sequence
encoding the
TCR polypeptide and a second nucleotide sequence encoding a P2A ribosome
skipping element
positioned downstream of the nucleotide sequence encoding the TCR polypeptide,
wherein the
first and second nucleotide sequences encoding the P2A ribosome skipping
elements are codon-
diverged relative to each other. In certain embodiments, the first and second
homology arms of
the polynucleotide are each from about 300 bases to about 2,000 bases in
length. In certain
embodiments, the polynucleotide further comprises: a nucleotide sequence
encoding for the
amino acid sequence Gly Ser Gly positioned immediately upstream of the
nucleotide sequences
encoding the 2A ribosome skipping elements; a nucleotide sequence encoding for
a Furin
cleavage site upstream of the second nucleotide sequence encoding a 2A
ribosome skipping
element; and a nucleotide sequence encoding for a human growth hormone signal
peptide
positioned upstream of the nucleotide sequence encoding the TCR. In certain
embodiments, the
polynucleotide further comprises a second nucleotide sequence encoding a TCR
polypeptide
sequence between the second nucleotide sequence encoding a P2A ribosome
skipping element
and the second homology arm. In certain embodiments, the polynucleotide
further comprises a
second nucleotide sequence encoding for a human growth hormone signal peptide
positioned
upstream of the second nucleotide sequence encoding the TCR polypeptide. In
certain
embodiments, the polynucleotide is a circular DNA.
In certain embodiments, the edited cell expresses an exogenous TCR gene
sequence
encoding for a TCR that recognizes a tumor antigen. In certain embodiments,
the population of
cells comprises a cell expressing an exogenous TCR gene sequence encoding for
a TCR that
recognizes a tumor antigen. In certain embodiments, the tumor antigen is a
neoantigen. In
certain embodiments, the tumor antigen is a patient specific neoantigen. In
certain embodiments,
the exogenous TCR gene sequence is a patient specific TCR gene sequence.
In certain embodiments, the transfecting comprises cleavage of an endogenous
locus by a
nuclease In certain embodiments, the nuclease is a Clustered Regularly
Interspaced Short
Palindromic Repeats (CRISPR) family nuclease or derivative thereof In certain
embodiments,
thenuclease further comprises an sgRNA. In certain embodiments, the editing
comprises a viral
infection. In certain embodiments, the editing comprises electroporation.
In certain embodiments, the cell is a primary cell, a lymphocyte, or a T cell.
In certain
embodiments, the population of cells comprises a primary cell, a lymphocyte, a
T cell, or a
combination thereof. In certain embodiments, the T cell is a CD8 or a CD4 T
cell. In certain
embodiments, the T cell is a young T cell. In certain embodiments, the young T
cell is
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CD45RA , CD62L CD28 , CD95-, CCR7 , and CD27 . In certain embodiments, the
young T
cell is CD45RA+, CD62L+, CD28+, CD95+, CCR7+, CD27+. In certain embodiments,
the young
T cell is CD45RW, CD62L+, CD28, CD95', CCR7', CD27, CD127-. In certain
embodiments,
the young T cell is a memory stem cell (Tmsc). In certain embodiments, the
young T cell is a
central memory cells (Tcm).
In certain embodiments, the population of cells comprises at least about 20%
of Tmsc and
Tcm collectively, at least about 25% Tmsc and Tcm collectively, at least about
30% Tmsc and Tcm
collectively, at least about 35% Tmsc and Tcm collectively, at least about 40%
Tmsc and Tcm
collectively, at least about 45% Tmsc and Tem collectively, at least about 50%
Tmsc and Tcm
collectively, at least about 55% Tmsc and Tcm collectively, at least about 60%
Tmsc and Tom
collectively or more than about 61% Tmsc and Tcm collectively.
In certain embodiments, the cell is obtained from a subject. In certain
embodiments, the
cell is obtained by leukapheresis. In certain embodiments, the cell is
obtained by a tissue sample.
In certain embodiments, the tissue sample is a tumor sample. In certain
embodiments, the cell is
cryopreserved.
In certain embodiments, the cell culture medium comprises interleukin 2 (IL2),
interleukin 7 (IL7), interleukin 15 (IL15), interleukin (IL21), or any
combination thereof. In
certain embodiments, the cell culture medium comprises 1L7 and 11-15. In
certain embodiments,
the cell culture medium comprises fibronectin, insulin, transferrin, or any
combination thereof.
In certain embodiments, the cell culture medium comprises glucose
concentration of at least
about 3.7 g/L. In certain embodiments, the culturing is performed within a
period of about 10
days, about 11 days, or about 12 days.
In certain embodiments, the activation reagent comprises an anti-CD3 antibody,
an anti-
CD2 antibody, an anti-CD28 antibody, or a combination thereof. In certain
embodiments, the
activation reagent comprises non-magnetic beads or magnetic beads. In certain
embodiments,
the activation reagent comprises an artificial APCs. In certain embodiments,
the contacting is
performed within a period of about 2 days, or about 3 days.
In certain embodiments, the method further comprises cryopreserving the edited
cell. In
certain embodiments, the method further comprises cryopreserving the
population of cells.
In certain embodiments, the edited cell or population of cells is
cryopreserved in a
phattnaceutical formulation. In certain embodiments, the pharmaceutical
formulation comprises
a cryopreservation medium, a serum albumin, a crystalloid solution, or a
combination thereof In
certain embodiments, the cryopreservation medium is at a final concentration
of about 50% v/v.
In certain embodiments, the serum albumin is at a final concentration of about
1% w/v. In
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certain embodiments, the crystalloid solution is at a final concentration of
about 46% v/v. In
certain embodiments, the pharmaceutical formulation comprises CryoStork C S10,
human serum
albumin, Plasma-Lyte A, or a combination thereof.
In certain embodiments, the closed system comprises a peristaltic pump. In
certain
embodiments, the edited cell is infused into a subject. In certain
embodiments, the population of
cells is infused into the subject.
In certain embodiments, the presently disclosed subject matter also provides a
composition comprising the edited cells obtained by the methods disclosed
herein. In certain
embodiments, the presently disclosed subject matter provides a composition
comprising the
population of cells obtained by the methods disclosed herein. In certain
embodiments, the
composition further comprises a pharmaceutical excipient. In certain
embodiments, the
composition comprises a therapeutically effective amount of cells. In certain
embodiments, the
composition comprises at least 1 x 106 cells/ml. In certain embodiments, the
composition
comprises at least 10 x 106 cells/ml. In certain embodiments, the composition
comprises at least
100 x 106 cells/ml. In certain embodiments, the composition comprises at least
about 4.0 x 108
gene-edited cells. In certain embodiments, the composition comprises at least
about 1.3 x 109
gene-edited cells. In certain embodiments, the composition comprises at least
about 4.0 x 109
gene-edited cells. In certain embodiments, the composition comprises at least
about 1.3 x 1010
gene-edited cells. In certain embodiments, the composition comprises at least
about 4.0 x 1010
gene-edited cells.
In certain embodiments, the presently disclosed subject matter further
provides methods
of treating a cancer comprising administering the edited cell obtained by the
methods disclosed
herein, the population of cells obtained by the methods disclosed herein, or
the compositions
disclosed herein to a subject in need thereof.
In certain embodiments, the presently disclosed subject matter provides the
edited cell
obtained by the methods disclosed herein, the population of cells obtained by
the methods
disclosed herein, or the compositions disclosed herein for use in the
treatment of a cancer. In
certain embodiments, the presently disclosed subject matter provides the use
of the edited cell
obtained by the methods disclosed herein, the population of cells obtained by
the methods
disclosed herein, or the compositions disclosed herein for the manufacture of
a medicament for
the treatment of cancer.
In certain embodiments, the presently disclosed subject matter provides a
method of
manufacturing NeoTCR Cells using Process 1, Process 2, Process 3, or Process 4
described
herein. In certain embodiments, the presently disclosed subject matter also
provides a
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composition comprising NeoCells manufactured using Process 1, Process 2,
Process 3, or
Process 4 described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1A-1C. Figures 1A-1C show an example of a NeoE TCR cassette and gene
editing methods that can be used to make NeoTCR Products. Figure 1A shows a
schematic
representing the general targeting strategy used for integrating neoantigen-
specific TCR
constructs (NeoTCRs) into the TCRa locus. Figures 1B and 1C show a neoantigen-
specific
TCR construct design used for integrating a NeoTCR into the TCRa locus wherein
the cassette is
shown with signal sequences ("SS"), protease cleavage sites ("P"), and 2A
peptides ("2A").
Figure 1B shows a target TCRa locus (endogenous TRAC, top panel) and its
CRISPR Cas9
target site (horizontal stripes, cleavage site designated by the arrow), and
the circular plasmid HR
template (bottom panel) with the polynucleotide encoding the NeoTCR, which is
located
between the left and right homology arms ("LHA" and "RHA" respectively) prior
to integration.
Figure 1C shows the integrated NeoTCR in the TCRa locus (top panel), the
transcribed and
spliced NeoTCR mRNA (middle panel), and translation and processing of the
expressed
NeoTCR (bottom panel).
Figure 2. Figure 2 shows a schematic for Process 1 as described in the
Examples.
Figure 3. Figure 3 shows a schematic for Process 2 as described in the
Examples.
Figure 4. Figure 4 shows a schematic for Process 2 as described in the
Examples as it
pertains to a 3 NeoTCR Product. Specifically, the middle column shows the
process steps for
each of the three different sublots for a 3 NeoTCR Product, while the left
column lists the
processing equipment utilized at each step. The right column illustrates QC
sampling during the
process.
Figures 5A and 5B. Figures 5A and 5B show the results of unit-operations-based
optimizations, T cell activation, and gene-editing efficiencies. Figure 5A
shows CD25
expression as measured by flow cytometry following two days of activation of
CD4/CD8 cells in
either Prodigy or G-Rex 100M flasks using the same ratio of TransAct. Figure
5B shows T cell
activation in G-Rex flasks results in similar gene-editing rates (% NeoTCR+)
as compared to T
cells activated in Prodigy at end of culture (day 13). WT= wild-type cells
(non-edited cells
expressing endogenous T cell receptor), KO: knock-out, i.e. cells in which
endogenous TCR was
knocked out, but no NeoTCR knock-in occurred, NeoTCR: cells expressing only
the neoantigen-
specific TCR.
Figures 6A and 6B. Figures 6A and 6B show the results of unit-operations-based
optimizations and T cell expansion and phenotype. For evaluation of
feasibility of cell expansion
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in G-Rex flasks, cells were activated in G-Rex 100M flask and following day 2
electroporation
cells were transferred to a G-Rex 100M flask for further cell expansion. On
day 9, cells were
transferred to G-Rex 500M (1:5 split) until end of culture on day 13. Cells
from the same donor
(split-run) were activated on the Prodigy, nucleofected on day 2, and expanded
in Prodigy
CentriCult chamber until day 13. Figure 6A shows that cells expanded in G-Rex
flasks have
enhanced cell expansion as compared to cells expanded within Prodigy
CentriCult unit. Figure
6B shows that cells expanded in G-Rex show similar T cell phenotype at the end
of culture (day
13) as compared to cells expanded in the Prodigy. Tn: naïve T cells, Tmsc+Tcm:
memory stem
cell T cells and central memory T cells, Ttm +Tem: transitional memory T cells
and effector
memory T cells, T eff: effector T cells.
Figure 7. Figure 7 shows the post-rebuffering cell recovery in the Rotea v.
the Prodigy.
Results show day 2 percent post rebuffering cell recovery using either current
Prodigy CAP
rebuffering process (Process 1) (n=7, 5 donors) or optimized process using
Rotea (Process 2)
(n=12, 6 donors). % Cell recovery was calculated based on total viable cell
counts pre- and post-
rebuffering into electroporation buffer. Cells used for Rotea rebuffering
process were activated
for two days in G-Rex 100M flasks, while cells rebuffered using Prodigy were
activated in
Prodigy.
Figure 8. Figure 8 shows the percent recovery after final formulation using
the Rotea
the Prodigy. Results show a comparison of % cell recovery post formulation
into Plasmalyte +
2% HSA using either current Prodigy harvest procedure (Process 1) (n=7, 4
different donors) or
Rotea formulation procedure (Process 2) (n=9, 6 donors). Cells used for Rotea
final formulation
process were activated and expanded in G-Rex flasks (Process 2), while cells
for Prodigy control
were activated and expanded in the Prodigy (Process 1). % Cell recovery was
calculated based
on total viable cell counts pre- and post-formulation into Plasmalyte + 2%
HSA.
Figure 9. Figure 9 shows post-thaw viability following cryopreservation. Post-
thaw
viability was measured for the NeoTCR Product formulated at a target cell
concentration of 10
million cells/mL cryopreserved in either CryoMACS 250 bags with a fill volume
of 35 mL or a
CryoMACS 500 bag with 70mL fill volume_ Cells used for final formulation were
manufactured
using either the current Prodigy manufacturing process (Process 1) or
optimized manufacturing
process (Process 2).
Figure 10. Figure 10 shows post-thaw cell concentration by final formulation
method.
Post-thaw cell concentration was measured for the NeoTCR Product formulated at
a target cell
concentration of 10 million cell s/mL cryopreserved in CryoMACS 250 bags with
a fill volume of
mL and CryoMACS 500 bags with 70mL fill volume. Cells used for final
formulation were
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manufactured using either the current Prodigy manufacturing process (Process
1) or optimized
manufacturing process (Process 2).
Figure 11. Figure 11 shows the percent NeoTCR+ cells (i.e., NeoTCR Cells) by
final
formulation method. The percentage of NeoTCR Cells was measured for the NeoTCR
Product
formulated at a target cell concentration of 10 million cells/mL cryopreserved
in CryoMACS 250
bags (35 mL fill volume), CryoMACS 500 bag (70mL fill volume), and QC sample
vial. Cells
used for final formulation were manufactured using either the current Prodigy
manufacturing
process (Process 1) or optimized manufacturing process (Process 2).
Figure 12. Figure 12 shows T cell phenotype by cryopreservation method. T cell
phenotype was assessed for NeoTCR Product formulated at a target cell
concentration of 10
million cells/mL cryopreserved in CryoMACS 250 bags (35 mL fill volume),
CryoMACS 500
bag (70mL fill volume), and QC sample vial. Cells used for final formulation
were manufactured
using either the current Prodigy manufacturing process (Process 1) or
optimized manufacturing
process (Process 2). Tnaive: naive T cells, Tcm+Tmsc: central memory T cells
and memory
stem cell T cells, Tem+Ttm: effector memory T cells and transitional memory T
cells, T eft':
effector T cells.
Figure 13. Figure 13 shows cell expansion for Process 2 as compared to Process
1.
Data shown are the results from five direct split comparison runs (same donor)
with fold
expansion (day 2 to end of study) shown in blue for Process 1 and red
(optimized Process 2).
Historical cell expansion data from original engineering and clinical
readiness runs at Miltenyi
(v1.0) are shown for comparison in black.
Figure 14. Figure 14 shows percentage of NeoTCR+ cells post gene-editing for
optimized process (Process 2) as compared to Process 1. Data shown are the
results from five
direct split comparison runs (same donor) with the % NeoTCR+ of final product
shown in blue
for Process 1 and red for the optimized Process 2. Historical percentage of
NeoTCR+ expression
levels from original engineering and clinical readiness runs at Miltenyi are
shown in black for
comparison.
Figure 15. Figure 15 shows NeoTCR+ cell yield for the optimized process
(Process 2)
as compared to Process 1. Data shown are the results from five direct split
comparison runs
(same donor) with total NeoTCR+ cell yield at end of study shown in full
circles for Process 1
and full diamonds for Process 2. Historical NeoTCR+ cell yield from original
engineering and
clinical readiness runs at Miltenyi are shown in empty circles for comparison.
Figure 16 Figure 16 shows percent viability of NeoTCR Cells for optimized
process
(Process 2) as compared to Process 1. Data shown are the results from five
direct split
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comparison runs (same donor) with % viability (NeoTCR Product, pre-
cryopreservation) shown
in blue for Process 1 and red for Process 2. Historical % Viability (NeoTCR
Product QC release
data) from original engineering and clinical readiness runs at Miltenyi are
shown for comparison.
Figure 17 Figure 17 shows IFN-gamma production of NeoTCR Cells for the
optimized
process (Process 2) as compared to Process 1. Data shown are the results for
NeoTCR Product
IFN-gamma production from four direct split comparison runs (same donor) shown
in blue for
Process 1 and red for Process 2. Samples for IFN-gamma analysis were taken pre-
harvest as per
quality control (QC) sampling plan for the current NeoTCR Product. Historical
IFN-gamma
production (NeoTCR Product QC release data) from original engineering and
clinical readiness
runs at Miltenyi are shown in black for comparison.
Figures 18A and 18B. Figures 18A and 18B show NeoTCR Cells generated using
either
the optimized process (Process 2) or Process 1 induced highly specific anti-
tumor cytotoxicity.
Data shown are results from IncuCytee killing assays, in which NeoTCR Cells
from Process 2,
Process 1 or media only as negative control were added to target cells
expressing the NeoTCR
target (Figure 18A) or wild-type cell line (Figure 181B) at a 5.1 ratio. These
cell lines only differ
in the expression of one amino acid which is altered in the neoE sequence. The
reduction in %
nRFP is a measure of target cell death.
Figure 19 Figure 19 shows a high level diagram of Process 2 and Process 1
Figures 20A and 20. Figures 20A and 20B show a comparison of activation
markers and
proliferation data for TexMACS media supplemented with 3% human serum compared
to Prime-
XV media supplemented with 2% Physiologix and shows that Prime XV media can
support
activation and proliferation in a huAB serum-free environment. Figure 20A
shows cell surface
markers CD25 and CD69 (associated with T cell activation) and KI-67 (an
intracellular marker of
proliferation) which were assessed by flow cytometry on day 2 from all the
experimental arms.
As shown, there was greater than 10% increases in all three markers for Prime-
XV media versus
the TexMACs media. Figure 20B shows the increased T cell growth kinetics for
the Prime-XV
media (correlating to the increase in activation markers shown in Figure 20A).
Figures 21A and 21B. Figures 21A and 21B show that in addition to the overall
support
of Prime XV medium +2% Physiologix for T cell activation and proliferation, a
5-fold increase
in NeoTCR Cells over that seen with TexMACS medium with 3 % huAB serum was
also
observed (Figure 21A), supported by the relative increase in Dex+ IP26+ T
cells. Figure 21B
shows that there was a slight decrease in the CD4/CD8 ratio. Based on this,
enhanced T cell
proliferation also supported expansion of the NeoTCR Cells. Figure 21A is
represented as
follows: Yellow Diamond: total viable NeoTCR Cells ( calculated as % NeoTCR+
(Dex+/1P26+)
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x total viable cells based on NC200; Green bar: % NeoTCR+ cells (Dex+/IP26+);
Black Bar: WT
cells not knockout cells, as they are still expressing TCR; Grey bar: %
knockout cells, which
neither express NeoTCR nor WT TCR. Overall, the use of Prime-XV supports
improved gene-
editing (partially due to better survival of gene-edited cells post el
ectroporati on and less huAB
residuals). As better editing in CD8 T cells in large scale was shown, skewing
the CD4/CD8
ratio towards CD8 will increase the total percent of NeoTCR Cells.
Figure 22. Figure 22 shows that improved expansion observed in PrimeXV media
did
not result in loss of CD62L expression (i.e., the increased proliferation and
better support of the
NeoTCR Cells did not promote further differentiation of the T cells).
Figures 23A and 23B. Figure 23A shows a 2 fold increase in gene editing
efficiency
without serum/serum replacement and 4 fold increase in total edited cells on
Day 13. Figure
23B shows a 3 fold increase in cell expansion with Prime-XV supplemented with
2%
Physiologix XF compared to TexMACs and improved cell expansion observed even
in the
absence of serum/serum replacement.
Figures 24A and 24B. Figure 24A shows the gene-editing efficiency at Day 8
with
different media using large-scale manufacturing and electroporation methods.
Figure 24B shows
the cell growth kinetics for the cells cultured under conditions presented in
Figure 24A. See
Figure 21A for a description of the bars and diamonds in Figure 24A.
Figure 25. Figure 25 shows a diagram of the custom-designed Gravity Drain to
harvest
cells. It was used on Day 2 of Process 2 to transfer activated cells from G-
Rex to a transfer pack
that was subsequently welded on the Rotea; used on Day 13 of Process 2 to
split culture
dependent on cell density; used on Day 13 of Process 2 to harvest cells from
the G-Rex into a
transfer pack that was welded on the Rotea. The same design was also used for
Process 3;
however, the timing of when the Gravity Drain was used (i.e., Days 2 and 13)
varied based on the
other manufacturing optionalities described for Process 3.
Figure 26. Figure 26 shows a diagram of the custom media removal setup that
was used
on Day 8 of Process 2 to reduce culture volume to ¨100mL for media
exchange/culture split and
on Day 13 of Process 2 to reduce culture volume for cell substance harvest and
formulation. The
same design was also used for Process 3; however, the timing of when the
custom media removal
setup was used (i.e., Days 8 and 13) varied based on the other manufacturing
optionalities
described for Process 3.
Figure 27 Figure 27 shows a diagram for the custom-designed post-
electroporation cell
transfer that was used on Day 2 after el ectroporation in Process 2. The cells
were transferred into
a G-Rex containing pre-warmed media. The same design was also used for Process
3; however,
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the timing of when the custom post-electroporation cell transfer was used
(i.e., Day2) varied
based on the type of activation agent used in the process.
Figure 28. Figure 28 shows the use of the WMFG 530S enables seamless closed
transfer
with accurate volume delivery (WMFG 5305 is an example of the closed transfer
machine
diagramed in Figure 19). The WMFG 530S was used in Process 2 for the following
steps:
Activation setup, Expansion setup, Day 8 Culture Split, Harvest, and CS 10
Addition.
Figure 29. Figure 29 shows that 40% of cells were washed out during the
processing of
cells using standard centrifugation as described in Example 5. In contrast,
the use of
counterflow centrifugation (using the Rotea) resulted in minimal cell loss and
resulting in
minimal cell loss.
Figure 30. Figure 30 shows glucose measurements obtained from cultures after
the
rebuffering procedures using standard centrifugation and washes/buffer
exchanges (the Prodigy)
compared to counterflow centrifugation (G-Rex, using the Rotea). This shows
that counterflow
centrifugation is able to significantly increase media removal compared to
standard
centrifugation methods.
Figure 31. Figure 31 shows two variations of counterflow centrifugation
methods:
Version 1 (V1) and Version 2 (V2). V1 was set at a centrifugal force of 2500g
with a fluid flow
rate of 30mL/min and V2 was set at a centrifugal force of 2700g with a fluid
flow rate of
10mL/min. As shown, V2 with an increased g force and slower fluid flow rate
resulted in a
significant increase in the rate of cell recovery.
Figure 32 Figure 32 shows variable T cell expansion in ENG runs The observed
variability prompted Medium A investigation. Medium A is TexMACs.
Figure 33 Figure 33 shows the study design for evaluation of incoming Medium A
lots.
Medium A is TexMACs.
Figure 34. Figure 34 shows activation markers of T cells cultured with
different cell
media. No significant differences were observed across media lots. Medium A is
TexMACs.
Medium B is PrimeXV.
Figure 35 Figure 35 shows cell expansion and viability on day 8. Cell
expansion and
viability of T cells had comparable results with ENG runs on day 8. Medium A
is TexMACs.
Medium B is PrimeXV.
Figure 36. Figure 36 shows gene editing efficiencies on day 8. Substantial
differences
in gene editing were observed on day 8 across medium A lots. Medium A is
TexMACs.
Medium B is PrimeXV.
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Figure 37. Figure 37 shows cell expansion and viability on day 13. The
differences in
cell expansion and viability were sustained at harvest on day 13. Medium A is
TexMACs.
Medium B is PrimeXV.
Figure 38 Figure 38 shows gene editing efficiencies on day 13. The differences
in gene
editing were maintained at harvest on day 13. Medium A is TexMACs. Medium B is
PrimeXV.
Figure 39. Figure 39 shows a summary of the data and results disclosed in
Figures 32-
38. Medium A is TexMACs. Medium B is PrimeXV.
Figure 40. Figure 40 shows capillary electrophoresis ¨ mass spectrometry (CE-
MS)
analysis of T cell media. Significant variability was observed across medium A
lots identified
through extensive screening. CE-MS analysis revealed absence of glutamine in
low-performance
lots. Medium A is TexMACs. Medium B is PrimeXV.
