NZ793724A - Restimulation of cryopreserved tumor infiltrating lymphocytes - Google Patents
Restimulation of cryopreserved tumor infiltrating lymphocytesInfo
- Publication number
- NZ793724A NZ793724A NZ793724A NZ79372417A NZ793724A NZ 793724 A NZ793724 A NZ 793724A NZ 793724 A NZ793724 A NZ 793724A NZ 79372417 A NZ79372417 A NZ 79372417A NZ 793724 A NZ793724 A NZ 793724A
- Authority
- NZ
- New Zealand
- Prior art keywords
- tils
- population
- cells
- days
- expansion
- Prior art date
Links
- 210000003171 Lymphocytes, Tumor-Infiltrating Anatomy 0.000 title claims 98
- 240000003670 Sesamum indicum Species 0.000 claims abstract 8
- 238000001802 infusion Methods 0.000 claims abstract 5
- 230000002503 metabolic Effects 0.000 claims abstract 3
- 210000004027 cells Anatomy 0.000 claims 17
- 230000001225 therapeutic Effects 0.000 claims 14
- 206010028980 Neoplasm Diseases 0.000 claims 13
- 210000000612 Antigen-Presenting Cells Anatomy 0.000 claims 11
- 108010002350 Interleukin-2 Proteins 0.000 claims 11
- 239000002609 media Substances 0.000 claims 8
- 239000006143 cell culture media Substances 0.000 claims 7
- 201000011510 cancer Diseases 0.000 claims 5
- 230000002255 enzymatic Effects 0.000 claims 5
- 230000001502 supplementation Effects 0.000 claims 5
- 239000003814 drug Substances 0.000 claims 3
- 210000003162 effector T lymphocyte Anatomy 0.000 claims 3
- 150000002500 ions Chemical class 0.000 claims 3
- 238000004519 manufacturing process Methods 0.000 claims 3
- 210000003071 memory T lymphocyte Anatomy 0.000 claims 3
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 claims 2
- 102000020504 Collagenase family Human genes 0.000 claims 2
- 108060005980 Collagenase family Proteins 0.000 claims 2
- 102000016911 Deoxyribonucleases Human genes 0.000 claims 2
- 108010053770 Deoxyribonucleases Proteins 0.000 claims 2
- 108009000344 Head and Neck Squamous Cell Carcinoma Proteins 0.000 claims 2
- 230000036091 Metabolic activity Effects 0.000 claims 2
- 208000002154 Non-Small-Cell Lung Carcinoma Diseases 0.000 claims 2
- 108009000071 Non-small cell lung cancer Proteins 0.000 claims 2
- 208000000102 Squamous Cell Carcinoma of Head and Neck Diseases 0.000 claims 2
- 229960002424 collagenase Drugs 0.000 claims 2
- 238000005138 cryopreservation Methods 0.000 claims 2
- 238000003306 harvesting Methods 0.000 claims 2
- 201000000459 head and neck squamous cell carcinoma Diseases 0.000 claims 2
- 230000001400 myeloablative Effects 0.000 claims 2
- 150000007523 nucleic acids Chemical class 0.000 claims 2
- 108020004707 nucleic acids Proteins 0.000 claims 2
- 230000002463 transducing Effects 0.000 claims 2
- 102100018910 B3GAT1 Human genes 0.000 claims 1
- 101710027071 B3GAT1 Proteins 0.000 claims 1
- 206010005003 Bladder cancer Diseases 0.000 claims 1
- 210000004369 Blood Anatomy 0.000 claims 1
- 206010006187 Breast cancer Diseases 0.000 claims 1
- 102100019459 CD27 Human genes 0.000 claims 1
- 101700056583 CD27 Proteins 0.000 claims 1
- 101700033362 CD28 Proteins 0.000 claims 1
- 102100019461 CD28 Human genes 0.000 claims 1
- 206010008342 Cervix carcinoma Diseases 0.000 claims 1
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 claims 1
- 229960004397 Cyclophosphamide Drugs 0.000 claims 1
- 241000701806 Human papillomavirus Species 0.000 claims 1
- 210000004072 Lung Anatomy 0.000 claims 1
- 108060004664 MDFI Proteins 0.000 claims 1
- 206010025650 Malignant melanoma Diseases 0.000 claims 1
- 206010027480 Metastatic malignant melanoma Diseases 0.000 claims 1
- 101700077124 NCAM1 Proteins 0.000 claims 1
- 102100007544 NCAM1 Human genes 0.000 claims 1
- 206010033128 Ovarian cancer Diseases 0.000 claims 1
- 208000006265 Renal Cell Carcinoma Diseases 0.000 claims 1
- 206010038389 Renal cancer Diseases 0.000 claims 1
- 108091008153 T cell receptors Proteins 0.000 claims 1
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 claims 1
- 210000001744 T-Lymphocytes Anatomy 0.000 claims 1
- 210000003411 Telomere Anatomy 0.000 claims 1
- 229940035295 Ting Drugs 0.000 claims 1
- 102000004965 antibodies Human genes 0.000 claims 1
- 108090001123 antibodies Proteins 0.000 claims 1
- 239000008280 blood Substances 0.000 claims 1
- 238000004113 cell culture Methods 0.000 claims 1
- 201000010881 cervical cancer Diseases 0.000 claims 1
- 230000003247 decreasing Effects 0.000 claims 1
- 229960000390 fludarabine Drugs 0.000 claims 1
- GIUYCYHIANZCFB-FJFJXFQQSA-N fludarabine phosphate Chemical compound C1=NC=2C(N)=NC(F)=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@@H]1O GIUYCYHIANZCFB-FJFJXFQQSA-N 0.000 claims 1
- 230000034659 glycolysis Effects 0.000 claims 1
- 201000010536 head and neck cancer Diseases 0.000 claims 1
- 238000001990 intravenous administration Methods 0.000 claims 1
- 201000010982 kidney cancer Diseases 0.000 claims 1
- 201000001441 melanoma Diseases 0.000 claims 1
- 238000000034 method Methods 0.000 claims 1
- 230000035812 respiration Effects 0.000 claims 1
- 230000000241 respiratory Effects 0.000 claims 1
- 230000029058 respiratory gaseous exchange Effects 0.000 claims 1
- 230000011664 signaling Effects 0.000 claims 1
- 229920000511 telomere Polymers 0.000 claims 1
- 201000005112 urinary bladder cancer Diseases 0.000 claims 1
- 230000000638 stimulation Effects 0.000 abstract 1
Abstract
The present disclosure provides methods for re-stimulating TIL populations that lead to improved phenotype and increased metabolic health of the TILs and provides methods of assaying for TIL populations to determine suitability for more efficacious infusion after re- stimulation.
Description
The present disclosure provides methods for re-stimulating TIL populations that lead to improved
phenotype and increased metabolic health of the TILs and provides methods of assaying for TIL
tions to determine suitability for more efficacious infusion after re- stimulation.
NZ 793724
RESTIMULATION OF CRYOPRESERVED TUMOR INFILTRATING
LYMPHOCYTES
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to U. S. Provisional Patent Application Nos.
62/413,283 and 62/413,387, filed October 26, 2016, entitled “Expansion of Tumor-
Infiltrating Lymphocytes and Methods of Using the Same,” and US. Provisional Patent
Application No. 62/415,452, filed r 31, 2016, entitled MULATION OF
ESERVED TUMOR INFILTRATING LYMPHOCYTES,” which are hereby
incorporated by reference in their entirety.
OUND OF THE INVENTION
ent of bulky, refractory cancers using adoptive transfer of tumor infiltrating
lymphocytes (TILs) represents a powerful approach to therapy for patients with poor
prognoses. Gattinoni, er al., Nat. Rev. Immunol. 2006, 6, 383-393. A large number of TILs
are required for successful immunotherapy, and a robust and reliable process is needed for
commercialization. This has been a challenge to achieve because of technical, ical, and
regulatory issues with cell expansion. ILbased TIL expansion followed by a “rapid
expansion process” (REP) has become a preferred method for TIL expansion because of its
Speed and efficiency. Dudley, er al., Science 2002, 298, 850-54, Dudley, et 61]., J. Clin.
Oncol. 2005, 23, 2346-57, Dudley, et 51]., J. Clin. Oncol. 2008, 26, 5233-39, l, er al.,
Science 1992, 257, 238-41; Dudley, et al., J. Immunolher. 2003, 26, 332-42. REP can result
in a 1,000-fold expansion of TILs over a 14-day period, gh it requires a large excess
(e. g., ld) of irradiated allogeneic peripheral blood mononuclear cells (PBMCs), often
from multiple donors, as feeder cells, as well as anti-CD3 antibody (OKT3) and high doses of
IL-2. Dudley, et 61]., J. Immunother. 2003, 26, 332-42.
TILs that have undergone an REP procedure have produced successful adoptive cell
therapy ing host immunosuppression in patients with melanoma. Current infusion
acceptance ters rely on readouts of the composition of TILs (e.g., CD28, CD8, or CD4
positivity) and on fold ion and viability of the REP product.
However, current REP protocols, as well as current TIL expansion protocols
generally, give little insight into the health of the TIL that will be infused into the patient. T
cells undergo a profound metabolic shift during the course of their maturation from naive to
effector T cells (see Chang, el al, Nat. Immunol. 2016, I 7, 364, hereby expressly
incorporated in its entirety, and in particular for the discussion and markers of anaerobic and
aerobic lism). For example, naive T cells rely on mitochondrial respiration to produce
ATP, while mature, y effector T cells such as TIL are highly glycolytic, relying on
aerobic ysis to provide the bioenergetics substrates they require for proliferation,
migration, activation, and anti-tumor efficacy.
In addition, these expanded cell populations can be cryopreserved, leading to ease
of use, long-term storage for multiple reinfusions into patients with recurrent disease, and
other considerations. However, current infusion acceptance parameters rely on readouts of
the composition of TILs and on fold-expansion and viability of the expanded TIL based
product, These measures give little insight into the health of the TIL that will be infused into
the patient, and little is known about the s of cryopreservation on TIL populations.
Accordingly, the present invention is directed to methods for expanding and re-
stimulating TIL populations that lead to improved phenotype and increased lic health
of the TILs and towards methods of assaying for TIL populations to determine suitability for
more efficacious infusion after re-stimulation.
BRIEF Y OF THE INVENTION
The present invention es s for expanding TILs in , sometimes
therapeutic, populations in combination with optional eservation.
According to the present disclosure, a method for expanding tumor rating
lymphocytes (TILs) into a therapeutic population of TILs comprising the following steps is
provided:
(i) obtaining a first population of TILs from a tumor resected from a patient;
(ii) performing a first expansion by culturing the first population of TILs in a cell
culture medium comprising IL—2 to produce a second population of TILs, and
(iii) performing a second expansion by supplementing the cell culture medium of the
second population of TILs with additional IL-2, OKT-3, and antigen presenting cells
(APCs), to produce a third population of TILs, wherein the third population of TILs is
at least d or lOO-fold greater in number than the second population of TILs, and
wherein the second expansion is performed for at least 14 days in order to obtain the
third population of TILs, wherein the third population of TILs is a therapeutic
WO 81473
population of TlLs which comprises an increased subpopulation of effector T cells
and/or central memory T cells relative to the second population of TILs.
In some embodiments, the method further comprises:
(iv) performing an additional second expansion by supplementing the cell e medium
of the third population of TILs with additional IL-2, additional OKT-3, and additional
APCs, wherein the additional second expansion is performed for at least 14 days to obtain
a larger therapeutic population of TILs than ed in step (iii), wherein the larger
therapeutic population of TILs comprises an increased subpopulation of effector T cells
and/or central memory T cells ve to the third population of TILs.
In some embodiments, after step (iii), the cells are removed from the cell culture
and cryopreserved in a storage medium prior to performing step (iv).
In some embodiments, the cells are thawed prior to performing step (iv).
In some embodiments, step (iv) is ed one to four times in order to obtain
sufficient TlLs in the therapeutic population of TILs for a eutically effective dosage of
the TILs.
In some embodiments, steps (i) through (iii) or (iv) are performed within a period of
about 40 days to about 50 days. In some embodiments, steps (i) through (iii) or (iv) are
performed within a period of about 42 days to about 48 days. In some ments, steps (i)
through (iii) or (iv) are performed within a period of about 42 days to about 45 days. In some
ments, steps (i) through (iii) or (iv) are med within about 44 days.
In some embodiments, the cells from steps (iii) or (iv) express CD4, CD8, and TCR
0t [3 at levels similar to freshly harvested cells.
In some embodiments, the antigen presenting cells are peripheral blood
mononuclear cells (PBMCs). In some embodiments, the PBMCs are added to the cell culture
on any of days 9 through 17 in step (iii).
In some embodiments, the effector T cells and/or central memory T cells in the
eutic population of TILs in step (iv) exhibit one or more characteristics selected from
the group consisting of expression of CD27, expression of CD28, longer telomeres, increased
CD57 expression, and decreased CD56 expression, relative to effector T cells and/or central
memory T cells in the third population of cells.
WO 81473
In some embodiments, the effector T cells and/or central memory T cells exhibit
increased CD57 expression and decreased CD56 expression.
In some embodiments, the APCs are artificial APCs (aAPCs).
In some embodiments, the method further comprises the step of transducing the first
population of TILs with an expression vector sing a nucleic acid encoding a high-
affinity T cell receptor.
In some embodiments, the method further ses the step of transducing the first
population of TILs with an expression vector comprising a nucleic acid encoding a chimeric
antigen receptor (CAR) sing a single chain variable fragment dy fused with at
least one endodomain of a T-cell signaling molecule.
In some embodiments, the eutic population of TILs are infused into a patient.
In some embodiments, step (iii) further comprises a step of removing the cells from
the cell culture medium.
In some ments, step (iii) is repeated one to four times in order to obtain
sufficient TILs in the therapeutic population of TILs for a therapeutically effective dosage of
the TILs.
In some embodiments, the number of TILs sufficient for a therapeutically effective
dosage is from about 2.3X1010 to about 13.7X1010.
The present disclosure also provides a population of expanded TILs made according
to the method of claim 1.
The present disclosure also provides a population of expanded TILs made according
to the method of claim 1, n the expanded TILs have at least a two—fold increase in
basal glycolysis as compared to thawed cryopreserved TILs.
The present disclosure also provides methods for assessing the metabolic activity of
a TIL cell tion made according to the methods described herein, comprising measuring
the basal glycolysis of the cells.
The present disclosure also provides methods for assessing the metabolic activity of
a TIL cell population made according to the methods described herein, comprising ing
the basal respiration of the cells.
The present disclosure also provides methods for assessing the metabolic ty of
a TIL cell population made according to the methods described herein, sing measuring
the spare respiratory capacity (SRC) of the cells.
The present disclosure also provides methods for assessing the metabolic activity of
a TIL cell tion made according to the methods bed herein, comprising measuring
the glycolytic reserve of the cells.
The present disclosure also provides a method for ing tumor infiltrating
lymphocytes (TILs) into a therapeutic population of TILs comprising:
(i) performing a first expansion by culturing a first population of TILs from a tumor
resected from a patient in a cell culture medium comprising IL-2 to obtain a second
population of TILs, and
(ii) performing a second expansion by supplementing the cell culture medium of the
second population of TILs with additional IL-2, OKT-3, and antigen presenting cells
(APCs) to obtain a third population of TILs, n the third population of TILs is at
least 50-fold or lOO-fold greater in number than the second population of TILs, and
wherein the second expansion is performed for at least 14 days in order to obtain the
third population of TILs, n the third population of TILs is a eutic
population of TILs which comprises an increased subpopulation of effector T cells
and/or central memory T cells relative to the second population of TILs.
In some embodiments, the method further comprises:
(iii) performing an additional second expansion of the third population of TILs by
supplementing the cell culture medium of the third population of TILs with additional
IL-2, onal OKT-3, and additional APCs, wherein the additional second
expansion is performed for at least 14 days to obtain a larger therapeutic population of
TILs than obtained in step (ii), wherein the larger therapeutic population of TILs
exhibits an increased subpopulation of effector T cells and/or central memory T cells
ve to the third tion of TILs.
In some embodiments, the cells from the cell culture medium in step (ii) are
removed and cryopreserved in a storage medium prior to step (iii).
In some embodiments, the cells are thawed prior to step (iii).
WO 81473
In some embodiments, step (ii) is repeated one to four times in order to obtain
sufficient TILs in the therapeutic tion of TILs for a therapeutically ive dosage of
the TILs.
In some embodiments, the number of TILs sufficient for a therapeutically effective
dosage is from about 2.3 >< 1010 to about l3.7><1010.
In some embodiments, the APCs are peripheral blood mononuclear cells (PBMCs).
In some embodiments, the effector T cells and/or central memory T cells exhibit
one or more characteristics selected from the group consisting of expression of CD27,
expression of CD28, longer telomeres, increased CD57 expression, and decreased CD56
expression, relative to effector T cells and/or central memory T cells in the third population
of cells.
In some embodiments, the effector T cells and/or l memory T cells exhibit
increased CD57 expression and decreased CD56 expression.
The t disclosure also provides a method for treating a subject with cancer
comprising administering expanded tumor infiltrating lymphocytes (TILs) comprising:
(i) obtaining a first population of TlLs from a tumor ed from a patient,
(ii) performing a first expansion by culturing the first population of TILs in a cell
culture medium comprising IL—2 to produce a second population of TILs,
(iii) performing a second expansion by supplementing the cell culture medium of the
second population of TILs with additional IL-2, OKT-3, and antigen presenting cells
(APCs), to produce a third population of TILs, wherein the third population of TILs is
at least 50-fold or lOO-fold greater in number than the second tion of TILs, and
wherein the second ion is performed for at least 14 days in order to obtain the
third population of TILs, wherein the third population of TILs is a therapeutic
population of TILs which ses an sed subpopulation of effector T cells
and/or central memory T cells relative to the second population of TILs, and
(iv) administering a therapeutically effective dosage of the third tion of TILs to
the patient.
In some embodiments, the method further comprises prior to step (iv) a step of
performing an additional second expansion by supplementing the cell culture medium of the
third population of TILs with additional IL—2, additional OKT—3, and additional APCs,
n the additional second expansion is performed for at least 14 days to obtain a larger
therapeutic population of TILs than obtained in step (iii), wherein the larger therapeutic
population of TILs comprises an increased subpopulation of effector T cells and/or central
memory T cells relative to the third population of TILs.
In some embodiments, after step (ii) the cells are removed from the cell culture
medium and cryopreserved in a storage medium prior to the onal second expansion
according to the methods described herein.
In some embodiments, the cells are thawed prior to the additional second expansion
of according to the methods described herein.
In some embodiments, step (iii) is repeated one to four times in order to obtain
sufficient TILs in the eutic population of TILs for a therapeutically effective dosage of
the TILs.
In some embodiments, the number of TILs sufficient for a therapeutically ive
dosage is from about 2.3 X 1010 to about 13.7X1010.
In some ments, the APCs are peripheral blood mononuclear cells (PBMCs).
In some embodiments, the effector T cells and/or l memory T cells exhibit
one or more characteristics selected from the group consisting of expression of CD27,
expression of CD28, longer telomeres, increased CD57 expression, and sed CD56
expression, relative to effector T cells and/or central memory T cells in the third population
of cells.
In some embodiments, the effector T cells and/or central memory T cells exhibit
increased CD57 expression and decreased CD56 expression.
In some embodiments, the cancer is selected from the group ting of
melanoma, cervical cancer, head and neck cancer, glioblastoma, ovaiian cancer, sarcoma,
pancreatic cancer, bladder cancer, breast cancer, triple negative breast cancer, and non-small
cell lung carcinoma.
The present disclosure also provides a method for treating a t with cancer
comprising administering expanded tumor ating lymphocytes (TILs) sing:
(i) performing a first expansion by culturing a first population of TILs from a tumor
resected from a patient in a cell culture medium comprising IL-2 to obtain a second
population of TILs,
(ii) performing a second expansion by menting the cell culture medium of the
second population of TILs with additional IL-2, OKT-3, and antigen presenting cells
(APCS) to obtain a third population of TILs, wherein the third population of TILs is at
least 50-fold or lOO-fold greater in number than the second tion of TILs, and
wherein the second expansion is performed for at least 14 days in order to obtain the
third population of TILs, wherein the third tion of TILs is a therapeutic
population of TILs which comprises an sed subpopulation of effector T cells
and/or central memory T cells relative to the second population of TILs; and
(iii) administering a therapeutically effective dosage of the therapeutic population of
TILs to the patient.
In some embodiments, the method further comprises prior to step (iii) a step of
performing an additional second expansion by supplementing the cell culture medium of the
third population of TILs with additional IL-2, additional OKT-3, and additional APCs,
wherein the onal second expansion is performed for at least 14 days to obtain a larger
therapeutic population of TILs than ed in step (ii), wherein the larger therapeutic
tion of TILs comprises an increased subpopulation of effector T cells and/or central
memory T cells relative to the third population of TILs.
In some embodiments, the cells from the cell culture medium in step (ii) are
removed and cryopreserved in a storage medium prior to the additional second expansion as
described .
In some embodiments, the cells are thawed prior to the additional second expansion
as described herein.
In some embodiments, step (ii) is ed one to four times in order to obtain
sufficient TILs in the therapeutic population of TILs for a eutically effective dosage of
the TILs.
In some embodiments, the number of TILs sufficient for a therapeutically effective
dosage is from about 2.3 X 1010 to about 13.7X1010.
In some embodiments, the APCS are peripheral blood mononuclear cells ).
In some embodiments, the effector T cells and/or central memory T cells exhibit
one or more characteristics selected from the group consisting of expression of CD27,
expression of CD28, longer telomeres, increased CD57 expression, and decreased CD56
expression, relative to effector T cells and/or central memory T cells in the third population
of cells.
In some embodiments, the effector T cells and/or central memory T cells exhibit
increased CD57 sion and sed CD56 expression.
In some embodiments, the cancer is selected from the group consisting of
melanoma, cervical cancer, head and neck cancer, glioblastoma, ovarian cancer, sarcoma,
pancreatic cancer, bladder cancer, breast cancer, triple negative breast cancer, and non-small
cell lung carcinoma.
The present invention also provides assay methods for determining TIL viability.
The present disclosure provides methods for assaying TILs for ity by expanding tumor
infiltrating lymphocytes (TILs) into a larger population of TILs comprising:
(i) obtaining a first population of TILs which has been previously expanded;
(ii) ming a first expansion by culturing the first population of TILs in a cell
e medium comprising IL-2 to produce a second population of TILs; and
(iii) performing a second expansion by supplementing the cell culture medium of the
second population of TILs with additional IL—2, OKT—3, and n presenting cells
(APCs), to produce a third population of TILs, wherein the third population of TILs is
at least 50-fold or lOO-fold greater in number than the second population of TILs, and
wherein the second expansion is performed for at least 14 days in order to obtain the
third population of TILs, wherein the third population of TILs comprises an increased
subpopulation of effector T cells and/or central memory T cells relative to the second
population of TILs, and wherein the third population is further assayed for ity.