Figure 41. Figure 41 shows that Medium B improves post EP recovery and gene
editing
efficiency. Significant improvement in gene editing efficiency was observed in
medium B
(u=24.5% vs. 14.6%). Medium B attenuated EP induced lag phase between day 2-8
and
improved overall expansion (approximately 2-fold). This evidence shows that
even greater
expansion can be achieved with additional media exchanges to mitigate lactate
accumulation.
Medium A is TexMACs. Medium B is PrimeXV.
Figure 42 Figure 42 shows increased NeoTCR-F cell yield with Medium B CDM at
clinical MFG scale. Medium A is TexMACs. Medium B is PrimeXV.
Figure 43. Figure 43 shows T cell immunophenotype characterization and
comparable
subset. Medium A is TexMACs. Medium B is PrimeXV
Figure 44. Figure 44 shows cell product characterization (IFNy secretion and
cytotoxicity). Medium A is TexMACs. Medium B is PrimeXV.
Figure 45. Figure 45 shows a summary of the medium B CDM implementation.
DETAILED DESCRIPTION
The present disclosure is directed to compositions and methods for the
manufacture of
cell therapeutics, e.g., autologous cell therapeutics such as NeoTCR Cells.
Non-limiting
embodiments of the compositions and methods for the manufacture of cell
therapeutics are
described by the present description and examples. For purposes of clarity of
disclosure and not
by way of limitation, the detailed description is divided into the following
subsections:
1. Definitions
2. Adoptive Cell Therapies
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3. Therapeutic Compositions and Methods of Manufacturing
4. Article of Manufacture
5. Methods of Treatment
6. Kits
1. Definitions
Unless defined otherwise, all technical and scientific terms used herein have
the meaning
commonly understood by a person skilled in the art. The following references
provide one of
skill with a general definition of many of the terms used in the present
disclosure: Singleton et
al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The
Cambridge
Dictionary of Science and Technology (Walker ed., 1988); The Glossary of
Genetics, 5th Ed., R.
Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper
Collins Dictionary
of Biology (1991). As used herein, the following terms have the meanings
ascribed to them
below, unless specified otherwise.
It is understood that aspects arid embodiments of the invention described
herein include
"comprising," " consisting, ' and "consisting essentially of aspects and
embodiments. The terms
"comprises" and "comprising" are intended to have the broad meaning ascribed
to them in U.S.
Patent Law and can mean "includes", "including" and the like.
As used herein, the term "about" or "approximately" means within an acceptable
error
range for the particular value as determined by one of ordinary skill in the
art, which will depend
in part on how the value is measured or determined, i.e., the limitations of
the measurement
system. For example, "about" can mean within 3 or more than 3 standard
deviations, per the
practice in the art. Alternatively, "about" can mean a range of up to 20%,
e.g., up to 10%, up to
5%, or up to 1% of a given value. Alternatively, particularly with respect to
biological systems
or processes, the term can mean within an order of magnitude, e.g., within 5-
fold or within 2-
fold, of a value.
As used herein, the term "cell culture medium" refers to a composition, e.g.,
liquid or gel,
designed to support the growth, maintenance, and/or differentiation of cells.
The composition
can include sources of energy and additional compounds regulating the cell
functions. In certain
non-limiting embodiments, the cell culture medium includes essential or non-
essential amino
acids (e.g., cysteine and glutamic acid), vitamins, inorganic salts, glucose,
serum, hormones (e.g.,
insulin), lipids, chemokines, and cytokines. In certain non-limiting
embodiments, the cell culture
medium is serum-free.
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As used herein, the term -cryopreservation medium" refers to a composition
comprising a
cryopreservative, e.g., DMSO. Non-limiting examples of cryopreservation medium
include
CryoStor C S10, PSC Cryopreservation Medium, CryoDefend Cell Lines Media,
and
HypoThermosol FRS.
As used herein, the term -culturing" refers to contacting a cell with a cell
culture medium
under conditions suitable to the growth, maintenance, and/or differentiation
of the cell.
As used herein, "Medium A" refers to TexMACs. As used herein, "Medium B"
refers to
PrimeXV.
As used herein, the terms "closed system" or "closed-system" refer to
processes that have
no exposure to the surrounding environment. Closed systems prevent the ingress
of microbes
from the environment and incorporates single-use disposable components for all
materials that
come into contact with the product, e.g., NeoTCR Cells. In certain
embodiments, all components
and reagents are presterilized by terminal or filter sterilization. In certain
embodiments, closed
systems enable the processing of multiple patient batches in parallel in the
same area. In certain
embodiments, closed systems eliminate the need for the processing of products
in a biosafety
cabinet (B SC) within a high-grade cleanroom (Grade B/Class 10,000).
"Dextramer" as used herein means a multimerized neoepitope-HLA complex that
specifically binds to its cognate NeoTCR.
The term "tumor antigen" as used herein refers to an antigen (e.g., a
polypeptide) that is
uniquely or differentially expressed on a tumor cell compared to a normal or
non-neoplastic cell.
In certain embodiments, a tumor antigen includes any polypeptide expressed by
a tumor that is
capable of activating or inducing an immune response via an antigen-
recognizing receptor or
capable of suppressing an immune response via receptor-ligand binding.
As used herein, the terms "neoantigen", -neoepitope" or "neoE" refer to a
newly formed
antigenic determinant that arises, e.g., from a somatic mutation(s) and is
recognized as "non-
self" A mutation giving rise to a "neoantigen", "neoepitope" or "neoE" can
include a frameshift
or non-frameshift indel, missense or nonsense substitution, splice site
alteration (e.g.,
alternatively spliced transcripts), genomic rearrangement or gene fusion, any
genomic or
expression alterations, or any post-translational modifications.
"TCR" as used herein means T cell receptor.
-NeoTCR- and "NeoE TCR- and "exogenous TCR- as used herein mean a neoepitope-
specific T cell receptor that is introduced into a T cell, e.g., by gene-
editing methods. As used
herein, the term "TCR gene sequence" refers to a NeoTCR gene sequence
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-NeoTCR cells" as used herein means one or more cells precision-engineered to
express
one or more NeoTCRs. In certain embodiments, the cells are T cells. In certain
embodiments,
the T cells are CD8+ and/or CD4+ T cells. In certain embodiments, the CD8+
and/or CD4+ T
cells are autologous cells from the patient for whom a NeoTCR Product will be
administered.
The terms "NeoTCR cells" and "NeoTCR-I- cells" and "NeoTCR-P1 T cells" and
"NeoTCR-P1
cells" are used interchangeably herein.
"Cell Product" as used herein means a gene-edited cell therapy wherein one or
more 2A
peptides are used in the gene-editing process. In certain embodiments, the
Cell Product is made
through the insertion of DNA wherein the gene of interest is inserted between
two 2A sequences
(see, e.g., Figure 2A). In certain embodiments, the DNA is linear or circular
(e.g., plasmid
DNA). In certain embodiments, the Cell Product is made through the insertion
of DNA wherein
the gene of interest is flanked on one side by a 2A peptide. In certain
embodiments, when there
is more than one 2A peptide sequence, such sequences are the same 2A peptides
(e.g., two P2A
sequences, two T2A sequences, two E2A sequences, or 2 F2A sequences). In
certain
embodiments, when there is more than one 2A peptide sequence, such sequences
are different 2A
peptides (e.g., but not limited to, one T2A and one P2A). In certain
embodiments, Cell Products
are made using viral gene-editing methods. In certain embodiments, Cell
Products are made
using targeted or non-targeted viral methods. In certain embodiments, Cell
Products are made
using non-viral gene-editing methods. Cell Products include but are not
limited to T cell
products, NK cell products HSCs, Tits, and cell products derived from HSCs.
Cell Products can
also include any other naturally occurring cell that can be edited using a 2A
peptide as part of the
gene-editing process. Cell Products can be used, for example, for the
treatment of autoimmune
diseases, neurological diseases and injuries (including but not limited to
Alzheimer's disease,
Parkinson's disease, spinal cord, and nerve injuries and/or damage), cancer,
infectious diseases,
joint disease (including but not limited to rebuilding damaged cartilage in
joints), improving the
immune system, cardiovascular disease and abnormalities, aging, immune
deficiencies (including
but not limited to multiple sclerosis and amyotrophic lateral sclerosis),
allergies, and genetic
disorders. Cell Products include NeoTCR Products and NeoTCR Viral Products.
"NeoTCR Product" as used herein means a pharmaceutical formulation comprising
one
or more NeoTCR cells. NeoTCR Product consists of autologous precision genome-
engineered
CD8+ and CD4+ T cells. Using a targeted DNA-mediated non-viral precision
genome
engineering approach, expression of the endogenous TCR is eliminated and
replaced by a
patient-specific NeoTCR isolated from peripheral CDS+ T cells targeting the
tumor-exclusive
neoepitope. In certain embodiments, the resulting engineered CD8+ or CD4+ T
cells express
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NeoTCRs on their surface of native sequence, native expression levels, and
native TCR function.
The sequences of the NeoTCR external binding domain and cytoplasmic signaling
domains are,
in certain embodiments, unmodified from the TCR isolated from native CD8+ T
cells.
Regulation of the NeoTCR gene expression is, in certain embodiments, driven by
the native
endogenous TCR promoter positioned upstream of where the NeoTCR gene cassette
is integrated
into the genome. Through such approaches, native levels of NeoTCR expression
are observed in
unstimulated and antigen-activated T cell states.
The NeoTCR Product manufactured for each patient represents a defined dose of
autologous CD8+ and/or CD4+ T cells that are precision genome engineered to
express a single
neoE-specific TCR cloned from neoE-specific CD8+ T cells individually isolated
from the
peripheral blood of that same patient.
As used herein, the term "CD8 Product" refers to a pharmaceutical composition
comprising one or more NeoTCR cells expressing an exogenous CD8. Additional
information
on the CD8 Products can be found in U.S. Patent Publication No. 2021/0085721,
the content of
which is incorporated by reference in its entirety.
"NeoTCR Viral Product" as used herein has the same definition of NeoTCR
Product
except that the genome engineering is performed using viral-mediated methods.
"Pharmaceutical Formulation" refers to a preparation that is in such form as
to permit the
biological activity of an active ingredient contained therein to be effective,
and which contains no
additional components which are unacceptably toxic to a subject to which the
formulation would
be administered For clarity, quantities of DMSO used in a Cell Product or a
NeoTCR Product
are not considered unacceptably toxic.
"Treat," "Treatment," and "treating" are used interchangeably and as used
herein mean
obtaining beneficial or desired results including clinical results. Desirable
effects of treatment
include, but are not limited to, preventing occurrence or recurrence of
disease, alleviation of
symptoms, diminishment of any direct or indirect pathological consequences of
the disease,
preventing metastasis, decreasing the rate of disease progression,
amelioration or palliation of the
disease state, and remission or improved prognosis. In some embodiments, the
NeoTCR Product
of the present disclosure is used to delay the development of a proliferative
disorder (e.g., cancer)
or to slow the progression of such disease.
A "subject," "patient," or an "individual" for purposes of treatment refers to
any animal
classified as a mammal, including humans, domestic and farm animals, and zoo,
sports, or pet
animals, such as dogs, horses, cats, cows, etc Preferably, the mammal is
human.
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The terms -Cancer" and "Tumor" are used interchangeably herein. As used
herein, the
terms "Cancer" or "Tumor" refer to all neoplastic cell growth and
proliferation, whether
malignant or benign, and all pre-cancerous and cancerous cells and tissues.
The terms are further
used to refer to or describe the physiological condition in mammals that is
typically characterized
by unregulated cell growth/proliferation. Cancer can affect a variety of cell
types, tissues, or
organs, including but not limited to an organ selected from the group
consisting of bladder, bone,
brain, breast, cartilage, glia, esophagus, fallopian tube, gallbladder, heart,
intestines, kidney,
liver, lung, lymph node, nervous tissue, ovaries, pancreas, prostate, skeletal
muscle, skin, spinal
cord, spleen, stomach, testes, thymus, thyroid, trachea, urogenital tract,
ureter, urethra, uterus,
and vagina, or a tissue or cell type thereof Cancer includes cancers, such as
sarcomas,
carcinomas, or plasmacytomas (malignant tumor of the plasma cells). Examples
of cancer
include, but are not limited to, those described herein. The terms "Cancer" or
"Tumor" and
"Proliferative Disorder" are not mutually exclusive as used herein.
"2A" and "2A peptide" are used interchangeably herein and mean a class of 18-
22 amino
acid long, viral, self-cleaving peptides that are able to mediate cleavage of
peptides during
translation in eukaryotic cells. Four well-known members of the 2A peptide
class are T2A, P2A,
E2A, and F2A. The T2A peptide was first identified in the Thosea asigna virus
2A. The P2A
peptide was first identified in the porcine teschovirus-1 2A. The E2A peptide
was first identified
in the equine rhinitis A virus. The F2A peptide was first identified in the
foot-and-mouth disease
virus. The self-cleaving mechanism of the 2A peptides is a result of ribosome
skipping the
formation of a glycyl-prolyl peptide bond at the C-terminus of the 2A
Specifically, the 2A
peptides have a C-terminal conserved sequence that is necessary for the
creation of steric
hindrance and ribosome skipping. The ribosome skipping can result in one of
three options. 1)
successful skipping and recommencement of translation resulting in two cleaved
proteins (the
upstream of the 2A protein which is attached to the complete 2A peptide except
for the C-
terminal proline and the downstream of the 2A protein which is attached to one
proline at the N-
terminal; 2) successful skipping but ribosome fall-off that results in
discontinued translation and
only the protein upstream of the 2A; or 3) unsuccessful skipping and continued
translation (i.e., a
fusion protein). The term "endogenous" as used herein refers to a nucleic acid
molecule or
polypeptide that is normally expressed in a cell or tissue.
The term "exogenous- as used herein refers to a nucleic acid molecule or
polypeptide that
is not endogenously present in a cell. The temi "exogenous" would therefore
encompass any
recombinant nucleic acid molecule or polypeptide expressed in a cell, such as
foreign,
heterologous, and over-expressed nucleic acid molecules and polypeptides. By -
exogenous"
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nucleic acid is meant a nucleic acid not present in a native wild-type cell;
for example, an
exogenous nucleic acid may vary from an endogenous counterpart by sequence, by
position/location, or both. For clarity, an exogenous nucleic acid may have
the same or different
sequence relative to its native endogenous counterpart; it may be introduced
by genetic
engineering into the cell itself or a progenitor thereof, and may optionally
be linked to alternative
control sequences, such as a non-native promoter or secretory sequence.
As used herein, the term "population of cells" refers to a group of two or
more cells. For
example, and without any limitation, a population of cells can include
different cell types, e.g., T
cells and NK cells, or different clones, e.g., NeoTCR cells expressing
different NeoTCRs. In
certain non-limiting embodiments, the population of cells includes at least
about 2, at least about
10, at least about 100, at least about 200, at least 500, at least 1000 cells,
at least about 1 x 104
cells, at least about 1 x 105 cells, at least about 1 x106 cells, at least
about 1 x 107 cells, or at least
about 1 x 108 cells.
"Young" or "Younger" or "Young T cell" as it relates to T cells means memory
stem
cells (Tmsc) and central memory cells (Tcm). These cells have T cell
proliferation upon specific
activation and are competent for multiple cell divisions. They also can
engraft after re-infusion,
to rapidly differentiate into effector T cells upon exposure to their cognate
antigen and target and
kill tumor cells, as well as to persist for ongoing cancer surveillance and
control.
2. Adoptive Cell Therapies
In one aspect, the present disclosure is directed to methods and compositions
relating to
the development and improvement of cell-based therapies, referred herein as
adoptive cell
therapies. As described in detail herein, adoptive cell therapies involve the
use of cells to target
and facilitate the elimination of diseased cells, e.g., cancer cells. In
certain non-limiting
embodiments, the present disclosure is directed to methods useful for the
manufacturing of
adoptive cell therapies. For example, without any limitation, the methods
disclosed herein allow
for the manufacturing of adoptive cell therapies in closed systems to minimize
the risk of
contamination.
As used herein, "adoptive cell therapy" refers to a therapy in which cells
from the
immune system are infused into a subject to help the body fight diseases. In
certain
embodiments, the adoptive cell therapy involves the use of cells taken from a
subject's own
immune system, expanded ex vivo, and then infused into the subject to help the
subject's immune
system fight a disease. In certain embodiments, the adoptive cell therapy
relates to the infusion
of cells that are engineered to improve their ability to target a cancer cell
In certain
embodiments, the adoptive cell therapy relates to the infusion of cells of the
immune system or
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hematopoietic stem cells. For example, without any limitation, adoptive cell
therapies can
involve the infusion of CDS+ T cells, CD4+ T cells, NK-cells, delta-gamma T-
cells, regulatory
T-cells, or tumor-infiltrating lymphocytes. In certain embodiments, the
adoptive cell therapy
involves the infusion of T cells. In certain embodiments, the adoptive cell
therapy involves the
infusion of NK cells. In certain embodiments, the adoptive cell therapy is
directed to the use of
Cell Products, as described in Section 2.1. In certain embodiments, the
adoptive cell therapy is
directed to the use of NeoTCR Products, described in Section 2.2. In certain
embodiments, the
adoptive cell therapy includes NeoTCR Viral Products, as described below.
2.1. Cell Products
Cell Products comprise cell therapies derived from cells that are gene-edited
using
constructs containing one or more 2A peptides. Such cell products can be made
using non-viral
or viral methods wherein a gene of interest is inserted into a genome using 2A
peptides in the
constructs. The use of the 2A peptide sequences enables the expression of
multiple proteins
within a single open reading frame through co-translational cleavage events
and can overcome
the problem of uneven expression of different proteins which overcomes a major
hurdle in gene
editing.
Cell Products include gene-edited cells that retain all or a portion of a 2A
peptide
sequence in the translated product such that the gene or genes of interest
inserted into a genome
retain all or a portion of the 2A peptide on one or both of the flanking ends
of the gene(s).
Cell Products also include gene-edited cells that fully cleave off the 2A
peptide from the
gene of interest during translation such that the gene or genes of interest
inserted into a genome
do not have all or a portion of the 2A peptide on either of the flanking ends
of the gene(s). In this
scenario, the inserted gene of interest does not contain any non-native
epitopes caused by one or
more amino acids of the 2A peptide including on either of the flanking ends of
the gene(s).
Cell Products include gene-edited cells that are edited using viral and/or non-
viral
methods.
2.2. NeoTCR Products
In certain embodiments, using the gene-editing technology and NeoTCR isolation
technology described in PCT/U S2020/17887 and PCT/US2019/025415, which are
incorporated
herein in their entireties, NeoTCRs are cloned in autologous CD8+ and CD4+ T
cells from the
same patient with cancer by precision genome engineered (using a DNA-mediated
(non-viral)
method as described in Figures 1A-1C) to express the NeoTCR. In other words,
the NeoTCRs
that are tumor-specific are identified in cancer patients, such NeoTCRs are
then cloned, and then
the cloned NeoTCRs are inserted into the cancer patient's own T cells. NeoTCR
expressing T
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cells are then expanded in a manner that preserves -young" T cell phenotypes,
resulting in a
NeoTCR Product in which the majority of the T cells exhibit T memory stem cell
and T central
memory phenotypes.
These 'young' or 'younger' or less-differentiated T cell phenotypes are
described to
confer improved engraftment potential and prolonged persistence post-infusion.
Thus, the
administration of NeoTCR Product, consisting significantly of 'young' T cell
phenotypes, has the
potential to benefit patients with cancer, through improved engraftment
potential, prolonged
persistence post-infusion, and rapid differentiation into effector T cells to
eradicate tumor cells
throughout the body.
Ex vivo mechanism-of-action studies were also performed with NeoTCR Product
manufactured with T cells from patients with cancer. Comparable gene editing
efficiencies and
functional activities, as measured by antigen-specificity of T cell killing
activity, proliferation,
and cytokine production, were observed demonstrating that the manufacturing
process described
herein is successful in generating product with T cells from patients with
cancer as starting
material.
In certain embodiments, the NeoTCR Product manufacturing process involves
electroporation of dual ribonucleoprotein species of CRISPR-Cas9 nucleases
bound to guide
RNA sequences, with each species targeting the genomic TCRa and the genomic
TCRp loci_ The
specificity of targeting Cas9 nucleases to each genomic locus has been
previously described in
the literature as being highly specific. Comprehensive testing of the NeoTCR
Product was
performed in vitro and in silico analyses to survey possible off-target
genomic cleavage sites,
using COSMID and GUIDE-seq, respectively. Multiple NeoTCR Products or
comparable cell
products from healthy donors were assessed for cleavage of the candidate off-
target sites by deep
sequencing, supporting the published evidence that the selected nucleases are
highly specific.
Further aspects of the precision genome engineering process have been assessed
for
safety. No evidence of genomic instability following precision genome
engineering was found in
assessing multiple NeoTCR Products by targeted locus amplification (TLA) or
standard FISH
cytogenetics. No off-target integration anywhere into the genome of the NeoTCR
sequence was
detected. No evidence of residual Cas9 was found in the cell product.
The comprehensive assessment of the NeoTCR Product and precision genome
engineering process indicates that the NeoTCR Product will be well tolerated
following infusion
back to the patient.
The genome engineering approach described herein enables highly efficient
generation of
bespoke NeoTCR T cells (i.e., NeoTCR Products) for personalized adoptive cell
therapy for
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patients with solid and liquid tumors. Furthermore, the engineering method is
not restricted to the
use in T cells and has also been applied successfully to other primary cell
types, including natural
killer and hematopoietic stem cells.
2 . 3. Pharmaceutical Formulations
Pharmaceutical formulations of the Cell Products are prepared by combining the
NeoTCR
cells in a solution that can preserve the 'young' phenotype of the cells in a
cryopreserved state.
Additional pharmaceutically acceptable carriers, buffers, stabilizers, and/or
preservatives
can also be added to the cryopreservation solution or the aqueous storage
solution. Any
cryopreservation agent and/or media can be used to cryopreserve the Cell
Product, including but
not limited to CryoStor, Cry oStor CS5, CELLBANKER, and custom
cryopreservation media that
optionally include DMSO.
In certain embodiments, the Cell Products are NeoTCR Products or NeoTCR Viral
Products, hi certain embodiments, the Cell Products are NeoTCR Viral Products.
In certain
embodiments, the Cell Products are NeoTCR Products.
2.4. Gene-Editing Methods
In certain embodiments, the present disclosure involves, in part, methods of
engineering
human cells, e.g., engineered T cells or engineered human stem cells. In
certain embodiments,
the present disclosure involves, in part, methods of engineering human cells,
e.g., NK cells, NKT
cells, macrophages, hematopoietic stem cells (HSCs), cells derived from HSCs,
or
dendritic/antigen-presenting cells. In certain embodiments, such engineering
involves genome
editing. For example, but not by way of limitation, such genome editing can be
accomplished
with nucleases targeting one or more endogenous loci, e.g., TCR alpha (TCRa)
locus and TCR
beta (TCRP) locus. In certain embodiments, the nucleases can generate single-
stranded DNA
nicks or double-stranded DNA breaks in an endogenous target sequence. In
certain
embodiments, the nuclease can target coding or non-coding portions of the
genome, e.g., exons,
introns. In certain embodiments, the nucleases contemplated herein comprise
homing
endonuclease, meganuclease, megaTAL nuclease, transcription activator-like
effector nuclease
(TALEN), zinc-finger nuclease (ZFN), and clustered regularly interspaced short
palindromic
repeats (CRISPR)/Cas nuclease. In certain embodiments, the nucleases can
themselves be
engineered, e.g., via the introduction of amino acid substitutions and/or
deletions, to increase the
efficiency of the cutting activity.
In certain embodiments, a CRISPR/Cas nuclease system is used to engineer human
cells.
In certain embodiments, the CRISPR/Cas nuclease system comprises a Cas
nuclease and one or
more RNAs that recruit the Cas nuclease to the endogenous target sequence,
e.g., single guide
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RNA. In certain embodiments, the Cas nuclease and the RNA are introduced in
the cell
separately, e.g. using different vectors or compositions, or together, e.g.,
in a polycistronic
construct or a single protein-RNA complex. In certain embodiments, the Cas
nuclease is Cas9 or
Casl 2a. In certain embodiments, the Cas9 polypeptide is obtained from a
bacterial species
including, without limitation, Streptococcus pyogene,s or Neisseria
menengitidis. Additional
examples of CRISPR/Cas systems are known in the art. See Adli, Mazhar. -The
CRISPR tool kit
for genome editing and beyond." Nature communications vol. 9,1 1911 (2018),
herein
incorporated by reference for all that it teaches.
In certain embodiments, genome editing occurs at one or more genome loci that
regulate
immunological responses. In certain embodiments, the loci include, without
limitation, TCR
alpha (TCRa) locus, TCR beta (TCRI3) locus, TCR gamma (TCRy), and TCR delta
(TCR).
In certain embodiments, genome editing is performed by using non-viral
delivery
systems. For example, a nucleic acid molecule can be introduced into a cell by
administering the
nucleic acid in the presence oflipofection (Feigner et al., Proc. Natl. Acad.
Sci. U.S.A. 84:7413,
1987; Ono et al., Neuroscience Letters 17.259, 1990; Brigham et al., Am. J.
Med. Sci. 298:278,
1989; Staubinger et al., Methods in Enzymology 101:512, 1983),
asialoorosomucoid-polylysine
conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu
etal., Journal of
Biological Chemistry 264:16985, 1989), or by micro-injection under surgical
conditions (Wolff
et al., Science 247:1465, 1990). Other non-viral means for gene transfer
include transfection in
vitro using calcium phosphate, DEAE dextran, electroporation, and protoplast
fusion. Liposomes
can also be potentially beneficial for delivery of DNA into a cell.
Transplantation of normal
genes into the affected tissues of a subject can also be accomplished by
transferring a normal
nucleic acid into a cultivatable cell type ex vivo (e.g., an autologous or
heterologous primary cell
or progeny thereof), after which the cell (or its descendants) are injected
into a targeted tissue or
are injected systemically.
In certain embodiments, genome editing is performed by using viral delivery
systems. In
certain embodiments, the viral methods include targeted integration (including
but not limited to
AAV) and random integration (including but not limited to lentiviral
approaches). In certain
embodiments, the viral delivery would be accomplished without integration of
the nuclease. In
such embodiments, the viral delivery system can be Lentiflash or another
similar delivery
system.
In certain embodiments, the gene-editing methods comprise insertion (i.e.,
knock-in) of a
NeoTCR into a cell in combination with the knockout of the endogenous TCR. In
certain
embodiments, the knock-in of a NeoTCR and knockout of the endogenous TCR gene-
edited cell
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further comprises a knock-in of an additional expression element. In certain
embodiments, the
knock-in of a NeoTCR and knockout of the endogenous TCR gene-edited cell
further comprises
a knockout of an additional endogenous element. Additional information
regarding the gene-
editing can be found in International Patent Application Nos. PCT/US20/031007,
PCT/US20/030818, and PCT/U S20/030704.
2.5. Homology Recombination Templates
In certain embodiments, the present disclosure provides genome editing of a
cell by
introducing and recombining homologous recombination (HR) template nucleic
acid sequence
into an endogenous locus of a cell. In certain embodiments, the Hit template
nucleic acid
sequence is linear. In certain embodiments, the HR template nucleic acid
sequence is circular.
In certain embodiments, the circular FIR template can be a plasmid, a
minicircle, or a
nanoplasmid. In certain embodiments, the HR template nucleic acid sequence
comprises first
and second homology arms. In certain embodiments, the homology arms can be of
about 300
bases to about 2,000 bases. For example, each homology arm can be 1,000 bases.
In certain
embodiments, the homology arms can be homologous to first and second
endogenous sequences
of the cell. In certain embodiments, the endogenous locus is a TCR locus. For
example, the first
and second endogenous sequences are within a TCR alpha locus or a TCR beta
locus. In certain
embodiments, the I-1R template comprises a TCR gene sequence. In non-limiting
embodiments,
the TCR gene sequence is a patient-specific TCR gene sequence. In non-limiting
embodiments,
the TCR gene sequence is tumor-specific. In non-limiting embodiments, the TCR
gene sequence
can be identified and obtained using the methods described in
PCT/US2020/017887, the content
of which is herein incorporated by reference. In certain embodiments, the HR
template
comprises a TCR alpha gene sequence and a TCR beta gene sequence.
In certain embodiments, the FIR template is a polycistronic polynucleotide. In
certain
embodiments, the HR template comprises sequences encoding for flexible
polypeptide sequences
(e.g., Gly-Ser-Gly sequence). In certain embodiments, the BR template
comprises sequences
encoding an internal ribosome entry site (IRES). In certain embodiments, the
fiR template
comprises a 2A peptide (e.g., P2A, T2A, E2A, and F2A). Additional information
on the BR
template nucleic acids and methods of modifying a cell thereof can be found in
International
Patent Application no. PCT/US2018/058230, the content of which is herein
incorporated by
reference.
2.6. Nucleic Acid Compositions and Vectors
The present disclosure provides compositions comprising cells (e.g., T cells)
disclosed
herein.
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In certain embodiments, the present disclosure provides nucleic acid
compositions
comprising a polynucleotide encoding a NeoTCR. In certain embodiments, the
nucleic acid
compositions disclosed herein comprise a polynucleotide encoding a NeoTCR and
one or more
additional expression elements. In certain embodiments, the nucleic acid
compositions disclosed
herein comprise a polynucleotide encoding a NeoTCR and one or more additional
elements to
knockdown or knockout the expression of an endogenous protein. Also provided
are cells
comprising such nucleic acid compositions.
In certain embodiments, the nucleic acid composition further comprises a
promoter that is
operably linked to the NeoTCR disclosed herein. In certain embodiments, the
nucleic acid
composition further comprises a promoter that is operably linked to one or
more additional
expression elements disclosed herein.
In certain embodiments, the promoter is endogenous or exogenous. In certain
embodiments, the exogenous promoter is selected from the group consisting of
an elongation
factor (EF)-1 promoter, a CMV promoter, a SV40 promoter, a PGK promoter, a
long terminal
repeat (LTR) promoter, and a metallothionein promoter. In certain embodiments,
the promoter is
an inducible promoter. In certain embodiments, the inducible promoter is
selected from the
group consisting of an NFAT transcriptional response element (TRE) promoter, a
CD69
promoter, a CD25 promoter, an 11_,2 promoter, an IL12 promoter, a p40
promoter, and a Bc1-xL
promoter.
The compositions and nucleic acid compositions can be administered to subjects
or
and/delivered into cells by suitable art-known methods or as described herein.
Genetic
modification of a cell (e.g., a T cell) can be accomplished by transducing a
substantially
homogeneous cell composition with a recombinant DNA construct. In certain
embodiments, a
retroviral vector (either a gamma-retroviral vector or a lentiviral vector) is
employed for the
introduction of the DNA construct into the cell. Non-viral vectors may be used
as well.
Possible methods of transduction also include direct co-culture of the cells
with producer
cells, e.g., by the method of Bregni, et al. (1992) Blood 80:1418-1422, or
culturing with viral
supernatant alone or concentrated vector stocks with or without appropriate
growth factors and
polycations, e.g., by the method of Xu, et al. (1994) Exp. Hemat. 22:223-230;
and Hughes, et al.
(1992)1 (/in. Invest. 89:1817.
Other transducing viral vectors can be used to modify a cell. In certain
embodiments, the
chosen vector exhibits high efficiency of infection and stable integration and
expression (see,
e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al.,
Current Eye Research
15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997;
Naldini et al.,
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Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A.
94:10319, 1997).
Other viral vectors that can be used include, for example, adenoviral,
lentiviral, and adena-
associated viral vectors, vaccinia virus, a bovine papilloma virus, or a
herpes virus, such as
Epstein-Barr Virus (also see, for example, the vectors of Miller, Human Gene
Therapy 15-14,
1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques
6:608-614, 1988;
Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The
Lancet 337:1277-
1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology
36:311-322, 1987;
Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991;
Miller et al.,
Biotechnology 7:980-990, 1989; LeGal La Salle et al., Science 259:988-990,
1993; and Johnson,
Chest 107:77S- 83S, 1995). Retroviral vectors are particularly well developed
and have been
used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990;
Anderson et al., U.S.
Pat. No. 5,399,346).
Non-viral approaches can also be employed for genetic modification of a cell.
For
example, a nucleic acid molecule can be introduced into a cell by
administering the nucleic acid
in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A.
84:7413, 1987; Ono et
al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci.
298:278, 1989;
Staubinger et al., Methods in Enzymology 101:512, 1983), asialoorosomucoid-
polylysine
conjugation (Wu et al , Journal of Biological Chemistry 263:14621, 1988; Wu et
al., Journal of
Biological Chemistry 264:16985, 1989), or by micro-injection under surgical
conditions (Wolff
et al., Science 247:1465, 1990). Other non-viral means for gene transfer
include transfection in
vitro using calcium phosphate, DEAE dextran, electroporation, and protoplast
fusion. Liposomes
can also be potentially beneficial for delivery of DNA into a cell.
Transplantation of normal
genes into the affected tissues of a subject can also be accomplished by
transferring a normal
nucleic acid into a cultivatable cell type ex- vivo (e.g., an autologous or
heterologous primary cell
or progeny thereof), after which the cell (or its descendants) are injected
into a targeted tissue or
are injected systemically.
Polynucleotide therapy methods can be directed from any suitable promoter
(e.g., the
human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein
promoters), and
regulated by any appropriate mammalian regulatory element or intron (e.g. the
elongation factor
la enhancer/promoter/intron structure). For example, if desired, enhancers
known to
preferentially direct gene expression in specific cell types can be used to
direct the expression of
a nucleic acid. The enhancers used can include, without limitation, those that
are characterized
as tissue- or cell-specific enhancers. Alternatively, if a genomic clone is
used as a therapeutic
construct, regulation can be mediated by the cognate regulatory sequences or,
if desired, by
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regulatory sequences derived from a heterologous source, including any of the
promoters or
regulatory elements described above.
The resulting cells can be grown under conditions similar to those for
unmodified cells,
whereby the modified cells can be expanded and used for a variety of purposes.
3. Therapeutic Compositions and Methods of Manufacturing
In another aspect, the present disclosure is directed to methods of
manufacturing the
adoptive cell therapeutic compositions described herein. Manufacture of
adoptive cell
therapeutic compositions (each being an adoptive cell therapy) is a highly
complex multi-step
process involving the isolation, activation, expansion, and formulation of
cells in preparation for
their subsequent infusion into patients. The present disclosure provides
methods for reducing not
only the complexity of the manufacturing process but also the risks for
contamination, while
simultaneously improving the quality and scalability of the manufacturing
processes.
3.1. Cell Collection and Isolation
In certain non-limiting embodiments, the present disclosure provides methods
for
obtaining a sample comprising cells, e.g., immune cells, for use in the
context of adoptive cell
therapy from a subject. In certain embodiments, the cells, e.g., immune cells,
can be obtained by
using suitable methods known in the art. For example, but without any
limitation, immune cells
can be obtained by leukapheresis. Leukapheresis refers to a specific apheresis
strategy where
blood is removed from a subject and white blood cells, i.e., leukocytes, are
separated and
collected and the remaining blood is returned to the subject. In certain
embodiments, the
leukapheresis can be autologous (i.e., the cells collected will be
subsequently infused into the
donor of the cells) or allogeneic (i.e., the cells collected will be
subsequently infused into a
patient that was not the donor).
In certain embodiments, leukapheresis is employed to produce a leukopak. A
leukopak
can comprise any suitable container, typically a flexible bag, for the
collection and storage of
leukocytes. In certain embodiments the leukopak while be configured to contain
a target volume
of about 100 mL up to about 400 mL (e.g., following addition of autologous
plasma).
In certain embodiments, the cells, e.g., immune cells, can be obtained from a
tumor
sample. For example, but without any limitation, immune cells can be tumor-
infiltrating
lymphocytes (TILs). In certain embodiments, the TILs cell and can be isolated
after dissection,
fragmentation, and isolation from a solid tumor sample.
In certain embodiments, the cells obtained from a subject can be stored at a
temperature
between about 2 C and about 8 C In certain embodiments, the cells are
processed within 24
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hours from collection. In certain embodiments, the cells can be optionally
frozen after collection
(e.g., cryopreserved) and stored before further processing.
Following collection, e.g., by leukapheresis, the cells can be isolated and
enriched for
their phenotype. In certain embodiments, but without any limitation, the cells
can be isolated by
their positivity for a suitable cell marker. In certain non-limiting
embodiments, the marker can
be CD3, CD4, CD8, CD45, or any combination thereof In certain embodiments, the
isolation
occurs by suitable methods known in the art. In certain embodiments, the cells
can be isolated by
using chromatography, magnetic beads, or fluorescence-activated cell sorting
(FACS),
centrifugation, filtration, and other methods known in the art. In certain
embodiments, the
isolation is performed by FACS using antibodies reacting against CD3, CD8,
CD4, CD45, or any
combination thereof. In certain embodiments, the isolation comprises
centrifugation. In certain
embodiments, the isolation comprises counterflow centrifugation. In certain
embodiments, the
isolation comprises counterflow centrifugation elutriati on.
In certain embodiments, following initial sampling for cell count/viability,
including but
not limited to cell count/viability and flow cytometry (for cell
characterization) a CliniMACS
Prodigy or other suitable system can be employed to facilitate the enrichment
process. For
example, but not by way of limitation, a leukopak can be loaded onto a
CliniMACS Prodigy
instrument, or other suitable system, e.g., by sterile welding to the sterile
single use disposable
Prodigy TS520 kit. In certain embodiments, CD4-- and CD8+ T cells can be
positively enriched
for further processing using the Prodigy's "T Cell Transduction Process
Program" or other
suitable enrichment program In certain embodiments, such enrichment will
involve discarding
other cell types/impurities. In certain embodiments, cells can again be
sampled for cell
count/viability and flow cytometry (cell characterization assays).
In certain embodiments, the target cell count after enrichment is < about 5 x
109 cells. In
certain embodiments, the target cell count after enrichment is from about 1 x
107 cells to about 5
x 109 cells. In certain embodiments, the target cell count after enrichment is
from about 1 x 108
cells to about 5 x 109 cells. In certain embodiments, the target cell count
after enrichment is from
about 1 x 109 cells to about 5 x 109 cells.
3.2. Cell Activation
In certain non-limiting embodiments, the present disclosure provides methods
including
activation of the cells, e.g., T cells. In certain embodiments, the methods
disclosed herein
include contacting the cells with an activation reagent. As used herein, an
"activation reagent"
refers to a composition capable of activating certain biological processes
When related to T
cells, an "activation reagent" refers to a composition capable of activating T
cells.
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In certain embodiments, the activation reagent includes an antigen-presenting
cell (APC).
In certain embodiments, the APC is a dendritic cell. In certain embodiments,
the APC is a B cell.
In certain embodiments, the APC presents an antigen in the major histone
compatibility complex
(MT-IC). In certain embodiments, the activation reagent includes a second cell
providing a
costimulatory signal. For example, but without any limitation, the second cell
can be a monocyte
expressing a B7 molecule.
In certain embodiments, the activation reagent includes an artificial antigen-
presenting
cell (aAPC). In certain embodiments, the aAPC comprises artificial lipids and
one or more
costimulatory molecules. Additional information on aAPC can be found in
Latouche and
Sadelain, Nat Biolechnol. 2000 Apr,18(4):405-9; or Perica et al., Biochhn
Biophys Ada.
2015;1853(4):781-790.
In certain embodiments, the activation reagent includes a costimulatory
ligand. A
costimulatory ligand can be soluble or provided on a cell surface. Non-
limiting examples of
costimulatory ligand include CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-
1BBL,
OX4OL, inducible costimulatory ligand (ICOS-L), intercellular adhesion
molecule (ICAM),
CD3OL, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor,
3/TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds Toll ligand
receptor and a ligand
that specifically binds with B7-H3.
In certain embodiments, the activation reagent includes one or more
antibodies. In
certain embodiments, the antibody is selected from the group consisting of
anti-CD2, anti-CD3,
anti-CD27, anti-CD28, anti-4-1BB, anti-0X40, anti-CD30, anti-CD40, anti-PD-1,
anti-ICOS,
anti-lymphocyte function-associated antigen-1 (LFA-1), anti-CD7, anti-LIGHT,
anti-NKG2C,
anti-B7-H3, anti-CD83, or a combination thereof In certain embodiments, the
activation reagent
includes an anti-CD3 antibody. Non-limiting examples of anti-CD3 antibodies
include OKT-3,
T3, CD3a, otelixizumab, teplizumab, and visilizumab. In certain embodiments,
the activation
reagent includes an anti-CD28 antibody. In certain embodiments, the activation
reagent includes
an anti-CD2 antibody. In certain embodiments, the activation reagent includes
an anti-CD2, an
anti-CD3, and an anti-CD28 antibody. In certain embodiments, the activation
reagent includes
an anti-CD3 and an anti-CD28 antibody.
In certain embodiments, the antibody is soluble. In certain embodiments, the
antibody is
bound to a surface. For example, without any limitation, the antibody is bound
to a polymeric
surface, a magnetic bead, a non-magnetic bead, or an agarose bead Non-limiting
examples of
antibodies used in the methods disclosed herein include T Cell TransActTm,
DynaheadsTM Human
T-Activator CD3/CD28, and ImmunoCultTM Human CD3/CD28 T Cell Activator.
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In certain embodiments, the activation reagent includes a CD3 and a CD28
agonist.
In certain embodiments, the activation reagent includes growth factors and/or
cytokines.
In certain embodiments, the activation reagent includes a cytokine selected
from the group
consisting of 1E2, 1E7, 1E10, FL12, 1E15, 1E21, or a combination thereof. In
certain
embodiments, the cytokine is IL2. In certain embodiments, the cytokine is 1L7.
In certain
embodiments, the cytokine is IL15.
In certain embodiments, the enriched cells are transferred to a G-Rex 100M-CS
flask, or
other suitable container, for activation. In certain embodiment, a non-bead-
based activation
strategy (TransAct, Miltenyi) will be employed. For example, but not by
limitation, T cells can
be activated by incubation with TransAct (aCD3/CD28 reagent). In certain
embodiments, the
aCD3/CD28 reagent will be employed at a ratio of 1:17.5, In certain
embodiments, the
aCD3/CD28 reagent will be contacted with the cells in a suitable medium, e.g.,
TexMACS
medium. In certain embodiments, the medium will comprise one or more suitable
supplements.
For example, but not by way of limitation, the media can be supplemented with
3% human AB
serum. In certain embodiments the media will be supplemented with additional
activation
reagents, e.g., IL7 and/or IL15. In certain embodiments, the media will be
supplemented with
about 12.5 ng/mL IL7 and/ about 12.5 ng/mL IL15. In certain embodiments, the
cells can be
cultured in the activation medium for about 48 to about 72 hours In certain
embodiments, the
activation will take place in an incubator at about 37 C and about 5% CO2
In certain embodiments, the activation reagent is removed and/or separated
from the cells.
For example, but without any limitation, the activation reagent is removed by
exchanging cell
culture media, by affinity chromatography, or by counterflow centrifugation.
In certain
embodiments, the cell activation occurs in a closed system.
3.3. Cell Editing and Transfection
In certain non-limiting embodiments, the present disclosure comprises methods
including
editing and/or transfection of a cell, e.g., T cell, to express an exogenous
nucleic acid and/or
protein, e.g., a NeoTCR. Various transfection methods can be used including,
but without any
limitation, viral infection, electroporation, membrane disruption, and
combination thereof
In certain embodiments, the transfection can occur with a viral vector. In
certain
embodiments, the viral vector can be a retrovirus. In certain embodiments, the
viral vector can
be a lentivirus. In certain embodiments, the viral vector can be an adena-
associated virus (AAV).
Additional information on the viral vectors contemplated by the present
disclosure and used by
the methods disclosed herein can he found in Section 2.6.
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In certain embodiments, the transfection can occur with non-viral vectors
and/or non-viral
methods. For example, without limitation, the cells can be transfected by
electroporation. In
certain embodiments, the electroporation is a large-scale electroporation. As
used herein, the
term "large-scale" refers to experimental and/or manufacturing conditions
using an amount of
cells of at least about 100 x 106 cells, at least about lx 107 cells, at least
about 10 x 107 cells, at
least about 100 x 107 cells, at least about 1 x 108 cells, at least about 10 x
108 cells, at least about
100 x 108 cells, at least about 1 x 109 cells, at least about 10 x 109 cells,
at least about 100 x 109
cells, or at least about 1 x 10' cells. In certain embodiments, the large-
scale electroporation
produces at least about 100 x 106 edited cells, at least about 1 x 10 edited
cells, at least about 10
x 107 edited cells, at least about 100 x 107 edited cells, at least about 1 x
108 edited cells, at least
about 10 x 108 edited cells, at least about 100 x 108 edited cells, at least
about 1 x 109 edited cells,
at least about 10 x 109 edited cells, at least about 100 x 109 edited cells,
or at least about 1 x 1010
edited cells. Additional information on the non-viral vectors and non-viral
methods
contemplated by the present disclosure can be found in Sections 2.4-2.6 above
and in
International Patent Application No. PCT/1JS2018/058230, the content of which
is incorporated
herein in its entirety.
In certain embodiments, after activation, the cell is collected using a
counterflow
centrifugation system and mixed with the transfection reagents, e.g., DNA
plasmid. Collection
of the cell and concentration in a small volume facilitates and increases the
transfection
efficiency. In certain embodiments, after transfection, the cell is
transferred in a gas-permeable
flask. In certain embodiments, the cell transfection occurs in a closed
system.
In certain embodiments, the transfected cell can be selected. For example, but
without
any limitation, the transfected cell can be selected for the expression of the
NeoTCR using
peptide-major histocompatibility complex (pMHC) multimers bound to dextramer.
Additional
information regarding selection of transfected cells can be found in
International Patent
Publication Nos. W02019195310A1, W02020056173A1, and W02020167918A1, the
content
of each of which is incorporated herein in its entirety. In certain
embodiments, the transfected
cell is not selected.
3.4. Cell Expansion
In certain non-limiting embodiments, the present disclosure provides methods
including
culturing the cell, e.g., T cell. In certain embodiments, the cell is edited.
In certain
embodiments, the cell is cultured to obtain a population of cells. In certain
embodiments, the cell
is cultured in a cell culture medium. In certain embodiments, the cells are
transferred to a cell
culture chamber for cell proliferation and expansion. In certain embodiments,
the cell culture is
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designed to promote the cells to maintain, develop, and/or retain a stem-like
state (i.e., T cells
that have a memory stem cell or stem cell (Tmsc or Tcm) phenotype).
For example, in certain experiments, the cells can be cultured in cell culture
chambers in
an incubator (5% CO,, 37 C) in culture medium. In certain experiments, the
cell culture
chambers is a G-Rex (Wilson Wolf) cell culture chambers, or other suitable
chamber. In certain
embodiments, alternative static gas exchange cell culture chambers can be used
based on such
static gas exchange cell culture chamber's ability to allow for sufficient
cell proliferation of gene
edited cells that possess a memory stem cell or stem cell (Tmsc or Tcm)
phenotype).
In certain experiments, the media used to culture the cells following
electroporation is a
chemically-defined, animal component-free medium shown to promote T cell
expansion while
maintaining T cell functionality and potency. In certain experiments, the
media can be PRIME-
XV Cell CDM (Irvine Scientific CDM), ImmunoCult XF (Stemcell),
ExCellerate (R&D
Systems), LumphoOne (Takara Bio), GT-T551 (Takara Bio), X-VIVO 15, AIM V, CTS
OpTmizer (Gibco), and any other suitable medias with similar physiological
attributes as those
described herein. Suitable medias are known to those of skill in the art that
are animal
component-free, that enable efficient T cell expansion without the addition of
serum or plasma,
and promote expansion and growth of T cells with a naïve phenotype (e.g., Tmsc
and Tern) can
be used in the medias and methods described herein.
Serum free substitute additives can also be used in the medias described
herein. For
example, but not by way of limitation, Physiologix (Nucleus Biologics), human
platelet lysate (a
growth factor-rich cell culture supplement derived from healthy donor human
platelets, Stem
Cell), CTS Immune Cell Serum Replacement (Gibco) are suitable supplements that
can be used
in the context of the methods of the present disclosure. Additional suitable
serum free substitutes
are known to those of skill in the art to enable efficient cell, e.g., T cell,
expansion without the
addition of serum or plasma, and promote expansion and growth of cells, e.g.,
T cells, with a
naïve phenotype (e.g., Tmsc and Tern) and thus can be used in the medias and
methods described
herein.