In some embodiments, the method further ses:
(iv) performing an additional second expansion by supplementing the cell culture
medium of the third population of TILs with additional IL-2, onal OKT-3, and
additional APCs, wherein the additional second expansion is performed for at least 14
days to obtain a larger population of TILs than obtained in step (iii), wherein the
larger tion of TILs comprises an increased subpopulation of effector T cells
and/or central memory T cells relative to the third population of TILs, and n
the third population is further assayed for ity.
In some embodiments, prior to step (i), the cells are cryopreserved.
In some embodiments, the cells are thawed prior to performing step (i).
In some ments, step (iv) is ed one to four times in order to obtain
sufficient TILs for analysis.
In some embodiments, steps (i) through (iii) or (iv) are performed within a period of
about 40 days to about 50 days.
In some embodiments, steps (i) h (iii) or (iv) are performed within a period of
about 42 days to about 48 days.
In some embodiments, steps (i) through (iii) or (iv) are performed within a period of
about 42 days to about 45 days.
In some embodiments, steps (i) through (iii) or (iv) are performed within about 44
days.
In some embodiments, the cells from steps (iii) or (iv) express CD4, CD8, and TCR
0t B at levels similar to freshly harvested cells.
In some ments, the antigen presenting cells are peripheral blood
mononuclear cells (PBMCs).
In some embodiments, the PBMCs are added to the cell culture on any of days 9
through 17 in step (iii).
In some embodiments, the effector T cells and/or central memory T cells in the
larger population of TILs in step (iv) exhibit one or more characteristics selected from the
group consisting of expression of CD27, expression of CD28, longer telomeres, increased
CD57 expression, and decreased CD56 expression, ve to effector T cells, and/or central
memory T cells in the third population of cells.
In some embodiments, the effector T cells and/or central memory T cells exhibit
increased CD57 expression and decreased CD56 expression.
In some embodiments, the APCs are artificial APCs (aAPCs).
In some embodiments, the method further ses the step of transducing the first
population of TILs with an expression vector comprising a nucleic acid encoding a finity
T cell receptor.
In some embodiments, the step of transducing occurs before step (i).
In some embodiments, the method further comprises the step of ucing the first
tion of TILs with an expression vector comprising a nucleic acid encoding a chimeric
antigen receptor (CAR) comprising a single chain variable fragment antibody fused with at
least one endodomain of a T-cell signaling molecule.
In some ments, the step of transducing occurs before step (i).
In some embodiments, the TILs are d for viability.
In some embodiments, the TILs are d for viability after cryopreservation.
In some embodiments, the TILs are assayed for viability after cryopreservation and
after step (iv).
According to the present disclosure, a method for assaying TILs for ity and/or
r use in administration to a subject. In some embodiments, the method for assay tumor
ratitng lymphocytes (TILs) comprises:
(i) obtaining a first population of TILs;
(ii) ming a first expansion by culturing the first population of TILs in a cell
culture medium comprising IL—2 to produce a second population of TILs, and
(iii) performing a second expansion by supplementing the cell culture medium of the
second population of TILs with additional IL-2, OKT-3, and antigen presenting cells
(APCs), to produce a third population of TILs, wherein the third population of TILs is
at least 50-fold greater in number than the second population of TILs,
(iv) harvesting, washing, and cryopreserving the third population of TILs;
(v) storing the eserved TILs at a cryogenic temperature,
(vi) thawing the third population of TILs to provide a thawed third population of
TILs, and
(vii) performing an additional second expansion of a portion of the thawed third
population of TILs by supplementing the cell culture medium of the third population
with IL-2, OKT-3, and APCs for a reREP period of at least 3 days, wherein the third
expansion is performed to obtain a fourth population of TILs, wherein the number of
TILs in the fourth population of TILs is compared to the number of TILs in the third
population of TILs to obtain a ratio;
(viii) determining based on the ratio in step (vii) whether the thawed population of
TILs is suitable for administration to a patient;
(ix) administering a therapeutically effective dosage of the thawed third tion of
TILs to the patient when the ratio of the number of TILs in the fourth population of
TILs to the number of TILs in the third tion of TILs is determined to be r
than 5:1 in step (viii).
In some embodiments, the reREP period is performed until the ratio of the number
of TILs in the fourth population of TILs to the number of TILs in the third population of TILs
is greater than 50:1.
In some embodiments, the number of TILs sufficient for a therapeutically effective
dosage is from about 2.3X1010 to about l3.7><1010.
In some embodiments, steps (i) through (vii) are performed within a period of about
40 days to about 50 days. In some embodiments, steps (i) through (vii) are performed within
a period of about 42 days to about 48 days. In some embodiments, steps (i) through (vii) are
performed within a period of about 42 days to about 45 days. In some embodiments, steps (i)
through (vii) are performed within about 44 days.
In some embodiments, the cells from steps (iii) or (vii) express CD4, CD8, and TCR
0t B at levels similar to freshly harvested cells. In some embodiments the cells are TILs.
In some embodiments, the antigen presenting cells are peripheral blood
mononuclear cells (PBMCs). In some embodiments, the PBMCs are added to the cell culture
on any of days 9 h 17 in step (iii).
In some embodiments, the effector T cells and/or l memory T cells in the
larger population of TILs in steps (iii) or (vii) exhibit one or more characteristics selected
from the group consisting of expression of CD27, expression of CD28, longer telomeres,
increased CD57 expression, and decreased CD56 expression, relative to effector T cells,
and/or central memory T cells in the third tion of cells.
In some embodiments, the effector T cells and/or central memory T cells exhibit
increased CD57 expression and sed CD56 expression.
In some embodiments, the APCs are artificial APCs (aAPCs).
In some embodiments, the step of transducing the first population of TILs with an
sion vector comprising a c acid encoding a high-affinity T cell receptor.
In some embodiments, the step of transducing occurs before step (i).
In some embodiments, the step of transducing the first population of TILs with an
expression vector comprising a nucleic acid encoding a chimeric antigen or (CAR)
comprising a single chain variable fragment antibody fused with at least one endodomain of a
T-cell signaling molecule.
In some embodiments, the step of transducing occurs before step (i).
In some ments, the TILs are assayed for viability after step (vii).
The present disclosure also provides further methods for assaying TILs. In some
embodiments, the disclosure provides a method for assaying TILs comprising:
(i) obtaining a portion of a first population of cryopreserved TILs,
(ii) thawing the portion of the first population of cryopreserved TILs,
(iii) performing a first expansion by culturing the portion of the first population of
TILs in a cell e medium comprising lL-2, OKT-3, and antigen presenting cells
(APCs) for a reREP period of at least 3 days, to e a second population of TILs,
wherein the n from the first population of TILs is compared to the second
population of TILs to obtain a ratio of the number of TILs, wherein the ratio of the
number of TILs in the second population of TILs to the number of TILs in the portion
of the first population of TILs is greater than 5: 1,
(iv) determining based on the ratio in step (iii) whether the first population of TILs is
suitable for use in therapeutic administration to a patient,
(v) determining the first population of TILs is suitable for use in therapeutic
administration when the ratio of the number of TILs in the second population of TILs
to the number of TILs in the first population of TILs is determined to be greater than
:1 in step (iv).
In some ments, the ratio of the number of TILs in the second population of
TILs to the number of TILs in the portion of the first tion of TILs is greater than 50:1.
In some embodiments, the method further ses performing expansion of the
entire first population of cryopreserved TILs from step (i) according to the methods as
described in any of the ments provided herein.
In some embodiments, the method further comprises administering the entire first
population of cryopreserved TILs from step (i) to the patient.
The present disclosure also provides further methods for assaying TILs. In some
embodiments, the disclosure provides a method for assaying TILs comprising:
(i) obtaining a n of a first population of cryopreserved TILs;
(ii) thawing the portion of the first population of cryopreserved TILs;
(iii) performing a first ion by culturing the portion of the first population of
TILs in a cell culture medium comprising 1L-2, OKT-3, and antigen presenting cells
(APCs) for a reREP period of at least 3 days, to produce a second population of TILs,
wherein the portion from the first population of TILs is compared to the second
population of TILs to obtain a ratio of the number of TILs, wherein the ratio of the
number of TILs in the second population of TILs to the number of TILs in the portion
of the first tion of TILs is r than 5:1,
(iv) determining based on the ratio in step (iii) r the first population of TILs is
suitable for use in therapeutic administration to a patient; and
(v) therapeutically administering the remainder of the first tion of TILs to the
patient when the ratio of the number of TILs in the second population of TILs to the
number of TILs in the first population of TILs is determined to be greater than 5:1 in
step (iv).
In some embodiments, the ratio of the number of TILs in the second population of
TILs to the number of TILs in the portion of the first tion of TILs is greater than 50:1.
In some embodiments, the method further ses performing expansion of the
entire first population of cryopreserved TILs from step (i) according to the methods of any of
the preceding claims.
In some embodiments, the method further comprises administering the entire first
population of cryopreserved TILs from step (i) to the patient.
] In some ments, the method further comprised the step of assessing the
metabolic health of the second population of TILs.
In some embodiments, the method further comprises the step of assessing the
phenotype of the second population of TILs.
In some embodiments, the antigen presenting cells are allogeneic peripherial blood
mononuclear cells.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Shows the results from Example 1. As the Table shows, following the
antigen restimulation rapid expansion protocol (“reREP”), the TILs exhibit a marked
enhancement in their glycolytic ation. SRC = spare respiratory capacity.
Figure 2: Composition of fresh vs. thawed TIL. TIL were stained for TCROLB and
CD56 to define T-cell and NK tions. The data shown are averages of 6 individual
TILs.
Figure 3: Memory phenotype is defined by CD45RA and CCR7 Expression. CD4
and CD8 TIL are mainly Effector Memory (EM) This remains the same in the thawed TIL.
Each point is one sample analyzed. No significant difference is found in a Wilcoxon
matched-pairs signed rank test.
] Figure 4: Pearson’s correlation of CD4, CD8, CD4+CD28+, and CD8+CD28+
frequency between fresh and thawed TIL. Cells were stained with above markers. Each dot
represents one individual with the fresh value on the x axis and the thawed value on the y
axis. The fit line was drawn using linear regression analysis.
Figure 5: Comparable Activation Markers on Fresh and Thawed TILs. No
significant difference in activation status of fresh vs. thawed TIL was found using a
on Matched-Pairs Rank Test. Each point represents one sample analyzed and is shown
as mean +/- SEM.
Figure 6: Maintenance of LAG-3 Staining Following Cryopreservation and Thaw.
A: LAG-3 staining of CD8 TIL. B: % frequency of regulatory les of the CD4 and
CD8 populations on fresh and thawed TIL. M-3+ and CD8+LAG—3+ thawed TIL
have a lower % than fresh TIL. Mann-Whitney statistical test.
Figure 7: Remarkably stable tumor-infiltrating cytes (TIL) for infusion
phenotype ing cryopreservation.
] Figure 8: Scatter plot showing phenotypic characterization of reREP TILs. Q1
shows 19.0% CD45RA+/CCR7'; Q2 shows 0.066% CD45RA+/CCR7+; Q4 shows 80.6%
CD45RA'/CCR7', and Q3 shows 0.36% '/CCR7+,
Figure 9: Diagram and data showing the phenotypic characterization of reREP
TILs, during the first and second expansion phases 0.08% CD45RA+/CCR7', 0.03%
CD45RA+/CCR7+, 73.97% CD45RA'/CCR7'; and 25.91% CD45RA'/CCR7+ at Day 14, after
the first expansion but prior to the second expansion. Proliferation of CM or EM TIL in the
repeat ReREP. Central Memory (CM) TIL and or Memory (EM) TIL were tested for
the proliferation capacity using repeat ReREP. , 1.3 x 106 Post REP TIL were co-
culture with 1.3 x 107 PBMC feeders (CFSE labelled), OKT3 (30 ng/nl) and rhIL-2 (3000
IU/ml), culture was ted for 14 days. On Day 14, central memory TIL and effector
memory TIL were gated for L/D Aqua-/CF SE-/TCROt/B +/CD45RA-/CCR7+ and L/D Aqua-
/CF SE—/TCR0L/B +/CD45RA—/CCR7— population respectively and flow cytometry sorted.
Purity of the cell population was 97%. 1 x 104 flow sorted CM or EM or unsorted TIL were
then cultured 1 x 106 PBMC feeders, OKT3 (30 ng/nl) and IL-2 (3 000 IU/ml) in triplicates
for 7 days. Cell were counted and recorded. Central memory TIL were more proliferative
when compared to Effector memory TIL. We are repeating this ment with more post
REP TIL lines.
Figure 10A and 10B: Phenotypic characterization of TILS during ReREP. Cells
were gated on Aqua-/TCR a/B+/CD4+ or CD8+ to show Central Memory TILs (CD45RA'
CCR7+) or Effector Memory TILs (CD45RA'CCR7') memory phenotype. Student “t” was
used to calculate tical significance. >“p < 0.05, ns non-significant.
Figure 11: Exemplary schematic of the TIL preparation process, sometimes
referred to herein as the 1C process.
] Figure 12: Successful expansion of TILs from non-melanoma tumors. Data shows
the distribution of TIL (CD4+/CD8+) in non-melanoma tumors.
] Figure 13: Non-melanoma TILs expressed CD27 and CD3 8, consistent with young
TILs.
] Figure 14: Activated TILs skew towards effector memory population.
Figure 15: Fresh versus reREP TIL phenotypes.
DETAILED DESCRIPTION OF THE ION
1. Introduction
Adoptive cell y ing TILs cultured ex vivo by the Rapid Expansion
Protocol (REP) has produced successful adoptive cell therapy following host
immunosuppression in patients with ma. Current infusion acceptance parameters rely
on readouts of the composition of TILs (e.g., CD28, CD8, or CD4 positivity) and on the
numerical folds of expansion and viability of the REP product.
t REP protocols give little insight into the health of the TIL that will be
infused into the patient. T cells undergo a nd metabolic shift during the course of their
maturation from naive to effector T cells (see Chang, et al., Nat. Immunol. 2016, 17, 364,
hereby expressly incorporated in its entirety, and in particular for the discussion and s
of anaerobic and aerobic metabolism). For e, naive T cells rely on ondrial
respiration to produce ATP, while , healthy effector T cells such as TIL are highly
glycolytic, relying on aerobic glycolysis to provide the bioenergetics substrates they require
for proliferation, migration, activation, and anti-tumor efficacy.
Previous papers report that limiting glycolysis and promoting mitochondrial
metabolism in TlLs prior to transfer is desirable as cells that are relying heavily on glycolysis
will suffer nutrient deprivation upon adoptive transfer which results in a majority of the
transferred cells dying. Thus, the art teaches that ing mitochondrial metabolism might
promote in vivo longevity and in fact suggests using inhibitors of glycolysis before ion
of the immune response. See Chang et a1. (Chang, et al., Nat. l. 2016, 17(3 64), 574-
The present invention is directed in preferred aspects to novel methods of
augmenting REPS with an additional restimulation protocol, sometimes referred to herein as a
“restimulation Rapid Expansion Protocol” or “reRE ”, which leads surprisingly to expanded
memory T cell subsets, including the central memory (CD45RA'CCR7+) or effector memory
(CD45RA'CCR7') ypes, and/or to marked enhancement in the glycolytic respiration as
compared to freshly harvested TILs or thawed cryopreserved TlLs for the restimulated TILs
(sometimes referred to herein as “reTILs”). That is, by using a reREP procedure (126., a
procedure comprising a first expansion and a second expansion) on cryopreserved TILs,
patients can e highly metabolically active, healthy TILs, leading to more favorable
outcomes.
The t invention is further directed in some embodiments to methods for
ting and quantifying this increase in metabolic health. Thus, the present invention
provides s of assaying the relative health of a TIL population using one or more
general evaluations of metabolism, including, but not limited to, rates and amounts of
glycolysis, oxidative phosphorylation, spare respiratory capacity (SRC) and glycolytic
reserve.
Furthermore, the present invention is further directed in some embodiments to
methods for evaluating and quantifying this increase in lic health. Thus, the present
invention provides methods of assaying the relative health of a TIL population using one or
more general evaluations of metabolism, including, but not limited to, rates and amounts of
glycolysis, ive phosphorylation, spare atory capacity (SRC), and glycolytic
reserve.
In addition, optional additional evaluations include, but are not limited to, ATP
production, mitochondrial mass and glucose uptake.
In some cases, the reREP cell population with increased metabolic health are
infused into a patient as is generally known in the art.
II. Definitions
] By “tumor infiltrating lymphocytes” or “TILs” herein is meant a population of cells
ally obtained as white blood cells that have left the bloodstream of a subject and
migrated into a tumor. TILs include, but are not limited to, CD8+ cytotoxic T cells
(lymphocytes), Th1 and Thl7 CD4+ T cells, natural killer cells, dendritic cells and M1
hages. TILs e both primary and secondary TILs. “Primary TILs” are those that
are obtained from patient tissue samples as outlined herein (sometimes referred to as “freshly
harvested”), and “secondary TILs” are any TIL cell populations that have been expanded or
proliferated as discussed , including, but not limited to bulk TILs, expanded TILs
(“REP TILs”) as well as “reREP TILs” as discussed herein.
] TILs can generally be defined either biochemically, using cell surface markers, or
functionally, by their ability to rate tumors and effect treatment. TILs can be generally
categorized by expressing one or more of the following biomarkers: CD4, CD8, TCR dB,
CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD—l, and CD25. Additionally, and
alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon
reintroduction into a patient. TILS may further be characterized by potency — for example,
TILS may be considered potent if, for e, interferon (IFN) release is greater than about
50 pg/mL, greater than about 100 pg/mL, greater than about 150 pg/mL, or greater than about
200 pg/mL. Interferon can include interferon gamma (IFNy).
By “cryopreserved TILs” herein is meant that TILs, either primary, bulk, or
expanded (REP TILs), are treated and stored in the range of about -lSO°C to -6OOC. l
methods for cryopreservation are also described elsewhere herein, ing in the Examples.
For clarity, “cryopreserved TILs” are distinguishable from frozen tissue samples which may
be used as a source of primary TILs.
By “thawed cryopreserved TILs” herein is meant a population of TILs that was
previously cryopreserved and then d to return to room temperature or , including
but not limited to cell culture temperatures or temperatures wherein TILs may be
administered to a patient.
By “population of cells” (including TILs) herein is meant a number of cells that
share common . In general, populations generally range from 1 X 106 to 1 X 1010 in
number, with different TIL populations comprising different s. For example, l
growth of primary TILs in the presence of IL-2 results in a population of bulk TILS of
roughly 1 X 108 cells. REP expansion is generally done to provide populations of 1.5 X 109 to
1.5 X 1010 cells for infusion.
In general, TILs are initially obtained from a patient tumor sample (“primary TILs”)
and then expanded into a larger population for further manipulation as described herein,
ally eserved, restimulated as outlined herein and optionally evaluated for
ype and metabolic parameters as an indication of TIL health.
In general, the harvested cell sion is called a “primary cell population” or a
“freshly harvested” cell tion.
In general, as discussed herein, the TILs are initially prepared by ing a
primary population of TILs from a tumor resected from a patient as discussed herein (the
“primary cell population” or “first cell population”). This is followed with an initial bulk
expansion utilizing a culturing of the cells with IL-2, forming a second population of cells
(sometimes referred to herein as the “bulk TIL population” or “second population”).
] The term “cytotoxic lymphocyte” includes cytotoxic T (CTL) cells (including CD8+
cytotoxic T lymphocytes and CD4+ T-helper lymphocytes), natural killer T (NKT) cells and
natural killer (NK) cells. Cytotoxic lymphocytes can include, for example, peripheral blood-
derived OL/BTCR-positive or a/BTCR-positive T cells activated by tumor associated antigens
and/or uced With tumor specific chimeric antigen receptors or T-cell receptors, and
tumor-infiltrating lymphocytes (TILs).
The term “central memory T cell” refers to a subset of T cells that in the human are
CD45RO+ and constitutively express CCR7 (CCR7 hi) and CD62L (CD62 hi). The surface
phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-lSR.
Transcription s for central memory T cells include BCL-6, BCL-6B, MBD2, and BMII.
Central memory T cells primarily secret IL-2 and CD4OL as effector molecules after TCR
triggering. l memory T cells are predominant in the CD4 compartment in blood, and in
the human are proportionally enriched in lymph nodes and tonsils.
The term tor memory T cell” refers to a subset of human or mammalian T
cells that, like central memory T cells, are CD45RO+, but have lost the constitutive
expression of CCR7 (CCR7lo) and are heterogeneous or low for CD62L expression
(CD62Llo). The surface phenotype of central memory T cells also includes TCR, CD3,
CD127 (IL—7R), and IL—lSR. Transcription factors for central memory T cells include
BLIMPl. Effector memory T cells y secret high levels of inflammatory cytokines
following antigenic ation, ing interferon-y, IL-4, and lL-S. Effector memory T
cells are predominant in the CD8 compartment in blood, and in the human are proportionally
enriched in the lung, liver, and gut. CD8+ effector memory T cells carry large amounts of
perforin. The term “closed system” refers to a system that is closed to the outside
environment. Any closed system appropriate for cell culture methods can be employed with
the methods of the t invention. Closed systems include, for example, but are not
d to closed G—containers. Once a tumor segment is added to the closed system, the
system is no opened to the outside environment until the TILs are ready to be administered to
the patient.
The terms “peripheral blood mononuclear cells” and “PBMCs” refers to a peripheral
blood cell having a round s, including lymphocytes (T cells, B cells, NK cells) and
monocytes. ably, the peripheral blood mononuclear cells are irradiated allogeneic
peripheral blood mononuclear cells.
The term “rapid expansion” means an increase in the number of antigen-specific
TILs of at least about 3-fold (or 4-, 5-, 6-, 7-, 8-, or 9-fold) over a period of a week, more
preferably at least about 10—fold (or 20—, 30—, 40—, 50—, 60—, 70—, 80—, or 90—fold) over a period
of a week, or most preferably at least about lOO-fold over a period of a week. A number of
rapid expansion protocols are described herein.
] In some embodiments, methods of the present disclosure further include a “pre-
REP” stage in which tumor tissue or cells from tumor tissue are grown in standard lab media
(including without limitation RPMI) and treated the with reagents such as irradiated feeder
cells and anti-CD3 antibodies to e a desired effect, such as increase in the number of
TILS and/or an enrichment of the population for cells containing desired cell surface markers
or other structural, biochemical or functional features. The pre-REP stage may utilize lab
grade ts (under the assumption that the lab grade reagents get diluted out during a later
REP stage), making it easier to incorporate alternative strategies for improving TIL
production. Therefore, in some embodiments, the disclosed TLR t and/or peptide or
peptidomimetics can be included in the e medium during the pre-REP stage. The pre-
REP culture can in some embodiments, e IL—2.