In certain embodiments, the addition of cytokines can also be used in the
context of the
medias and methods described herein. In certain experiments, the media can be
supplemented
with or otherwise contain IL2. In certain experiments, the media can be
supplemented with or
otherwise contains IL7. In certain experiments, the media can be supplemented
with or
otherwise contains 11,15. In certain experiments, the media can be
supplemented with or
otherwise contains IL21. In certain experiments, the media does not contain or
is not
supplemented with IL2.
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In addition to supplementation with IL2, 11L7, IL15, and/or 1L21 described
above as single
agents or combinations thereof for the supplementation of media, IL12, alpha
interferon, or beta
interferon can be used alone or in combination with each other or with the
IL2, IL7, IL15, and/or
IL21. Furthermore, any cytokine or chemokine that is involved in lymphocyte
proliferation and
differentiation can be added to any single IL2, IL7, IL12, IL15, IL21, alpha
interferon or beta
interferon, or any combination thereof. The concentration and ratios of each
of the cytokines
and/or chemokines can be adjusted based on the single agent use or combination
use and titrated
based on the lymphocyte proliferation and differentiation desired.
In certain embodiments, the media does not contain IL2. In certain
embodiments, the
media contains one or more cytokines but does not contain IL2. In certain
embodiments, the
media contains IL7 but does not contain IL2. In certain embodiments, the media
contains IL15
but does not contain IL2. In certain embodiments, the media contains IL7 and
IL15 but does not
contain IL2.
In certain embodiments, the cytokine is present in the cell culture medium at
a
concentration between about 0.05 ng/ml to about 100 ng/ml. For example, but
without any
limitation, the cytokine is present in the cell culture medium at a
concentration of about 0.05
ng/ml, about 0.1 ng/ml, about 0.5 ng/ml, about 1 ng/ml, about 2 ng/ml, about 5
ng/ml, about 8
ng/ml, about 10 ng/ml, about 20 ng/ml, about 30 ng/ml, about SO ng/ml, about
60 ng/ml, about 80
ng/ml, or about 100 ng/ml.
In addition to the addition of serum free substitute additives and/or
chemokines and/or
cytokines as described herein, the addition of fatty acids can be beneficial
in achieving the
desired proliferation and differentiation.
In certain embodiments, fibronectin, insulin, and/or transferrin can be
included in the
media. In certain embodiments, the transferrin used is recombinant
transferrin. In certain
embodiments, the transferrin used is non-recombinant transferrin. In certain
embodiments, it can
be useful to increase the concentration of transferrin when recombinant
transferrin is used
compared to non-recombinant transferrin in order to achieve the same benefits
of lymphocyte
proliferation and differentiation to achieve T cells in culture with a naïve
phenotype (e.g., Tmsc
and Tern).
In certain embodiments, different concentrations of glucose in the cell medias
can be
used. For example, but not by way of limitations, the glucose concentration
can be less than
about 3.7 g/L glucose. In certain embodiments, the glucose concentration is
between about 3.7 ¨
about 4.0 g/L glucose In certain embodiments, the glucose concentration is
between about 4.0 ¨
about 4.2 g/L glucose. In certain embodiments, the glucose concentration is
between about 4.2 -
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about 4.5 g/L glucose. In certain embodiments, the glucose concentration is
between about 4.3 ¨
about 4.4 g/L glucose. In certain embodiments, the glucose concentration is
between about 4.4 ¨
about 4.5 g/L glucose. In certain embodiments, the glucose concentration is
greater than about
4.5 g/L glucose. As cell density in culture increases, so can the
concentration of glucose. For
example, for a high density cell culture the glucose concentration can be
increased up to 100 g/L.
In certain embodiments, antioxidants can be added to the media to promote
lymphocyte
proliferation and differentiation to achieve T cells in culture with a naive
phenotype (e.g., Tmsc
and Tern).
In certain embodiments, reducing agents can be added to the media to promote
lymphocyte proliferation and differentiation to achieve T cells in culture
with a naive phenotype
(e.g., Tmsc and Tem).
In order to promote automation of the manufacturing processes, e.g., NeoTCR
Product
manufacture, stir bioreactors can be used to culture the cells instead of a
static gas exchange cell
culture chamber. Such stir bioreactors allow for real-time analytics and
reaction to changes in
conditions. For example, a stir bioreactor can be designed to have in line
bioanalytics to measure
cell mass, lactate, etc., in a closed system without manual sampling.
Alternatively, in order to promote automation of the manufacturing processes,
e.g.,
NeoTCR Product manufacture, shaking/rotating bioreactors can be used to
culture the cells
instead of a static gas exchange cell culture chamber. Such shaking/rotating
bioreactors allow for
real-time analytics and reaction to changes in conditions. For example, a
shaking/rotating
bioreactor can be designed to have in line bioanalytics to measure cell mass,
lactate, etc., in a
closed system without manual sampling.
Furthermore, bioreactors (e.g., stir, shanking, rotating, etc.) can be
designed and
programmed to automatically add media supplements to the culture in order to
increase or
decrease the concentration of certain components in the media. For example,
the bioreactor can
be designed and programmed to detect lactate levels in the cell culture and
add in glucose in
order to keep the glucose: lactate levels optimal for lymphocyte proliferation
and differentiation
to achieve cell, e.g., T cells, in culture with a naive phenotype (e.g., Tmsc
and Tcm). In other
examples, the bioreactors can be designed and programmed to remove lactate
during the culture
process in order to promote lymphocyte proliferation and differentiation to
achieve cells, e.g., T
cells, in culture with a naive phenotype (e.g., Tmsc and Tern). Another
example of the use of a
bioreactor is to design and program the bioreactor to detect dissolved oxygen
as a negative
indicator of a desired cell environment.
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In certain experiments, cell counts can be taken throughout the culture
period. In certain
experiments, the cells are taken from the static gas exchange culture chambers
(e.g., a G-Rex
flask) at the half-way point of cell culture (i.e., the halfway point between
the time of
electroporation and the time when the cells are cryopreserved, e.g., as a
NeoTCR Product) and
split into two new static gas exchange culture chambers with fresh media.
In certain embodiments, the cell culture medium includes fibronectin. In
certain
embodiments, the cell culture medium includes insulin. In certain embodiments,
the cell culture
medium includes transferrin.
In certain non-limiting embodiments, the cell culture medium induces
controlled growth
and proliferation of the cells in order to increase the total cell number. In
certain non-limiting
embodiments, the total cell number is at least about 1 x 106 cells, at least
about 3 x 106 cells, at
least about 5 x 106 cells, at least about 7 x 106 cells, at least about 9 106
cells, at least about 1
107 cells, at least about 5 " 107 cells, at least about 1 " 108 cells, at
least about 5 " 108 cells, at least
about 1 109 cells, at least about 3 " 109 cells, or at least about 5 " 109
cells.
In certain embodiments, the culturing occurs in a total volume of from about
0.1 L to
about 5 L, from about 0.1 L to about 2 L, or from about 0.2 L to about 2 L. In
certain
embodiments, the total volume is of about 0.1 L, about 0.2 L, about 0.3 L,
about 0.4 L, about 0.5
L, about 0.6 L, about 0.7 L, about 0 g L, about 0.9 L or about 1 0 L. In
certain non-limiting
embodiments, as illustrated in the Example section, the total volume can vary
based on the total
number of cells.
In certain embodiments, the culturing includes obtaining a young T cell. In
certain
embodiments, the young T cell is CD45RA-', CD62L-', CD28, CD95-, CCR7-', and
CD27-'. In
certain embodiments, the young T cell is CD45RA , CD621i, CD28h, CD95 ,
In certain embodiments, the young T cells is CD45R0+, CD62L-P, CD28+, CD95+,
CCRTP,
CD274, CD1274. In certain embodiments, the young T cell is a T memory stem T
cells (Tmsc).
In certain embodiments, the young T cell is a T central memory T cells (Tcm).
Additional
information regarding young T cells and methods for producing, obtaining,
and/or culturing them
can be found in International Patent Application No. PCT/US2020/025758, the
content of which
is incorporated herein in its entirety.
In certain embodiments, the culturing includes obtaining a population of
cells. In certain
embodiments, the population of cells comprises Tmsc and Tcm. In certain
embodiments, the
population of cells comprises at least about 20% of Tmsc and Tcm collectively,
at least about 25%
Tmsc and Tcm collectively, at least about 30% Tmsc and Tcm collectively, at
least about 35%
Tmsc and Tcm collectively, at least about 40% Tmsc and Tcm collectively, at
least about 45%
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Tmsc and Tcm collectively, at least about 50% Tmsc and Tcm collectively, at
least about 55%
Tmsc and Tcm collectively, at least about 60% Tmsc and Tcm collectively or
more than about 61%
Tmsc and Tcm collectively. In certain embodiments, the population of cells
comprises at least
about 65% Tmsc and Tcm collectively, at least about 70% Tmsc and Tcm
collectively, at least
about 75% Tmsc and Trm collectively, at least about 80% Timm- and Tcm
collectively, at least
about 85% Tmsc and Tcm collectively, at least about 90% Tmsc and Tcm
collectively, or at least
about 95% Tmsc and Tcm collectively.
In certain embodiments, the culturing occurs in a closed system. In certain
embodiments,
the culturing occurs in a gas permeable system Non-limiting examples of gas
permeable system
include G-rex , Cell Factory, MACS GIV1P Cell Expansion Bag, VueLife, and
Evolve. In
certain embodiments, the culturing includes counterflow centrifugation.
In certain embodiments, the cells are cultured for a period of time sufficient
to obtain a
particular total cell number. In certain embodiments, the cells are expanded
for about 10 days.
In certain embodiments, the cells are expanded for about 11 days. In certain
embodiments, the
cells are expanded for about 12 days.
In certain embodiments, after transfection, the cells are cultured in flasks
at about 37 C
and about 5% CO2 to facilitate expansion. In certain embodiments, the culture
can make use of
any suitable media In certain embodiments, the suitable media is TexMACS GMP
medium. In
certain embodiments the suitable media is Prime XV media. In certain
embodiments the suitable
media is Prime XV media supplemented with PhysiologixTM XF SR. In certain
embodiments,
the PhysiologixTM XF SR is supplemented at a concentration of 2% or
approximately 2%. In
certain embodiments, the suitable media is a media with substantially the same
components as
Prime XV media. In certain embodiments, the suitable media is a media with
equivalent
components as Prime XV media. In certain embodiments, the suitable media is a
media with
substantially the same or equivalent components as Prime XV media and is
supplemented with
PhysiologixTM XF SR. In certain embodiments, the suitable media is a media
with substantially
the same or equivalent components as Prime XV media and is supplemented with a
serum free
additive that is substantially the same or equivalent to PhysiologixTM XF SR.
In certain embodiments, the media will comprise supplements. For example, but
not by
way of limitation, the media can be supplemented with about 3% human AB serum
or other
suitable serum. For example, but not by way of limitation, the media can be
supplemented with a
serum free additive. In certain embodiments, the serum free additive is
PhysiologixTM XF SR or
a supplement that is substantially similar or equivalent to PhysiologixTM XF
SR In certain
embodiments, the media can comprise one or more cytokine supplements. For
example, but no
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by way of limitation, the media can comprise IL7 (at about 12.5ng/mL) and/or
IL15 (at about
12.5 ng/mL).
In certain embodiments, the cell density of the culture will be monitored to
ensure
appropriate expansion. For example, but not limitation, on a suitable day post-
transfecti on, e.g.,
on day 8, a cell count can be performed. Based on the cell number obtained,
the cells can be split
into one or more flasks to allow further expansion.
3.5. Harvesting
In certain non-limiting embodiments, the present disclosure provides methods
including
harvesting the cell, e.g., T cell. In certain embodiments, the cell is edited.
In certain
embodiments, the present disclosure provides methods including harvesting the
population of
cells.
In certain embodiments, the cells can be harvested once a particular total
cell number has
been achieved. In certain embodiments, the population cells can be harvested
once a particular
total cell number has been achieved. In certain non-limiting embodiments, the
total cell number
is at least about 1 106 cells, at least about 3 x 106 cells, at least about 5
x 106 cells, at least about
7 x 106 cells, at least about 9 106 cells, at least about 1 x 107 cells, at
least about 5 x 107 cells, at
least about 1 x 108 cells, at least about 5 x 108 cells, at least about 1 109
cells, at least about 3
109 cells, or at least about 5 " 109 cells.
In certain embodiments, harvesting can include one or more of centrifugation,
filtration,
e.g., TFDF, acoustic wave separation, flocculation, and cell removal
technologies. In certain
embodiments, harvesting includes counterflow centrifugation. In certain
embodiments,
harvesting occurs in a closed system.
3.6. Transferring in Infusion Bags and Final Formulation
In certain non-limiting embodiments, the present disclosure provides methods
including
transferring the cell, e.g., harvested cell, to a container for use in
administration to a patient (e.g.,
"infusion bag"). In certain embodiments, the present disclosure provides
methods including
transferring the population of cells to a container for use in administration
to a patient.
In certain embodiments, the cell is transferred to the container in a closed
system. For
example, but without any limitation, the cell can be transferred using a
peristaltic pump. In
certain embodiments, the cell is collected before transferring via
centrifugation. In certain
embodiments, the centrifugation is a counterflow centrifugation. In certain
embodiments, the
cell is prepared for being cryopreserved. In certain embodiments, the cell is
prepared for
administration to a patient_
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In certain embodiments, the population of cells is transferred to the
container in a closed
system. For example, but without any limitation, the population of cells can
be transferred using
a peristaltic pump. In certain embodiments, the population of cells is
collected before
transferring via centrifugation. In certain embodiments, the centrifugation is
a countertlow
centrifugation. In certain embodiments, the population of cells is prepared
for being
cryopreserved. In certain embodiments, the population of cells is prepared for
administration to a
patient.
In certain non-limiting embodiments, the container comprises a formulation. In
certain
embodiments, the formulation is a pharmaceutical formulation. In certain
embodiments, the
formulation is suitable for administration to a patient.
In certain embodiments, the pharmaceutical formulation comprises at least
about 1 x 106
gene-edited cells, at least about 3 x 106 gene-edited cells, at least about 5
106 gene-edited cells,
at least about 7 x 106 gene-edited cells, at least about 9 x 106 gene-edited
cells, at least about 1 x
107 gene-edited cells, at least about 5 107 gene-edited cells, at least about
1 108 gene-edited
cells, at least about 5 x 108 gene-edited cells, or at least about 1 109 gene-
edited cells. In certain
embodiments, the pharmaceutical formulation comprises about 4.0 108 gene-
edited cells, about
1.3 x 109 gene-edited cells, about 4.0 X 109 gene-edited cells, approxim about
ately 1.3 X 1010
gene-edited cells, or about 4.0 1010 gene-edited cells. In certain
embodiments, the
pharmaceutical formulation comprises about 4.0 x 108 gene-edited cells. In
certain embodiments,
the pharmaceutical formulation comprises about 1.3 x 109 gene-edited cells. In
certain
embodiments, the pharmaceutical formulation comprises about 4.0 109 gene-
edited cells. In
certain embodiments, the pharmaceutical formulation comprises about 1.3 x 1010
gene-edited
cells. In certain embodiments, the pharmaceutical formulation comprises about
4.0 x 1010 gene-
edited cells.
In certain embodiments, the pharmaceutical formulation comprises a crystalloid
solution.
As used herein, the term "crystalloid solution" refers to an intravenous
solution useful in clinical
setting. In certain non-limiting embodiments, crystalloid solutions can be
used for intravenous
medication delivery. Non-limiting examples of crystalloid solution include
0.9% sodium
chloride (NaCl), Hartmann's (or Ringer's lactate or compound sodium lactate)
solution, or
PlasmaLyte. In certain embodiments, the crystalloid solution is PlasmaLyte. In
certain
embodiments, the crystalloid solution is present in the formulation at a final
concentration from
about 30% v/v to about 60 % v/v, from about 35% v/v to about 60% v/v, from
about 40% v/v to
about 60 % v/v, from about 45% v/v to about 60 % v/v, from about 50% v/v to
about 60 A v/v,
from about 40% v/v to about 50 % v/v, or from about 45% v/v to about 50 % v/v.
In certain
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embodiments, the crystalloid solution is present in the formulation at a final
concentration of
about 46% v/v.
In certain embodiments, the pharmaceutical formulation comprises a serum
albumin. In
certain embodiments, the serum albumin is human. In certain embodiments, the
serum albumin
is present in the formulation at a final concentration from about 0.1% w/v to
about 5% w/v, from
about 0.2% w/v to about 5% w/v, from about 0.3% w/v to about 5% w/v, from
about 0.5% w/v to
about 5% w/v, from about 0.7% w/v to about 5% w/v, from about 0.8% w/v to
about 5% w/v,
from about 0.9% w/v to about 5% w/v, from about 1.0% w/v to about 5% w/v, from
about 1.5%
w/v to about 5% w/v, from about 2% w/v to about 5%, from about 3% w/v to about
5% w/v, or
from about 4% w/v to about 5% w/v. In certain embodiments, the serum albumin
is present in
the formulation at a final concentration of about 1% w/v.
In certain embodiments, the pharmaceutical formulation comprises a
cryopreservation
medium. In certain embodiments, the cryopreservation medium is CryoStor CS10.
In certain
embodiments, the cryopreservation medium is present in the formulation at a
final concentration
from about 10% v/v to about 70 % v/v, from about 20% v/v to about 70% v/v,
from about 30%
v/v to about 70 % v/v, from about 40% v/v to about 70 % v/v, from about 500/a
v/v to about 70 c,'<3
v/v, from about 60% v/v to about 70 % v/v, or from about 45% v/v to about 55 %
v/v. In certain
embodiments, the cryopreservation medium is present in the formulation at a
final concentration
of about 50% v/v.
In certain embodiments, the final pharmaceutical formulation contains 5%
dimethyl
sulfoxide (DMSO), human serum albumin, and Plasma-Lyte. In certain
embodiments, the final
pharmaceutical formulation contains the list of components provided in Table
1.
Table 1. Composition of Exemplary Pharmaceutical Formulations
Component Specification/Grade
Total nucleated NeoTCR cells cGMP manufactured
Plasma-Lyte A USP
Human Serum Albumin in 0.02 - 0.08 M USP
sodium caprylate and sodium tryptophanate
CryoStor CS10 eGNIP manufactured with USP
grade
materials
3.7. Quality Control and Additional Features
In certain embodiments, the methods of the present disclosure include the
monitoring of
parameters on-line (e.g., by direct connection to an analyzer) or off-line
(e.g., by user
intervention).
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In certain embodiments, the monitored parameters include temperature, pH,
glucose,
lactate, glucose/lactate ratio, oxygen, carbon dioxide, cell count, viability,
gene and/or protein
expression, complete blood count, potency, mycoplasma, functional features
(e.g., cytokine
secretion), sterility, endotoxins, and/or cell characterization
In certain embodiments, the methods disclosed herein include adjustment of the
parameters. For example, but without any limitation, if the monitored oxygen
level is too low to
achieve the cell expansion, the oxygen level is increased by introducing an
oxygenated cell
culture medium or by replacing the cell culture medium with an oxygenated cell
culture medium.
In certain embodiments, the adjustment is performed manually. In certain
embodiments, the
adjustment is performed automatically.
In certain embodiments, the methods disclosed herein further include
characterization of
the edited cell or the population of cells. In certain embodiments, the
characterization includes
determining the expression levels of an exogenous nucleic acid. For example,
but without any
limitation, the expression levels of a NeoTCR are determined by gene
expression analysis or by
FACS analysis. In certain embodiments, the characterization includes
determining the gene
knockout levels in the adoptive cell therapy. For example, but without any
limitation, the
expression levels of an endogenous gene, e.g., endogenous TCR, can be
determined. In certain
embodiments, the characterization includes genetic tests and sequencing of the
cell's genome.
Non-limiting examples of genetic tests include Targeted Locus Amplification
(TLA), Next
Generation Sequencing (NGS), deep sequencing, targeted deep sequences, and
Fluorescence In
Situ Hybridization (FISH).
In certain embodiments, the characterization includes determining the cell
subtypes in the
adoptive cell therapy. For example, but without any limitation, the percentage
of young T cells is
determined.
In certain non-limiting embodiments, the present disclosure comprises the use
of flasks
and vessels for cell culturing. In certain embodiments, the flasks and vessels
are gas permeable.
In certain embodiments, the cell activation can be performed in flasks or gas-
permeable bags. In
certain embodiments, the cell activation can be performed in gas-permeable
flasks. In certain
embodiments, the cell expansion can be performed in flasks or gas-permeable
bags. In certain
embodiments, the cell expansion can be performed in gas-permeable flasks. In
certain non-
limiting embodiments, the present disclosure comprises methods comprising the
use of a process
and/or apparatus for aseptically concentrating and washing T cells. In certain
embodiments, the
method comprises centrifugation. In certain embodiments, the method comprises
counterflow
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centrifugation separation technology. Non-limiting examples of counterflow
centrifugation
include Gibco Rotea and Ksep System.
In certain non-limiting embodiments, the present disclosure comprises methods
using an
instrument or device that can pump a solution comprising cells in a sterile
and/or closed system
environment, allowing for continuous flow and cell processing. In certain
embodiments, the
instrument or device can perform cell separation, washing, fluid exchange,
concentration, and/or
other cell processing steps in a closed, sterile system.
4. Articles of Manufactnre
In certain embodiments, the present disclosure provides articles of
manufacture
comprising the adoptive cell therapeutics disclosed herein. In certain
embodiments, the articles
of manufacture comprising adoptive cell therapeutics are obtained by the
methods disclosed
herein. In certain embodiments, the adoptive cell therapeutic is a Cell
Product. In certain
embodiments, the adoptive cell therapeutic is a NeoTCR Product.
The Cell Products can be used in combination with articles of manufacture.
Such articles
of manufacture can be useful for the prevention or treatment of proliferative
disorders (e.g.,
cancer). Examples of articles of manufacture include but are not limited to
containers (e.g.,
infusion bags, bottles, storage containers, flasks, vials, syringes, tubes,
and IV solution bags) and
a label or package insert on or associated with the container. The containers
may be made of any
material that is acceptable for the storage and preservation of the NeoTCR
cells within the Cell
Products. In certain embodiments, the container may be an intravenous solution
bag or a vial
having a stopper pierceable by a hypodermic injection needle. For example, the
container may
be a CryoMACS freezing bag. The label or package insert indicates that the
Cell Products are
used for treating the condition of choice and the patient of origin The
patient is identified on the
container of the Cell Product because the Cell Product is made from autologous
cells and
engineered as a patient-specific and individualized treatment.
The article of manufacture may comprise: 1) a first container with a Cell
Product
contained therein.
The article of manufacture may comprise: 1) a first container with a Cell
Product
contained therein; and 2) a second container with the same Cell Product as the
first container
contained therein. Optionally, additional containers with the same Cell
Product as the first and
second containers may be prepared and made. Optionally, additional containers
containing a
composition comprising a different cytotoxic or otherwise therapeutic agent
may also be
combined with the containers described above.
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The article of manufacture may comprise: 1) a first container with a Cell
Product
contained therein; and 2) a second container with a composition contained
therein, wherein the
composition comprises a further cytotoxic or otherwise therapeutic agent.
The article of manufacture may comprise: 1) a first container with two Cell
Products
contained therein; and 2) a second container with a composition contained
therein, wherein the
composition comprises a further cytotoxic or otherwise therapeutic agent.
The article of manufacture may comprise: 1) a first container with a Cell
Product
contained therein; 2) a second container with a second Cell Product contained
therein; and 3)
optionally a third container with a composition contained therein, wherein the
composition
comprises a further cytotoxic or otherwise therapeutic agent. In certain
embodiments, the first
and second Cell Products are different Cell Products. In certain embodiments,
the first and
second Cell Products are the same Cell Products.
The article of manufacture may comprise: 1) a first container with three Cell
Products
contained therein; and 2) optionally a second container with a composition
contained therein,
wherein the composition comprises a further cytotoxic or otherwise therapeutic
agent.