The present invention is directed in preferred aspects to novel methods of
augmenting REPs with an additional restimulation protocol, sometimes referred to herein as a
“restimulation Rapid Expansion ol” or “reRE ”, which leads surprisingly to expanded
memory T cell subsets, including the memory effector T cell subset, and/or to marked
enhancement in the glycolytic respiration as compared to freshly harvested TILs or thawed
cryopreserved TILs for the restimulated TILs (sometimes referred to herein as “reTlLs”).
That is, by using a reREP procedure on cryopreserved TILs, patients can e highly
metabolically active, healthy TILs, leading to more favorable outcomes. Such restimulation
protocols, also ed to herein as additional “expansions” of the cell populations, are
described in r detail herein.
The terms “fragmenting,” “fragment,” and “fragmented,” as used herein to describe
ses for disrupting a tumor, es mechanical fragmentation methods such as
crushing, slicing, ng, and morcellating tumor tissue as well as any other method for
disrupting the physical structure of tumor tissue. The term “in vivo” refers to an event that
takes place in a subject's body.
] The term “in vitro” refers to an event that takes places outside of a subject's body. In
vitro assays encompass cell-based assays in which cells alive or dead are employed and may
also encompass a cell-free assay in which no intact cells are employed.
The term “anti-CD3 antibody” refers to an dy or variant f, e. g., a
monoclonal antibody and including human, humanized, chimeric or murine antibodies which
are directed against the CD3 receptor in the T cell n receptor of mature T cells. Anti-
CD3 antibodies include OKT-3, also known as muromonab, and UCHT-l. Other anti-CD3
antibodies include, for example, otelixizumab, teplizumab, and visilizumab.
The term “OKT-3” (also referred to herein as “OKT3”) refers to a monoclonal
antibody or ilar or variant thereof, including human, zed, ic, or murine
antibodies, directed against the CD3 receptor in the T cell antigen receptor of mature T cells,
and includes commercially-available forms such as OKT-3 (30 ng/mL, MACS GMP CD3
pure, Miltenyi Biotech, Inc, San Diego, CA, USA) and muromonab or variants, conservative
amino acid substitutions, glycoforms, or biosimilars thereof The amino acid sequences of the
heavy and light chains of nab are given in Table 1 (SEQ ID N01 and SEQ ID
NO:2). A hybridoma capable of producing OKT—3 is deposited with the American Type
Culture tion and assigned the ATCC accession number CRL 8001. A hybridoma
capable of producing OKT-3 is also deposited with European Collection of Authenticated
Cell Cultures (ECACC) and assigned Catalogue No. 86022706.
TABLE 1. Amino acid ces of muromonab.
ce (One-Letter Amino Acid Symbols)
SEQ ID N021 QVQLQQSGAE SVKM SC<ASGYTFT RYTMHWVKQR PGQGLEWIGY INPSRGYTNY
Muromonab heavy NQKFKDKATL STAY MQJSSLTSED SAVYYCARYY DDHYCLDYWG QGTTLTVSSA
Chain KTTAPSVYPL APVCGGTTGS SVTLGCLVKG YFPEPVTLTW NSGSLSSGVH TFPAVLQSDL
YTLSSSVTVT SSTWPSQSIT CNVAHPASST KVDKKIEPRP KSCDKTHTCP PCPAPELLGG
PSVFLFPPKP KDTLMISRTP VDVS HEDPEVKFNW YVDGVEVHNA EQYN
STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE
LTKNQVSLTC LVKGFYPSDI AVEWESNGQP TPPV LDSDGSFFLY SKLTVDKSRW
QQGNVFSCSV MHEALHNHYT QKSLSLSPGK
SEQ ID NO:2 QIVLTQSPAI MSASPGEKVT MTCSASSSVS YMNWYQQKSG TSPKRWIYDT SKLASGVPAH
Muromonab light FRGSGSGTSY SLTISGMEAE DAATYYCQQW SSNPFTFGSG TKLEINRADT APTVSIFPPS
chain GGAS VVCFLNNFYP KDINVKWKID GSERQNGVLN SWTDQDSKDS TYSMSSTLTL
TKDEYERHNS YTCEATHKTS TSPIVKSFNR NEC
The term “IL-2” (also referred to herein as “IL2”) refers to the T cell growth factor
known as interleukin-2, and includes all forms of IL-2 including human and mammalian
forms, conservative amino acid substitutions, glycoforms, biosimilars, and ts thereof.
IL-2 is described, e.g., in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev.
Immunol. 2008, 26, 453-79, the disclosures of which are incorporated by reference herein.
The amino acid sequence of recombinant human IL-2 suitable for use in the invention is
given in Table 2 (SEQ ID NO:3). For example, the term IL-2 encompasses human,
recombinant forms of IL-2 such as aldesleukin (PROLEUKIN, available commercially from
multiple suppliers in 22 million IU per single use vials), as well as the form of recombinant
IL-2 commercially supplied by CellGeniX, Inc., Portsmouth, NH, USA (CELLGRO GMP) or
ProSpec-Tany TechnoGene Ltd, East Brunswick, NJ, USA (Cat. No. CYTb) and other
commercial equivalents from other vendors. Aldesleukin (des-alanyl-l, serine-125 human 1L-
2) is a nonglycosylated human recombinant form of IL-2 with a molecular weight of
approximately 15 kDa. The amino acid sequence of aldesleukin le for use in the
invention is given in Table 2 (SEQ ID N04). The term IL-2 also encompasses pegylated
forms of IL-2, as described herein, including the pegylated IL2 prodrug NKTR-214, available
from Nektar Therapeutics, South San Francisco, CA, USA. NKTR-214 and ted IL-2
suitable for use in the invention is described in US. Patent Application Publication No. US
2014/0328791 A1 and International Patent Application Publication No.
the disclosures of which are incorporated by reference . Alternative forms of
conjugated IL-2 suitable for use in the invention are described in U. S. Patent Nos. 4,766,106,
,206,344, 5,089,261 and 4902,502, the disclosures of which are incorporated by reference
herein. Formulations of IL-2 suitable for use in the invention are described in US. Patent No.
6,706,289, the disclosure of which is incorporated by reference herein.
TABLE 2. Amino acid sequences of interleukins.
Identifier Sequence (One-Letter Amino Acid s)
SEQ ID NO:3 MAPTSSSTKK TQLQLEHLLJ DLQMILNGIN LTRM LTEKEYMPKK ATELKHLQCL
recombinant LEEV LNLAQSKNEI ISNI NVIVLELKGS ETTEMCEYAD ETATIVEELN
human ILi2 RWITECQSII STLT
(rhILi2)
SEQ ID NO:4 PTSSSTKKTQ LQLEHLLLDJ QMILNGINNY RMLT EKFYMPKKAT ELKHLQCLEE
Aidesieukin ELKPLEEVLN LAQSKNEHLR PRDLISNINV IVLELKGSET ADET ATIVEELNRW
ITESQSIIST LT
SEQ ID NO:5 TLQE IIKTLNSLTE QKTLCTELTV SKNT TEKETECRAA TVLRQEYSHH
recombinant EKDTRCLGAT HKQS IRELKRLDRN LWGLAGLNSC PVKEANQSTL ENELERLKTI
human ILi4 MREKYSKCSS
(rhIL74)
SEQ ID N026 MDCDIEGKDG KQYESVLMVS IDQLLDSMKE IGSNCLNNEF NFFKRHICDA NKEGMFLFRA
recombinant ARKLRQELKM NSTGDEDLHu LKVSEGTTIL LNCTGQVKGR KPAALGEAQP TKSLEENKSL
human ILi7 KEQKKLNDLC ELKRLLQEI< TCWNKILMGT KEH
(rhIL77)
SEQ ID NO:7 ISDL IQSM HIDATLYTES DVHPSCKVTA MKCFLLELQV ISLESGDASI
recombinant HDTVENLIIL ANNSLSSNGN VTESGCKECE ELEEKNIKEE IVQM EINTS
human ILelS
SEQ ID NO:8 MQDRHMIRMR QLIDIVDQLK NYVNDLVPEE LPAPEDVETN CEWSAESCEQ KAQLKSANTG
recombinant NNERIINVSI KKLKRKPPST NAGRRQKHRL TCPSCDSYEK KPPKEELERF KSLLQKMIHQ
human ILi2l HLSSRTHGSE DS
(rhILi21)
The term “IL-4” (also referred to herein as “IL4”) refers to the cytokine known as
interleukin 4, which is produced by Th2 T cells and by eosinophils, ils, and mast cells.
IL-4 tes the differentiation of naive helper T cells (Th0 cells) to Th2 T cells. Steinke
and Borish, Respir. Res. 2001, 2, 66-70. Upon activation by IL—4, Th2 T cells subsequently
produce additional IL-4 in a positive feedback loop. IL—4 also stimulates B cell proliferation
and class II MHC expression, and induces class switching to IgE and IgG1 sion from B
cells Recombinant human IL-4 suitable for use in the invention is commercially available
from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ,
USA (Cat. No. CYT-2l l) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human
IL-lS recombinant protein, Cat. No. Gibco CTP0043). The amino acid sequence of
recombinant human IL—4 le for use in the ion is given in Table 2 (SEQ ID NO:5).
The term “IL-7” (also referred to herein as “IL7”) refers to a glycosylated tissue-
derived cytokine known as interleukin 7, which may be obtained from stromal and epithelial
cells, as well as from dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can
stimulate the development of T cells. IL—7 binds to the IL-7 or, a heterodimer
consisting of IL-7 receptor alpha and common gamma chain receptor, which in a series of
signals important for T cell development within the thymus and al within the ery.
Recombinant human IL-4 suitable for use in the invention is commercially ble from
multiple suppliers, including ProSpec-Tany TechnoGene Ltd, East Brunswick, NJ, USA
(Cat. No. CYT—254) and ThermoFisher ific, Inc., Waltham, MA, USA (human IL-15
recombinant protein, Cat. No. Gibco PHC0071). The amino acid sequence of recombinant
human IL-7 suitable for use in the invention is given in Table 2 (SEQ ID NO:6).
The term “IL-15” (also referred to herein as “IL15”) refers to the T cell growth
factor known as interleukin-15, and includes all forms of IL-2 including human and
ian forms, conservative amino acid tutions, orms, biosimilars, and
variants thereof. IL-15 is described, e.g., in Fehniger and Caligiuri, Blood 2001, 97, 14-32,
the disclosure of which is incorporated by reference herein. IL-lS shares [3 and y signaling
receptor subunits with IL-2. Recombinant human IL-15 is a single, non-glycosylated
polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a
molecular mass of 12.8 kDa. Recombinant human IL-15 is commercially available from
multiple suppliers, including ProSpec-Tany TechnoGene Ltd, East Brunswick, NJ, USA
(Cat. No. CYTb) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-lS
recombinant protein, Cat. No. 3482). The amino acid sequence of recombinant human
IL-15 suitable for use in the invention is given in Table 2 (SEQ ID NO:7).
The term “IL-21” (also referred to herein as “IL2l”) refers to the pleiotropic
ne protein known as interleukin-21, and includes all forms of IL-21 including human
and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and
variants thereof. IL-21 is described, e.g., in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014,
13, 379-95, the disclosure of which is incorporated by reference herein. IL-21 is primarily
produced by natural killer T cells and activated human CD4+ T cells. inant human
IL-21 is a single, non-glycosylated polypeptide chain containing 132 amino acids with a
molecular mass of 15.4 kDa. Recombinant human IL-21 is commercially ble from
multiple suppliers, including ProSpec-Tany TechnoGene Ltd, East Brunswick, NJ, USA
(Cat. No. CYTb) and ThermoFisher Scientific, Inc., m, MA, USA (human IL-21
recombinant protein, Cat. No. 1480). The amino acid sequence of recombinant human
IL-2l le for use in the invention is given in Table 2 (SEQ ID N08).
] When “an anti-tumor effective amount77 (C
an tumor-inhibiting effective amount”, or
“therapeutic amount” is indicated, the precise amount of the compositions of the t
invention to be administered can be determined by a physician with consideration of
individual differences in age, weight, tumor size, extent of infection or metastasis, and
condition of the patient (subject). It can generally be stated that a pharmaceutical composition
comprising the genetically modified cytotoxic lymphocytes described herein may be
administered at a dosage of 104 to 1011 cells/kg body weight (e.g., 105 to 106, 105 to 1010, 105
to 10“, 106 to 1010, 106 to 1011,107 to 10“, 107 to 1010, 108 to 10“, 108 to 1010, 109 to 10“, or
109 to 1010 cells/kg body ), including all integer values within those ranges.
Genetically modified cytotoxic cytes compositions may also be administered multiple
times at these dosages. The genetically d cytotoxic lymphocytes can be administered
by using on techniques that are commonly known in immunotherapy (see, e.g.,
Rosenberg et al., New Eng. J. ofMed. 319: 1676, 1988). The optimal dosage and treatment
regime for a ular patient can readily be determined by one d in the art of medicine
by monitoring the patient for signs of disease and adjusting the treatment accordingly.
The term ological malignancy” refers to mammalian s and tumors of
the hematopoietic and lymphoid tissues, including but not limited to tissues of the blood,
bone marrow, lymph nodes, and lymphatic system. logical malignancies are also
referred to as “liquid tumors.” Hematological malignancies include, but are not limited to,
acute lymphoblastic ia (ALL), chronic lymphocytic lymphoma (CLL), small
lymphocytic lymphoma (SLL), acute myelogenous leukemia (AML), chronic myelogenous
leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-
Hodgkin's lymphomas. The term “B cell hematological malignancy” refers to hematological
malignancies that affect B cells.
The term “solid tumor” refers to an abnormal mass of tissue that usually does not
contain cysts or liquid areas. Solid tumors may be benign or malignant. The term “solid
tumor cancer refers to malignant, neoplastic, or cancerous solid tumors. Solid tumor cancers
include, but are not limited to, sarcomas, carcinomas, and lymphomas, such as cancers of the
lung, breast, prostate, colon, rectum, and bladder. The tissue structure of solid tumors
includes interdependent tissue compartments including the parenchyma (cancer cells) and the
supporting stromal cells in which the cancer cells are dispersed and which may provide a
supporting nvironment.
] The term “liquid tumor” refers to an abnormal mass of cells that is fluid in nature.
Liquid tumor cancers include, but are not limited to, leukemias, as, and lymphomas,
as well as other hematological ancies. TILs obtained from liquid tumors may also be
referred to herein as marrow infiltrating lymphocytes (MILs).
The term “microenvironment,” as used herein, may refer to the solid or
hematological tumor microenvironment as a whole or to an individual subset of cells within
the microenvironment. The tumor nvironment, as used herein, refers to a complex
mixture of “cells, e factors, signaling molecules, extracellular matrices, and mechanical
cues that promote neoplastic transformation, support tumor growth and invasion, protect the
tumor from host ty, foster therapeutic resistance, and provide niches for nt
metastases to thrive,” as bed in Swartz, et al, Cancer Res, 2012, 72, 2473. Although
tumors express antigens that should be recognized by T cells, tumor clearance by the immune
system is rare because of immune suppression by the microenvironment.
In an embodiment, the invention es a method of treating a cancer with a
population of rTILs, wherein a patient is pre-treated with non-myeloablative chemotherapy
prior to an infusion of rTILs according to the invention. In some embodiments, the tion
of rTILs may be provided with a population of eTils, wherein a patient is eated with
nonmyeloablative chemotherapy prior to an infusion of rTILs and eTils according to the
invention. In an embodiment, the non-myeloablative chemotherapy is cyclophosphamide 60
mg/kg/d for 2 days (days 27 and 26 prior to rTIL infusion) and fludarabine 25 mg/m2/d for 5
days (days 27 to 23 prior to rTIL on). In an embodiment, after eloablative
chemotherapy and rTlL infusion (at day 0) according to the invention, the patient receives an
intravenous infusion of IL-2 intravenously at 720,000 IU/kg every 8 hours to physiologic
tolerance.
] Experimental findings indicate that lymphodepletion prior to adoptive transfer of
specific T lymphocytes plays a key role in enhancing treatment efficacy by eliminating
regulatory T cells and ing elements of the immune system (“cytokine sinks”).
Accordingly, some embodiments of the invention utilize a lymphodepletion step (sometimes
also referred to as “immunosuppressive conditioning”) on the patient prior to the introduction
of the rTILs of the invention.
The terms “co-administration, 77 cc co-administering, )7 (C administered in combination
with,” “administering in ation with,” “simultaneous,” and “concurrent,” as used
herein, ass administration of two or more active pharmaceutical ingredients (in a
red embodiment of the present invention, for example, at least one potassium channel
agonist in combination with a plurality of TILs) to a subject so that both active
pharmaceutical ingredients and/or their metabolites are present in the subject at the same
time. Co—administration includes aneous administration in separate compositions,
administration at different times in separate compositions, or administration in a composition
in which two or more active pharmaceutical ingredients are present. Simultaneous
administration in separate compositions and administration in a composition in which both
agents are present are preferred.
The term “effective amount” or “therapeutically effective ” refers to that
amount of a compound or combination of compounds as described herein that is sufficient to
effect the intended application including, but not limited to, disease treatment. A
therapeutically effective amount may vary depending upon the intended application (in vitro
or in vivo), or the subject and disease condition being treated (e.g, the weight, age and
gender of the subject), the severity of the disease condition, or the manner of administration.
The term also applies to a dose that will induce a ular response in target cells (e.g., the
reduction of platelet adhesion and/or cell ion). The c dose will vary depending
on the particular compounds chosen, the dosing regimen to be followed, r the
nd is administered in combination with other compounds, timing of administration,
the tissue to which it is administered, and the al delivery system in which the
compound is carried.
The terms “treatment”, “treating”, “treat”, and the like, refer to obtaining a desired
pharmacologic and/or physiologic effect. The effect may be lactic in terms of
completely or partially preventing a disease or symptom thereof and/or may be therapeutic in
terms of a partial or complete cure for a disease and/or adverse effect attributable to the
disease. “Treatment”, as used herein, covers any treatment of a disease in a mammal,
particularly in a human, and includes: (a) preventing the disease from occurring in a subject
which may be predisposed to the disease but has not yet been diagnosed as having it,
(b) inhibiting the disease, i.e., arresting its development or progression; and (c) relieving the
disease, i.e., causing regression of the e and/or relieving one or more disease
symptoms. “Treatment” is also meant to ass delivery of an agent in order to provide
for a pharmacologic effect, even in the absence of a disease or ion. For example,
“treatment” encompasses delivery of a composition that can elicit an immune response or
confer immunity in the absence of a disease condition, e.g., in the case of a e.
The term “heterologous” when used with reference to portions of a nucleic acid or
protein tes that the nucleic acid or n comprises two or more subsequences that are
not found in the same relationship to each other in . For instance, the nucleic acid is
typically recombinantly produced, having two or more sequences from unrelated genes
ed to make a new functional nucleic acid, e.g., a promoter from one source and a
coding region from another source, or coding regions from ent sources. Similarly, a
heterologous n indicates that the protein comprises two or more subsequences that are
not found in the same relationship to each other in nature (e. g., a fusion protein).
The terms “sequence identity, 77 capercent identity,” and “sequence percent identity”
(or synonyms thereof, e.g., “99% identical”) in the context of two or more nucleic acids or
polypeptides, refer to two or more sequences or subsequences that are the same or have a
specified percentage of nucleotides or amino acid residues that are the same, when compared
and d (introducing gaps, if ary) for maximum correspondence, not considering
any conservative amino acid substitutions as part of the sequence identity. The percent
identity can be measured using ce ison software or algorithms or by visual
inspection. Various algorithms and software are known in the art that can be used to obtain
alignments of amino acid or nucleotide sequences. Suitable programs to determine percent
sequence identity include for example the BLAST suite of ms available from the U. S.
Government’ 5 National Center for Biotechnology ation BLAST web site.
Comparisons between two sequences can be carried using either the BLASTN or BLASTP
algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to
compare amino acid sequences. ALIGN, ALIGN-Z (Genentech, South San Francisco,
California) or MegAlign, available from DNASTAR, are additional publicly available
software programs that can be used to align sequences. One skilled in the art can determine
appropriate parameters for maximal alignment by particular alignment software. In certain
embodiments, the default parameters of the alignment software are used.
As used herein, the term “variant” encompasses but is not limited to antibodies or
fusion proteins which comprise an amino acid sequence which differs from the amino acid
sequence of a reference antibody by way of one or more tutions, deletions and/or
additions at certain positions within or adjacent to the amino acid sequence of the reference
antibody. The variant may comprise one or more conservative substitutions in its amino acid
sequence as compared to the amino acid sequence of a reference antibody. Conservative
substitutions may involve, e. g., the substitution of similarly charged or uncharged amino
acids. The variant retains the ability to specifically bind to the antigen of the reference
dy. The term variant also es ted dies or proteins.
] The term “in viva” refers to an event that takes place in a subject's body.
The term “in vitro” refers to an event that takes places outside of a t's body. In
vitro assays encompass cell-based assays in which cells alive or dead are employed and may
also encompass a cell-free assay in which no intact cells are employed.
The term “rapid ion” means an increase in the number of antigen-specific
TILs of at least about 3-fold (or 4-, 5-, 6-, 7-, 8-, or 9-fold) over a period of a week, more
preferably at least about 10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold) over a period
of a week, or most preferably at least about lOO-fold over a period of a week. A number of
rapid expansion protocols are outlined below.
III. Restimulation of Cyropreserved TILs
As discussed , the present invention relates to the restimulation of
cryopreserved TILs to increase their metabolic activity and thus relative health prior to
transplant into a patient, and methods of testing said metabolic health. As generally outlined
herein, TILs are generally taken from a patient sample and lated to expand their
number prior to transplant into a patient. In some embodiments, the TILs may be optionally
genetically manipulated as sed below, and then cryopreserved. Once thawed, they are
then restimulated to increase their metabolism prior to infusion into a patient.
The “Step” Designations A, B, C, etc, below are in nce to Figure 11. The
ordering of the Steps below and in Figure 11 is ary and any combination or order of
steps, as well as additional steps, repetition of steps, and/or omission of steps is contemplated
by the present application and the methods disclosed herein.
A. STEP A: Obtain Patient Tumor Sample
In general, TILs are initially obtained from a t tumor sample (“primary TILs”)
and then expanded into a larger population for further manipulation as described herein,
optionally cryopreserved, restimulated as outlined herein and optionally evaluated for
phenotype and metabolic parameters as an indication of TIL health.
A patient tumor sample may be ed using s known in the art, generally
via surgical resection, needle biopsy or other means for obtaining a sample that contains a
mixture of tumor and TIL cells. In general, the tumor sample may be from any solid tumor,
including primary tumors, invasive tumors or metastatic . The tumor sample may also
be a liquid tumor, such as a tumor obtained from a hematological malignancy. The solid
tumor may be of any cancer type, including, but not limited to, , pancreatic, prostate,
colorectal, lung, brain, renal, h, and skin (including but not limited to squamous cell
carcinoma, basal cell carcinoma, and melanoma). In some embodiments, useful TILs are
obtained from malignant melanoma tumors, as these have been reported to have particularly
high levels of TILs.
The term “solid tumor” refers to an abnormal mass of tissue that usually does not
contain cysts or liquid areas. Solid tumors may be benign or malignant. The term “solid
tumor cancer refers to malignant, neoplastic, or ous solid tumors. Solid tumor cancers
include, but are not d to, sarcomas, carcinomas, and lymphomas, such as cancers of the
lung, breast, triple negative breast cancer, prostate, colon, rectum, and r. In some
embodiments, the cancer is selected from al cancer, head and neck ,
glioblastoma, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple
negative breast cancer, and non-small cell lung carcinoma. The tissue structure of solid
tumors includes interdependent tissue compartments including the parenchyma (cancer cells)
and the supporting l cells in which the cancer cells are dispersed and which may
provide a supporting microenvironment.
The term “hematological malignancy” refers to mammalian cancers and tumors of
the hematopoietic and lymphoid tissues, including but not limited to s of the blood,
bone marrow, lymph nodes, and lymphatic . Hematological malignancies are also
referred to as “liquid tumors.” Hematological ancies include, but are not limited to,
acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small
cytic lymphoma (SLL), acute myelogenous ia (AML), c myelogenous
leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-
Hodgkin's lymphomas. The term “B cell logical malignancy” refers to hematological
malignancies that affect B cells.
Once obtained, the tumor sample is generally nted using sharp dissection into
small pieces of between 1 to about 8 mm3, with from about 2-3 mm3 being particularly
useful. The TILs are cultured from these fragments using enzymatic tumor digests. Such
tumor digests may be produced by incubation in enzymatic media (e.g, Roswell Park
Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, lO mcg/mL gentamicine, 30
units/mL of DNase and 1.0 mg/mL of collagenase) followed by mechanical dissociation (e.g.,
using a tissue dissociator). Tumor digests may be produced by placing the tumor in
enzymatic media and mechanically dissociating the tumor for approximately 1 minute,
followed by incubation for 30 minutes at 37 °C in 5% C02, followed by repeated cycles of
mechanical dissociation and tion under the foregoing conditions until only small tissue
pieces are present. At the end of this process, if the cell suspension contains a large number
of red blood cells or dead cells, a density gradient separation using FICOLL branched
hydrophilic polysaccharide may be med to remove these cells. Alternative methods
known in the art may be used, such as those described in US. Patent Application Publication
No. 2012/0244133 Al, the sure of which is orated by reference herein. Any of
the foregoing methods may be used in any of the embodiments described herein for methods
of expanding TILs or s treating a cancer.
In some embodiments, fragmentation includes physical fragmentation, including for
example, dissection as well as digestion. In some embodiments, the fragmentation is physical
ntation. In some embodiments, the fragmentation is dissection. In some embodiments,
the fragmentation is by digestion. In some embodiments, TILs can be initially cultured from
enzymatic tumor digests and tumor fragments obtained from patients.
In some embodiments, where the tumor is a solid tumor, the tumor undergoes
physical fragmentation after the tumor sample is obtained, for example such as in Step A of
Figure 11. In some embodiments, the fragmentation occurs before eservation. In some
embodiments, the fragmentation occurs after cryopreservation. In some embodiments, the
fragmentation occurs after obtaining the tumor and in the absence of any cryopreservation. In
some embodiments, the tumor is fragmented and 2, 3, or 4 fragments or pieces are placed in
each container for the first expansion. In some embodiments, the tumor is fragmented and 3
or 4 fragments or pieces are placed in each container for the first expansion. In some
embodiments, the tumor is fragmented and 4 fragments or pieces are placed in each container
for the first expansion,
In some embodiments, the TILs are obtained from tumor fragments. In some
embodiments, the tumor fragment is obtained sharp dissection. In some embodiments, the
tumor fragment is n about 1 mm3 and 10 mm3. In some embodiments, the tumor
fragment is between about 1 mm3 and 8 mm3. In some embodiments, the tumor nt is
about 1 mm3. In some embodiments, the tumor fragment is about 2 mm3. In some
embodiments, the tumor fragment is about 3 mm3. In some embodiments, the tumor fragment
is about 4 mm3. In some embodiments, the tumor fragment is about 5 mm3. In some
embodiments, the tumor fragment is about 6 mm3. In some embodiments, the tumor fragment
is about 7 mm3. In some embodiments, the tumor fragment is about 8 mm3. In some
embodiments, the tumor fragment is about 9 mm3. In some embodiments, the tumor fragment
is about 10 mm3.
In some embodiments, the TILs are obtained from tumor digests. In some
embodiments, tumor s were generated by incubation in enzyme media, for example but
not limited to RPMI 1640, 2 mM GlutaMAX, 10 mg/mL gentamicin, 3O U/mL DNase, and
1.0 mg/mL enase, followed by ical dissociation (GentleMACS, Miltenyi
Biotec, Auburn, CA). After placing the tumor in enzyme media, the tumor can be
mechanically dissociated for approximately 1 . The solution can then be incubated
for 30 minutes at 37°C in 5% C02 and it then mechanically disrupted again for
approximately 1 minute. After being incubated again for 30 minutes at 37°C in 5% C02, the
tumor can be mechanically disrupted a third time for approximately 1 minute. In some
embodiments, after the third mechanical disruption if large pieces of tissue were present, 1
or 2 additional mechanical dissociations were applied to the sample, with or without 30
onal minutes of tion at 37°C in 5% C02. In some embodiments, at the end of
the final tion if the cell suspension contained a large number of red blood cells or
dead cells, a density gradient separation using Ficoll can be performed to remove these
cells.
In some embodiments, the harvested cell suspension prior to the first ion step
is called a “primary cell population” or a “freshly ted” cell population.
In some embodiments, cells can be optionally frozen after sample harvest and stored
frozen prior to entry into Step B, which is described in further detail below.
B. STEP B: First Expansion
In some ments, a first expansion of TILs (also referred to as a first expansion
or first TIL expansion) may be performed using an l bulk TIL expansion step (for
example, Step B as indicated in Figure ll or a first expansion step, this can include an
expansion step referred to as preREP) as described below and herein, followed by a second
expansion step (for example, Step D as indicated in Figure 11; which can include as an
example what is referred to as a rapid expansion protocol (REP) step) as described below and
, followed by optional cryopreservation (for example, after Step D as indicated in
Figure 11), and followed by an additional second expansion (for example, a second Step D,
as indicated in Figure 11, which can include what is mes referred to as a restimulation
REP step) as described below and herein. The TILs obtained from this process may be
optionally characterized for phenotypic characteristics and metabolic parameters as described
herein. In some embodiments, the TILs are frozen (116., cryopreserved) after the first
expansion (for example, Step B as ted in Figure 11) and stored until phenotyped for
selection then thawed prior to ding to one or more second expansion steps (for
example, one or more expansion according to Step D as indicated in Figure 11).
In some embodiments, where the cells are frozen after obtained from the tumor
sample (such as, for e, during in Step A as indicated in Figure 11), the cells are
thawed prior to the first expansion (for example, Step B as indicated in Figure 11).
In embodiments where TIL cultures are initiated in 24-well plates, for example,
using Costar 24-well cell culture r, flat bottom (Corning Incorporated, Corning, NY,
each well can be seeded with lxlO6 tumor digest cells or one tumor fragment in 2 mL of
complete medium (CM) with IL-2 (6000 IU/mL, Chiron Corp, ille, CA). In some
embodiments, the tumor fragment is between about 1mm3 and 10 mm3.
After ation of the tumor fragments, the resulting cells (i.e., fragments) are
cultured in serum containing IL—2 under conditions that favor the growth of TILs over tumor
and other cells. In some embodiments, the tumor digests are incubated in 2 mL wells in
media comprising inactivated human AB serum (or, in some cases, as outlined herein, in the
presence of aAPC cell population) with 6000 IU/mL of IL-2. This primary cell population is
cultured for a period of days, generally from 10 to 14 days, resulting in a bulk TIL
population, generally about 1 X 108 bulk TIL cells. In some embodiments, the growth media
during the first expansion comprises IL-2 or a variant thereof. In some embodiments, the IL
is recombinant human [L-2 (rh[L-2). In some embodiments the IL-2 stock solution has a
specific ty of 20-30x106 IU/mg for a 1 mg vial. In some embodiments the IL-2 stock
solution has a specific activity of 20-x106 IU/mg for a 1 mg vial. In some embodiments the
IL-2 stock solution has a specific activity of 25x106 IU/mg for a 1 mg vial. In some
embodiments the IL-2 stock on has a specific ty of 30x106 IU/mg for a 1 mg vial.
In some embodiments, the IL- 2 stock solution has a final concentration of 4-8x106 IU/mg of
IL-2. In some embodiments, the IL- 2 stock solution has a final concentration of 6
IU/mg of IL-2. In some embodiments, the IL- 2 stock solution has a final concentration of
6x106 IU/mg of IL-2. In some embodiments, the IL-2 stock solution is prepare as described in
Example 4. In some embodiments, first expansion culture media comprises about 10,000
IU/mL of IL-2, about 9,000 IU/mL of IL-2, about 8,000 IU/mL of IL—2, about 7,000 IU/mL
of IL—2, about 6000 IU/mL of IL-2 or about 5,000 IU/mL of IL-2. In some embodiments, first
expansion culture media ses about 9,000 IU/mL of IL-2, to about 5,000 IU/mL of IL-
2. In some embodiments, first expansion culture media comprises about 8,000 IU/mL of IL-2,
to about 6,000 IU/mL of IL-2. In some embodiments, first expansion culture media comprises
about 7,000 IU/mL of IL-2, to about 6,000 IU/mL of IL-2. In some embodiments, first
ion culture media comprises about 6,000 IU/mL of IL-2. In an embodiment, the cell
culture medium further comprises IL-2. In some embodiments, the cell culture medium
comprises about 3000 IU/mL of IL-2. In an ment, the cell culture medium comprises
about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000
IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000 IU/mL, about
5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL,
or about 8000 IU/mL of IL—2. In an embodiment, the cell culture medium comprises n
1000 and 2000 IU/mL, between 2000 and 3000 IU/mL, between 3000 and 4000 IU/mL,
between 4000 and 5000 IU/mL, n 5000 and 6000 IU/mL, between 6000 and 7000
IU/mL, between 7000 and 8000 IU/mL, or between 8000 IU/mL of IL-2.
In some embodiments, the first expansion culture medium is referred to as “CM”, an
abbreviation for culture media. In some embodiments, it is referred to as CMl (culture
medium 1). In some embodiments, CM consists ofRPMI 1640 with GlutaMAX,
supplemented with 10% human AB serum, 25mM Hepes, and 10 mg/mL gentamicin. In
embodiments where cultures are initiated in rmeable flasks with a 40 mL capacity and
a 10cm2 gas-permeable silicon bottom (for example, G-Rex10, Wilson Wolf Manufacturing,
New Brighton, MN) (Fig. 1), each flask was loaded with 10—40x 106 Viable tumor digest cells
or 5—30 tumor fragments in 10—40mL of CM with IL-2. Both the G-RexlO and 24-well plates
were incubated in a fied incubator at 37°C in 5% C02 and 5 days after culture
initiation, half the media was removed and replaced with fresh CM and IL-2 and after day 5,
half the media was changed every 2—3 days. In some embodiments, the CM is the CM]
described in the Examples, see, Example 5. In some ments, the first expansion occurs
in an initial cell e medium or a first cell culture medium. In some embodiments, the
initial cell culture medium or the first cell culture medium comprises 1L-2.
] In some embodiments, the first TIL expansion can proceed for 11 days, 12 days, 13
days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or 21 days. In some
embodiments, the first TIL expansion can proceed for 11 days to 21 days. In some
embodiments, the first TIL expansion can proceed for 12 days to 21 days. In some
embodiments, the first TIL expansion can proceed for 13 days to 21 days. In some
embodiments, the first TIL expansion can proceed for 14 days to 21 days. In some
embodiments, the first TIL expansion can proceed for 15 days to 21 days. In some
embodiments, the first TIL expansion can proceed for 16 days to 21 days. In some
ments, the first TIL expansion can proceed for 17 days to 21 days. In some
ments, the first TIL expansion can proceed for 18 days to 21 days. In some
embodiments, the first TIL expansion can proceed for 19 days to 21 days. In some
embodiments, the first TIL expansion can proceed for 20 days to 21 days. In some
embodiments, the first TIL ion can proceed for 21 days.
C. STEP C: First Expansion to Second Expansion Transition
In some embodiments, the TILs ed from the first expansion (for example,
from Step B as indicated in Figure 11) are stored until phenotyped for selection. In some
embodiments, the TILs obtained from the first expansion are cryopreserved after the first
expansion and prior to the second ion. In some embodiments, the TILs are
cryopreserved as part of the first expansion to second expansion transition. For example, in
some embodiments, the TILs are cryopreserved after Step B and before Step D as indicated in
Figure 11. In some embodiments, the TILs are cryopreserved and thawed as part of the first
expansion to second ion transition. For e, in some embodiments, the TILs are
cryopreserved after Step B then thawed prior to proceeding to Step D (as provided in Figure
11). In some embodiments, the transition from the first expansion to the second expansion
occurs at about 22 days, 23, days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, or 30
days from when fragmentation occurs. In some embodiments, the transition from the first
expansion to the second expansion occurs at about 22 days to 30 days from when
fragmentation occurs. In some embodiments, the transition from the first expansion to the
second expansion occurs at about 24 days to 30 days from when fragmentation occurs. In
some embodiments, the transition from the first expansion to the second ion occurs at
about 26 days to 30 days from when fragmentation occurs. In some embodiments, the
transition from the first expansion to the second expansion occurs at about 28 days to 30 days
from when fragmentation occurs. In some embodiments, the transition from the first
ion to the second ion occurs at about 30 days from when ntation occurs.
D. STEP D: Second Expansion
In some embodiments, the second expansion or second TIL ion (which can
include expansions sometimes referred to as REP) of TIL can be performed using any TIL
flasks or containers known by those of skill in the art. In some embodiments, the second TIL
expansion can proceed for 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21
days, or 22 days. In some embodiments, the second TIL expansion can proceed for about 14
days to about 22 days. In some embodiments, the second TIL expansion can proceed for
about 14 days to about 20 days. In some embodiments, the second TIL expansion can
proceed for about 14 days to about 18 days. In some embodiments, the second TIL expansion
can proceed for about 14 days to about 16 days. In some embodiments, the second TIL
ion can proceed for about 14 days.
In some embodiments, the second expansion occurs in a supplemented cell culture
medium. In some embodiments, the supplemented cell culture medium comprises lL-2, OKT-
3, and antigen-presenting feeder cells. In some ments, the second cell culture medium
comprises IL—2, OKT—3, and antigen—presenting cells (APCs, also referred to as antigen—
presenting feeder cells).
In some embodiments, the second expansion (which can e expansions referred
to as REP) of TILs can be performed using T-175 flasks and gas-permeable bags as
previously bed (Tran KQ, Zhou J, Durflinger KH, et al., 2008, JImmunother., 31 :742—
751, and Dudley ME, Wunderlich JR, Shelton TE, et al. 2003, JImmunother., 26:332—342)
or gas-permeable G-Rex flasks. In some embodiments, the second expansion is performed
using flasks. In some embodiments, the second expansion is performed using gas-permeable
G—Rex flasks. For TIL the second expansion in T-175 flasks, about 1 x 106 TIL are suspended
in about 150 mL of media and this is added to each T-175 flask. The TIL are cultured with
ated (50 Gy) allogeneic PBMC as “feeder” cells at a ratio of 1 to 100 and the cells were
cultured in a 1 to 1 mixture of CM and AIM-V medium (50/50 medium), supplemented
with 3000 IU/mL of IL-2 and 30 ng/mL of anti-CD3. The T-175 flasks are incubated at
37°C in 5% C02. In some embodiments, half the media is changed 5 days into the second
expansion using 50/50 medium with 3000 IU/mL of IL-2. In some ments, on day 7,
cells from 2 T-175 flasks are combined in a 3 L bag and 300 mL of AIM-V with 5%
human AB serum and 3000 IU/mL of IL-2 is added to the 300 mL of TIL suspension. The
number of cells in each bag can be counted every day or two and fresh media can be added
to keep the cell count between about 0.5 and about 2.0 x 106 cells/mL.
In some embodiments, the second expansion (which can include ions referred
to as REP) of TIL can be performed in 500 mL capacity gas permeable flasks with 100 cm2
gas-permeable silicon s (G—Rex 100, commercially available from Wilson Wolf
Manufacturing Corporation, New Brighton, MN, USA) (Fig. 1), about 5 x 106 or 10 x 106
TIL are ed with irradiated allogeneic PBMC at a ratio of 1 to 100 in 400 mL of 50/50
medium, supplemented with 3000 IU/mL of IL-2 and 30 ng/ mL of anti- CD3 (OKT3). The
G—RexlOO flasks can be incubated at 37°C in 5% C02. In some ments, 5 days into the
second expansion, 250 mL of supernatant is removed and placed into centrifuge bottles and
centrifuged at 1500 rpm (491 x g) for 10 minutes. The TIL pellets can then be resuspended
with 150 mL of fresh medium with 5% human AB serum, 3000 IU per mL of IL-2 and added
back to the original G—RexlOO flasks. In embodiments where TILs are expanded serially in
OO flasks, on day 7 the TIL in each G-RexlOO are suspended in the 300 mL of media
present in each flask and the cell suspension was divided into three 100 mL aliquots that can
be used to seed three G—RexlOO flasks. Then 150 mL of AIM—V with 5% human AB serum
and 3000 IU per mL of IL-2 can be added to each flask. The G—RexlOO flasks can be
incubated at 37°C in 5% CO2 and after 4 days in to the second expansion, 150 mL of AIM-V
with 3000 IU per mL of IL-2 can be added to each G—Rex100 flask. In some ments,
the cells are harvested on day 14 of culture.
In some embodiments, the second expansion (which can include expansions ed
to as REP) of TIL can be performed in a gas permeable container. For example, TILs can be
rapidly expanded using non-specific T-cell receptor stimulation in the presence of
interleukin-2 (IL-2) or interleukin-15 (IL-15). In an embodiment, expansion of the number of
TILs uses about 1 x 109 to about 1 x 1011 antigen-presenting feeder cells. The non-specific T-
cell receptor us can include, for example, about 30 ng/ml of OKT3, a mouse
monoclonal anti-CD3 antibody rcially available from McNeil, Raritan, NJ or
yi Biotech, Auburn, CA). TILs can be rapidly expanded fiirther stimulation of the TILs
in vitro with one or more antigens, including antigenic portions thereof, such as epitope(s), of
the cancer, which can be optionally expressed from a vector, such as a human leukocyte
antigen A2 (HLA-AZ) binding peptide, e.g., 0.3 uM MART-l :26-35 (27 L) or gpl 00:209—
217 (210M), optionally in the presence of a T-cell growth factor, such as 300 IU/mL IL-2 or
IL-15. Other suitable ns may include, e.g., NY-ESO-l, TRP-l, TRP-Z, tyrosinase
cancer antigen, MAGE—A3, SSX—2, and VEGFRZ, or antigenic portions f. TIL may
also be rapidly expanded by re-stimulation with the same antigen(s) of the cancer pulsed onto
HLA-AZ-expressing antigen-presenting cells. Alternatively, the TILs can be further re-
stimulated with, e.g., example, irradiated, autologous lymphocytes or with irradiated HLA—
A2+ allogeneic cytes and IL-2.
In some embodiments, the second expansion (which can include expansions referred
to as REP) of TIL can be performed in 500 mL capacity gas permeable flasks with 100 cm
rmeable silicon bottoms (G—Rex 100, commercially available from Wilson Wolf
Manufacturing Corporation, New Brighton, MN, USA), 5 X 106 or 10 X 106 TIL may be
cultured with aAPCs at a ratio of l to 100 in 400 mL of 50/50 medium, supplemented with
% human AB serum, 3000 IU per mL of IL-2 and 30 ng per ml of anti-CD3 (OKT3). The
G—Rex 100 flasks may be incubated at 37°C in 5% C02. On day 5, 250 mL of supernatant
may be removed and placed into fuge s and centrifuged at 1500 rpm (491 X g) for
minutes. The TIL pellets may be re-suspended with 150 mL of fresh medium with 5%
human AB serum, 3000 IU per mL of IL-2, and added back to the original G—Rex 100 flasks.
When TIL are expanded serially in G—Rex 100 flasks, on day 7 the TIL in each G—Rex 100
may be suspended in the 300 mL of media present in each flask and the cell suspension may
be divided into 3 100 mL aliquots that may be used to seed 3 G—Rex 100 flasks. Then 150 mL
of AIM-V with 5% human AB serum and 3000 IU per mL of IL-2 may be added to each
flask. The G—Rex 100 flasks may be incubated at 37° C in 5% C02 and after 4 days 150 mL
of AIM-V with 3000 IU per mL of IL-2 may be added to each G—RexlOO flask. The cells may
be harvested on day 14 of culture.
In one ment, the second ion (including expansions referred to as REP)
is performed in flasks with the bulk TILs being mixed with a 100- or ZOO-fold excess of
inactivated feeder cells, 30 mg/mL OKT3 anti-CD3 dy and 3000 IU/mL IL-2 in 150 ml
media. Media replacement is done (generally 2/3 media replacement via ation with
fresh media) until the cells are transferred to an alternative growth chamber. Alternative
growth chambers e GRex flasks and gas permeable containers as more fully discussed
below.
] In another ment, the second expansion (including expansions referred to as
REP) is performed and further comprises a step wherein TILs are selected for superior tumor
reactivity. Any selection method known in the art may be used. For example, the methods
described in US. Patent Application Publication No. 2016/0010058 Al, the disclosures of
which are incorporated herein by reference, may be used for selection of TILs for superior
tumor reactivity.
Optionally, a cell viability assay can be performed after the second expansion
(including expansions referred to as the REP expansion), using standard assays known in the
art. For example, a trypan blue exclusion assay can be done on a sample of the bulk TILs,
which selectively labels dead cells and allows a viability assessment. In some embodiments,
TIL samples can be counted and ity ined using a Cellometer K2 automated cell
counter (Nexcelom Bioscience, Lawrence, MA). In some embodiments, viability is
determined according to the Cellometer K2 Image Cytometer Automatic Cell Counter
protocol described, for e, in Example 2.
In some embodiments, cells are grown for 7 days, 8 days, 9 days, 10 days, or 11
days of the total second expansion time before being split into more than one container or
flask.
In some embodiments, the second expansion culture medium (e. g., sometimes
ed to as CM2 or the second cell culture medium), comprises IL-2, OKT-3, as well as
the antigen—presenting feeder cells (APCs), as discussed in more detail below.
In some embodiments, the antigen-presenting feeder cells are PBMCS. In some
embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells.
In an embodiment, the ratio of TILs to antigen-presenting feeder cells in the second
expansion is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150,
about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300,
about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500. In an
embodiment, the ratio of TILs to antigen-presenting feeder cells in the second ion is
n 1 to 50 and l to 300. In an embodiment, the ratio of TILs to antigen-presenting
feeder cells in the second expansion is between 1 to 100 and l to 200.