The article of manufacture may comprise: 1) a first container with a Cell
Product
contained therein; 2) a second container with a second Cell Product contained
therein; 3) a third
container with a third Cell Product contained therein; and 4) optionally a
fourth container with a
composition contained therein, wherein the composition comprises a further
cytotoxic or
otherwise therapeutic agent. In certain embodiments, the first, second, and
third Cell Products are
different Cell Products. In certain embodiments, the first, second, and third
Cell Products are the
same Cell Products. In certain embodiments, two of the first, second, and
third Cell Products are
the same Cell Products.
The article of manufacture may comprise: 1) a first container with four Cell
Products
contained therein; and 2) optionally a second container with a composition
contained therein,
wherein the composition comprises a further cytotoxic or otherwise therapeutic
agent.
The article of manufacture may comprise: 1) a first container with a Cell
Product
contained therein; 2) a second container with a second Cell Product contained
therein; 3) a third
container with a third Cell Product contained therein; 4) a fourth container
with a fourth Cell
Product contained therein; and 5) optionally a fifth container with a
composition contained
therein, wherein the composition comprises a further cytotoxic or otherwise
therapeutic agent. In
certain embodiments, the first, second, third, and fourth Cell Products are
different Cell Products.
In certain embodiments, the first, second, third, and fourth Cell Products are
the same NeoTCR
Products. In certain embodiments, two of the first, second, third, and fourth
Cell Products are the
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same NeoTCR Products. In certain embodiments, three of the first, second,
third, and fourth Cell
Products are the same Cell Products.
The article of manufacture may comprise: 1) a first container with five or
more Cell
Products contained therein; and 2) optionally a second container with a
composition contained
therein, wherein the composition comprises a further cytotoxic or otherwise
therapeutic agent.
The article of manufacture may comprise: 1) a first container with a Cell
Product
contained therein; 2) a second container with a second Cell Product contained
therein; 3) a third
container with a third Cell Product contained therein; 4) a fourth container
with a fourth Cell
Product contained therein; 5) a fifth container with a fifth Cell Product
contained therein; 6)
optionally a sixth or more additional containers with a sixth or more Cell
Product contained
therein; and 7) optionally an additional container with a composition
contained therein, wherein
the composition comprises a further cytotoxic or otherwise therapeutic agent.
In certain
embodiments, all of the containers of Cell Products are different Cell
Products. In certain
embodiments, all of the containers of Cell Products are the same Cell
Products. In certain
embodiments, there can be any combination of same or different Cell Products
in the five or
more containers based on the availability of detectable Cells in a patient's
tumor sample(s), the
need and/or desire to have multiple Cell Products for the patient, and the
availability of any one
Cell Product that may require or benefit from one or more container.
The article of manufacture may comprise: 1) a first container with a Cell
Product
contained therein; 2) a second container with a second Cell Product contained
therein; 3) a third
container with a third Cell Product contained therein.
The article of manufacture may comprise: 1) a first container with a Cell
Product
contained therein; 2) a second container with a second Cell Product contained
therein; 3) a third
container with a third Cell Product contained therein; 4) optionally a fourth
container with a
fourth Cell Product contained therein.
The article of manufacture may comprise: 1) a first container with a Cell
Product
contained therein; 2) a second container with a second Cell Product contained
therein; 3) a third
container with a third Cell Product contained therein; 4) a fourth container
with a fourth Cell
Product contained therein; 5) optionally a fifth container with a fourth Cell
Product contained
therein.
The article of manufacture may comprise a container with one Cell Product
contained
therein. The article of manufacture may comprise a container with two Cell
Products contained
therein The article of manufacture may comprise a container with three Cell
Products contained
therein. The article of manufacture may comprise a container with four Cell
Products contained
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therein. The article of manufacture may comprise a container with five Cell
Products contained
therein.
The article of manufacture may comprise 1) a first container with one Cell
Product
contained therein, and 2) a second container with two Cell Products contained
therein. The
article of manufacture may comprise 1) a first container with two Cell
Products contained
therein, and 2) a second container with one Cell Product contained therein. In
the examples
above, a third and/or fourth container comprising one or more additional Cell
Products may be
included in the article of manufacture. Additionally, a fifth container
comprising one or more
additional Cell Products may be included in the article of manufacture.
Furthermore, any container of Cell Product described herein can be split into
two, three,
or four separate containers for multiple time points of administration and/or
based on the
appropriate dose for the patient.
In certain embodiments, the Cell Products are provided in a kit. The kit can,
by means of
non-limiting examples, contain package insert(s), labels, instructions for
using the Cell
Product(s), syringes, disposal instructions, administration instructions,
tubing, needles, and
anything else a clinician would need in order to properly administer the Cell
Product(s).
In certain embodiments, the Cell Products used in the methods of manufacture
disclosed
herein are NeoTCR Products or NeoTCR Viral Products_ In certain embodiments,
the Cell
Products used in the methods of manufacture disclosed herein are NeoTCR
Products. In certain
embodiments, the Cell Products used in the methods of manufacture disclosed
herein are
NeoTCR Viral Products.
5. Methods of Treatment
The present disclosure provides methods for inducing and/or increasing an
immune
response in a subject in need thereof. In certain embodiments, the methods
include administering
the adoptive cell therapies disclosed herein. In certain embodiments, the
methods include
administering the adoptive cell therapies obtained by the methods disclosed
herein. In certain
embodiments, the adoptive cell therapy comprises a Cell Product. In certain
embodiments, the
adoptive cell therapy comprises a NeoTCR Product.
The Cell Products can be used for treating and/or preventing a cancer in a
subject. The
Cell Products can be used for prolonging the survival of a subject suffering
from a cancer. The
Cell Products can also be used for treating and/or preventing a cancer in a
subject. The Cell
Products can also be used for reducing tumor burden in a subject. Such methods
comprise
administering the Cell Products in an amount effective or a composition (e.g.,
a pharmaceutical
composition) comprising thereof to achieve the desired effect, be it
palliation of an existing
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condition or prevention of recurrence. For treatment, the amount administered
is an amount
effective in producing the desired effect. An effective amount can be provided
in one or a series
of administrations. An effective amount can be provided in a bolus or by
continuous perfusion.
In certain embodiments, the Cell Products can be used for treating viral or
bacterial
diseases. In certain embodiments, the Cell Products can be used for treating
autoimmune
diseases.
In certain embodiments, an effective amount of the Cell Products are delivered
through
IV administration In certain embodiments, the Cell Products are delivered
through IV
administration in a single administration. In certain embodiments, the Cell
Products are
delivered through IV administration in multiple administrations. In certain
embodiments, the
Cell Products are delivered through IV administration in two or more
administrations. In certain
embodiments, the Cell Products are delivered through IV administration in two
administrations.
In certain embodiments, the Cell Products are delivered through IV
administration in three
administrations.
The present disclosure provides methods for treating and/or preventing cancer
in a
subject. In certain embodiments, the method comprises administering an
effective amount of the
Cell Products to a subject having cancer.
Non-limiting examples of cancer include blood cancers (e_g leukemias,
lymphomas, and
myelomas), ovarian cancer, breast cancer, bladder cancer, brain cancer, colon
cancer, intestinal
cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin
cancer, stomach cancer,
glioblastoma, throat cancer, melanoma, neuroblastoma, adenocarcinoma, glioma,
soft tissue
sarcoma, and various carcinomas (including prostate and small cell lung
cancer). Suitable
carcinomas further include any known in the field of oncology, including, but
not limited to,
astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma,
ependymoma,
medulloblastoma, primitive neural ectodermal tumor (PNET), chondrosarcoma,
osteogenic
sarcoma, pancreatic ductal adenocarcinoma, small and large cell lung
adenocarcinomas,
chordoma, angiosarcoma, endotheliosarcoma, squamous cell carcinoma,
bronchoalveolarcarcinoma, epithelial adenocarcinoma, and liver metastases
thereof,
lymphangiosarcoma, lymphangioendotheliosarcoma, hepatoma, cholangiocarcinoma,
synovioma,
mesothelioma, Ewing's tumor, rhabdomyosarcoma, colon carcinoma, basal cell
carcinoma, sweat
gland carcinoma, papillary carcinoma, sebaceous gland carcinoma, papillary
adenocarcinoma,
cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell
carcinoma, bile
duct carcinoma, choriocarcinom a, s ern i n om a, embryonal carcinoma, Wilms'
tumor, testicular
tumor, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,
hemangioblastoma,
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acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma,
retinoblastoma, leukemia,
multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease,
breast tumors
such as ductal and lobular adenocarcinoma, squamous and adenocarcinomas of the
uterine
cervix, uterine and ovarian epithelial carcinomas, prostatic adenocarcinom as,
transitional
squamous cell carcinoma of the bladder, B and T cell lymphomas (nodular and
diffuse)
plasmacytoma, acute and chronic leukemias, malignant melanoma, soft tissue
sarcomas and
leiomyosarcomas. In certain embodiments, the neoplasia is selected from the
group consisting of
blood cancers (e.g. leukemias, lymphomas, and myelomas), ovarian cancer,
prostate cancer,
breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer,
liver cancer, lung
cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer,
glioblastoma, and throat
cancer. In certain embodiments, the presently disclosed young T cells and
compositions
comprising thereof can be used for treating and/or preventing blood cancers
(e.g., leukemias,
lymphomas, and myelomas) or ovarian cancer, which are not amenable to
conventional
therapeutic interventions.
In certain embodiments, the cancer is a solid cancer or a solid tumor. In
certain
embodiments, the solid tumor or solid cancer is selected from the group
consisting of
glioblastoma, prostate adenocarcinoma, kidney papillary cell carcinoma,
sarcoma, ovarian
cancer, pancreatic adenocarcinoma, rectum adenocarcinoma, colon
adenocarcinoma, esophageal
carcinoma, uterine corpus endometrioid carcinoma, breast cancer, skin
cutaneous melanoma,
lung adenocarcinoma, stomach adenocarcinoma, cervical and endocervical cancer,
kidney clear
cell carcinoma, testicular germ cell tumors, and aggressive B-cell lymphomas
The subjects can have an advanced form of disease, in which case the treatment
objective
can include mitigation or reversal of disease progression, and/or amelioration
of side effects. The
subjects can have a history of the condition, for which they have already been
treated, in which
case the therapeutic objective will typically include a decrease or delay in
the risk of recurrence.
Suitable human subjects for therapy typically comprise two treatment groups
that can be
distinguished by clinical criteria. Subjects with "advanced disease" or "high
tumor burden" are
those who bear a clinically measurable tumor. A clinically measurable tumor is
one that can be
detected on the basis of tumor mass (e.g., by palpation, CAT scan, sonogram,
mammogram, or
X-ray; positive biochemical or histopathologic markers on their own are
insufficient to identify
this population). A pharmaceutical composition is administered to these
subjects to elicit an anti-
tumor response, with the objective of palliating their condition. Ideally,
reduction in tumor mass
occurs as a result, but any clinical improvement constitutes a benefit
Clinical improvement
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includes decreased risk or rate of progression or reduction in pathological
consequences of the
tumor.
In certain embodiments, the Cell Products used in the methods of treatment
disclosed
herein are NeoTCR Products or NeoTCR Viral Products. In certain embodiments,
the Cell
Products used in the methods of treatment disclosed herein are NeoTCR
Products. In certain
embodiments, the Cell Products used in the methods of treatment disclosed
herein are NeoTCR
Viral Products.
6. Kits
In certain embodiments, the present disclosure provides kits for obtaining
adoptive cell
therapies disclosed herein.
The present disclosure provides kits for inducing and/or enhancing immune
response
and/or treating and/or preventing cancer or a pathogen infection in a subject.
In certain
embodiments, the kit comprises an effective amount of presently disclosed
cells or a
pharmaceutical composition comprising thereof In certain embodiments, the kit
comprises a
sterile container, such containers can be boxes, ampules, bottles, vials,
tubes, bags, pouches,
blister-packs, or other suitable container forms known in the art. Such
containers can be made of
plastic, glass, laminated paper, metal foil, or other materials suitable for
holding medicaments.
In certain non-limiting embodiments, the kit includes an isolated nucleic acid
molecule encoding
a presently disclosed HR template.
If desired, the cells and/or nucleic acid molecules are provided together with
instructions
for administering the cells or nucleic acid molecules to a subject having or
at risk of developing
cancer or pathogen, or immune disorder. The instructions generally include
information about the
use of the composition for the treatment and/or prevention of cancer or
pathogen infection. In
certain embodiments, the instructions include at least one of the following:
description of the
therapeutic agent; dosage schedule and administration for treatment or
prevention of a neoplasia,
pathogen infection, or immune disorder or symptoms thereof; precautions;
warnings; indications;
counter-indications; over-dosage information; adverse reactions; animal
pharmacology; clinical
studies; and/or references. The instructions may be printed directly on the
container (when
present), or as a label applied to the container, or as a separate sheet,
pamphlet, card, or folder
supplied in or with the container. The resulting cells can be grown under
conditions similar to
those for unmodified cells, whereby the modified cells can be expanded and
used for a variety of
purposes.
In certain embodiments, the present disclosure provides kits for performing
the methods
disclosed herein. In certain embodiments, the kits include reagents (e.g.,
activation reagent,
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DNA plasmid), materials (e.g., electroporation cells, gas-permeable flasks,
infusion bags), and
instructions for carrying out the methods disclosed herein.
EXAMPLES
The following are examples of methods and compositions of the invention It is
understood that various other embodiments may be practiced, given the general
description
provided above.
Example I Generation of NeoTCR Products
Neoepitope-specific TCRs identified by the imPACT Isolation Technology
described in
PCT/US2020/17887 (which is herein incorporated by reference in its entirety)
were used to
generate homologous recombination (HR) DNA templates. These 1-11t templates
were transfected
into primary human T cells in tandem with site-specific nucleases (see Figures
1A-1C). The
single-step non-viral precision genome engineering resulted in the seamless
replacement of the
endogenous TCR with the patient's neoepitope-specific TCR, expressed by the
endogenous
promoter. The TCR expressed on the surface is entirely native in sequence.
The precision of NeoTCR-T cell genome engineering was evaluated by targeted
Locus
Amplification (TLA) for off-target integration hot spots or translocations,
and by next generation
sequencing based off-target cleavage assays and found to lack evidence of
unintended outcomes.
As shown in Figures 1A-1C, constructs containing genes of interest were
inserted into
endogenous loci. This was accomplished with the use of homologous repair
templates containing
the coding sequence of the gene of interest flanked by left and right HR arms.
In addition to the
HR arms, the gene of interest was sandwiched between 2A peptides, a protease
cleavage site that
is upstream of the 2A peptide to remove the 2A peptide from the upstream
translated gene of
interest, and signal sequences (Figure 1B). Once integrated into the genome,
the gene of
interested expression gene cassette was transcribed as single messenger RNA.
During the
translation of this gene of interest in messenger RNA, the flanking regions
were unlinked from
the gene of interest by the self-cleaving 2A peptide and the protease cleavage
site was cleaved for
the removal of the 2A peptide upstream from the translated gene of interest
(Figure 1C). In
addition to the 2A peptide and protease cleavage site, a gly-ser-gly (GSG)
linker was inserted
before each 2A peptide to further enhance the separation of the gene of
interest from the other
elements in the expression cassette.
It was determined that P2A peptides were superior to other 2A peptides for
Cell Products
because of its efficient cleavage. Accordingly, two (2) P2A peptides and codon
divergence were
used to express the gene of interest without introducing any exogenous
epitopes from remaining
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amino acids on either end of the gene of interest from the P2A peptide. The
benefit of the gene
edited cell having no exogenous epitopes (i.e., no flanking P2A peptide amino
acids on either
side of the gene of interest) is that immunogenicity is drastically decreased
and there is less
likelihood of a patient infused with a Cell Product containing the gene edited
cell to have an
immune reaction against the gene edited cell.
As described in PCT/US/2018/058230, NeoTCRs were integrated into the TCRa
locus of
T cells. Specifically, a homologous repair template containing a NeoTCR coding
sequence
flanked by left and right FIR Arms was used. In addition, the endogenous TCRP
locus was
disrupted leading to the expression of only TCR sequences encoded by the
NeoTCR construct.
The general strategy was applied using circular I-1R templates as well as with
linear templates.
The target TCRa locus (Ca) is shown along with the plasmid HR template, and
the
resulting edited sequence and downstream mRNA/protein products in Figures 1B
and 1C. The
target TCRa locus (endogenous TRAC) and its CRISPR Cas9 target site
(horizontal stripe,
cleavage site designated by arrow) are shown (Figures 1A-1C). The circular
plasmid HR
template with the polynucleotide encoding the NeoTCR is located between left
and right
homology arms ("LHA" and "RHA" respectively). The region of the TRAC
introduced by the
HR template that was codon optimized is shown (vertical stripe). The TCRP
constant domain
was derived from TRBC2, which is indicated as being functionally equivalent to
TR_BC1. Other
elements in the NeoTCR cassette include: 2A = 2A ribosome skipping element (by
way of non-
limiting example, the 2A peptides used in the cassette are both P2A sequences
that are used in
combination with codon divergence to eliminate any otherwise occurring non-
endogenous
epitopes in the translated product); P = protease cleavage site upstream of 2A
that removes the
2A tag from the upstream TCR p protein (by way of non-limiting example the
protease cleavage
site can be a furin protease cleavage site); SS = signal sequences (by way of
non-limited example
the protease cleavage site can be a human growth hormone signal sequence). The
FIR template of
the NeoTCR expression gene cassette includes two flanking homology arms to
direct insertion
into the TCRa genomic locus targeted by the CRISPR Cas9 nuclease RNP with the
TCRa guide
RNA. These homology arms (LHA and RHA) flank the neoE-specific TCR sequences
of the
NeoTCR expression gene cassette. While the protease cleavage site used in this
example was a
furin protease cleavage site, any appropriate protease cleavage site known to
one of skill in the
art could be used. Similarly, while HGH was the signal sequence chosen for
this example, any
signal sequence known to one of skill in the art could be selected based on
the desired trafficking
and used.
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Once integrated into the genome (Figure 1C), the NeoTCR expression gene
cassette is
transcribed as a single messenger RNA from the endogenous TCRct promoter,
which still
includes a portion of the endogenous TCRa polypeptide from that individual T
cell (Figure 1C).
During ribosomal polypeptide translation of this single NeoTCR messenger RNA,
the NeoTCR
sequences are unlinked from the endogenous, CRISPR-disrupted TCRot polypeptide
by self-
cleavage at a P2A peptide (Figure 1C). The encoded NeoTCRct and NeoTCRI3
polypeptides are
also unlinked from each other through cleavage by the endogenous cellular
human furin protease
and a second self-cleaving P2A sequence motifs included in the NeoTCR
expression gene
cassette (Figure 1C). The NeoTCRct and NeoTCR E polypeptides are separately
targeted by
signal leader sequences (derived from the human growth hormone, HGH) to the
endoplasmic
reticulum for multimer assembly and trafficking of the NeoTCR protein
complexes to the T cell
surface. The inclusion of the furin protease cleavage site facilitates the
removal of the 2A
sequence from the upstream TCR i3 chain to reduce potential interference with
TCR I3 function.
Inclusion of a gly-ser-gly linker before each 2A (not shown) further enhances
the separation of
the three polypeptides.
Additionally, three repeated protein sequences are codon diverged within the
HR template
to promote genomic stability. The two P2A are codon diverged relative to each
other, as well as
the two HGH signal sequences relative to each other, within the TCR gene
cassette to promote
stability of the introduced NeoTCR cassette sequences within the genome of the
ex vivo
engineered T cells. Similarly, the re-introduced 5' end of TRAC exon 1
(vertical stripe) reduces
the likelihood of the entire cassette being lost over time through the removal
of intervening
sequence of two direct repeats.
In-Out PCR was used to confirm the precise target integration of the NeoE TCR
cassette.
Agarose gels show the results of a PCR using primers specific to the
integration cassette and site
generate products of the expected size only for cells treated with both
nuclease and DNA
template (KOKI and KOKIKO), demonstrating site-specific and precise
integration.
Furthermore, Targeted Locus Amplification (TLA) was used to confirm the
specificity of
targeted integration Crosslinking, ligation, and use of primers specific to
the NeoTCR insert
were used to obtain sequences around the site(s) of integration. The reads
mapped to the genome
are binned in 10 kb intervals. Significant read depths were obtained only
around the intended site
the integration site on chromosome 14, showing no evidence of common off-
target insertion
sites.
Antibody staining for endogenous TCR and peptide-HLA staining for NeoTCR
revealed
that the engineering results in high frequency knock-in of the NeoTCR, with
some TCR- cells
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and few WT T cells remaining. Knock-in is evidenced by NeoTCR expression in
the absence of
an exogenous promoter. Engineering was carried out multiple times using the
same NeoTCR
with similar results. Therefore, efficient and consistent expression of the
NeoTCR and knockout
of the endogenous TCR in engineered T cells was achieved.
Example 2. Example of Process 1
The NeoTCR Product was manufactured with Process 1 using a fully enclosed and
programmable manufacturing process on the CliniMACS Prodigy instrument
(Miltenyi). Open
manipulations were only required for media, buffer, and reagent preparation,
as well as during
final formulation of NeoTCR Product, which were performed in an ISO 5
biosafety cabinet.
Changeover procedures and utilization of disposable materials were in place to
avoid cross-
contamination.
A schematic of the complete manufacturing process, from patient leukopak
through
NeoTCR Product, is shown in Figure 2. The leukopak was collected at the
clinical site and
shipped to manufacturing facility overnight at 2-8 C. Depending on whether the
NeoTCR
Product was manufactured to comprise one or more NeoTCRs, the initial leukopak
was split into
one or more bags (i.e., one bag per NeoTCR) with each bag loaded onto a
separate CliniMACS
Prodigy unit. Using the Prodigy's "T Cell Transduction Process Program" CD4
and CD8 T cells
were positively enriched for further processing, and other cell
types/impurities were discarded.
After CD8 and CD4 T cell enrichment, the designated number of cells were
activated in the
CentriCult chamber of the Prodigy using non-bead-based activation (TransAct,
Miltenyi) and
cultured for 48 hours. On day 2, the cells were precision-genome engineered to
express the
NeoTCR by electroporation using Lonza 4DNucleofectorTM LV. For this purpose,
cells were
concentrated by centrifugation within the CentriCult of the Prodigy,
resuspended in
electroporation buffer and pumped to a custom-made reservoir using a
specifically developed
Prodigy Custom Application Program (CAP). The reservoir was connected by
sterile welding to
the electroporation system. Reagents (DNA plasmid and RNPs) were transferred
to a sterile
single use pouch within a biosafety cabinet (B SC), which was connected to the
tubing set of the
electroporator cuvette. Cells were mixed together with subject-specific
plasmid DNA and
ribonucleoproteins (RNPs) reagents within the nucleocuvette immediately prior
to
electroporation for precision genome engineering to knock out the endogenous
TCR and replace
with the NeoTCR. Following electroporation, cells are pumped back from the
output reservoir to
the Prodigy CentriCult chamber using a Prodigy Custom Application Program.
Jsing the Prodigy's "T Cell Transduction Process Program", the cells were
cultured in
the Prodigy's CentriCult chamber in TexMACS GMP medium supplemented with 3%
human
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AB serum, IL7 (12.5ng/mL) and 1L15 (12.5 ng/mL) for the remainder of the
manufacturing
period until harvest on day 13. Starting from process day 4 (2 days post
electroporation), daily
media changes were performed to maintain optimal growth conditions until
harvest on day 13.
During the harvest, the cells were washed in 2% HSA (w/v) in Plasm a-Lyte A,
concentrated and
eluted into a target cell bag (part of Miltenyi Prodigy TS520 tubing set) in
approximately 100 mL
total volume of 2% HSA in Plasma-Lyte A. Following harvest, cells were
formulated into two
CroMACS250 bags filled with 35mL of cells in final formulation medium (46%
Plasma-Lyte A,
1% HSA (w/v), 50%CryoStor CS10). A separate set of CroMACS250 bag was used for
each
NeoTCR¨depending on how many NeoTCRs were used for that product. For example,
if there
is one NeoTCR per NeoTCR Product, two CroMACS250 bags would be used; splitting
the total
cells into two bags. Alternatively, if there are three NeoTCRs per NeoTCR
Product, six
CroMACS250 bags would be used. Cells were then cryopreserved in controlled
rate freezer and
stored in vapor phase liquid nitrogen until shipment to clinical site for
infusion.
Material information for the final formulated and filled NeoTCR Product is
provided in
Table 2.
Table 2. Patient-specific NeoTCR Product Information
Material Name Patient-specific NeoTCR Product
Concentration 10 x106 -100 x 106 cells/mL
Formulation 46% P1 asmalyte A (v/v), 4% HSA (v/v, 1% w/v), 50%
CryoStor CS10
(v/v)
Fill Volume 2 x 35 mL
Container Closure CryoMACS 250 bags (Miltenyi, Cat#200-074-401)
Storage Vapor phase liquid nitrogen (below -120 'V)
Retest Date 6 months from date of manufacture
Lenkopak collection. The leukopak was collected at the clinical site according
to
medically acceptable protocols and shipped to the manufacturing facility
overnight using a
qualified shipper with temperature monitoring to maintain temperature between
2-8 C during
transport. Upon arrival at the manufacturing site, the leukopak was inspected
following standard
operating procedures to ensure the leukopak met quality requirements and
matched study
participant identification requirements prior to transport into controlled
environment room for
initiation of the manufacturing process. The leukopak details are provided in
Table 3.
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Table 3. Starting Material Parameters
aVerati ortg-7,7"--77¨ Parann
"'INFErri!
......... , : ...
... .
Leukopak collection Blood Volume TBD
Anti-Coagulant ACD-A
Container 1 Blood
Collection Bag
Collection Volume 100 mL target
volume up to
400 mL (following addition of
autologous plasma)
Leukopak shipment Temperature 2-8 C
Duration <24 hours
Shipping Container C3 shipper
(Cryoport) or
comparable validated shipper
CD4/C1)8 Enrichment. Following initial sampling for cell count/viability,
including but
not limited to cell count/viability and flow cytometry (for cell
characterization), the leukopak was
removed from biosafety cabinet and then loaded onto the CliniMACS Prodigy
instrument by
sterile welding to the sterile single use disposable Prodigy TS520 kit. Using
the Prodigy's "T
Cell Transduction Process Program" CD4 and CD8 T cells were positively
enriched for further
processing, and other cell types/impurities were discarded. Following
enrichment, cells were
again sampled for cell count/viability and flow cytometry (cell
characterization assays). The
CD4/CD8 election parameters are provided in Table 4.
Table 4. CD4/CD8 Enrichment Parameters
Operation !12!::!!!!!:!!!!1!!!!.:;.7.;.::::!!!.!!!.!!.:!!!..::::::.:!.1!.:!1!
:
CD4/CD8 Selection Prodigy Tubing Set TS520
Process Buffer CliniMACS buffer
+ .05%
HSA
Prodigy Program T Cell
Transduction Process
Program
CD4 Reagent Vial 1
CD8 Reagent Vial 1
Target cells for Enrichment <5 x 109
# Selection Cycles 3 - 5
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T cell activation. After CD8 and CD4 T cell enrichment, the designated number
of cells
were activated in the CentriCult chamber of the Prodigy using non-bead-based
activation
(TransAct, Miltenyi). Specifically, T cells were activated by incubation with
TransAct
(CD3/CD28 reagent at a ratio of 1:17.5) in TexMACS medium supplemented with 3%
human
AB serum, 12.5 ng/mL IL7 and 12.5 ng/mL1L15. Culture occurred for 48 hours in
the Prodigy
CentriCult chamber at 37 C and 5% CO2 (Programmed Prodigy setting of 39 C
translates to
actual desired temperature of 37 C).
Electroporation. On day 2, the cells were precision-genome engineered to
express the
NeoTCR. For this purpose, cells were concentrated by centrifugation within the
CentriCult of
the Prodigy, resuspended in electroporation buffer and pumped to a custom-made
reservoir (Saint
Gobain) using a product specific Prodigy Custom Application Program (CAP). The
reservoir
was connected to the Prodigy by aseptic tube welding to maintain a closed
single-patient system
(see, e.g., Figure 25). Following rebuffering the cells in the rebuffering
solution were detached
from the Prodigy using a heat sealer and connected to the cell input (upper
line) of the LV
Nucleocuvette cartridge tubing set of the electroporation system (Lonza
4DNucleofectorTM LV).
Reagents (DNA plasmid and RNPs) were aseptically transferred to a sterile
single use pouch
within a biosafety cabinet (BSC), which was then connected to the reagent
input (lower line) of
the electroporator cuvette tubing set Upon start of the Lanza electroporation
program, the cells
were mixed together with subject-specific plasmid DNA and ribonucleoproteins
(RNPs) reagents
(i.e. GMP Cas9, sgRNA TRACI and sgRNA TRBC2) by the electroporator immediately
prior to
electroporation within the nucleocuvette. This process allowed precision
genome engineering to
knock out the endogenous TCR and replace with the NeoTCR. Following
electroporation, the
electroporated cells were pumped into the output reservoir. The output
reservoir with the cells
was then detached from the LV Nucleocuvette cartridge tubing set and welded
back onto the
Prodigy kit. Cells were then pumped back from the output reservoir to the
Prodigy CentriCult
chamber using Prodigy CAP for cell expansion.
Table 5. Electroporation Conditions
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F.. 0 p eniiiim:MONNOWpaninieter'0E!!!RnpTirget
P3 Primary Nucleofector Solution
Electroporation Buffer
(Lonza)
Prodigy CAP P001 Rebuffering
Weldable Reservoir
Container Type
(Saint-Gobain)
Rebuffering Rebuffering Cell Volume 10 mL
Cell Concentration for Not currently
controlled:
Electroporation 50 x 106 cells/mL
(desired)
20 A or less
Residual Media Volume
(current Range 20-50%)
48 hr. (44hr -60 hr.)
Rebuffering start
post T cell activation
G e RNAs RNPa sgRNA TRACI and RNPI3
uid
sgRNA TRBC2
Plasmid DNA Preparation 5-7 min Incubation at 56
C
Patient-Specific NeoTCR encoding
Reagent Plasm id DNA Plasmid
Preparation (300 g/m1 reaction
volume)
83ng Cas9 complexed with 3 nmol
Cas9 RNPa or RNPI3 sgRNA/m1
reaction
volume
Total reagent volume 2.5 mL reagents/10m1 cell
suspension
Electroporator Lonza 4D device
4D Nucleofector Cartridge (weldable
Electroporation Cartridge
tubing)
Time from start of
Electroporation electroporation (first pulse) 10 min
to cell dilution with media
Total volume 70mL in TexMACS with
Cell dilution with media in
30/0 human AB serum, 1L7 (12.5ng/m1),
Prodigy
IL15 (12.5 ng/m1)
Techl expansion. Using the Prodigy's "T Cell Transduction Process Program",
the cells
were cultured in the Prodigy's CentriCult chamber in TexMACS GM? medium
supplemented
with 3% human AB serum, IL7 (12.511g/mL) and IL15 (12.5 ng/mL) for the
remainder of the
manufacturing period until harvest on day 13. Starting from process day 4 (2
days post
electroporation), daily media changes were performed to maintain optimal
growth conditions
until harvest on day 13. Cell growth was monitored by sampling for cell count
and viability at
regular intervals (day 3, day 6, day 8, day10).
Table 6. T Cell Culture Parameters
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opera ti otiUMKP ra eterpn!!!p qFpQMOUP,"targei-
ORUNIFir
TexMACS with 3% human AB
Media Type
serum, 1L7 (12.5ng/m1), IL15
(12.5 ng/ml)
Container Type Prodigy
Centricult chamber
37 2 C
Incubation Temperature
Culture Conditions
(entered value 39 C)
CO2% 5%
Culture Volume 70 n--11 to
250 mL
11 days
Culture Duration
(Process day 2 to day 13)
Cell Count/Viability Sampling Days 3, 6, 8, 10 and 13
Feed (+ 4 hours) 200 mL
Activate Shaker (+ 5 hours) Shake Type 1
-125m1/+ 175mL (total culture
Media Exchange (Day 2)
volume 250 ml)
Prodigy Activity Activate Shaker
Matrix (times post (Day 2, 1 hour post
media Shake Type 3
restart of TCT exchange)
program) Media Exchange (Day 3,
Day 4, -150m1/+ 150mL (total culture
Day 5, Day 6) volume 250
ml)
Media Exchange (Day 7, Day 8, -180m1/+ 180mL (total culture
Day 9, Day 10) volume 250
ml)
End of Culture (day 11) End Culture
Harvest and Final Formulation. Prior to harvest, the cell culture was sampled
to collect
cells for potency, a final product release test, in process cell count and
viability, as well as for
characterization tests. Cells were washed with 2% HSA in Plasma-Lyte, then
harvested into
.5 target cell bag of Prodigy TS520 tubing system in a total volume of
approximately 100 mL in 2%
HSA in Plasma-Lyte. Cells in the target cell bag (cell substance) was sealed
off from the Prodigy
kit and transported into the BSC in preparation for final formulation and
cryopreservation of final
cell product (drug product manufacturing).
Within the BSC, cells were transferred to sterile conical tubes, centrifuged
and the
supernatant was discarded. Cells were resuspended in Plasmalyte/2%HSA and
transferred to a
CryoMACS250 bag. For final formulation, the cells in Plasmalyte/2%HSA were
mixed with an
equal volume of cold Cryostor CS10 and distributed into two CryoMACS bags (35
ml total
volume per bag post QC sampling).
Table 7 Harvest and Final Parameters
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4)peratioif nm l'argef
Prodigy Harvest Prodigy Program
TCT Program Harvest
Wash/Elution buffer
Plasmalyte + 2% HSA
Elution Volume ¨ 100 mL
Volume Reduction Container Type 50 ml
Tubes
Centrifugation Speed 300x g
Centrifugation Time 10 min
Final Resuspension Medium Plasmalyte + 2% HSA
Formulation/Filling Ciyopreservation Medium CryoStor C
S10
Final Formulation 1:1 addition of cryopreservation
medium to resuspended cells
Final cell concentration
106-1006 total viable cells/ml
Container Type CryoMACS 250 bags
Fill Volume 35 mL/bag
Batch size
2 bags (1-TCR product)
Cryopreservation Storage. The final formulated cell product (i.e., the NeoTCR
Product)
in the cryopreservation bags was placed into metal storage cassettes and
frozen in the controlled
rate freezer using an optimized freezing program. The sample temperature
profile in the
controlled rate freezer was monitored and data filed with the batch records.
Once cryopreserved,
cell product was stored in vapor phase liquid nitrogen at below -120 C until
shipment to clinical
site. Furthermore, the NeoTCR Product remained below ¨120 C during transport
to and storage
at the clinical site.
Hold Times. The processing time limits and in-process material hold times were
provided
to ensure consistent manufacturing operations and performance (see Table 8).
Table 8. Hold Times
process
Bow
Description A Teritp.
. .õ Tinie.
..Thne
Incoming fresh leukapheresis 2 ¨ 8 C <
24 hr.
TBD
Cells in electroporation buffer (overall duration) ambient
(<2 hrs.)
Plasmid DNA (post Thermomixer incubation) ambient
TBD
Cas9/sgRNA complex ambient
TBD
Nucleofection Mastermix (Cas9/sgRNA/Plasmid
DNA) ambient
TBD
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Cells post addition of cryopreservation medium until
2 ¨ 8 C TBD
start of cryopreservation
Example 3. Example of Process 2
The cell manufacturing Process 2 encompasses the process from receipt of
patient
leukapheresis in the GM? manufacturing facility, enrichment and activation of
CD4/CD8 T cells
from leukapheresis, followed by gene-editing using a NeoTCR plasmid, expansion
of NeoTCR
Cells, harvest, final formulation and cryopreservation, as well as QC release
of final NeoTCR
Product.
The NeoTCR Product was manufactured under Process 2 using a fully enclosed and
programmable manufacturing process.
A schematic of the complete manufacturing process, from patient leukopak
through the
final cell product, is shown in Figures 2 and 3. The leukopak was collected at
the clinical site
and shipped to manufacturing facility overnight using a qualified shipper at 2-
8 C. Following
inspection, the leukopak was loaded onto the Prodigy. Using the Prodigy's "T
Cell Transduction
Process Program" CD4 and CD8 T cells were positively enriched for further
processing, and
other cell types/impurities are discarded (specifically aiming to remove the
monocytes and NK
cells from the CD4+ and CD8+ T cells, along with other non- CD4+ and CD8+
cells). After CD8
and CD4 T cell enrichment, the designated number of cells was activated in a G-
Rex 100M-CS
flask per NeoTCR-sublot using non-bead-based activation (in this example,
TransActTm,
Miltenyi) and cultured for 48 hours. On day 2, the cells were precision-genome
engineered
according to the process described in Example 1, to express the NeoTCR by
electroporation
using Lonza 4DNucleofectorTM LV. For this purpose, cells were harvested from
the G-REX
100-CS flask using peristaltic pump and then rebuffered in electroporation
buffer and pumped to
a custom-designed reservoir using the Rotea Counterflow Centrifugation System
(Thermofisher)
(Figure 25). The reservoir was connected by sterile welding to the
electroporation system.
Reagents (DNA plasmid and RNPs) were transferred to a sterile single use pouch
within a
biosafety cabinet (B SC), which was then connected to the tubing set of the
electroporator cuvette.
Cells were then mixed together with subject-specific plasmid DNA and
ribonucleoproteins
(RNPs) reagents within the nucleocuvette immediately prior to electroporation
for precision
genome engineering to knock out the endogenous TCR and replace with the
NeoTCR. Following
electroporation, the cells were pumped from the output reservoir to a new G-
REX-100M-CS
flask for further expansion (Figure 27).
"[he cells were then cultured in the G-Rex in an incubator (5% CO2, 37 C) in
culture
medium (TexMACS GMP medium supplemented with 3% human AB serum, 1L7
(12.5ng/mL)
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and IL15 (12.5 ng,/mL)). On day 8, a cell count was performed, and cells were
split into an
appropriate number of G-Rex flasks (G-Rex 100M-CS or G-REX 500M-CS) using
fresh culture
medium and cultured until day 13 (Figure 26). On day 13, the cells were
collected from the G-
Rex flasks using a peristaltic pump into a collection bag, which was then
loaded onto the Rotea
for further processing (Figure 26). Using the Rotea "final formulation
program", the cells were
washed in 2% HSA (w/v) in Plasma-Lyte A, concentrated and eluted into one
CryoMACS500
bag in 1/2 of target final formulation volume (Figure 25). Following closed
system addition of an
equal volume cold CryoStor CS10 using the peristaltic pump, approximately 1/2
of final
formulated cell suspension was transferred into a second CryoMACS500 bag.
CryoMACS 500
bags filled with 70mL of cells in final formulation medium (46% Plasma-Lyte A,
1% HSA (w/y),
50% CryoStor CS10) were then cryopreserved in controlled rate freezer and
stored in vapor
phase liquid nitrogen until shipment to clinical site for infusion (see Table
9).
Table 9. Patient-specific NeoTCR Product Information
Material Name Patient-specific NeoTCR Product
Concentration 10 x106 -100 x 106 cells/mL
Formulation 46% Plasmalyte A (v/v), 4% HSA (v/v, 1% w/v), 50%
CryoStor CSIO (v/v)
Fill Volume 2 x 70 mL/ TCR sublot
Container Closure CryoMACS 500 bags (Miltenyi, Cat#200-074-402)
Storage Vapor phase liquid nitrogen (below -120 'V)
Retest Date TBD: expected 6 months from DOM
The leukopak was collected at the clinical site and shipped to the
manufacturing facility
overnight using a qualified shipper with temperature monitoring to maintain
temperature between
2-8 C during transport.
Lenkopak collection. The leukopak was collected as described in Example 2
except the
collection volume of blood was 200mL target volume up to 400mL (following
addition of
autologous plasma).
CD4/CD8 Enrichment. The CD4/CD8 enrichment was performed as described in
Example 2 except the number of target cells for enrichment was < 5 x 109.
T Cell Activation. After CD8 and CD4 T cell enrichment, the designated number
of cells
were transferred to a G-Rex 100M-CS flask using non-bead-based activation
(TransAct,
Miltenyi). Specifically, T cells were activated by incubation with TransAct
(aCD3/CD28 reagent
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at a ratio of 1:17.5) in TexMACS medium supplemented with 3% human AB serum,
12.5 ng/mL
IL7 and 12.5 ng/mL IL15. The cells were culture in the activation medium for
48 hours in an
incubator at 37 C and 5% CO2 (Table 10).
Table 10. T Cell Activation Specifications
ParameteArl. RiiirliPMTarget 1E00
... .
TexMACS with 3% human AB
Media Type serum, IL7
(12.5ng/m1), IL15
(12.5 ng/ml)
Transact Volume 14.3 mL (Ratio 1:17.5)
Container Type G-REX 100M-CS
T cell Activation T cell number 715 x 106
enriched T cells
Incubation Temperature 37 2 C
CO2% 5%
Culture Duration 48 hr. (44 hr. -60 hr.)
Culture Volume 250 mL
Electroporation. On day 2, the cells were precision-genome engineered to
express the
NeoTCR (see Table 11). For this purpose, cells were harvested from the G-Rex
100M-CS flask
using a peristaltic pump into a bag, which was then connected to a Rotea
single-use disposable
tubing set (1 set per each NeoTCR) by sterile tube welding to maintain closed
system. Using the
Rotea's automated rebuffering program, the cells were concentrated by counter-
flow
centrifugation, resuspended in electroporation buffer and pumped to a custom-
made reservoir
(Saint Gobain). Following rebuffering, the cells in the rebuffering solution
were detached from
the Rotea kit using a heat sealer and connected to the cell input (upper line)
of the LV
Nucleocuvette cartridge tubing set of the electroporation system (Lonza 4D-
NucleofectorTM LV).
Reagents (DNA plasmid and RNPs) were aseptically transferred to a sterile
single use pouch,
which was then connected to the reagent input (lower line) of the
electroporator cuvette tubing
set. Upon start of the Lonza electroporation program, the cells were mixed
together with subject-
specific plasmid DNA and ribonucleoproteins (RNPs) reagents (i.e. GMP Cas9,
sgRNA TRACI
and sgRNA TRBC2) by the electroporator immediately prior to electroporation
within the
Nucleocuvette. This process allowed precision genome engineering to knock out
the endogenous
TCR and replace with the NeoTCR. Following electroporation, the electroporated
cells were
pumped into the output reservoir. The output reservoir with the cells was then
detached from the
LV Nucleocuvette cartridge tubing set and welded onto a custom tube adapter,
which was
connected to the tubing of a new G-Rex 100M-CS flask and culture media bag.
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Cells were then diluted with culture media and pumped back from the output
reservoir to
the G-Rex 100M-CS filled with culture medium. In the case of a 2-NeoTCR or 3-
NeoTCR
process (or any NeoTCR Product with greater than 3 NeoTCRs), this process was
repeated for
each TCR-sublot. While the same Rotea kit may be used for all three sublots,
separate reagents
and materials are required for electroporation and transfer to G-Rex 100M
flasks.
Table 11. T Cell Nucleofection Parameters
Operation Parameter 'Target
Electroporation Buffer P3 Primary Nucleofector Solution (Lonza)
Rotea Program
Weldable Reservoir
Container Type
(Saint-Gobain)
Rebuffering Cell Volume 10 mL
Rebuffering Cell Concentration for Not
currently controlled:
Electroporation 50 x 106 cells/mL
(desired)
20% or less
Residual Media Volume (current Range 20-50% as
measured by
glucose conc.)
Rebuffering start 48 hr. (44 hr. -60
hr.)
post T cell activation
Guide RNAs
RNPa sgRNA TRACI and RNPI3 sgRNA
TRBC2
Plasmid DNA Preparation 5-7 min Incubation at
56 C
Reagent Patient-Specific NeoTCR encoding Plasmid
Plasmid DNA
Preparation (300 giml reaction
volume)
83 pg Cas9 complexed with 3 nmol RNPa or
Cas9
RNP13 sgRNA/m1 reaction volume
Total reagent volume 2.5 mL reagents/10m1 cell suspension
Electroporator Lonza 4D device
4D Nucleofector Cartridge (weldable
Electroporation Cartridge
tubing)
Time from start of
Electroporation electroporation (first pulse) 10 min
to cell dilution with media
Total volume 1L in TexMACS with 3%
Cell dilution with media in
human AB serum, IL7 (12.5ng/m1), IL15
G-Rex 1001\4-CS
(12.5 ng/ml)
Peristaltic pump Watson Marlow 530S
Peristaltic
Peristaltic pump speed 30 mL/min
pump transfer
Transfer volume 40 mL
T Cell Expansion. The cells were cultured in G-Rex-100M-CS flasks in tissue
culture
incubators at 37 C and 5% CO2 using 1L TexMACS GI\SP medium supplemented with
3%
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human AB serum, IL7 (12.511g/mL) and IL15 (12.5 ng/mL). On day 8, a cell count
was
performed. Depending on cell numbers, cells were split into one or more G-Rex
culture vessels
to maintain optimal growth conditions. Glucose/Lactate was monitored by
sampling at regular
intervals (day 3, day 6, day 8, day 10). See Table 12,
Table 12. T Cell Culture Parameters
'
pa ram eterm.,,a, arget
TexMACS with 3% human
Media Type AB serum, 1L7
(12.5ng/m1),
IL15 (12.5 ng/ml)
one G-REX 100M-CS per
Container Type (day 2 - day 8)
TCR sublot
Incubation Temperature 37 2 'V
Culture Conditions CO2 % 5%
1000 mL/G-REX 100M-CS
Culture Volume
flask
Total 11 days
Culture Duration
(day 2 to day 13)
Glucose/Lactate Sampling Days 3, 6, 8, 10
and 13
Cell count/viability sampling Day 8
Selected based on total viable
cell count day 8 (per TCR
sublot):
<1x109: 1 G-Rex-100M-CS
>1x109 and < 3x109: 2 G-Rex-
Culture vessel and No/TCR
1001\4-CS
Day 8 Split sublot
>3x109 and, <5x109: 3 G-Rex-
100M-CS
>>5x109: adjust splitting of G-
Rex chambers accordingly
Target seeding concentration 0.5-1 x107
cells/cm2
Harvest and Final Formulation. Cells from the G-Rex flasks were combined into
a
single collection bag per TCR sublot using a peristaltic pump after reducing
the volume to
approximately 100 mL. The combined culture was then sampled to collect cells
for potency, a
final product release test, in process cell count and viability, as well as
for characterization tests.
The bag with the cell suspension was then connected to a Rotea tubing set for
further processing.
Using Rotea's "final formulation program", cells were washed with 2% HSA in
Plasma-Lyte,
then harvested into one CryoMACS 500 bag in a total volume of approximately 75
mL in 2%
HSA in Plasma-Lyte. Cells in the CryoMACS 500 bag (cell substance) was sealed
off from the
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Rotea kit and connected to custom single use disposable adapter and bag with
cryopreservation
medium (CryoStor CS10) by sterile welding. The peristaltic pump was then used
to dispense an
equal amount of cold CryoStor CS10 to the cell suspension (final formulation
46%Plasmalyte A+
1% HSA (w/v) + 50% CryoStor CS10; the NeoTCR Product)
Approximately 1/2 of the cell suspension was transferred to second CryoMACS
500 bag.
See Table 13 for the harvest and final formulation parameters.
Table 13. Harvest and Final Formulation
Operation Parameter
Collection of cells Peristaltic pump Watson Marlow
530S
from G-Rex flasks
Pump speed 70 mL/min
300 mL Transfer Pack
Collection bag 300 mL Transfer
Pack
Rotea formulation Rotea program TrifeCtaR Final
Formulation
Rotea tubing set A45130
Resuspension Medium Plasmalyte + 2%
HSA
Target output volume 75 mL
Collection bag CryoMACS 500
bag
Final formulation/fill Cryopreservation Medium CryoStor CS10
Final Formulation 1:1 addition of
cryopreservation
medium to resuspended cells in
Plasmalytc/2%HSA
Peristaltic pump transfer
parameters
Final cell concentration 106-1006 total
viable cells/ml
Container Type CryoMACS 500
bags
Fill Volume 70 mL/bag
Batch size 2 bags (1-NeoTCR
NeoTCR
Product); 4 bags (2-NeoTCR
NeoTCR Product); 6 bags (3-
NeoTCR NeoTCR Product); etc.