] In an embodiment, the TIL expansion ures described herein require an excess
of feeder cells during the second expansion ding for example, expansions referred to as
REP TIL expansions). In many embodiments, the feeder cells are peripheral blood
mononuclear cells (PBMCs) obtained from standard whole blood units from healthy blood
donors. The PBMCs are obtained using standard methods such as Ficoll-Paque gradient
separation. In an embodiment, artificial antigen-presenting (aAPC) cells are used in place of
PBMCS.
In general, the allogenic PBMCs are inactivated, either via ation or heat
treatment, and used in the REP procedures.
In some embodiments, the growth media during the first expansion comprises lL-2
or a variant thereof. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In
some embodiments the IL-2 stock solution has a specific ty of 20-30x106 IU/mg for a 1
mg vial. In some embodiments the IL-2 stock on has a specific activity of 20-xlO6
IU/mg for a 1 mg vial. In some embodiments the IL-2 stock solution has a specific activity of
25x106 IU/mg for a 1 mg vial. In some embodiments the IL-2 stock solution has a specific
activity of 30x106 IU/mg for a 1 mg vial. In some ments, the IL- 2 stock solution has a
final concentration of 4-8x106 IU/mg of IL-2. In some embodiments, the IL- 2 stock solution
has a final concentration of 5-7x106 IU/mg of IL-2. In some embodiments, the IL- 2 stock
solution has a final concentration of 6x106 IU/mg of [L-2. In some embodiments, the IL-2
stock solution is e as described in Example 4. In some ments, first expansion
culture media comprises about 10,000 IU/mL of IL-2, about 9,000 IU/mL of IL-2, about
8,000 IU/mL of IL-2, about 7,000 IU/mL of IL-2, about 6000 IU/mL of IL-2 or about 5,000
IU/mL of IL-2. In some embodiments, first expansion culture media comprises about 9,000
IU/mL of IL-2, to about 5,000 IU/mL of IL-2. In some embodiments, first expansion culture
media comprises about 8,000 IU/mL of IL-2, to about 6,000 IU/mL of IL-2. In some
embodiments, first expansion culture media comprises about 7,000 IU/mL of IL-2, to about
6,000 IU/mL of IL-2. In some embodiments, first expansion culture media comprises about
6,000 IU/mL of IL-2. In an embodiment, the cell culture medium r comprises IL-2. In
some embodiments, the cell culture medium comprises about 3000 IU/mL of IL-2. In an
embodiment, the cell culture medium comprises about 1000 IU/mL, about 1500 IU/mL,
about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000
IU/mL, about 4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about
6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL, or about 8000 IU/mL of IL-2. In an
embodiment, the cell culture medium comprises between 1000 and 2000 IU/mL, between
2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000 IU/mL,
between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between 7000 and 8000
IU/mL, or between 8000 IU/mL of IL-2.
In some embodiments, the second expansion cell culture media also includes an
anti-CD3 antibody. In some embodiment, the cell culture medium comprises OKT3 antibody.
In some embodiments, the cell e medium comprises about 30 ng/mL of OKT3
dy. In an embodiment, the cell culture medium ses about 0.1 ng/mL, about 0.5
ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL,
about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about
40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90
ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1 ug/mL of OKT3
antibody. In an ment, the cell culture medium comprises between 0.1 ng/mL and 1
ng/mL, between 1 ng/mL and 5 ng/mL, between 5 ng/mL and 10 ng/mL, between 10 ng/mL
and 20 ng/mL, between 20 ng/mL and 30 ng/mL, between 30 ng/mL and 40 ng/mL, between
40 ng/mL and 50 ng/mL, and between 50 ng/mL and 100 ng/mL of OKT3 antibody.
In some embodiments, an anti-CD3 antibody in combination with IL—2 s T
cell activation and cell division in the TIL population. This effect can be seen with full length
antibodies as well as Fab and F(ab’)2 fragments, with the former being generally preferred;
see, e.g., Tsoukas et 61]., J. Immunol. 1985, 135, 1719, hereby incorporated by nce in its
entirety. As will be appreciated by those in the art, there are a number of suitable uman
CD3 antibodies that find use in the invention, including anti-human CD3 polyclonal and
onal antibodies from various mammals, including, but not d to, murine, human,
primate, rat, and canine antibodies. In particular embodiments, the OKT3 anti-CD3 antibody
is used rcially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech,
Auburn, CA).
In some embodiment, the cells in the second expansion are grown in a culture media
with high doses of a cytokine, in particular lL-Z, as is known in the art.
Alternatively, using combinations of nes for the second expansion of TILS is
additionally possible, with ations of two or more of IL—2, TL-lS and 11—21 as is
generally ed in International Publication No. WC 2015/ l 893 56 and International
Publication No.
entirety. Thus, possible combinations include IL—2 and IL—15, IL—2 and IL—21, IL—15 and IL-
21 and IL-2, IL-lS and IL-21, with the latter finding particular use in many embodiments.
The use of combinations of cytokines specifically favors the generation of lymphocytes, and
in particular T—cells as described therein.
E. al Repeats of Step D: Second Expansion
In some embodiments, the second expansion is performed one or more times, 1.6.,
the second expansion is repeated. For example, in some embodiments the Step D second
expansion as indicated in Figure 11 is repeated one or more times. In some embodiments, the
second expansion is referred to as an onal second expansion. In some embodiments
where the second expansion is performed more than once (i.e., where the second expansion is
repeated), this can e procedures referred to as a TIL Rapid Expansion Protocol. In some
embodiments, the TIL cell population is expanded in number after harvest and first
expansion. This process is generally referred to in the art as a rapid expansion process (REP)
and the repeated second expansion can include expansion referred to as reREP. This overall
protocol can be generally accomplished using culture media comprising a number of
ents, including feeder cells, a cytokine source, and an anti-CD3 antibody, in a gas-
permeable container. In some embodiments, one or more subsequent second expansion(s) are
performed as described above. In some embodiments, one or more subsequent second
ions are performed as provided in under Step D in Figure 11 and prior to Step E as
provide in Figure 11. In some embodiments, one, two, three, four or more second expansions
are med as described above. In some ments, one, two, three, four or more
second expansions are performed as provided in Step D of Figure ll before Step E of Figure
11. In some embodiments, two second expansions are performed as described above. In some
embodiments, two second expansions are performed as provided in Step D of Figure 11
before Step E of Figure 11. In some embodiments, three second expansions are performed as
described above. In some embodiments, three second expansions are performed as ed
in Step D of Figure 11 before Step E of Figure 11. In some embodiments, four second
ions are performed as described above. In some embodiments, four second expansions
are performed as provided in Step D of Figure 11 before Step E of Figure 11.
In some ments, the repeat of the second expansion of the TILS (such as for
example in Step D of Figure 11) can be referred to as a restimulation of TILs. In some
embodiments, the t invention includes a restimulation step, i.e., a repeat of the second
expansion (for example, a repeat of the second expansion from Step D of Figure 11). In some
embodiments, the repeated second expansion (which can e an expansion referred to as
a restimulation step (“reRE ”)) is performed on cells that have been eserved. In some
embodiments, the TILS are eserved after Step D. In some embodiments, after an initial
second expansion in Step D,, the cells may be cultured in regular media, e.g. a “resting”
media, and then one or more second expansions steps are performed. In some embodiments,
the resting media comprises IL—2. In some embodiments, the resting media does not comprise
IL-2. In some embodiments, the g media is a standard cell culture media known in the
art. In some embodiments, the resting media is AHVI—V, DMEM, DMEM/F 12, MEM, RPMI,
OptilVIEM, IlVEDM, or any other standard media that is known in art, including commercially
available media. In some embodiments, the resting media is AIM-V.
In l, as discussed herein, the TILs are initially prepared by obtaining a
primary population of TILs from a tumor resected from a patient as discussed herein (the
“primary cell population” or “first cell population”). This is followed with an initial bulk
expansion utilizing a culturing of the cells with IL-2, forming a second population of cells
(sometimes referred to herein as the “bulk TIL population” or “second population”). In some
embodiments, this is also referred to as the initial or first expansion.
The bulk TIL population (for example, the population ed from for e
Step A in Figure 11) is then subjected to a REP step, sometimes referred to as a first
expansion (for example, the first expansion as described in Step B of Figure 11) in a cell
culture media comprising IL-2, OKT-3, and antigen presenting feeder cells (APCs), wherein
the APCs lly comprise peripheral blood mononuclear cells (PBMCs, or, atively
as discussed herein, using antigen presenting cells), wherein the rapid expansion (for
example, the second expansion as provide in Step D of Figure 11) is performed for at least 14
days. As discussed herein, the media may also contain combinations of IL-2, IL-15 and/or IL-
23 rather than IL-2 alone. In some embodiments, this post second ion (for example,
post Step D of Figure ll) expanded TIL population is at least 50 —fold or lOO-fold greater in
number than the second population of TILs (for example, the population of TILs obtained
from Step B of Figure 11). In some embodiments, the population of TILs obtained after the
second expansion in Step D of Figure ll are 50-fold or lOO-fold greater in number than the
TILs obtained from the first expansion in Step B of Figure 11. TILs are measured by cell
counting methods known in the art, including those methods described in the es
provided herewith, including Examples 1, 2, and 3. In some embodiments, a K2 cell counter
is employed to count the TILs. In some embodiments, a Cellometer 1C2 Image cytometer is
employed to count the TILs.
In some embodiments, as sed herein, the TIL tion obtained after the
second expansion (sometimes referred to as a third TIL population or a REP cell population)
is removed from the supplemented cell culture media (for example, the culture media used in
Step D of Figure 11 or the media referred to as CM2 in the Examples) and optionally
cryopreserved in a storage media (for example, media containing 5% DMSO) prior to
performing and additional second expansion step.
] Optionally, the TILs can be cryopreserved after a second expansion and before an
onal second expansion. In some embodiments, the TILs are cryopreserved after
performing Step D of Figure 11 and before performing an additional Step D of Figure 11. In
some embodiments, the cryopreserved TILs are thawed prior to performing the additional
second expansion. In some embodiments, the cryopreserved TILs are thawed prior to
performing the additional Step D as provided in Figure 11. In some embodiments, the TILs
are cryopreserved in 5% DMSO. In some embodiments, the TILs are cryopreserved in cell
culture media plus 5% DMSO. Alternatively, the cells are removed from the supplemented
cell culture media (for example, the culture media used in Step D of Figure 11) and ed
in a resting media. Such media include those that are described in Examples 1 and 5, as well
as the other Examples provided herewith. In some embodiments, resting media can include
media with IL—2. In some embodiments, the resting media can be the media referred to as
CMl in the examples.
The additional second expansion ding ions referred to as reREP) is
done on either the thawed cells or g cells, using a mented cell culture medium
(for e, a medium as provide in Step D of Figure 11) comprising IL-2, OKT-3, and
feeder cells (for example, antigen presenting cells), generally comprising peripheral blood
mononuclear cells (PBMCs; or, atively as discussed herein, using antigen presenting
cells), wherein the onal second expansion is performed for at least 14 days. As
discussed herein, the media may also contain combinations of IL-2, IL-15 and/or IL-23 rather
than IL-2 alone.
This results in an ed tion of TILs that are characterized in that these
expanded TILs exhibits an increased subpopulation of effector T cells and/or central memory
T cells relative to the second population of TILs (e.g., the bulk starting TILS). In some
embodiments, these expanded TILs are the TILs obtained from Step D of Figure 11.
In some embodiments the memory T cells are those cells that constitutively CCR7
and CD62L. See, Sallusto, el 51]., Annu. Rev. Immunol, 2004, -763, incorporated by
reference herein in its entirety.
Thus, the present invention provides methods for the restimulation of cryopreserved
TILs upon thawing, based on haw methods that result in increases of metabolic health
such as glycolysis and respiration. In some embodiments, method comprises providing a
tion of thawed cryopreserved TILs that are then treated to increase their metabolic
health to allow for l treatment upon infusion into patients.
F. STEP E: Harvest TILS from Step D
After the second expansion step, cells can be harvested. In some embodiments the
TILs are harvested after one, two, three, four or more second expansion steps. In some
embodiments, the TILs are harvested after one, two, three, four or more second expansion
steps according to Step D as provided in Figure ll.
TILs can be ted in any appropriate and sterile manner, including for example
by centrifugation. Methods for TIL harvesting are well known in the art and any such know
methods can be employed with the present process.
G. STEP F: Final Formulation and/or Transfer to on Bag
After Steps A through E as provided in an exemplary order in Figure 11 and as
ed in detailed above and herein are complete, cells are transferred to a container for use
in administration to a patient. In some embodiments, once a therapeutically sufficient number
of TILs are obtained using the expansion methods described above, they are transferred to a
container for use in administration to a t.
In an embodiment, TILs expanded using APCs of the present disclosure are
administered to a patient as a ceutical composition. In an embodiment, the
pharmaceutical composition is a suspension of TILs in a e buffer. TILs expanded using
PBMCs of the present sure may be administered by any suitable route as known in the
art. In some embodiments, the T-cells are administered as a single arterial or
intravenous infusion, which preferably lasts approximately 30 to 60 minutes. Other suitable
routes of administration e eritoneal, intrathecal, and intralymphatic.
1. Pharmaceutical Compositions, Dosages, and Dosing Regimens
In an embodiment, TlLs expanded using APCs of the present disclosure are
administered to a patient as a pharmaceutical composition. In an ment, the
pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs expanded using
PBMCs of the present disclosure may be administered by any suitable route as known in the
art. In some embodiments, the T-cells are stered as a single arterial or
intravenous infusion, which preferably lasts approximately 30 to 60 minutes. Other suitable
routes of administration include intrapelitoneal, intrathecal, and intralymphatic
administration.
Any suitable dose of TILs can be administered. In some embodiments, a
therapeutically sufficient number of TILs are needed for a suitable . In some
embodiments, from about 2.3 X 1010 to about 13.7X1010 TILs are administered, with an average
of around 7.8X1010 TILs, particularly if the cancer is melanoma. In an embodiment, about
1.2X 1010 to about 4.3X 1010 of TILs are administered. In some embodiments, about 3X 1010 to
about 12X1010 TILs are administered. In some embodiments, about 4X 1010 to about 10X 1010
TILs are administered. In some embodiments, about 5X 1010 to about 8X 1010 TILs are
administered. In some embodiments, about 6X 1010 to about 8X1010 TILs are administered. In
some embodiments, about 7X 1010 to about 8X1010 TILs are administered. In some
embodiments, the therapeutically effective dosage is about 2.3 X 1010 to about l3.7X1010. In
some embodiments, the therapeutically effective dosage is about 7 .8X 1010 TILs, particularly
of the cancer is melanoma. In some embodiments, the eutically effective dosage is
about 1.2X 1010 to about 4.3X1010 of TILs. In some embodiments, the therapeutically effective
dosage is about 3 X 1010 to about 12X 1010 TILs. In some ments, the therapeutically
effective dosage is about 4X 1010 to about 10X 1010 TILs. In some embodiments, the
therapeutically effective dosage is about 5X 1010 to about 8X1010 TILs. In some embodiments,
the therapeutically effective dosage is about 6>< 1010 to about 8X1010 TILs. In some
embodiments, the eutically effective dosage is about 7X 1010 to about 8X1010 TILs.
In some ments, the number of the TILs provided in the pharmaceutical
compositions ofthe ion is about 1X106, 2X106, 38106, 4><106, 5X106, 6X106, 7><106,
8><106,9X106,1><107,2X107,3X107,4X107,5><107,6X107,7X107,8X107,9><107,1X108,2X108,
3x108,4x108,5x108,6x108,7x108,8x103,9x108,1x109,2x109,3x109,4x109,5x109,6x109,
8X109,9X109,1><101°,2><1010,3X1010,4X1010,5X1010,6X1010,7X1010,8X1010,9X1010,
1x1011,2x1011,3x1011,4x1011,5x1011,6x1011,7x1011,8x1011,9x1011,1x1012,2x1012,
3x1012,4x1012,5x1012,6x1012,7x1012,8x1012,9x1012,1x1013,2x1013,3x1013,4x1013,
,6X1013, 7X 10”, 8X10”, and 9X10”. In an embodiment, the number ofthe TILs
provided in the pharmaceutical compositions of the invention is in the range of 1><106 to
5x106, 5x106 to 1x107, 1x107 to 5x107, 5x107 to 1x108, 1x108 to 5x108, 5x108 to 1x109,
1x109 to 5x109, 5x109 to 1x10“), 1x1010 to ,5><1010to1X1011,5X1011t01X1012,
1X1012 to 5X10”, and 5X1012 to 1X10”. In some embodiments, the therapeutically effective
dosage is about 1x106, 2x106, 3x106, 4x106, 5x106, 6X106, 7x106, 8x106, 9x106, 1x107,
2X107,3><107,4><107,5X107,6X107,7><107,8><107,9X107,1X108,2><108,3><103,4X108,5X108,
6X108,7X108,8X108,9X108,1X109,2X109,3X109,4X109,5X109,6X109,7X109,8X109,9X109,
1X1010,2X1010,3X1010,4X1010,5X1010,6X1010,7X1010,8X1010,9><1010,1X10“,2X10“,
3x1011,4x1011,5x1011,6x1011,7x1011,8x1011,9x1011, 1x1012,2x1012,3x1012,4x1012,
5X1012,6><1012,7><1012,8X1012,9X1012,1X1013,2X1013,3X1013,4X1013,5X1013,6X1013,
7><1013,8X1013,and 9x10”.
In some embodiments, the concentration of the TILs provided in the pharmaceutical
compositions of the invention is less than, for example, 100%, 90%, 80%, 70%, 60%, 50%,
40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%,
%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%,
0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%,
0.002%, 0.001%, 0.0009%, %, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%,
0,0002% or 0,0001% w/W, W/v or v/v of the pharmaceutical composition.
In some embodiments, the concentration of the TILs provided in the pharmaceutical
compositions of the invention is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%,
19.75%, 19.50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25%
17%, 16.75%, , 16.25% 16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%,
14.25% 14%, 13.75%, 13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, ,
11.50%, 11.25% 11%, 10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%,
8.50%, 8.25% 8%, 7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5,75%, 5.50%,
.25% 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%,
2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%,
0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%,
0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, %,
0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v, or v/v of the pharmaceutical
composition.
In some ments, the concentration of the TILs provided in the pharmaceutical
itions of the invention is in the range from about % to about 50%, about
0.001% to about 40%, about 0.01% to about 30%, about 0.02% to about 29%, about 0.03% to
about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about
%, about 0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%,
about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to
about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%,
about 0.8% to about 14%, about 0.9% to about 12% or about 1% to about 10% W/w, w/v or
v/v of the pharmaceutical composition.
In some embodiments, the concentration of the TILs provided in the pharmaceutical
compositions of the invention is in the range from about 0.001% to about 10%, about 0.01%
to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about
3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about
0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% W/W, W/v or v/v of
the pharmaceutical composition.
In some embodiments, the amount of the TILs provided in the pharmaceutical
compositions ofthe invention is equal to or less than 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0
g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g,
0.85 g, 08 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 04 g, 0.35 g, 0.3 g, 0.25 g, 0.2
g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g,
0.008 g, 0.007 g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g, 0.0008 g,
0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g, or 0.0001 g.
In some embodiments, the amount of the TILs provided in the pharmaceutical
compositions of the invention is more than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g,
0.0006 g, 0.0007 g, 0.0008 g, 0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0,003 g, 0.0035
g, 0.004 g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085
g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g,
0.055 g, 0.06 g, 0.065 g, 0.07 g, 0,075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 02 g,
0.25 g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, 0.5 g, 0.55 g, 0.6 g, 0.65 g, 0.7 g, 0.75 g, 0.8 g, 0.85 g, 0.9
g, 0.95 g, lg, 1.5 g, 2 g, 2.5, 3 g, 3.5, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9
g, 9.5 g, or 10 g.
] The TILs ed in the pharmaceutical compositions of the invention are effective
over a wide dosage range. The exact dosage will depend upon the route of administration, the
form in which the compound is administered, the gender and age of the t to be treated,
the body weight of the subject to be treated, and the ence and experience of the
attending physician. The clinically-established dosages of the TILs may also be used if
appropriate. The amounts of the pharmaceutical compositions administered using the
methods herein, such as the dosages of TILs, will be dependent on the human or mammal
being treated, the severity of the disorder or condition, the rate of administration, the
disposition of the active pharmaceutical ingredients and the discretion of the prescribing
physician.
In some embodiments, TILs may be administered in a single dose. Such
administration may be by injection, e.g, intravenous injection. In some embodiments, TILs
may be administered in multiple doses. Dosing may be once, twice, three times, four times,
five times, six times, or more than six times per year. Dosing may be once a month, once
every two weeks, once a week, or once every other day. Administration of TILs may continue
as long as necessary.
In some embodiments, an effective dosage of TILs is about 1X106, 2><106, 3X106,
4X106,5X106,6X106,7><106,8X106,9X106,1X107,2><107,3X107,4X107,5X107,6><107,7X107,
9x107,1x108,2x108,3x108,4x108,5x108,6><108,7x108,8x108,9x108,1x109,2x109,
3x109,4x109,5x109,6x109,7x109,8x109,9x109,1x101°,2x1010,3x1010,4x1010,5x1010,
6X1010,7X1010, 8X1010,9X1010, 1x1011,2x1011,3x1011,4x1011, 5x1011,6x1011,7x1011,
8x1011,9x1011, 1x1012,2x1012,3x1012,4x1012,5x1012,6x1012,7x1011,8x1012,9x1012,
1x1013,2x1013,3x1013,4x1013, 5x1013,6x1013,7x1013, 8><1013,and 9x1013.1n some
ments, an effective dosage of TILs is in the range of 1X106 to 5X106, 5>< 106 to 1X107,
1x107 to 5x107, 5x107 to 1x108, 1x108 to 5x108, 5x108 to 1x109, 1x109 to 5x109, 5x109 to
1x1010,1x1010to 5x10“), 5x1010 to 1x10“, 5x1011 to 1x10”, 1x1012 to 5x10”, and 5x1012
to 1x10”.
] In some embodiments, an effective dosage of TILs is in the range of about 0.01
mg/kg to about 4.3 mg/kg, about 0.15 mg/kg to about 3.6 mg/kg, about 0.3 mg/kg to about
3.2 mg/kg, about 0.35 mg/kg to about 2.85 mg/kg, about 0.15 mg/kg to about 2.85 mg/kg,
about 0.3 mg to about 2.15 mg/kg, about 0.45 mg/kg to about 1.7 mg/kg, about 0.15 mg/kg to
about 1.3 mg/kg, about 0.3 mg/kg to about 1.15 mg/kg, about 0.45 mg/kg to about 1 mg/kg,
about 0.55 mg/kg to about 0.85 mg/kg, about 0.65 mg/kg to about 0.8 mg/kg, about 0.7
mg/kg to about 0.75 mg/kg, about 0.7 mg/kg to about 2.15 mg/kg, about 0.85 mg/kg to about
2 mg/kg, about 1 mg/kg to about 1.85 mg/kg, about 1.15 mg/kg to about 1.7 mg/kg, about 1.3
mg/kg mg to about 1.6 mg/kg, about 1.35 mg/kg to about 1.5 mg/kg, about 2.15 mg/kg to
about 3.6 mg/kg, about 2.3 mg/kg to about 3.4 mg/kg, about 2.4 mg/kg to about 3.3 mg/kg,
about 2.6 mg/kg to about 3.15 mg/kg, about 2.7 mg/kg to about 3 mg/kg, about 2.8 mg/kg to
about 3 mg/kg, or about 2.85 mg/kg to about 2.95 mg/kg.