Example 4 Process 2 Has Superior Properties to Process I
These experiments demonstrate that changes from manufacturing Process 1 to
Process 2
allows for improved yields and efficiency without changing the qualitative
nature of the product.
The changes to the manufacturing Process 2 compared to Process 1 enhanced the
viable cell yield
and increase manufacturing efficiency necessary to meet the manufacturing
demands required for
the NeoTCR Product
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The experiments in Example 4 demonstrates that the NeoTCR Product manufactured
using Process 2 allows for increased NeoTCR cell yields whilst being of the
same quality
(identity, safety and potency) as cell product produced by Process 1.
NeoTCR Process 2 was developed in order to overcome limitations of the current
clinical
manufacturing process with regard to NeoTCR cell yield and improve scalability
and
manufacturing capacity. The current clinical manufacturing process utilizes
the closed,
automated CliniMACS Prodigy for T cell selection, T cell activation, T cell
rebuffering prior to
day 2 electroporation on the Lonza nucleofector and T cell expansion (day 2 to
day 13). Final
formulation was performed manually within an ISO 5 BSC. Overview of current 3
NeoTCR
manufacturing process is shown in Figure 4.
The limited capacity of the CentriCult chamber of the Prodigy is the primary
limitation
of obtaining higher cell yields. Therefore, the optimized process replaced the
CentriCult chamber
with a closed system that is easily scalable, namely sterile single-use G-Rex
flasks, either G-Rex
100M-CS or G-Rex 500M-CS as the culture vessel during T cell activation and T
cell expansion.
Another benefit of using the G-Rex system is that G-Rex flasks are placed into
standard tissue
culture incubators, which can easily be monitored and maintained and are
generally less
susceptible to failure than more complex instrumentation. Furthermore, gas
exchange is
facilitated through a membrane at the bottom of the device allowing the cells
to be undisturbed
for longer amounts of time (rather than shaking and constant media exchanges
necessary in the
CentriCult). Importantly, no changes were made with respect to T cell
activation reagent
(including same ratio of cells to reagent and volume) or the culture medium to
ensure similar T
cell activation levels as compared to manufacturing Process 1.
Due to the change in culture vessel, the closed system transfer method was
changed from
Prodigy to the Rotea Counterflow Centrifugation System (Thermofisher) for
initial seeding of G-
Red flasks for T cell activation as well as for rebuffering of the cells into
electroporation buffer
for nucleofection. The Rotea is a counterflow centrifugation device that
allows gentle
concentration of the cells into small volumes with minimal cell loss. The
Rotea utilizes GMP
sterile single-use disposable kits Due to the higher accuracy of the Rotea at
small volumes
(approximately +1- lml as opposed to +I- 5m1 of Prodigy), this change resulted
in improved
consistency in cell suspension volume and cell concentration for the
nucleofection process. No
changes were introduced to the gene-editing process using the Lonza
nucleofector.
As an additional safety improvement, a closed system harvest and final
formulation
process was developed to replace the current manual open final formulation
within BSC For this
purpose, the cells were harvested from the single use disposable sterile G-Rex
culture vessels
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using a peristaltic pump then formulated into defined volume of PlasmaLyte +2%
HSA using the
Rotea. Closed system addition of CryoStor CS10 was performed using a
peristaltic pump. Due
to increased cell yield using the optimized process, the final product
container was changed from
CryoMACS 250 bag with a fill volume of 35 nil to two CryoMACS 500 with a final
fill volume
of 70m1 to keep same number of bags and same cell concentration as produced in
the
manufacturing Process 1. An overview of optimized manufacturing Process 2 is
shown in
Figure 4, while Table 14 summarizes changes between NeoTCR Product
manufacturing
versions.
Table 14. Comparison of Process 1 and Process 2
Operation:: = Process e "==='=]:]: "" Process
CD4/C-138 Enrichment on Prodigy Enrichment on Prodigy
enrichment = no changes
T cell activation T cell activation in Prodigy T cell
activation in G-Rex
= Closed-system transfer to G-Rex
using Rotea
= No changes to reagents and
volumes
Electroporation Gene-Modification using Gene-Modification
using Lonza XL-LV
Lonza XL-LV = no changes to
procedure
Closed-system transfer to/from Lonza
Closed-system transfer XL-LV using Rotea
to/from Lonza XL-LV using
Prodigy CAP
Culture Expansion Culture expansion in Prodigy Culture expansion in G-Rex
CentriCult chamber with = split on day 8
multiple media exchanges = No changes to media
formulation
Final Formulation/ Harvest/Wash using Prodigy, Harvest/Wash and closed system
final
Cryopreservation Manual formulation within formulation using
peristaltic pump and
BSC Rotea
= Cryopreservation in 2
Cryopreservation in 2 CryoMACS 500 bags
with 70
CryoMACS 250 bags with ml fill volume
using controlled
35 ml fill volume using rate freezer (same
CRF program)
controlled rate freezer
As part of the process evaluation, the individual unit operations were
initially evaluated
separately and compared to Process 1.
o evaluate the feasibility of ri cell activation in G-Rex flasks the same
number of
CD4/CD8 enriched cells were activated in the CentriCult unit of the Prodigy or
in a G-Rex 100M
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flask using same ratio of TransAct for two days followed by nucleofection and
small-scale
expansion (6M G-Rex). Cells from both conditions showed similar expression of
activation
markers (Figure 5A), as well as similar levels of % NeoTCR+ gene-editing
efficiency (Figure
5B).
In parallel, to evaluate T cell activation in G-Rex flasks, experiments to
compare T cell
expansion in the Prodigy CentriClut or G-Rex flasks were performed. These
experiments showed
that cell expansion in G-Rex flasks resulted in higher-fold expansion (Figure
6A), but similar T
cell phenotype (Figure 6B) as compared to cell expansion in the Prodigy
CentriCult unit.
Subsequently the G-Rex seeding density and appropriate time for cell split was
evaluated for
robust expansion across multiple donors. Based on these results, a day 8 split
with a target
seeding density between 5x106to 1x107cellsicm2 was selected.
As a result of moving the T cell activation into the G-Rex, a new closed
system
rebuffering process was developed to concentrate and resuspend cells into
electroporation buffer
prior to nucleofection. The Rotea Counterflow Centrifugation System was used
for this purpose.
While cell recovery was variable, average cell recovery was generally higher
as compared to the
Prodigy CAP rebuffering process (Figure 7).
The performance of the Rotea during final formulation of the NeoTCR Cells into
formulation buffer (Plasmalyte A+ 2% human serum albumin (HSA)) was compared
to
formulation using the Prodigy. Rotea formulation resulted in a higher average
% of cell recovery
as compared to the Prodigy with an average cell recovery of 91% Rotea vs 81%
cell recovery in
the Prodigy (Figure 8).
Due to the overall higher T cell yield using the optimized Process 2 (Figures
6A and 6B
and Figure 15), the final formulation container was switched from a CryoMACS
250 bag with a
35 mL fill volume to a CryoMACS 500 bag with a 70 mL fill volume. No changes
were
implemented with regard to final formulation or controlled rate freezer
program.
In order to assess whether this change would impact the NeoTCR Product
quality, post
thaw QC release attributes of NeoTCR Cells using Process 1 cryopreserved in
CyroMACS250
bags (35 mL fill volume), NeoTCR Cells using Process 2 formulated in
CyroMACS250 bags (35
mL fill volume) and NeoTCR Cells using Process 2 formulated using Rotea and
cryopreserved
in CyroMACS 500 bags (70 mL fill volume) were compared. Bags were filled with
a target cell
concentration of 10 Million cell s/mL. All conditions showed similar post thaw
viability (Figure
9) and post thaw cell concentration (Figure 10) suggesting that change in
final product container
does not negatively impact NeoTCR Product quality.
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In addition, similar post thaw % NeoTCR+ expression was observed in cells
cryopreserved in QC (quality control) vials, CryoMACS 250 bags (35 mL fill
volume) or
CryoMACS 500 bags (70mL fill volume). In this particular run, a NeoTCR Product
obtained
using Process 2 had slightly lower %NeoTCR+ as compared to a NeoTCR Product
obtained
using Process 1 due to normal variability of the Lonza nucleofector
electroporation method, but
no impact on %NeoTCR+ was noticed as a result of formulation or
cryopreservation method
(Figure 11).
No significant changes to T cell phenotype were observed as a result of the
cryopreservation method, as cells manufactured using optimized Process 2
showed similar T cell
phenotype regardless of whether cells were cryopreserved in QC vials, CryoMACS
250 bags (35
mL fill volume) or CryoMACS 500 bags (70mL fill volume) (Figure 12).
Similarly, no changes
in phenotype were noted for cells generated using Process 1 and cryopreserved
in QC vials or
CryoMACS 250 bag with 35 ml fill volume (Figure 12).
Following evaluation of the individual unit operations, side-by side
feasibility studies
from the same donor were performed to evaluate Process 2 at scale in
comparison to
manufacturing Process 1 using upgraded Prodigy software with TCT version 2Ø
Data
comparisons were performed to historical data from engineering runs and
clinical readiness runs.
A total of five comparison runs were completed with last two runs utilizing a
fully closed system
Earlier runs were performed using few open steps while all major unit
operations (T cell
selection, T cell activation, Rotea rebuffering, expansion in G-Rex 100M) were
in place.
Data from the studies show that using G-Rex as the culture vessel successfully
increased
overall T cell expansion as compared to Process 1 in the Prodigy (Figure 13).
As the gene-editing procedure remained unchanged between with both processes
utilizing
the Lonza LV-XL nucleofector and same reagents, percentages of NeoTCR Cells in
the final
product of Process 2 remained within normal variability of manufacturing
Process 1 with the
average percentage of NeoTCR Cells trending slightly higher (Figure 14). The
positive trend
towards improved gene-editing is due both to the higher cell concentration and
reduced residual
media post rebuffering using the Rotea as well as increased survival of
electroporated cells in the
static culture conditions of the G-Rex.
Due to the improved cell expansion, overall yield of NeoTCR Cells using
Process 2 was
significantly increased (Figure 15) as compared to manufacturing Process 1
(average number of
NeoTCR Cells of 1.4 x109 cells for a single TCR). The increase allows higher
dose levels (1.4
x109 NeoTCR Cells per TCR would meet dose the required number of cells for a 1
NeoTCR, 2
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NeoTCR, or 3 NeoTCR Product (including the ability to expand to have 4 or more
NeoTCR
Products)).
Whilst the NeoTCR Cell yield was significantly increased for Process 2, %
viability of
the NeoTCR Product (based on post-harvest, pre-cryopreservati on sample) is
similar between the
two processes (Figure 16) with average % viability above 90% for either
process.
In addition, the NeoTCR Cells from Process 2 retain the ability to secrete IFN-
gamma at
a similar level to manufacturing Process 1, confirming that the cells are
functional (Figure 17).
Levels of IFN-gamma secreted for Process 2 trend slightly higher in a direct
comparison using
the same donor.
Cytotoxic activity of the NeoTCR Product from a split comparison run was
analyzed
using an IncuCyte killing assay as an additional comparability measure. The
NeoTCR Product
was generated using a NeoTCR for which a matching tumor cell line expressing
cognate
neoantigen and a WT control cell line are available. The cells were then used
as target cells for
the killing assay, while wild-type tumor cells lacking respective neoantigen
expression served as
assay control. Based on IncuCyte killing assay data, the NeoTCR Product from
Process 2 and
Process 1 induced specific cytotoxicity in cell line expressing the NeoTCR
target at similar
levels. No killing activity was observed in target cells not expressing the
cognate neoE target
(Figures 18A and 18B)
Example 5. Process 3
Leukopak. For Process 3, the leukopak was gathered pursuant to standard
acceptable
medical procedures and it was shipped to the manufacturing site no later than
overnight from the
date of acquisition.
Enclosed manufacturing process. Process 3 comprises programable manufacturing
process that is either substantially or fully enclosed. Open manipulations are
only permitted for
media, buffer, and reagent preparation, as well as during final formulation of
NeoTCR Product.
Open manipulations, if any, were performed in an ISO 5 biosafety cabinet or
substantially similar
sterile environment.
A schematic of the complete manufacturing process, starting from the loading
of the
leukopack onto a device to select and isolate the CD4+ and CD8+ cells from the
leukopak
through the cryopreservation of the finished NeoTCR Product, is provided in
Figure 19. As
shown, following CD8+ and CD4+ T cell selection and isolation, the designated
number of cells
are transferred to and activated in a cell culture chamber that is capable of
expanding T cells.
Ideal cell culture chambers provide a gas permeable membrane In certain
instances, the gas
permeable membrane used in Process 3 was a static gas exchange cell culture
chamber. Such
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ideal cell culture chambers allow for cell expansion with minimal disturbance
of the T cells. In
certain instances, the cell culture chambers provide a gas permeable membrane
and require nor
more than two (2) media changes per week. In certain instances, the cell
culture chambers
provide a gas permeable membrane and requires one (1) media change during the
manufacturing
process. In certain instances, the cell culture chambers provide a gas
permeable membrane and
require no media changes during the manufacturing process. An example of a
cell culture
chamber used in Process 3 is the G-Rex (Wilson Wolf) which, as described as an
element above,
allows for cell expansion with minimal disturbance of the T cells in culture.
The activation of the CD4+ and CD8+ T cells occurred using non-magnetic beads.
One (1) to three (3) days following activation, the T cells were precision-
genome
engineered to express a NeoTCR using the methods described herein. In certain
experiments, the
T cells were precision genome engineered approximately two (2) days following
activation. In
certain experiments the T cells were precision genome engineered two (2) days
following
activation. Given the large volume of activated cells needed for
electroporation in order to
produce a clinical or commercial product, large scale electroporation devices
were used. Certain
devices that were used include chamber-based electroporation systems (e.g.,
chambers that hold
approximately 0.5mL ¨ 1.5mL cell suspension). In certain experiments, chamber-
based
electroporation systems that hold approximately lmL of cell suspension were
used In other
experiments, certain devices that were used include flow-through
electroporation devices
(wherein suspended cells are passed through a chamber or device using a pump-
based or
microfluidic device system).
The cells were transferred from the cell culture chambers to the
electroporation device
using peristaltic pump. Prior to the transfer of the cells from the cell
culture chambers to the
electroporation device, the cells were rebuffered in electroporation buffer
and pumped to a
custom-designed reservoir using a centrifugation system. In certain
experiments, the
centrifugation was a performed using a traditional centrifugation system. In
certain experiments,
the centrifugation was performed using a counter flow centrifugation system.
It was found that
the counter flow centrifugation system provided less agitation to the cells
and resulted in a more
complete replacement of the activation media with the electroporation media
(Figure 30). Such
replacement of media was found to be an important factor in the
electroporation efficiency of the
cells. The custom-designed reservoir was connected by sterile welding to the
electroporation
system. Furthermore, another benefit that was discovered with counter flow
centrifugation was
the ability to declump the cultured T cells without additional force and
agitation. Such force and
agitation which was previously used to declump the cells prior to
electroporation was shown to
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adversely affect the health and stability of the cells which in turn resulting
in lower cell editing
rate and lower cell survival rate following the electroporation.
It was shown that the counterflow centrifugation provided increased cell
recovery
efficiencies over traditional centrifugation (Figure 29). As shown, the
recover was not
insubstantial; rather it was shown cells could be washed and concentrated with
minimal cell loss
compared to traditional centrifugation which resulted in up to 40% loss of
cells. Furthermore,
additional experiments using counterflow centrifugation were performed in
order to determine
the optimal centrifuge speeds and times. Experiments were performed using
centrifuge speed
from ranging from 2500g(V1) to 2700g(V2) during cells washing and pump speeds
ranging from
10mL/min to 30mL/min. In a specific experiment (data shown in Figure 31), two
versions of a
counterflow centrifugation procedure were performed: Version 1 (V1) and
Version 2 (V2). V1
was set at a centrifugal force of 2500g with a fluid flow rate of 30mL/min and
V2 was set at a
centrifugal force of 2700g with a fluid flow rate of 10mL/min.
In prior experiments, VI had shown improvement over the former cell wash and
harvest
program used on the Prodigy. The particles comprising the fluidized bed within
the counterflow
centrifugation chamber experience two forces, the centripetal force generated
by rotation around
the chamber's axis, and the fluidic drag force generated by the fluid flow.
These two forces are at
a balance if the particle is stabilized within the fluidized bed Therefore,
there was a question of
whether compacting the fluidized bed by increasing the centripetal force via
increased centrifuge
speed would yield better cell recoveries during processing. Furthermore, there
was a question of
whether reducing the fluidic drag force would also compound to cell retention
within the CFC
chamber. Accordingly, V1 and V2 were run in parallel to determine it would be
possible to
reduce cell loss from centrifugation. In fact, it was shown that V2 resulted
in reduced cell loss by
over 30%.
Furthermore, the traditional centrifugation procedures that used a double
centrifugation
step to first rid the culture of residual spent media, then performed a wash
using a buffered
solution, and finally harvested the cells into a reservoir for processing.
After such processing the
cell viability was shown to be reduced simply due to handling_ Thus, selecting
counterflow
centrifugation was key to being able to effectively create a manufacturing
process that is
clinically and commercially relevant in order to produce sufficient number of
gene edited cells
and harvest such cells at a high efficiency without substantial cell loss and
without damaging the
cells.
As shown in Figure 19, following el ectroporati on the cells were pumped from
the output
reservoir to a new cell culture chamber (as described above) for further
expansion.
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Following the electroporation, the cells were transferred to cell culture
chambers for cell
proliferation and expansion that promoted the cells to maintain, develop,
and/or retain a stem-like
state (i.e., T cells that have a memory stem cell or stem cell (Tmsc or Tcm)
phenotype).
In certain experiments, the cells were cultured in cell culture chambers in an
incubator
(5% CO), 37 C) in culture medium. In certain experiments, the cell culture
chambers that were
used were the G-Rex (Wilson Wolf) cell culture chambers. Alternatively,
another static gas
exchange cell culture chamber could be used based on such static gas exchange
cell culture
chamber's ability to allow for sufficient cell proliferation of gene edited
cells that possess a
memory stem cell or stem cell (Tmsc or Tcm) phenotype).
The preferred culture media allows for cell expansion and for cells to
maintain, develop,
and/or retain a stem-like state (i.e., T cells that have a memory stern cell
or stem cell (Tmsc or
Tcm) phenotype). In certain experiments, the media that was used to culture
the cells following
electroporation was the TexMACS GMP medium supplemented with 3% human AB
serum, 1L7
(12.5ng/mL) and IL15 (12.5 ng/mL)). In certain experiments, the media that was
used to culture
the cells following electroporation was a chemically-defined, animal component-
free medium
that was shown to promote T cell expansion while maintaining T cell
functionality and potency.
An example of the increased benefit of using a chemically-defined, animal
component-free
medium that was shown to promote T cell expansion while maintaining T cell
functionality and
potency is provided in Figures 20A-24B. In certain experiments, the media that
was used to
culture the cells following electroporation was PRIME-XV T Cell CDM (Irvine
Scientific
CDM). In certain experiments, the media that was used to culture the cells
following
electroporation was ImmunoCult XF (Stemcell). In certain experiments, the
media that was used
to culture the cells following electroporation was ExCellerate (R&D Systems).
Additional non-
limiting examples of T cell medias that can be used to promote T cell
expansion while
maintaining T cell functionality and potency include LumphoOne (Takara Bio),
GT-T551
(Takara Bio), X-VIVO 15, AIM V, CTS OpTmizer (Gibco), and all other medias
with similar
physiological attributes as those described herein. Additional medias known to
one of skill in the
art that are animal component-free, that enable efficient T cell expansion
without the addition of
serum or plasma, and promote expansion and growth of T cells with a naïve
phenotype (e.g.,
Tmsc and Tern) can be used in the medias and methods described herein.
Serum free substitute additives were also used in the medias and experiments
described
herein. In certain experiments, Physiologix (Nucleus Biologics) was a media
supplement used in
the media In certain experiments, human platelet lysate (a growth factor-rich
cell culture
supplement derived from healthy donor human platelets; Stem Cell) can be used
as a media
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supplement for the serum-free medias. In certain experiments, CTS Immune Cell
Serum
Replacement (Gibco) was a media supplement used in the media. Additional serum
free
substitutes known to one of skill in the art that enable efficient T cell
expansion without the
addition of serum or plasma, and promote expansion and growth of T cells with
a naïve
phenotype (e.g., Tmsc and Tom) can be used in the medias and methods described
herein.
In addition to the medias and serum free substitutes, the addition of
cytokines can also be
used in the medias and methods described herein. In certain experiments, the
media was
supplemented or contained IL2. In certain experiments, the media was
supplemented or
contained 1L7. In certain experiments, the media was supplemented or contained
IL15. In
certain experiments, the media was supplemented or contained IL21. In certain
experiments, the
media did not contain or was not supplemented with IL2. In certain
experiments, the media did
not contain or was not supplemented with IL2 and did contain or was
supplemented with IL7 and
IL15. In certain experiments, the media did not contain or was not
supplemented with IL2 and
did contain or was supplemented with IL7, IL15, and/or IL21. In certain
experiments, the media
was supplemented or contained IL2, 11,7, IL15, and 11,21. In certain
experiments, the media was
supplemented or contained IL2, IL7, and 11.15. In certain experiments, the
media was
supplemented or contained IL7, IL15, and IL21.
In addition to the IL2, IL7, IL15, and IL21 described above as single agents
or
combinations thereof for the supplementation of media, IL12, alpha interferon,
or beta interferon
can be used alone or in combination with each other or with the IL2, IL7,
IL15, and IL21.
Furthermore, any cytokine or chemokine that is involved in lymphocyte
proliferation and
differentiation can be added to any single IL2, 11.7, IL12, IL15, IL21, alpha
interferon or beta
interferon, or any combination thereof. The concentration and ratios of each
of the cytokines
and/or chemokines should be adjusted based on the single agent use or
combination use and
titrated based on optimizing lymphocyte proliferation and differentiation.
In addition to the addition of serum free substitute additives and/or
chemokines and/or
cytokines as described herein, in certain experiments the addition of fatty
acids was shown to be
beneficial for the optimization of proliferation and differentiation.
In certain experiments, fibronectin, insulin, and/or transferrin were included
in the media.
In certain experiments, the transferrin used was recombinant transferrin. In
certain experiments,
the transferrin used was non-recombinant transferrin. In certain experiments
it was determined
that it was beneficial to increase the concentration of transferrin when
recombinant transferrin
was used compared to non-recombinant transferrin in order to achieve the same
benefits of
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lymphocyte proliferation and differentiation to achieve T cells in culture
with a naïve phenotype
(e.g., Tmsc and Tem).
In certain experiments, different concentrations of glucose in the cell medias
were tested.
It was determined that increased glucose concentrations resulted in improved T
cell activation
and gene editing efficiency. In certain experiments, the increased glucose
concentration was less
than 3.7 g/L glucose. In certain experiments, the increased glucose
concentration was between
3.7 ¨ 4.0 g/L glucose. In certain experiments, the increased glucose
concentration was between
4.0 ¨ 4.2 g/L glucose. In certain experiments, the increased glucose
concentration was between
4.2-4.5 g/L glucose. In certain experiments, the increased glucose
concentration was between 4.3
- 4.4 g/L glucose. In certain experiments, the increased glucose concentration
was between 4.4 -
4.5 g/L glucose. In certain experiments, the increased glucose concentration
was greater than 4.5
g/L glucose. As cell density in culture increases, so can the concentration of
glucose. For
example, for a high density cell culture the glucose concentration can be
increased up to 100 g/L.
In certain experiments, antioxidants were added to the media to promote
lymphocyte
proliferation and differentiation to achieve T cells in culture with a naïve
phenotype (e.g., Tmsc
and Tem).
In certain experiments, reducing agents were added to the media to promote
lymphocyte
proliferation and differentiation to achieve T cells in culture with a naïve
phenotype (e.g., Tmsc
and Tcm). In certain experiments, reducing agents were not added to the media.
In order to promote automation of the NeoTCR Product manufacture, stir
bioreactors can
be used to culture the cells instead of a static gas exchange cell culture
chamber. Such stir
bioreactors allow for real-time analytics and reaction to changes in
conditions. For example, a
stir bioreactor can be designed to have in line bioanalytics to measure cell
mass, lactate, etc., in a
closed system without manual sampling.
Alternatively, in order to promote automation of the NeoTCR Product
manufacture,
shaking/rotating bioreactors can be used to culture the cells instead of a
static gas exchange cell
culture chamber. Such shaking/rotating bioreactors allow for real-time
analytics and reaction to
changes in conditions. For example, a shaking/rotating bioreactor can be
designed to have in line
bioanalytics to measure cell mass, lactate, etc., in a closed system without
manual sampling.
Furthermore, bioreactors (e.g., stir, shanking, rotating, etc.) can be
designed and
programmed to automatically add media supplements to the culture in order to
increase or
decrease the concentration of certain components in the media. For example,
the bioreactor can
be designed and programmed to detect lactate levels in the cell culture and
add in glucose in
order to keep the glucose: lactate levels optimal for lymphocyte proliferation
and differentiation
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to achieve T cells in culture with a naive phenotype (e.g., Tmsc and Tom). In
other examples,
the bioreactors can be designed and programmed to remove lactate during the
culture process in
order to promote lymphocyte proliferation and differentiation to achieve T
cells in culture with a
naïve phenotype (e.g., Tmsc and Tem). Another example of the use of a
bioreactor is to design
and program the bioreactor to detect dissolved oxygen as a negative indicator
of an optimal cell
environment.
In certain experiments, cell counts are taken throughout the culture period
following
electroporation. In certain experiments, the cells are taken from the static
gas exchange culture
chambers (e.g., a G-Rex flask) at the half-way point of post-electroporation
culture (i.e., the
halfway point between the time of electroporation and the time when the cells
are cryopreserved
as a NeoTCR Product) and split into two new static gas exchange culture
chambers with fresh
media.
At the end of the post-electroporation cell culture, the cells were collected
from the static
gas exchange chambers using peristaltic pump into a collection bag, which is
then loaded onto a
counterflow centrifugation system and the cells were washed in 2% HSA (w/v) in
Plasma-Lyte
A, and concentrated and eluted into two freezing bags (equal volume of cells
in each bag) that
have EVA tubing, leur connectors, roller clamps and an injection port(s). An
equal volume of a
cryopreservation media (e_g_, CryoStor CS10) was added to the freezing bags
(the final volume
of the freezing bag is 1/2 cell suspension and 1/2 cryopreservation media).
The final bags of cells
were then cryopreserved in controlled rate freezer and stored in vapor phase
liquid nitrogen until
shipment to clinical site for infusion.
CD4/CD8 Enrichment. The CD4/CD8 enrichment was performed using a matrix to
positively select for CD4+ and CD8+ cells in order to minimize the number of
other blood cells
prior to activation and electroporation. Specifically, the CD4+ and CD8+
selection was
performed to remove NK cells and B cells.
T Cell Activation. After CD8 and CD4 T cell enrichment, the designated number
of cells
are transferred to a static gas exchange culture chamber and activated using
non-magnetic beads.
Magnetic beads (and any metal-based matrix) was avoided because metal has a
sturdy
architecture that was shown to stress and/or harm the cells. Furthermore,
electroporation
efficiency and gene edited cell expansion was shown to be dependent on the
health of the cells at
the time of electroporation. Accordingly, activation reagents and/or media
that allow for easy
detachment from the T cells were preferred and utilized. In certain
experiments, TransAct
(aCD3/CD28 reagent at a ratio of 1:17.5) was used as an activation reagent
Alternatively,
agonists to CD3, CD2, and/or CD28 can be used to activate the T cells.
Examples of such
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reagents include but are not limited to ImmunoCult Human CD3/CD28 T Cell
Activator (Stem
Cell), Cloudz Cell Activation (R&D Systems), and any other CD3, CD2, and/or
CD28 reagent
that is gentle on T cells and that does not interfere with electroporation
efficiencies. Table 10.
Electroporation. Following the activation, the cells were precision-genome
engineered to
express the NeoTCR. For this, the cells are harvested from cell culture
chamber where the
activation occurred, washed to remove all activation agents and cell media,
concentrated using a
counter-flow centrifugation, and resuspended in electroporation buffer. The
cells were then
added to a custom-made reservoir (Saint Gobain). The cells were then
transferred into an
electroporation chamber. Given the large volume of activated cells needed for
electroporation in
order to produce a clinical or commercial product, large scale electroporation
devices were used.
Certain devices that were used include chamber-based electroporation systems
(e.g., chambers
that hold approximately 0.5mL ¨ 1.5mL cell suspension). In certain
experiments, chamber-based
electroporation systems that hold approximately lmL of cell suspension were
used. In other
experiments, certain devices that were used include flow-through
electroporation devices
(wherein suspended cells are passed through a chamber or device using a pump-
based or
microfluidic device system). In certain experiments, the chamber-based system
was a cuvette-
style vessel.
For the electroporation, the cells were mixed together with subject-specific
plasmid DNA
and ribonucleoproteins (RNPs) reagents (i.e. GMP Cas9, sgRNA TRACI and sgRNA
TRBC2)
by the electroporator immediately prior to electroporation within the
electroporation chamber.
This process allowed precision genome engineering to knock out the endogenous
TCR and
replace with the neoE targeted TCR as described in Example 1. Following
electroporation, the
electroporated cells were pumped into the output reservoir. The output
reservoir and then
transferred to a new static gas exchange culture chamber.
T Cell Expansion. The cells are cultured as described above in this example.
Harvest and Final Formulation. The harvest and final formulation was performed
as
described above in this example.
Example 6. Cell Products
The methods and processes described in Examples 2-5 can be modified to be
applicable
to all Cell Products. Specifically, while Examples 2-5 describe gene editing
that is accomplished
using non-viral methods to yield a NeoTCR Product, the electroporation step
can be substituted
with a viral transduction step. Specifically, as it applies to Examples 3-5,
Process 2 and Process
3 can he modified for a viral transduction step instead of a non-viral
electroporation step while
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maintaining the cell culture and manufacturing steps following the
electroporation described
therein.
Lenkopak collection. The leukopak is collected as described in Examples 2-5.
The
collection volume of blood is at least 100mL.
CD4/CD8 Enrichment. The CD4/CD8 enrichment is performed as described in
Example
2 with target cells for enrichment of less than 5 x 109.
T Cell Activation. After CD8 and CD4 T cell enrichment, the cells are
transferred to a
flask as described in Examples 2-5. In particular, the T cells are activated
by incubation with
TransAct (aCD3/CD28 reagent at a ratio of 1:17.5) in TexMACS medium
supplemented with 3%
human AB serum, 12.5 ng/mL IL7 and 12.5 ng/mL 1L15. The cells are cultured in
the activation
medium for 48 hours in an incubator at 37 C and 5% CO2 (Table 10).
Transfection. On day 2, the cells are engineered to express the NeoTCR.
Briefly, the
cells are harvested from the flask using a peristaltic pump into a bag, which
is connected to a
Rotea single-use disposable tubing set (1 set per each NeoTCR) by sterile tube
welding to
maintain a closed system. The cells are concentrated by counter-flow
centrifugation and
incubated with retrovirus comprising the NeoTCR construct. After incubation
with the viral
construct, the cells are concentrated by counter-flow centrifugation and
diluted with culture
media and pumped back to the flask. All the reagents and materials used during
the transfection
are sterile.
T Cell Expansion. After transfection, the cells are cultured in flasks at 37 C
and 5% CO2
using TexMACS GMP medium supplemented with 3% human AB serum, 1L7 (12.511g/mL)
and
IL15 (12.5 ng/mL). On day 8, a cell count is performed. Based on the cell
number, the cells are
split into one or more flasks to allow further expansion.
Harvest and Final Formulation. The cells from the flasks are combined into a
single
collection bag per TCR sublot. The cells can be transferred using a
peristaltic pump. The cells
are tested for quality control analysis such as determination of potency,
viral contamination,
viability, cell counts, as well as other characterization tests. The cell
suspension is centrifuged
and washed with 2% HSA in Plasma-Lyte, then harvested into one CryoMACS 500
bag in a
Plasma-Lyte solution comprising 2% HAS. Preferably, the cell suspension is
centrifuged using a
counter-flow centrifugation system, e.g., Rotea. The cell suspension is
further diluted for
cryopreservation by adding an equal amount of cold CryoStor C S10 (final
formulation 46%
Plasmalyte A+ 1% HSA (w/v) + 50% CryoStor C S10, the NeoTCR Product). The
final
formulation is described in Examples 2-5
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Example 7. Process 4
Introduction. Effective personalized autologous cell therapy is dependent on
optimal cell
culture methods that enable ex vivo activation and expansion. Furthermore, the
NeoTCR
Products described herein are also dependent on delivery of a patient-specific
NeoTCR plasmid
via electroporation with RNEs. Various commercial cell culture media
formulations, including
serum-free media, have been developed to improve the expansion of human T
cells building on
common formulations such as RPMI 1640, Isocove's modified Dulbecco's medium
(EVIDM),
DMEM, and F12. An often-overlooked aspect of cell manufacturing is
understanding how the
composition of the growth media medium impacts the functionality and efficacy
of a cell
product.
The area of immunometabolism is a critical aspect of adoptive cell therapy and
significant
progress has been made on understanding the unique metabolic requirements of
human T cells.
Upon activation through co-stimulatory signaling via CD3/CD28 domains, T cells
rewire their
metabolism from primarily oxidative phosphorylation toward aerobic glycolysis
in order to
satisfy increased cellular demands. Required for de novo synthesis of
macromolecules including
proteins, lipids, and nucleic acids, T cells are reliant on an exogenous
source of nutrients and
metabolites. For example, glucose, glutamine, and serine are instrumental in
promoting this
metabolic adaptation and are essential for T cell function and proliferation
(van der Wint et al
2012; Olenchock et al. 2017; O'Sullivan et al. 2019). As exogeneous nutrients
in the
microenvironment impact activation, proliferation, phenotype, and propensity
towards homology
directed repair via electroporation with plasmid and RNPs, the overall outcome
of ex vivo
processing of T cells is highly dependent on the properties of the growth
medium used.
Manufacturing of NeoTCR Products using TexMACS media as described herein for T
cell activation and expansion requires supplementation with 3% human AB serum.
Although
TexMACS is labeled as a serum-free media by the vendor, it is evident that
omission of serum
from the formulation results in significant cell losses following
electroporation and poor cell
expansion of NeoTCR Cells. The use of huAB in clinical manufacturing is less
than desirable as
it suffers from supply concerns around rapidly increased global demand and lot-
to-lot variability.
Furthermore, significant variability across TexMACS lots was observed. In
light of these
limitations, there was an identified need to develop a manufacturing process
and determine
optimal media and growth conditions for NeoTCR Cells to generate NeoTCR
Products.
Prime-XV T cell medium (Irvine Scientific / FujiFilm) is a chemically defined,
cGMP-
grade, animal component-free medium for T cell culture that has been optimized
for consistent
expansion of human T cells while maintaining functionality and potency.
Supplementation of
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Prime-XV with human AB serum is not required for cell expansion. However, the
addition of 2%
(v/v) Physiologix XF serum replacement (Nucleus Biologics) following
electroporation was
found to improve cell recovery and expansion. In addition to the removal of
human AB serum
from the media as described in Processes 1-3 above, Process 4 features an
additional media
exchange which was discovered to be needed for optimal cell growth and
expansion to make a
NeoTCR Product.
Summary. On average, Prime-XV showed 35-fold expansion from day 2 to day 13 as
compared to 22-fold expansion observed in cultures expanded with TexMACS in
large-scale split
comparison runs (n=5). With the additional media exchange on day 6, an average
of 47-fold
expansion was achieved (n=3). Comparable cell viability (>95%) was maintained
in cell
substance following expansion in Prime-XV CDM. Significant improvement in % of
NeoTCR+
expression was measured on day 13 in cells activated and expanded in Prime-XV
T cell CDM
relative to TexMACS, 26.4% and 20.2% respectively (n=5). Increasing the final
fill volume by
switching from the CryoMACS250 bags with 35 mL to CryoMACS500 with a 70 mL
final fill
volume did not have a significant impact on post-thaw viability, cell
concentration, % of
NeoTCR expression by dextramer staining, and T cell phenotype. A 2-fold
increase in total
NeoTCR+ cell yield was obtained with Prime-XV relative to TexMACS (4.6 x 109
and 2.3 x 109
NeoTCR+ cells respectively) (n=5). Additional media exchange on day 6 resulted
in a further
increase in NeoTCR+ cell yield with an average of 6.2 x 109 (n=3). Cells
expanded in Prime-XV
maintained comparable T-cell phenotype and functional activity in comparison
with TexMACS
Materials and Methods. All split comparison runs described in this study were
executed
according to their respective research procedures. For these runs,
leukapheresis was collected
from healthy donors (HemaCare, Cat # PBOO1F-1), one donor per comparability
run or
intermediate-scale development experiment, and shipped to manufacturing
facility via controlled
shipper at 2-8 C.
The CliniMACS Prodigy was used to enrich healthy donor Leukopaks for CD4+ and
CD8+ T cells using the TS520 tubing set, standard TCT protocol, and the
Miltenyi MACS
separation columns according to the Manufacturer's instructions. The enriched
target cells were
eluted in culture media (TexMACSTm with 3% human AB serum [Valley Medical] and
IL-7 and
IL-15 [12.5 ng/mL each]). For all conditions cultured in Prime-XV, 715x 106 or
71.5 x106 cells,
for large-scale and intermediate-scale experiments respectively, were
dispensed into 50 mL
conical tubes, centrifuged at 300 x g for 10 minutes, and resuspended in Prime-
XV CDM with
IL-7 and IL-15 [1 2. 5 ng/mL each] prior to activation Following enrichment,
TransACTTm was
added to the cell suspension at (1:17.5) in either 25 or 250 mL of culture
medium with a G-
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RexOw plate or Cf-Rex1UUM-CS. Forty-eight hours after activation, cells were
collected from the
G-Rex and either manually buffer exchanged and concentrated via centrifugation
at 300 x g for
minutes or in the context of large-scale experiment, buffer exchanged and
concentrated using
the CTS Rotea
5
Electroporation was performed on a Lonza 4D NucleofectorTM device either in LV
XL
cartridges for large-scale processing, as detailed in RP031: Lonza LV XL Setup
or RP030: Lonza
HRNucleofection using pre-complexed RNPs. Cells were electroporated with RNPs
and plasmid
DNA that carries the Neo12 or PACT035 TCR cassettes in a WT TCR backbone.
sgRNA
(TRAC-1 and TRBC-2) and Cas9 Nuclease protein were previously complexed into
RNPs. Cells
10 were electroporated using the EO-115 pulse code on the Lonza
nucleofector unit for all the
samples.
After a 10-minute rest period, cells were either manually transferred out of
the Lonza LV
lmL cartridge via pipette into 100 mL of pre-warmed culture medium plated in a
G-Rex6M plate
or via peristaltic pump into a G-Rex100M-CS with a total volume of 1L for
large-scale split runs.
For all Prime-XV conditions the culture medium following electroporation
contains 2%
Physiologix XF SR (Nucleus Biologics), IL-7, and IL-15 [12.5 ng/mL]. The cells
were cultured
in the G-REX100M-CS until Day 8 at which the cells were split if the total
viable cell count
exceeded 1x109 cells to maintain an optimal surface density between 5-1
0x106cells/cm2 Cells
were harvested on Day 13, washed in 2% HSA (w/v) in Plasma-Lyte A and
concentrated in 70
mL using the CTS Rotea. Following formulation of the cell substance in
PlasmaLyte A
supplemented with 2% HSA, an equal volume of CryoStor CS10 is added to the
CryoMACS500
bag and the contents are split via the peristaltic pump into a secondary bag
with a final fill
volume of 70 mL in each bag resulting in a final viable cell concentration
between 10x106-
100x106 cells/mL The final product bags were placed in storage cassette and
frozen in a
controlled rate freezer using the optimized freezing program.
Results from Research Scale Manufacturing. These studies suggest that
expansion in
Prime-XV results in improved cell proliferation following electroporation
relative to TexMACS,
regardless of whether 2% Physiologix XF SR was added to the culture. Prime-XV
showed
greater than 30-fold expansion as compared to only 6.7-fold expansion with
TexMACS.
However, addition of 2% Physiologix XF SR at 2% throughout the culture (37.2-
fold expansion)
or only during T cell expansion post electroporation (41.6-fold expansion)
further improved cell
expansion as compared to Prime-XV without any serum supplements except IL-7
and IL-15
(30.6-fold expansion).
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While presence of 2% Physiologix XF SR during the T cell expansion phase post
electroporation improved overall cell expansion, optimal gene-editing rates
were observed if
Prime-XV without Physiologix XF SR was used during T cell activation phase
from Day 0 to
Day 2(26.5% and 17.5% respectively compared to 12.8% using TexMACS). Omission
of 2%
Physiologix XF SR altogether resulted in a decrease in NeoTCR+ expression
(21.1%) relative to
the condition where 2% Phx was added post electroporation. Based on these
experiments, it was
determined that Physiologix XF SR plays an important role in attenuating an
electroporation
induced lag phase and thus recovery of NeoTCR Cells. With regard to addition
of Physiologix
XF SR during the activation phase, the observed decrease in NeoTCR Cell
expression was due to
presence of residuals that alter gene-editing outcomes.
Among the tested Prime-XV supplementation strategies, Prime-XV without
Physiologix
XF SR during T cell activation, followed by addition of 2% Physiologix XF SR
to the media
during the expansion phase, resulted in a significant increase in NeoTCR+ cell
yield, as
compared to the control.
As cell expansion of cells from cancer patients generally trends lower as
compared to
cells from healthy donors with an average 8-fold expansion in patients (n=17
sublots, 7 different
patients) as compared to an average 12-fold expansion for healthy donors (n=15
sublots, 8
different donors), experiments were designed and performed to interrogate the
ability of Prime-
XV to expand cells from cancer patients. Interestingly and unexpectedly, the
results showed that
the use of Prime-XV T cell media to expand cells from cancer patients
significantly increased
cell expansion with an average of 18-fold expansion between day 2 and day 13
as compared to
the control (with a mean 8-fold expansion). Including an additional media
exchange on Day 6
with Prime-XV medium resulted in a slight further increase in cell expansion
with an average 20-
fold expansion between day 2 and day 13.
Results from Large/Clinical Scale Manufacturing. Following initial research
experiments
aimed at optimizing the media formulation and feeding schedule, top conditions
including Prime-
XV with 2% Phx added following electroporation and the day 6 medium exchange
were
evaluated in several large-scale split runs. Extracellular glucose and lactate
concentrations were
monitored at various time points of the culture to confirm the optimum feeding
schedule
identified using the scale-down model. Aside from evaluation of parameters
directly contributing
to NeoTCR Cell yield, including %NeoTCR+, cell viability, and cell expansion,
NeoTCR
Product from split large-scale development runs were further characterized to
assess potential
changes in T cell phenotype and function. Furthermore, NeoTCR Products
generated using
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Prime-XV were assessed for functionality via an IFN-y release assay as well as
using IncuCyteg
killing assay to determine cytotoxicity.
NeoTCR Cells expanded in Prime-XV showed significantly increased cell
expansion with
an average 35-fold expansion from day 2 to day 1 3 as compared to the 22-fold
expansion
observed in TexMACS cultures (p=0.0048). Metabolite analysis using the Cedex
Bioanalyzer
throughout the culture duration revealed that the relatively fast growth
kinetics obtained using
Prime-XV resulted in increased lactate concentrations in Prime-XV cultures.
This increase in
lactate production due to metabolic remodeling toward anabolic growth and
biomass
accumulation was mediated by ex vivo activation via binding of costimulatory
aCD3/CD28
antibodies. Upon stimulation, T cells rely primarily rely on aerobic
glycolysis in which glucose is
converted into lactate and in turn generates various metabolic intermediates
that are essential for
cell proliferation. Although this glycolytic phenotype is a hallmark of T cell
activation, lactate
accumulation has been shown to significantly inhibit proliferation of T-cells.
In addition to
acidification of the culture medium, lactic acid represses cytokine
production, particularly with
respect to IL-2 and EFN-y expression. Furthermore, production of these
cytokines is entirely
ablated in the presence of 20 mM lactic acid.
As high lactate concentrations are known to negatively impact cell expansion
and
functionality of T cells, it was determined through experimentation that an
additional media
exchange on day6 to minimize lactate build-up and further improve cell
expansion was
beneficial. Indeed, the average cell expansion for this condition was highest
with an average fold
expansion of 47-fold. Differences in cell expansion as compared to TexMACS
were statistically
significant (paired Student's t-test (n=3 runs) p=0.0080 and unpaired
Student's t-test, p=0.0040
(n=5 TexMACS, 11=3 Prime-XV with additional media exchange)) Percent viability
of resulting
NeoTCR Product at end of culture was comparable regardless of expansion medium
used.
Comparison of percent NeoTCR+ expression via dextramer binding between NeoTCR
Cells generated using TexMACS, Prime-XV and Prime-XV with additional media
exchange on
day 6 showed improved gene-editing with an average % NeoTCR+ of 20.2 for
TexMACS, 26.4
for Prime-XV and 26.2 for Prime-XV with day 6 feed. Differences were
statistically significant
for TexMACS and Prime-XV based on paired Student's t-test with a p-value of
0.0004 (n= 5
runs), but not for TexMACS and Prime-XV with day 6 likely as a result of
having a smaller
sample size (n=3 runs) with a p-value of 0.0538 (paired Student's t-test).
Comparison of overall NeoTCR Cell yield showed that the highest average number
of
NeoTCR+ cells was obtained through cultivation in Prime-XV medium with a day 6
feed
yielding an average of 6.2 x 109 NeoTCR+ cells as compared to 4.6 x 109
NeoTCR+ cells using
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Prime-XV without the additional media exchange and 2.3 x 109 NeoTCR+ cells
using
TexMACS. This significant improvement in T cell expansion, particularly of the
NeoTCR+
population, supports the use of Prime-XV to achieve at least 4x109 gene-edited
cells per
manufacturing run. Differences were statistically significant for TexMACS and
Prime-XV
(p=0.0319, paired Student's t-test, n=5 runs), but not for TexMACS and Prime-
XV with day 6
feed (p=0.0839, paired Student's t-test, n=3 runs) likely a result of having a
smaller sample size
Example 8. Process 4 Improvements to Electroporation Efficiencies
Introduction. In order to further improve NeoTCR Cell yield, different
electroporation
devices and conditions were tested. It was determined that a large-scale
cuvette electroporation
device was able to improve NeoTCR Cell yield.
Pulse Protocols. Given that there was no data on gene editing efficiency of
primary T
cells using RNPs and plasmid DNA, multiple electroporation protocols were
tested. Two of the
top protocols tested were as follows: (1) Voltage (V)=2500, Pulse Width
(ms)=3, Pulses=4, and
Energy Density=30000; and (2) Voltage (V)=2500, Pulse Width (ms)=4, #
Pulses=4, and Energy
Density=40000. It was determined that both (1) and (2) described above
increased the total
edited cells and that (1) was the optimal pulse protocol. These protocols
using a large-scale
cuvette-based electroporation device yielded an increased efficiency of gene
editing compared to
the gene-editing efficiencies of Processes 1-4 described above when they used
the Lonza
electroporation units. It was surprising to see intermediate scale runs (runs
smaller than the ones
used to generate NeoTCR Products for infusion into patients) consistently
yielding
approximately 30% gene-edited cells (2500V, 4 pulses, 500ms rest interval)
with 4.24-7.35 x 109
gene-edited cells. Furthermore, the large-scale cuvette-based electroporation
device was shown
to consistently produce 1.3x1e gene-edited cells per electroporation run which
is more than
twice the amount of total edited cells produced using the Lonza unit.
Conclusion. Based on the data generated it was determined that large-scale
cuvette-based
electroporation devices are capable of producing high numbers of gene-edited
primary T cells
that are sufficient for treating patients with NeoTCR Products.
While the present invention has been described at some length and with some
particularity with respect to the several described embodiments, it is not
intended that it should
be limited to any such particulars or embodiments or any particular
embodiment, hut it is to he
construed with references to the appended claims so as to provide the broadest
possible
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interpretation of such claims in view of the prior art and, therefore, to
effectively encompass the
intended scope of the invention.
All publications, patent applications, patents, and other references mentioned
herein are
incorporated by reference in their entirety, In case of conflict, the present
specification,
including definitions, will control. In addition, section headings, the
materials, methods, and
examples are illustrative only and not intended to be limiting.
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