In some embodiments, an effective dosage of TILs is in the range of about 1 mg to
about 500 mg, about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 25 mg to
about 200 mg, about 1 mg to about 50 mg, about 5 mg to about 45 mg, about 10 mg to about
40 mg, about 15 mg to about 35 mg, about 20 mg to about 30 mg, about 23 mg to about 28
mg, about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 70 mg to about 130
mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg, or about 95 mg to about
105 mg, about 98 mg to about 102 mg, about 150 mg to about 250 mg, about 160 mg to about
240 mg, about 170 mg to about 230 mg, about 180 mg to about 220 mg, about 190 mg to
about 210 mg, about 195 mg to about 205 mg, or about 198 to about 207 mg.
An effective amount of the TILs may be administered in either single or le
doses by any of the accepted modes of administration of agents having similar utilities,
including intranasal and ermal routes, by intra-arterial injection, intravenously,
intraperitoneally, parenterally, intramuscularly, subcutaneously, topically, by transplantation,
or by inhalation.
H. Optional Cell Viability Analyses
Optionally, a cell viability assay can be performed after the Step B first expansion,
using standard assays known in the art. For example, a trypan blue exclusion assay can be
done on a sample of the bulk TILs, which ively labels dead cells and allows a viability
assessment. Other assays for use in g viability can include but are not limited to the
Alamar blue assay, and the MTT assay.
1. Cell Counts, Viability, Flow Cytometry
In some ments, cell counts and/or viability are measured. The expression of
markers such as but not limited CD3, CD4, CD8, and CD56, as well as any other disclosed or
described herein, can be measured by flow cytometry with antibodies, for example but not
limited to those commercially available from BD Bio-sciences (BD Biosciences, San Jose,
CA) using a FACSCantoTM flow cytometer (BD Biosciences). The cells can be counted
ly using a disposable c-chip hemocytometer (VWR, Batavia, IL) and viability can be
assessed using any method known in the art, ing but not limited to trypan blue ng.
In some cases, the bulk TIL population can be cryopreserved immediately, using the
protocols discussed below. Alternatively, the bulk TIL population can be subjected to REP
and then cryopreserved as discussed below. Similarly, in the case where cally modified
TILs will be used in therapy, the bulk or REP TIL populations can be subjected to genetic
modifications for suitable treatments.
2, Cell Cultures
In an embodiment, a method for expanding TILs may include using about 5,000 mL
to about 25,000 mL of cell medium, about 5,000 mL to about 10,000 mL of cell medium, or
about 5,800 mL to about 8,700 mL of cell medium, In an ment, expanding the number
of TILs uses no more than one type of cell culture medium. Any suitable cell culture medium
may be used, e. g., AIM-V cell medium (L-glutamine, 50 uM streptomycin sulfate, and 10 [1M
gentamicin sulfate) cell culture medium (Invitrogen, Carlsbad CA), In this regard, the
inventive methods advantageously reduce the amount of medium and the number of types of
medium required to expand the number of TIL. In an embodiment, expanding the number of
TIL may comprise adding fresh cell culture media to the cells (also referred to as feeding the
cells) no more frequently than every third or fourth day. ing the number of cells in a
gas ble container simplifies the procedures necessary to expand the number of cells by
reducing the feeding frequency necessary to expand the cells.
In an embodiment, the cell medium in the first and/or second gas permeable
container is unfiltered. The use of ered cell medium may simplify the procedures
WO 81473
ary to expand the number of cells. In an embodiment, the cell medium in the first
and/or second gas permeable container lacks beta-mercaptoethanol (BME).
In an embodiment, the duration of the method comprising obtaining a tumor tissue
sample from the mammal; culturing the tumor tissue sample in a first gas permeable
ner containing cell medium therein; obtaining TILs from the tumor tissue sample;
expanding the number of TILs in a second gas permeable container containing cell medium
therein using aAPCs for a duration of about 14 to about 42 days, e.g, about 28 days.
] In an embodiment, TILs are expanded in gas-permeable containers. Gas-permeable
containers have been used to expand TILs using PBMCs using methods, compositions, and
devices known in the art, including those described in US. Patent Application Publication
No. 106717 Al, the disclosures of which are incorporated herein by reference. In an
embodiment, TILs are expanded in gas—permeable bags. In an embodiment, TILs are
expanded using a cell ion system that expands TILs in gas permeable bags, such as the
Xuri Cell Expansion System W25 (GE Healthcare). In an embodiment, TILs are expanded
using a cell expansion system that expands TILs in gas permeable bags, such as the WAVE
Bioreactor System, also known as the Xuri Cell ion System W5 (GE Healthcare). In
an embodiment, the cell expansion system includes a gas permeable cell bag with a volume
selected from the group consisting of about 100 mL, about 200 mL, about 300 mL, about 400
mL, about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L,
about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, and
about 10 L. In an embodiment, TILs can be expanded in G—Rex flasks (commercially
available from Wilson Wolf Manufacturing). Such embodiments allow for cell populations to
expand from about 5x105 cells/cm2 to between 10x106 and 30x106 cells/cmz. In an
embodiment this expansion is conducted without adding fresh cell culture media to the cells
(also referred to as feeding the cells). In an embodiment, this is t feeding so long as
medium resides at a height of about 10 cm in the GRex flask. In an embodiment this is
t feeding but with the addition of one or more cytokines. In an embodiment, the
cytokine can be added as a bolus without any need to mix the ne with the medium.
Such containers, devices, and methods are known in the art and have been used to expand
TILs, and e those described in US. Patent Application Publication No. US
2014/0377739Al, International ation No.
Application Publication No. us 2013/0115617 A1, International Publication No. WO
2013/188427 Al, U. S. Patent Application Publication No. US 2011/0136228 A1, U. S. Patent
WO 81473
No. US 8,809,050 B2, International publication No.
Application Publication No. US 2016/0208216 A1, US. Patent Application Publication No.
US 2012/0244133 A1, International Publication No.
ation Publication No. US 2013/0102075 A1, US. Patent No. US 8,956,860 B2,
International Publication No.
US 2015/0175966 Al, the disclosures of which are incorporated herein by reference. Such
processes are also described in Jin er al., J. therapy, 2012, 35:283-292. Optional
Genetic Engineering of TILs
In some embodiments, the TILs are optionally genetically engineered to include
additional functionalities, including, but not limited to, a ffinity T cell receptor (TCR),
e.g., a TCR targeted at a tumor-associated antigen such as MAGE-l, HER2, or NY-ESO-l, or
a chimeric antigen receptor (CAR) which binds to a tumor-associated cell surface molecule
(e.g., mesothelin) or lineage—restricted cell surface molecule (e.g., CD19).
I. Optional Cryopreservation of TILs
As discussed above in Steps A through E, cryopreservation can occur at numerous
points throughout the TIL expansion process. In some embodiments, the bulk TIL population
after the first expansion according to Step B or the expanded tion of TILs after the one
or more second expansions according to Step D can be cryopreserved. eservation can
be generally accomplished by g the TIL population into a freezing solution, e.g., 85%
complement inactivated AB serum and 15% dimethyl sulfoxide (DMSO). The cells in
solution are placed into cryogenic vials and stored for 24 hours at -80 CC, with al
transfer to gaseous nitrogen freezers for cryopreservation. See, Sadeghi, er al., Acta
Oncologica 2013, 52, 978-986. In some ments, the TILs are cryopreserved in 5%
DMSO. In some embodiments, the TILs are cryopreserved in cell culture media plus 5%
DMSO. In some embodiments, the TILs are cryopreserved according to the s
provided in Examples 8 and 9.
] When appropriate, the cells are removed from the r and thawed in a 37 °C
water bath until approximately 4/5 of the solution is thawed. The cells are generally
resuspended in complete media and optionally washed one or more times. In some
embodiments, the thawed TILs can be counted and assessed for viability as is known in the
J. Phenotypic Characteristics of Expanded TILs
In some embodiment, the TILs are analyzed for expression of numerous phenotype
markers after expansion, including those described herein and in the Examples. In an
embodiment, sion of one or more phenotypic markers is examined. In some
embodiments, the phenotypic characteristics of the TILs are analyzed after the first expansion
in Step B. In some embodiments, the phenotypic characteristics of the TILs are analyzed
during the transition in Step C. In some embodiments, the phenotypic characteristics of the
TILs are analyzed during the tion according to Step C and after cryopreservation. In
some embodiments, the phenotypic characteristics of the TILs are analyzed after the second
expansion ing to Step D, In some embodiments, the ypic characteristics of the
TILs are analyzed after two or more expansions according to Step D. In some embodiments,
the marker is selected from the group ting of TCRab, CD57, CD28, CD4, CD27
CD56, CD8a, CD45RA, CD8a, CCR7, CD4, CD3, CD38, and HLA-DR. In some
embodiments, the marker is selected from the group consisting of TCRab, CD57, CD28,
CD4, CD27, CD56, and CD8a. In an ment, the marker is selected from the group
consisting of CD45RA, CD8a, CCR7, CD4, CD3, CD3 8, and HLA-DR. In some
embodiments, expression of one, two, three, four, five, six, seven, eight, nine, ten, eleven,
twelve, thirteen, or fourteen markers is examined. In some embodiments, the expression from
one or more markers from each group is examined. In some embodiments, one or more of
HLA-DR, CD3 8, and CD69 expression is maintained (i.e., does not exhibit a statistically
significant difference) in fresh TILs as compared to thawed TILs. In some embodiments, the
activation status of TILs is maintained in the thawed TILs.
] In an embodiment, sion of one or more regulatory markers is ed. In
some embodiments, the regulatory marker is ed from the group ting of CD137,
CD8a, Lag3, CD4, CD3,PD1, TIM-3, CD69, CD8a, TIGIT, CD4, CD3,KLRG1, and
CD154. In some embodiments, the regulatory marker is selected from the group consisting of
CD137, CD8a, Lag3, CD4, CD3, PD], and THVI-3. In some embodiments, the regulatory
marker is selected from the group consisting of CD69, CD8a, TIGIT, CD4, CD3, KLRGl,
and CD154. In some embodiments, regulatory molecule expression is decreased in thawed
TILs as compared to fresh TILs. In some embodiments, expression of regulatory molecules
LAG-3 and TIM-3 is decreased in thawed TILs as compared to fresh TILs. In some
embodiments, there is no significant difference in CD4, CD8, NK, TCRdB expression. In
some embodiments, there is no significant difference in CD4, CD8, NK, TCROLB expression,
and/or memory s in fresh TILs as compared to thawed TILs.
In some ments the memory marker is selected from the group consisting of
CCR7 and CD62L
In some embodiments, the viability of the fresh TILs as compared to the thawed
TILs is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at
least 98%. In some embodiments, the viability of both the fresh and thawed TILs is greater
than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater
than 95%, or greater than 98%. In some embodiments, the viability of both the fresh and
thawed product is greater than 80%, greater than 81%, greater than 82%, greater than 83%,
greater than 84%, r than 85%, greater than 86%, r than 87%, greater than 88%,
greater than 89%, or greater than 90%. In some embodiments, the viability of both the fresh
and thawed product is greater than 86%.
In an embodiment, restimulated TILs can also be evaluated for cytokine release,
using cytokine release assays. In some embodiments, TILs can be evaluated for eron-7
(LEN-7) ion in response to stimulation either with OKT3 or co—culture with autologous
tumor digest. For example, in embodiments employing OKT3 stimulation, TILs are washed
extensively, and duplicate wells are prepared with 1 x 105 cells in 0.2 mL CM in 96-well flat-
bottom plates precoated with 0.1 or 1.0 ug/mL of OKT3 diluted in phosphate-buffered saline.
After overnight incubation, the supernatants are harvested and [EN—7 in the supernatant is
measured by ELISA (Pierce/Endogen, Woburn, MA). For the co-culture assay, 1 x 105 TIL
cells are placed into a 96-well plate with autologous tumor cells. (1 :1 ratio). After a 24-hour
incubation, supernatants are harvested and [EN-7 release can be quantified, for example by
ELISA.
Flow cytometric analysis of cell surface biomarkers: TIL samples were aliquoted for
flow cytometric analysis of cell surface s see, for Example see Examples 7, 8, and 9.
In some embodiments, the TILs are being evaluated for various regulatory markers.
In some embodiments, the regulatory marker is selected from the group consisting of TCR
U/B, CD56, CD27, CD28, CD57, CD45RA, CD45RO, CD25, CD127, CD95, IL-2R—, CCR7,
CD62L, KLRGl, and CD122. In some embodiments, the regulatory marker is TCR OL/B. In
some embodiments, the tory marker is CD56. In some embodiments, the tory
marker is CD27. In some ments, the regulatory marker is CD28. In some
ments, the regulatory marker is CD57. In some embodiments, the regulatory marker is
CD45RA. In some ments, the regulatory marker is . In some embodiments,
the regulatory marker is CD25. In some embodiments, the regulatory marker is CD127. In
some embodiments, the regulatory marker is CD95. In some embodiments, the regulatory
marker is IL-2R—. In some embodiments, the regulatory marker is CCR7. In some
embodiments, the regulatory marker is CD62L. In some embodiments, the regulatory marker
is KLRGl. In some embodiments, the regulatory marker is CD122.
K. Metabolic Health of Expanded TILs
The ulated TILs are characterized by significant enhancement of basal
glycolysis as compared to either freshly harvested TILs and/or post-thawed TILs.
Spare respiratory capacity (SRC) and glycolytic reserve can be evaluated for TILs
expanded with aEM3 aAPCs in comparison to PBMC feeders. The Seahorse XF Cell Mito
Stress Test measures mitochondrial function by directly measuring the oxygen consumption
rate (OCR) of cells, using modulators of respiration that target components of the electron
transport chain in the mitochondria. The test compounds mycin, FCCP, and a mix of
rotenone and antimycin A, described below) are ly injected to measure ATP production,
maximal respiration, and non-mitochondrial respiration, respectively. Proton leak and spare
respiratory capacity are then calculated using these parameters and basal ation. Each
modulator s a specific component of the electron transport chain. Oligomycin inhibits
ATP synthase (complex V) and the decrease in OCR following injection of oligomycin
correlates to the mitochondrial respiration associated with cellular ATP production. Carbonyl
cyanide—4 (trifluoromethoxy) phenylhydrazone (FCCP) is an uncoupling agent that ses
the proton gradient and disrupts the mitochondrial membrane potential. As a result, electron
flow through the electron ort chain is bited and oxygen is maximally consumed
by complex IV. The FCCP—stimulated OCR can then be used to calculate spare respiratory
capacity, defined as the difference n maximal respiration and basal respiration. Spare
respiratory ty (SRC) is a measure of the ability of the cell to respond to increased
energy demand. The third injection is a mix of rotenone, a complex I inhibitor, and antimycin
A, a complex III tor. This combination shuts down mitochondrial respiration and
enables the calculation of nonmitochondrial respiration driven by processes outside the
mitochondria.
In some embodiments, the metabolic assay is basal respiration. In general, second
expansion TILs or second additional ion TILs (such as, for example, those described
in Step D of Figure 11, including TILs referred to as reREP TILs) have a basal respiration
rate that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%
of the basal respiration rate of freshly harvested TILs. In some embodiments, the basal
respiration rate is from about 50% to about 99% of the basal respiration rate of freshly
harvested TILs. In some ments, the basal respiration rate is from about 60% to about
99% of the basal respiration rate of freshly ted TILs. In some embodiments, the basal
respiration rate is from about 70% to about 99% of the basal respiration rate of freshly
harvested TILs. In some embodiments, the basal respiration rate is from about 80% to about
99% of the basal ation rate of freshly harvested TILs. In some embodiments, the basal
respiration rate is from about 90% to about 99% of the basal respiration rate of freshly
ted TILs. In some embodiments, the basal respiration rate is from about 95% to about
99% of the basal respiration rate of y harvested TILs. In some embodiments, the second
expansion or second additional expansion TILs (such as, for example, those described in Step
D of Figure ll, including TILs referred to as reREP TILs) have a basal respiration rate that is
not statistically cantly different than the basal respiration rate of freshly harvested
TILs.
In general, second expansion TILs or onal second expansion TILs, such as
those in Step D ding, for example, TILs referred to as reREP which have one an
additional second expansion) TILs have a spare respiratory capacity that is at least is at least
50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the basal
respiration rate of freshly harvested TILs. In some embodiments, the spare respiratory
capacity is from about 50% to about 99% of the basal respiration rate of freshly harvested
TILs. In some embodiments, the spare respiratory capacity is from about 50% to about 99%
of the basal respiration rate of freshly harvested TILs. In some embodiments, the spare
respiratory capacity is from about 60% to about 99% of the basal respiration rate of freshly
harvested TILs. In some embodiments, the spare respiratory ty is from about 70% to
about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the
spare respiratory capacity is from ab out 80% to about 99% of the basal respiration rate of
freshly harvested TILs. In some embodiments, the spare respiratory capacity is from about
90% to about 99% of the basal respiration rate of freshly harvested TILs. In some
embodiments, the spare respiratory capacity is from about 95% to about 99% of the basal
respiration rate of freshly harvested TlLs. In some embodiments, the second expansion TILs
or second onal expansion TILs (such as, for example, those described in Step D of
Figure ll, including TILs referred to as reREP TILs) have a spare respiratory ty that is
not statistically significantly different than the basal respiration rate of freshly harvested
TlLs.
In l, the second expansion TlLs or second additional expansion TlLs (such
as, for example, those described in Step D of Figure 11, including TILs referred to as reREP
TILs) have a spare respiratory capacity that is at least is at least 50%, at least 55%, at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 97%, at least 98%, or at least 99% of the basal respiration rate of freshly
harvested TILs. In some embodiments, the metabolic assay measured is glycolytic reserve. In
some embodiments, the metabolic assay is glycolytic reserve. In some embodiments, the
metabolic assay is spare respiratory ty. To measure cellular (respiratory) metabolism
cells were treated with inhibitors of mitochondrial respiration and glycolysis to determine a
metabolic profile for the TIL consisting of the following measures: baseline oxidative
phosphorylation (as measured by OCR), spare respiratory capacity, ne glycolytic
activity (as ed by ECAR), and glycolytic reserve. Metabolic profiles were performed
using the Seahorse Combination Mitochondrial/Glycolysis Stress Test Assay (including the
kit commercially available from Agilent®), which allows for determining a cells’ capacity to
perform glycolysis upon blockage of mitochondrial ATP production. In some embodiments,
cells are d of glucose, then glucose is injected, followed by a stress agent. In some
embodiments, the stress agent is selected from the group consisting of oligomycin, FCCP,
rotenone, antimycin A and/or 2-deoxyglucose (2-DG), as well as ations thereof. In
some embodiments, ycin is added at 10 mM. In some embodiments, FCCP is added at
mM. In some embodiments, rotenone is added at 2.5 mM. In some embodiments,
antimycin A is added at 2.5 mM. In some ments, 2—deoxyglucose (2-DG) is added at
500 mM. In some embodiments, glycolytic capacity, glycolytic reserve, and/or non-glycolytic
acidification are measured. In general, TILs have a ytic reserve that is at least 50%, at
least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the basal respiration
rate of freshly harvested TILs. In some embodiments, the glycolytic reserve is from about
50% to about 99% of the basal respiration rate of y harvested TILs. In some
embodiments, the glycolytic reserve is from about 60% to about 99% of the basal respiration
rate of freshly harvested TILs. In some embodiments, the glycolytic reserve is from about
70% to about 99% of the basal respiration rate of freshly harvested TILs. In some
embodiments, the glycolytic reserve is from about 80% to about 99% of the basal respiration
rate of freshly harvested TILs. In some embodiments, the glycolytic reserve is from about
90% to about 99% of the basal respiration rate of freshly harvested TILs. In some
ments, the glycolytic reserve is from about 95% to about 99% of the basal respiration
rate of freshly harvested TILs.
In some embodiments, the lic assay is basal glycolysis. In some
embodiments second expansion TILs or additional second expansion TILs, such as those in
Step D (including, for example, TILs referred to as reREP which have undergone an
additional second expansion) have an increase in basal glycolysis of at least two-fold, at least
three-fold, at least four-fold, at least five-fold, at least six-fold, at least 7-fold, at least eight-
fold, at least nine—fold, or at least ten—fold. In some embodiments, the second expansion TILs
or additional second ion, such as those in Step D (including TILs referred to as reREP
TILs) have an se in basal glycolysis of about two-fold to about ten-fold. In some
embodiments, the second expansion TILs or onal second expansion, such as those in
Step D ding TILs referred to as reREP TILs) have an increase in basal ysis of
about two-fold to about eight-fold. In some embodiments, the second expansion TILs or
additional second expansion, such as those in Step D ding TILs ed to as reREP
TILs) have an increase in basal glycolysis of about three-fold to about seven-fold. In some
embodiments, the second expansion TILs or additional second expansion, such as those in
Step D (including TILs referred to as reREP TILs) have an increase in basal glycolysis of
about two-fold to about four-fold. In some embodiments, the second expansion TILs or
onal second expansion, such as those in Step D (including TILs referred to as reREP
TILs) have an increase in basal glycolysis of about two—fold to about three—fold.
In general, second expansion TILs or additional second expansion, such as those in
Step D (including, for example, TILs referred to as reREP which have undergone an
additional second expansion) TILs have a glycolytic reserve that is at least 50%, at least 55%,
at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at least 97%, at least 98%, or at least 99% of the basal ation rate of
freshly harvested TILs. In some embodiments, the glycolytic reserve is from about 50% to
about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the
glycolytic reserve is from about 60% to about 99% of the basal respiration rate of freshly
WO 81473
harvested TILs. In some embodiments, the glycolytic reserve is from about 70% to about
99% of the basal respiration rate of freshly ted TILs. In some embodiments, the
glycolytic reserve is from about 80% to about 99% of the basal respiration rate of freshly
harvested TILs. In some embodiments, the glycolytic reserve is from about 90% to about
99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the
glycolytic e is from about 95% to about 99% of the basal respiration rate of freshly
harvested TILs. In some embodiments, the second expansion TILs or second additional
expansion TILs (such as, for example, those described in Step D of Figure 11, including TILs
referred to as reREP TILs) have a spare respiratory capacity that is not statistically
significantly different than the basal respiration rate of freshly harvested TILs.
] Granzyme B Production: Granzyme B is another measure of the ability of TIL to
kill target cells. Media supernatants restimulated as described above using antibodies to CD3,
CD28, and CD137/4—1BB were also evaluated for their levels of Granzyme B using the
Human Granzyme B DuoSet ELISA Kit (R & D Systems, Minneapolis, MN) according to the
manufacturer’s instructions. In some embodiments, the second expansion TILs or second
additional expansion TILs (such as, for e, those described in Step D of Figure 11,
including TILs referred to as reREP TILs) have increased Granzyme B production. In some
embodiments, the second expansion TILs or second additional expansion TILs (such as, for
example, those bed in Step D of Figure 11, including TILs ed to as reREP T]Ls)
have increased cytotoxic activity.
In some embodiments, the present methods include an assay for assessing TIL
viability, using the methods as described above. In some embodiments, the TILs are
expanded as discussed above, including for example as ed in Figure 11. In some
embodiments, the TILs are cryopreserved prior to being assessed for viability. In some
embodiments, the viability ment includes thawing the TILs prior to performing a first
expansion, a second expansion, and an additional second expansion. In some embodiments,
the present methods provide an assay for assessing cell proliferation, cell toxicity, cell death,
and/or other terms related to viability of the TIL population. Viability can be measured by
any of the TIL metabolic assays described above as well as any methods know for assessing
cell viability that are known in the art. In some ments, the t methods provide as
assay for assessment of cell proliferation, cell toxicity, cell death, and/or other terms d
to viability of the TILs expanded using the s described herein, including those
exemplified in Figure ll.
The present invention also es assay methods for determining TIL viability.
The present disclosure provides methods for assaying TILs for viability by expanding tumor
infiltrating lymphocytes (TILs) into a larger population of TILs sing:
(i) obtaining a first population of TILs which has been previously expanded;
(ii) ming a first expansion by culturing the first population of TILs in a cell
culture medium comprising IL-2 to produce a second population of TILs, and
(iii) performing a second expansion by supplementing the cell culture medium of the
second population of TILs with additional IL-2, OKT-3, and antigen presenting cells
(APCs), to produce a third population of TILs, wherein the third population of TILs is
at least 50-fold or lOO-fold greater in number than the second population of TILs, and
wherein the second expansion is performed for at least 14 days in order to obtain the
third population of TILs, wherein the third population of TILs ses an increased
subpopulation of effector T cells and/or central memory T cells relative to the second
population of TILs, and wherein the third population is further assayed for viability.
In some embodiments, the method further comprises:
(iv) ming an additional second expansion by supplementing the cell e
medium of the third population of TILs with additional IL-2, additional OKT-3, and
additional APCs, wherein the additional second expansion is performed for at least 14
days to obtain a larger population of TILs than obtained in step (iii), wherein the
larger tion of TILs comprises an increased subpopulation of effector T cells
and/or central memory T cells relative to the third population of TILs, and wherein
the third population is further assayed for viability.
In some ments, prior to step (i), the cells are cryopreserved.
In some embodiments, the cells are thawed prior to performing step (i).
In some embodiments, step (iv) is ed one to four times in order to obtain
sufficient TILs for analysis.
In some ments, steps (i) h (iii) or (iv) are performed within a period of
about 40 days to about 50 days.
In some embodiments, steps (i) through (iii) or (iv) are performed within a period of
about 42 days to about 48 days.
WO 81473
In some embodiments, steps (i) through (iii) or (iv) are performed within a period of
about 42 days to about 45 days.
In some embodiments, steps (i) through (iii) or (iv) are performed within about 44
days.
In some embodiments, the cells from steps (iii) or (iv) express CD4, CD8, and TCR
0t [3 at levels similar to freshly harvested cells.
In some embodiments, the antigen presenting cells are peripheral blood
clear cells (PBMCs).
In some embodiments, the PBMCS are added to the cell culture on any of days 9
through 17 in step (iii).
] In some embodiments, the or T cells and/or central memory T cells in the
larger population of TILs in step (iv) exhibit one or more characteristics selected from the
group consisting of expression of CD27, expression of CD28, longer telomeres, increased
CD57 expression, and decreased CD56 expression, ve to effector T cells, and/or central
memory T cells in the third population of cells.
In some embodiments, the effector T cells and/or central memory T cells exhibit
increased CD57 sion and decreased CD56 expression.
In some embodiments, the APCS are artificial APCs (aAPCs).
In some embodiments, the method further comprises the step of transducing the first
tion of TILs with an expression vector comprising a nucleic acid encoding a high-
y T cell receptor.
In some embodiments, the step of transducing occurs before step (i).
In some embodiments, the method further ses the step of transducing the first
population of TILs with an expression vector comprising a nucleic acid encoding a chimeric
antigen receptor (CAR) comprising a single chain le fragment antibody fused with at
least one endodomain of a T-cell signaling molecule.
In some embodiments, the step of transducing occurs before step (i).
In some embodiments, the TILs are assayed for viability.
In some embodiments, the TILs are assayed for viability after cryopreservation.
In some embodiments, the TILs are d for viability after cryopreservation and
after step (iv).
According to the present disclosure, a method for assaying TILs for viability and/or
further use in administration to a subject. In some embodiments, the method for assay tumor
infiltratitng lymphocytes (TILs) comprises:
(i) obtaining a first population of TILs;
(ii) performing a first expansion by culturing the first population of TILs in a cell
culture medium comprising IL-2 to produce a second population of TILs, and
(iii) performing a second expansion by supplementing the cell culture medium of the
second population of TILs with additional IL-2, OKT-3, and antigen presenting cells
(APCs), to produce a third population of TILs, wherein the third population of TILs is
at least d r in number than the second population of TILs;
(iv) harvesting, washing, and cryopreserving the third tion of TILs;
(v) g the cryopreserved TILs at a cryogenic temperature,
(vi) thawing the third population of TILs to provide a thawed third population of
TILs, and
(vii) performing an additional second expansion of a portion of the thawed third
population of TILs by supplementing the cell culture medium of the third population
with IL—2, OKT-3, and APCs for a reREP period of at least 3 days, wherein the third
expansion is performed to obtain a fourth population of TILs, wherein the number of
TILs in the fourth population of TILs is compared to the number of TILs in the third
population of TILs to obtain a ratio,
(viii) ining based on the ratio in step (vii) whether the thawed tion of
TILs is suitable for administration to a patient,
(ix) administering a therapeutically effective dosage of the thawed third tion of
TILs to the patient when the ratio of the number of TILs in the fourth population of
TILs to the number of TILs in the third population of TILs is determined to be greater
than 5:1 in step (viii).
In some ments, the reREP period is performed until the ratio of the number
of TILs in the fourth population of TILs to the number of TILs in the third population of TILs
is greater than 50:1.
In some embodiments, the number of TILs sufficient for a therapeutically effective
dosage is from about 2.3X1010 to about 13.7X1010.
In some embodiments, steps (i) through (vii) are performed within a period of about
40 days to about 50 days. In some embodiments, steps (i) through (vii) are performed within
a period of about 42 days to about 48 days. In some embodiments, steps (i) through (vii) are
performed within a period of about 42 days to about 45 days. In some embodiments, steps (i)
through (vii) are performed within about 44 days.
In some embodiments, the cells from steps (iii) or (vii) express CD4, CD8, and TCR
0t [3 at levels similar to y harvested cells. In some embodiments the cells are TILs.
In some embodiments, the antigen presenting cells are peripheral blood
mononuclear cells (PBMCs). In some embodiments, the PBMCs are added to the cell e
on any of days 9 through 17 in step (iii).
In some embodiments, the effector T cells and/or central memory T cells in the
larger population of TILs in steps (iii) or (vii) exhibit one or more characteristics selected
from the group consisting of expression of CD27, expression of CD28, longer telomeres,
increased CD57 sion, and decreased CD56 expression, relative to effector T cells,
and/or central memory T cells in the third population of cells.
In some ments, the effector T cells and/or central memory T cells exhibit
increased CD57 sion and decreased CD56 sion.
In some embodiments, the APCs are artificial APCs (aAPCs).
In some embodiments, the step of transducing the first population of TILs with an
expression vector comprising a nucleic acid encoding a high-affinity T cell receptor.
In some embodiments, the step of transducing occurs before step (i).
In some embodiments, the step of transducing the first population of TILs with an
expression vector comprising a nucleic acid encoding a ic antigen receptor (CAR)
comprising a single chain variable fragment antibody fused with at least one endodomain of a
T-cell ing le.
In some embodiments, the step of ucing occurs before step (i).
In some ments, the TILs are assayed for viability after step (vii).
] The present disclosure also provides further methods for assaying TILs. In some
embodiments, the disclosure provides a method for assaying TILs comprising:
(i) obtaining a n of a first population of cryopreserved TILs,
(ii) thawing the n of the first population of eserved TILs;
(iii) performing a first expansion by ing the portion of the first population of
T]Ls in a cell culture medium comprising 11—2, OKT-3, and antigen presenting cells
(APCs) for a reREP period of at least 3 days, to produce a second population of TILs,
wherein the portion from the first population of TILs is compared to the second
population of TILs to obtain a ratio of the number of TILs, wherein the ratio of the
number of TILs in the second population of TILs to the number of TILs in the portion
of the first population of TILs is greater than 5: 1,
(iv) determining based on the ratio in step (iii) whether the first population of TILs is
suitable for use in therapeutic administration to a patient;
(v) determining the first population of TILs is suitable for use in therapeutic
administration when the ratio of the number of TILs in the second population of TILs
to the number of TILs in the first tion of TILs is determined to be greater than
:1 in step (iv).
In some embodiments, the ratio of the number of TILs in the second population of
TILs to the number of TILs in the portion of the first population of TILs is r than 50:1.
In some ments, the method further comprises ming expansion of the
entire first population of cryopreserved TILs from step (i) according to the methods as
described in any of the embodiments provided .
In some embodiments, the method further comprises administering the entire first
population of cryopreserved TILs from step (i) to the patient.
In some embodiments, the cryopreserved TILs are thawed and a second expansion
performed to determine if the cells expand sufficiently. If the cells expand to a ratio of at
least 5: l, the TILs are sufficiently viably for administration to the patient. If the cells expand
to a ratio of at least 10:1, the TILs are sufficiently viably for administration to the patient. If
the cells expand to a ratio of at least 15:1, the TILs are sufficiently viably for administration
to the patient. If the cells expand to a ratio of at least 20:1, the TILs are sufficiently viably for
administration to the patient. If the cells expand to a ratio of at least 25:1, the TILs are
sufficiently viably for administration to the patient. If the cells expand to a ratio of at least
:], the TILs are sufficiently viably for administration to the patient. If the cells expand to a
ratio of at least 35:1, the TILs are sufficiently viably for administration to the patient. If the
cells expand to a ratio of at least 40: l, the TILs are sufficiently viably for administration to
the patient. If the cells expand to a ratio of at least 45:1, the TILs are sufficiently viably for
administration to the patient. If the cells expand to a ratio of at least 5:], the TILs are
sufficiently viably for administration to the patient.
The present disclosure also provides further methods for assaying TILs. In some
ments, the sure provides a method for assaying TILs comprising:
(i) obtaining a portion of a first population of cryopreserved TILs,
(ii) thawing the portion of the first population of cryopreserved TILs,
(iii) performing a first expansion by ing the portion of the first population of
TILs in a cell culture medium comprising IL-2, OKT-3, and antigen presenting cells
(APCs) for a reREP period of at least 3 days, to produce a second population of TILs,
n the portion from the first population of TILs is compared to the second
population of TILs to obtain a ratio of the number of TILs, wherein the ratio of the
number of TILs in the second population of TILs to the number of TILs in the portion
of the first population of TILs is greater than 5: 1;
(iv) determining based on the ratio in step (iii) whether the first population of TILs is
suitable for use in therapeutic administration to a patient; and
(v) eutically administering the remainder of the first population of TILs to the
patient when the ratio of the number of TILs in the second population of TILs to the
number of TILs in the first population of TILs is determined to be greater than 5:1 in
step (iv).
In some embodiments, the ratio of the number of TILs in the second tion of
TILs to the number of TILs in the portion of the first population of TILS is r than 50:1.
In some embodiments, the method further comprises performing expansion of the
entire first tion of eserved TILs from step (i) according to the methods of any of
the preceding claims.
In some embodiments, the method further ses administering the entire first
population of cryopreserved TlLs from step (i) to the patient.
In some embodiments, the method further comprised the step of ing the
metabolic health of the second population of TILs.
In some embodiments, the method further comprises the step of assessing the
phenotype of the second population of TILs.
In some embodiments, the antigen presenting cells are allogeneic erial blood
mononuclear cells.
L. Methods of Treating Patients
Methods of treatment begin with the initial TIL collection and culture of TILs. Such
s have been both described in the art by, for example, Iin el al. (J. Immunolhempy,
2012, 35(3):283—292), incorporated by reference herein in its entirety. As well as described
throughout the Examples section below.
The present invention provides novel methods for TIL generation that have not been
previously described, e. g, TILs produced ing to Steps A through F. The expanded
TILs produced according to Steps A through F above or as ise produced as described
herein find particular use in the treatment of patients with cancer. General methods of using
TILs for the treatment of cancer have been described in Goff, et al., J. Clinical Oncology,
2016, 34(20):2389-239, as well as the supplemental content; incorporated by reference herein
in its entirety.) Similarly, the TILs produced ing to the present invention can also be
used for the treatment of cancer. In some embodiments, TIL were grown from resected
deposits of metastatic melanoma as previously described (see, Dudley, et al., JImmunother.,
2003, 26:332-342, incorporated by reference herein in its entirety). Fresh tumor can be
dissected under e conditions. A representative sample can be collected for formal
ogic analysis. Single fragments of 2 mm3 to 3 mm3. In some embodiments, 5, 10, 15,
, 25 or 30 samples per patient are obtained. In some embodiments, 20, 25, or 30 samples
per patient are obtained. In some ments, 20, 22, 24, 26, or 28 samples per patient are
obtained, In some embodiments, 24 s per t are obtained. Samples can be placed
in individual wells of a 24-well plate, maintained in growth media with high-dose IL-2 (6,000
IU/mL), and monitored for destruction of tumor and/or proliferation of TIL. Any tumor with
viable cells remaining after processing can be enzymatically digested into a single cell
suspension and cryopreserved, as described herein.
In some embodiments, expanded TILs can be sampled for phenotype analysis (CD3,
CD4, CD8, and CD56) and tested against autologous tumor when ble. TILs can be
considered reactive if overnight co-culture yielded interferon-gamma (IFN—y) levels > 200
pg/mL and twice background. (Goff, et al., JImmunother., 2010, 33:840-847, incorporated
by reference herein in its entirety). In some ments, cultures with evidence of
autologous reactivity or sufficient growth patterns can be ed for a second expansion (for
example, a second expansion as ed in according to Step D of Figure 11), including
second expansions that are sometimes ed to as rapid expansion (REP). In some
embodiments, expanded TILs with high autologous vity (for example, high proliferation
during a second expansion), are selected for an additional second expansion. In some
ments, TILs with high autologous reactivity (for example, high proliferation during
second expansion as provided in Step D of Figure 11), are selected for an additional second
expansion according to Step D of Figure 11.
In some embodiments, the patient is not moved directly to ACT (adoptive cell
transfer), for example, in some embodiments, after tumor harvesting and/or a first expansion,
the cells are not utilized immediately. In such ments, TILs can be cryopreserved and
thawed 2 days before the second expansion step (for example, in some embodiments, 2 days
before a step referred to as a REP step). In such embodiments, TILs can be cryopreserved and
thawed 2 days before the second expansion step (for example, in some embodiments, 2 days
before a Step D as provided in Figure 11). As described in various embodiments throughout
the present application, the second expansion (including ses referred to as REP) used
OKT3 (anti-CD3) antibody (Miltenyi h, San Diego, CA) and IL-2 (3,000 IU/mL,
Prometheus, San Diego, CA) in the presence of irradiated feeder cells, gous when
possible, at a 100:1 ratio (see, Dudley, et al., J Immunother., 2003, 26:332-342; incorporated
by reference herein in its entirety). In some embodiments, the TILs can be cryopreserved and
thawed 5 days before the second ion step. In some embodiments, the TILs can be
cryopreserved and thawed 4 days before the second expansion step. In some embodiments,
the TILs can be cryopreserved and thawed 3 days before the second expansion step. In some
embodiments, the TILs can be cryopreserved and thawed 2 days before the second expansion
step. In some embodiments, the TILs can be eserved and thawed 1 day before the
second expansion step. In some embodiments, the TILs can be cryopreserved and thawed
immediately before the second expansion step.
Cell phenotypes of cryopreserved samples of infusion bag TIL can be analyzed by
flow cytometry (FlowJo) for surface markers CD3, CD4, CD8, CCR7, and CD45RA (BD
BioSciences), as well as by any of the methods described herein. Serum cytokines were
measured by using standard -linked immunosorbent assay techniques. A rise in serum
IFN-g was defined as >100 pg/mL and greater than 4 3 baseline levels.
1. Optional depletion Preconditioning of Patients
Experimental findings indicate that lymphodepletion prior to adoptive transfer of
tumor-specific T lymphocytes plays a key role in enhancing treatment efficacy by eliminating
regulatory T cells and competing elements of the immune system (‘cytokine sinks’).
Accordingly, some ments of the invention utilize a lymphodepletion step imes
also referred to as “immunosuppressive conditioning”) on the patient prior to the introduction
of the second expansion TILs or second additional ion TILs (such as, for example,
those described in Step D of Figure ll, including TILs referred to as reREP TILs) of the
invention.
In general, lymphodepletion is done using fludarabine and/or cyclophosphamide
(the active form being ed to as mafosfamide) and combinations thereof. Such methods
are described in Gassner et al. (Cancer Immunol Immunother. 2011, 60(1):75—85, Muranski,
et al., Nat Clin Pracl Oncol, 2006 3(l2):668—68l, Dudley, et al., J Clin Oncol 2008,
26:5233-5239, and Dudley, et al., JClz'n Oncol. 2005, 23(10):2346—23 57, all ofwhich are
incorporated by reference herein in their entireties.
In some embodiments, the fludarabine is at a concentration of 0.5 [lg/ml -10 ug/ml
fludarabine (Sigma-Aldrich, MO, USA). In some embodiments, the fludarabine is at a
concentration of l ug/ml fludarabine (Sigma-Aldrich, MO, USA). In some embodiments, the
fludarabine treatment is for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more.
In some embodiments, the fludarabine is administered at a dosage of 10 mg/kg/day,
mg/kg/day, 20 day, 25 mg/kg/day, 30 day, 35 mg/kg/day, 40 mg/kg/day, or
45 day. In some ments, the fludarabine treatment is for 2-7 days at
mg/kg/day. In some embodiments, the fludarabine treatment is for 4-5 days at
mg/kg/day. In some embodiments, the fludarabine treatment is for 4-5 days at
mg/kg/day.
In some embodiments, the mafosfamide, the active form of cyclophosphamide, is at
a concentration of 0.5 ug/ml -10 ug/ml. In some embodiments, the mafosfamide, the active
form of hosphamide, is at a concentration of 1 ug/ml. In some embodiments, the
cyclophosphamide treatment is for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or
more. In some embodiments, the cyclophosphamide is stered at a dosage of
100 mg/mZ/day, 150 mg/mZ/day, 175 mg/m2/day, 200 mg/mZ/day, 225 day, 250
mg/mZ/day, 275 mg/mz/day, or 300 mg/mZ/day. In some embodiments, the cyclophosphamide
is administered intravenously (7.6., i.v.) In some embodiments, the cyclophosphamide
treatment is for 2-7 days at 35 mg/kg/day. In some embodiments, the cyclophosphamide
treatment is for 4-5 days at 250 mg/mZ/day iv. In some embodiments, the cyclophosphamide
treatment is for 4 days at 250 mg/mZ/day i.v.
In some embodiments, the fludarabine and the cyclophosphamide are administered
together to a t. In some embodiments, bine is administered at 25 mg/mZ/day iv.
and cyclophosphamide is administered at 250 mg/mZ/day i.v. over 4 days.
This protocol includes administration of fiudarabine (25 mg/mZ/day iv.) and
cyclophosphamide (250 day iv.) over 4 days.
2, Exemplary Treatment Embodiments
] In some embodiments, the present disclosure provides a method of treating a cancer
with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of (a)
obtaining a first population of TILs from a tumor resected from a patient; (b) performing an
initial expansion of the first population of TILs in a first cell culture medium to obtain a
second population of TILs, wherein the second population of TILs is at least 5-fold greater in
number than the first population of TILs, and wherein the first cell culture medium comprises
IL-2, (c) performing a rapid expansion of the second population of TILs using a population of
d artificial antigen presenting cells (myeloid aAPCs) in a second cell culture medium
to obtain a third tion of TILs, wherein the third population of TILs is at least 50-fold
greater in number than the second population of TILs after 7 days from the start of the rapid
expansion; and wherein the second cell culture medium comprises IL-2 and OKT-3; (d)
administering a therapeutically effective portion of the third population of TILs to a patient
with the cancer. In some embodiments, the IL-2 is present at an initial concentration of about
3000 IU/mL and OKT-3 dy is present at an initial concentration of about 30 ng/mL in
the second cell culture medium. In some ments, first expansion is performed over a
period not greater than 14 days. In some embodiments, the first expansion is performed using
a gas permeable container. In some embodiments, the second expansion is performed using a
gas permeable ner. In some embodiments, the ratio of the second population of TILs to
the population of aAPCs in the rapid expansion is between 1 to 80 and l to 400. In some
embodiments, the ratio of the second population of TILs to the population of aAPCs in the
rapid expansion is about 1 to 300. In some embodiments, the cancer for treatment is selected
from the group consisting of melanoma, ovarian , cervical cancer, non-small-cell lung
cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human
papilloma virus, head and neck cancer, renal cancer, and renal cell carcinoma. In some
embodiments, the cancer for treatment is selected from the group consisting of ma,
ovarian cancer, and cervical cancer. In some embodiments, the cancer for treatment is
melanoma. In some embodiments, the cancer for treatment is n cancer. In some
embodiments, the cancer for treatment is cervical cancer. In some embodiments, the method
of treating cancer further comprises the step of treating the patient with a non-myeloablative
lymphodepletion regimen prior to administering the third population of TILs to the patient. In
some embodiments, the non-myeloablative lymphodepletion regimen ses the steps of
administration of cyclophosphamide at a dose of 60 mg/m2/day for two days followed by
stration of fludarabine at a dose of 25 mg/mZ/day for five days. In some ments,
the high dose IL-2 regimen ses 600,000 or 720,000 IU/kg of aldesleukin, or a
biosimilar or variant thereof, administered as a ute bolus intravenous infusion every
eight hours until tolerance.
3. Methods of co-administration
In some embodiments, the TILs produced as described herein in Steps A
h F can be administered in combination with one or more immune checkpoint
regulators, such as the antibodies bed below. For example, antibodies that target PD-l
and which can be inistered with the TILs of the present invention include, e.g, but are
not limited to nivolumab (EMS-93 6558, Bristol-Myers Squibb; Opdivo®), pembrolizumab
(lambrolizumab, MK03475 or MIC-3475, Merck, Keytruda®), humanized D-l antibody
J8001 (ShangHai JunShi), monoclonal anti—PD—l antibody TSR-042 o, Inc.),
Pidilizumab (anti-PD-l mAb CT-Ol l, Medivation), anti-PD-l monoclonal Antibody BGB-
A317 (BeiGene), and/or D-l antibody SHR-lZlO (ShangHai HengRui), human
monoclonal antibody REGN2810 (Regeneron), human monoclonal dy MDX—1106
(Bristol-Myers Squibb), and/or humanized anti-PD-l IgG4 antibody PDROOl (Novartis). In
some embodiments, the PD-l antibody is from clone: RMPl-l4 (rat IgG) - BioXcell cat#
BPOl46. Other suitable antibodies suitable for use in co-administration methods with TILs
produced according to Steps A through F as bed herein are anti-PD-l antibodies
disclosed in US. Patent No. 8,008,449, herein incorporated by reference. In some
embodiments, the antibody or antigen-binding portion thereof binds specifically to PD-Ll
and ts its ction with PD-l, thereby increasing immune activity. Any antibodies
known in the art which bind to PD-Ll and disrupt the ction n the PD-l and PD-
L1, and stimulates an anti- tumor immune response, are suitable for use in co-administration
methods with TILs produced according to Steps A through F as described herein. For
example, antibodies that target PD-Ll and are in clinical trials, e EMS-936559
(Bristol-Myers Squibb) and MPDL3280A (Genentech). Other suitable antibodies that target
PD-Ll are disclosed in US. Patent No. 7,943,743, herein incorporated by reference. It will be
understood by one of ordinary skill that any antibody which binds to PD-l or PD-Ll, disrupts
the PD-l/PD-Ll interaction, and stimulates an anti-tumor immune response, are suitable for
use in inistration methods with TILs produced according to Steps A through F as
described herein. In some ments, the subject administered the combination of TILs
produced according to Steps A through F is co-administered with a and anti-PD-l antibody
when the patient has a cancer type that is refractory to administration of the anti-PD-l
antibody alone. In some embodiments, the patient is administered TILs in combination with
and anti-PD-l when the patient has ory melanoma. In some embodiments, the t is
administered TILs in combination with and anti-PD-l when the patient has non-small cell
lung carcinoma (NSCLC).
4. Adoptive Cell Transfer
ve cell transfer (ACT) is a very effective form of immunotherapy and
involves the transfer of immune cells with antitumor activity into cancer patients. ACT is a
treatment approach that involves the identification, in vitro, of lymphocytes with antitumor
activity, the in vitro expansion of these cells to large numbers and their infusion into the
cancer-bearing host. Lymphocytes used for adoptive transfer can be derived from the stroma
of resected tumors (tumor infiltrating lymphocytes or TILs). TILs for ACT can be prepared
as described . In some ments, the TILs are prepared, for example, according to a
method as described in Figure 11. They can also be derived or from blood if they are
genetically engineered to express mor T—cell receptors (TCRs) or chimeric antigen
receptors (CARS), enriched with mixed lymphocyte tumor cell cultures (MLTCs), or cloned
using gous antigen presenting cells and tumor derived peptides. ACT in which the
lymphocytes originate from the cancer-bearing host to be infused is termed autologous ACT.
U. S. Publication No. 2011/0052530 relates to a method for performing adoptive cell therapy
to promote cancer regression, primarily for treatment of patients ing from metastatic
melanoma, which is incorporated by reference in its entirety for these methods.
In some embodiments, TILs can be stered as described herein. In some
embodiments, TILs can be administered in a single dose. Such administration may be by
injection, e.g., intravenous injection. In some embodiments, TILs and/or cytotoxic
lymphocytes may be stered in multiple doses. Dosing may be once, twice, three times,
four times, five times, six times, or more than six times per year. Dosing may be once a
month, once every two weeks, once a week, or once every other day. Administration of TILs
and/or cytotoxic lymphocytes may continue as long as necessary.
I. Exemplary Embodiments
In an embodiment, the invention provides a method for expanding tumor
infiltrating cytes (TILs) comprising:
(a) obtaining a first population of TILs from a tumor resected from a patient;
(b) performing an initial expansion of the first population of TILs in a first cell culture
medium to obtain a second tion of TILs, wherein the first cell culture medium
comprises IL-2,
(c) performing a rapid expansion of the second population of TlLs, wherein the third
population of TlLs is at least lOO-fold greater in number than the second population
of TILs, and wherein the second cell culture medium comprises IL-2, OKT-3, and
peripheral blood mononuclear cells ), wherein the rapid expansion is
performed for at least 14 days,
(d) removing the cells from the second cell culture medium and optionally
cryopreserving the cells in a storage medium to obtain a third population of cells;
(e) optionally thawing the third tion of cells, and
(f) ming a second rapid expansion of the third population of TlLs in a third cell
culture , wherein the third cell e medium comprises IL—2, OKT-3, and
peripheral blood mononuclear cells (PBMCs), wherein the second rapid expansion is
performed for at least 14 days, to obtain a fourth population of TILs, wherein the
fourth population of cells exhibits an sed subpopulation of effector T cells
and/or central memory T cells relative to the second population of TILs; and
g) optionally, ing step f) one or more times.
In an embodiment, the invention provides that said restimulated cells express
CD4, CD8 and TCR 0t [3 at levels similar to freshly harvested cells.
In an embodiment, the invention provides that said reREP medium comprises
eral blood mononuclear cells (PBMCs).
] In an embodiment, the ion provides that said PBMCs are added to the
TILs on any of days 9 through 17. In some embodiments, the invention provides that said
PBMCs are added to the TlLs on days 9, 10, ll, 12, l3, 14, 15, 16, and/or 17.
In an embodiment, the invention es that said reREP medium comprises
aAPCs.
In an embodiment, the invention es that the cryopreserved TILs were
transduced with an expression vector comprising a nucleic acid encoding a high-affinity T
cell receptor.
In an embodiment, the invention provides that the cryopreserved TILs were
transduced with an expression vector comprising a nucleic acid encoding a chimeric antigen
receptor (CAR) comprising an immunoglobulin light chain fused with an endodomain of a T-
cell signaling molecule.
In an embodiment, the invention es that restimulated TILs are infused
into a patient.
In an embodiment, the invention provides that step (1) further comprises
removing the cells from the second cell culture medium.
In an embodiment, the invention provides that step i) is repeated a sufficient
number of times in order to obtain sufficient TILs for a therapeutic dosage of said TILs.
In an embodiment, the invention provides a population of restimulated TILs
made ing to the methods described above and .
In an embodiment, the invention provides a population of ulated TILs
made according to the method of claim 1 wherein said restimulated TILs have at least a two-
fold increase in basal glycolysis as compared to said thawed cryopreserved TILs.
In an embodiment, the invention provides a method for ing the
metabolic activity of a TIL cell population comprising measuring the basal glycolysis of said
cells
In an embodiment, the invention provides a method for assessing the
metabolic ty of a TIL cell tion comprising measuring the basal respiration of said
cells.
In an embodiment, the ion es a method for assessing the
metabolic activity of a TIL cell population comprising measuring the spare respiratory
capacity (SRC) of said cells.
In an ment, the invention provides a method for assessing the
metabolic activity of a TIL cell tion comprising measuring the glycolytic reserve of
said cells.
In an embodiment, the invention provides a method of treating cancer in a
patient with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
a) obtaining a primary TIL population from said patient,
b) rapidly ing said y TIL population to form an expanded TIL
population,
c) cryopreserving said expanded population to form a cryopreserved TIL population;
(1) thawing said cryopreserved TIL population,
6) culturing said cryopreserved TIL population in media comprising IL-2 and anti-
CD3 antibody to form a reREP TIL population, and
f) administering a therapeutically effective amount of reREP TIL cells to said patient.
In an embodiment, the invention provides a method for expanding tumor
infiltrating lymphocytes (TILs) comprising:
(a) obtaining a first population of TILs from a tumor ed from a patient
(b) performing an initial expansion of the first population of TILs in a first cell culture
medium to obtain a second population of TILs, wherein the first cell culture medium
comprises IL—2;
(c) performing a rapid expansion of the second population of TlLs, wherein the third
population of TlLs is at least lOO-fold greater in number than the second population
of TILs, and wherein the second cell culture medium comprises IL-2, OKT-3, and
peripheral blood mononuclear cells (PBMCs), wherein the rapid expansion is
performed for at least 14 days;
(d) ng the cells from the second cell culture medium and optionally
cryopreserving the cells in a storage medium to obtain a third population of cells;
(e) optionally thawing the third population of cells;
(f) performing a second rapid expansion of the third population of TlLs in a third cell
e medium, wherein the third cell culture medium comprises IL-2, OKT-3, and
peripheral blood mononuclear cells (PBMCs), wherein the second rapid ion is
performed for at least 14 days, to obtain a fourth population of TILs, wherein the
fourth population of cells exhibits an increased subpopulation of effector T cells
and/or central memory T cells relative to the second population of TILs, and
(g) administering a therapeutically effective amount of reREP TIL cells to said
patient.
In an embodiment, the invention provides that step d) further comprises
removing the cells from the second cell culture medium.
In an embodiment, the invention provides that step f) is repeated a sufficient
number of times in order to obtain sufficient TILs for a eutic dosage of said TILs.
EXAMPLES
EXAMPLE 1: RESTIMULATION PROTOCOL
As discussed herein, a restimulation protocol and assay were ped utilizing
fresh antigen restimulation ing harvest or thaw of TILs grown in a REP.
The purpose of this example was to test the proliferation/expansion of post REP
Tumor Infiltrating Lymphocytes in a Re—stimulation assay. Post REP TIL (post Step D TIL
according to Figure 11) were be restimulated with allogeneic PBMC feeder cells, anti-CD3
(clone OKT3) antibody, and interleukin-2 . Viable cells were counted on Day 7 and
The post REP TIL (post Step D TIL according to Figure 11) were infused into the
patients who were previously lymphodepleted to facilitate TIL survival and expansion in
viva. Once the TIL were re-infused into the patient, they encountered n, resulting in the
activation of the TIL, but the TIL were ultimately short-lived. Re-stimulation of the TIL
through antigen contact er with exposure to IL-2 during ACT may result in TIL
proliferation and tumor control or may lead to deletion through apoptosis (activation induced
cell death) or induction of a non-proliferative (anergic) state due to lack of appropriate co-
stimulation. Without being bound by theory, restimulation of post REP TIL (restimulation of,
for e post Step D TIL according to Figure 11) with allogeneic PBMC feeder cells may
mimic the in vivo process by providing antigen stimulation and necessary cytokines for TIL
expansion. Post REP TIL (post Step D TIL according to Figure 11) were activated through
membrane receptors on the feeder MNCS that bind to anti-CD3 (clone OKT3) antibody and
crosslink to TIL in the REP flask, stimulating the TIL to expand.
Proliferation/Expansion of Post REP Tumor Infiltrating Lymphocytes in a Re-
stimulation assay
Post REP (post Step D TIL according to Figure ll) TIL were restimulated with
allogeneic PBMC feeder cells, anti-CD3 (clone OKT3) antibody, and interleukin-2 (IL-2).
Viable cells were d on Day 7 and recorded.
In some embodiments, this procedure can also be d to test or validate the
current REP protocol.
Table 3: DEFINITIONS AND ABBREVIATIONS
AOPI Acridine Oran_e Pro.idium Iodide
BSC Biolo_ical Safet Cabinet
BSL2 Biosafet Level 2
CM1 te Medium for TIL, #1
CM2 Com .lete Medium for TIL, #2; 50:50 mixture of CMl and AIM-V
GMP Good Manufacturin; Processin_
—_IPA Iso..ro 1 alcohol
LN2 Licuid n
MNC; PBMC clear Cells; Peri .heral Blood clear Cells
m1 Milliliter
NA Not a..1icab1e
NR Not recuired
MACS® GMP CD3 .ure clone OKT3 antibod
Personal .rotective e . ui . ment
Initial TIL cultures ori_inatin from tumor frag
Ra . id EX . ansion Protocol
San Die_o Blood Bank
Tumor Infiltratin_ L m.hoc te
Table 4: Materials
GleOTM/Llfe 087-0112DK
Technolo;
Cellometer ViaStainTM
NA Nexcelom CS2-0106 2-8°C
AOPI Stainin_ Solution
Disposable
NA Nexcelom CP2-001 RT
Hemac r
Prepared as per
CMl NA NA 2-8°C
Exam 1e 5
GMP recombinant 6 x 106 IU/ml stock
human 1L-2 (rh1L-2) solution prepared as per CellGenix 1020—1000 -20°C
Example 4
MACS® GMP CD3 pure
GMP Miltenyi Biotec 170116 2-8°C
clone OKT3 antibod
50ml conical tubes sterile Any in use RT
transfer pipets sterile Any in use RT
SCGPUOSRE or
500ml filter system EMD/Millipore
sterile RT
or ecuivalent ecuivalent
24—well tissue culture r or 662160 or
sterile RT
.lates e . uivalent e . nt
5ml, 10ml serological sterile
————_
———_—
Table 5: SPECIMENS
Cryopreserved and Gamm Stored freezer B N
irradiated MNC Feeder lots
Post-REP TIL cells Fresh or Frozen in Iovance NA NA
freezer Biotechnoloies
] The post REP (post Step D TIL according to Figure 11) TIL were infused into the
patients who were prior lymphodepleted to tate TIL survival and expansion in vivo.
Once the TIL were re-infused into the t, they encountered antigen, resulting in the
tion of the TIL, but the TIL were tely short-lived. Re-stimulation of the TIL
through antigen t together with exposure to IL-2 during ACT may result in TIL
proliferation and tumor control or may lead to deletion through apoptosis (activation induced
cell death) or induction of a non-proliferative (anergic) state due to lack of appropriate co-
stimulation. Our hypothesis was that restimulation of post REP TIL with allogeneic PBMC
feeder cells mimicked the in vivo process by providing antigen stimulation and necessary
nes for TIL expansion. Post REP TIL were activated through membrane receptors on
the feeder MNCs that bind to anti-CD3 (clone OKT3) antibody and crosslink to TIL in the
REP flask, stimulating the TIL to expand.
PROCEDURE
Either fresh post-REP (post Step D TIL according to Figure 11) or frozen post-REP
(post Step D TIL according to Figure 11) TIL that was thawed, was washed once in CMl
media. The Re-REP (repeat of Step D according to Figure 11) was set up in a 24 well tissue
e plate with 2 X 106 MNC feeder cells, 30 ng/ml OKT3, l x 104 post-REP TIL plus
3,000 IU/ml rhIL-2 in CMZ. The cultures were incubated for seven days in a 5% C02, 37°C
humidified incubator at which point viable cell recovery and viability was determined. The
fold expansion of TIL was calculated based on the viable cell counts.
ReREP- Day 0
Prepare TIL
TILs were obtained from fresh post REP or frozen post REP. TIL cultures were d
from the incubator and transferred to the BSC. Next, 200ul was removed for a cell count
using the Cellometer K2. Counts were recorded.
e Feeder Cells
For this protocol a minimum of 20 X 106 feeder cells were . Each 1 ml vial
frozen by SDBB had 100 X 106 viable cells upon freezing. Assuming a 50% recovery upon
thaw from LN2 storage, it was recommended to thaw at least two vials of feeder cells per lot
giving an estimated 100 X 106 viable cells for each REP. Before thawing feeder cells,
approximately 50ml of CM2 was rmed without rhIL—2 for each feeder lot that was
tested. The designated feeder lot vials were removed from LN2 e and placed on ice.
Vials were transferred to the tissue culture room. Vials were thawed in a 37°C water bath.
Vials were transferred to BSC and sprayed or wiped with 70% EtOH or IPA. Using a transfer
pipette, the contents of feeder vials was immediately transferred into 50 mL of warm CM2 in
a 50-mL conical tube. 200 pl was removed for cell counting using the Cellometer K2. Counts
were ed. Cells were centrifuged at 350 X g for 10 minutes. The supernatant and
resuspended cells were aspirated in a desired volume at 2 X 106cells/ml in warm CM2 plus
3000 IU/ml rhIL-2.
Prepare CM2 + 3000 IU/ml working solution
A sufficient amount of CM2 was ed for the conditions needed. Each well
contained 2 ml of CM2. Each well was supplemented the CM2 with 3000 IU/mL of rhIL—2.
From the stock of 6 XlO6 IU/mL, 50 ul was needed for each 100 ml of CM2.
Prepare MACS® GMP CD3 pure (OKT3) working solution
Stock solution of OKT3 (1 mg/ml) was taken out of the 4°C refrigerator. A final
concentration of 30 ng/ml OKT3 was used in the REP. 60 ng of OKT3 were needed for 2 ml
of CM2 medium in each 24 well. TIL + Feeders, TIL alone and Feeders alone conditions
were cultured in triplicates. For each feeder lot tested, 1000 ul of a 1:1000 dilution of
lmg/ml OKT3 for a working concentration of lug/ml (1,000 ng/ml) was made. For 9 wells,
1000 ul ofa 1:1000 dilution of lmg/ml OKT3. 1 pl 1 mg/ml OKT3 + 999 pl of CM2 with
3000 IU/ml IL-2 was made.
e 24 Well plate and Coculture.
Each ReREP tested required 9 wells of 24 well plate.
Each plate was labeled with Experiment Name, Feeder Lot #, post—REP TIL
designation, date, and operator initials. Each plate was filled with components as listed in
Table 8. Each component was added and each well filled with a total of 2 ml and place the
plates into 37°C incubator. Plates were mixed carefully 3 times using 1 ml pipette.
Table 6: REP set-up in 24 well plate
Order of addition to single well of 24 TIL + Feeders + TIL Feeders +
well plate OKT3 +OKT3 OKT3
TIL cells (1 x 104/05 ml) in CM2+1L-2 500 pl 500 pl _
PBMC feeder cells (2x106/l ml) in 1000 ul 1000 ul
CM2+IL-2
OKT3 (1000 ng/ml) in CM2+IL-2 60 “1 60 pl 60 pl
CM2 +1L-2 440 ul 1440 pl 940 pl
Total Volume 2000 ul 2000 ul 2000 ul
Media exchange — Day 5
CM2 was ed with 3000 IU/ml rhIL-2. 10ml was needed. 1 ml of the media
was removed from each well and discarded. With a 1 ml pipette, 1 ml warm CM2 with 3000
IU/mL rhIL-2 was transferred to each well, The plates were returned to the tor.
Harvest — Day 7
Using a 1 ml serological pipet, each well was mixed to break up any clumps of
cells. After thoroughly mixing cell suspension by pipetting, 200ul was removed for cell
counting using the Cellometer K2. All the ions were counted and recorded for TIL
+Feeders +OKT3, TIL +OKT3, and FEEDERS +OKT3.
In addition to 24 well ReREP, separate reREP were set up in 4 upright T25 tissue
culture flasks with 1.3 x 107 MNC feeder cells, 30 ng/ml OKT3, 0.65 x 105 pre-REP TIL plus
3,000 IU/ml rhIL-2 in CM2. Note: Please refer to Evaluation of Irradiated Allogeneic Feeder
Cells for Rapid Expansion Protocol of LN—144 (Example 6).
tion of cells for onal assays:
Table 7: Assay
Flow Phenotyping 106
Potency — P815effLuc-eGFP 406
For restimulation assay to Granzyme-B, IFN—
_amma
Metabolism 26
TCR Sequencing 16
Store culture supernatant of TIL+ s and
feeders alone for Multi lex ELISA
EVALUATION/ACCEPTANCE CRITERIA
Table 8: Acceptance Criteria Used
At least a 50fold ex-ansion of Post REP TIL With s
No expansmn and at least 20%) reduction in the total V1able. 0 . . .
PBMC Feeders cells alone
number of feeder cells
Reference Procedures — Included in Examples below
Table 9: Reference Procedures
Determination of Cell Count and Example 2
Viability of TIL es Using the
Cellometer K2 Cell Counter
-_—CellGenix
_CMMedia Formulation e 5
-Evaluation ofIrradiated AllogeneicFeeder Cells for Rapid ExpansionProtocol ofLN— 144
infiltratin_ L m hoc te after Post REP
-Validating the post REP cryofrozenTIL oroduct
EXAMPLE 2: DETERMINATION OF CELL COUNT AND VIABILITY OF TIL
CULTURES USING THE CELLOMETER K2 CELL COUNTER
This example provides exemplary ctions for how the operation of the
Cellometer K2 Image Cytometer automatic cell counter was carried out.
Scope: Determination of the total cell count and viability of cell cultures.
Table 10. Definitions
Microliter
Acridine Orane Pro.idium Iodine
Biolo_ical Safe Cabinet
Dulbecco’s Phos hate Buffered Saline
Milliliter
Mononuclear Blood Cells
Not A. .licable
Peri .heral Blood Mononuclear Cells
PPE Personal Protective ent
Pre-REP Initial TIL culture before Rapid Expansion Protocol of e
REP Rapid Expansion Protocol
TIL Tumor Infiltrating Lymphocytes
PROCEDURE
Cell suspension preparation
Tflpan Blue Preparation
The final Trypan blue concentration was 0.1%. The manufacturer recommended
preparing a stock solution of 0.2%. When using Trypan blue on the Cellometer K2, the stock
(0.4 %) with PBS was diluted to 0.2 %. The Trypan blue was filtered with a 0.2-0.4 micron
filter and aliquoted in small volumes into labeled, capped tubes. The cell suspension was
mixed at 1:1 with 0.2 % trypan blue.
AOPI Preparation
] When using AOPI on the eter K2, the AOPI solution was ed. Cell
samples were stained at 1:1 with AOPI solution. NOTE: When counting high tration
cultures, the cell samples were diluted in cell culture medium prior to the final 1:1 dilution
with Trypan Blue or AOPI. The cturer’s suggested range of counting was used to
determine the best dilution to use.
Cellometer K2 Set—Up
The eter K2 equipment was turned on. The Cellometer Image Cytometer
icon was selected on the associated computer monitor. On the main screen of the software,
one of the Assays listed in the dropdown box was selected. When selecting the appropriate
Assay, the Cell Type and Image Mode opulated. Under “Sample” section, Set
User/Sample ID was clicked to open another screen to input operator’s ation for
specimen. “User ID” was entered. This consisted of the user’s three letter initials. Enter
“Sample ID”. The sample ID was derived from ng specimen information.
Setup dilution parameters
When no other dilution was made besides the 1:1 mixture, the dilution factor was 2.
When a dilution was made prior to the final 1:] mixture, the dilution factor was 2 times of the
prior dilution. The dilution factor was d according to the mixture used.
Cell ng
The plastic backing was removed from both sides of a Cellometer counting chamber
slide (SDlOO) and placed on top of a clean, ree wipe. After ing the cell
suspension, a small aliquot of the sample was removed and transferred into a well of a
multiwell cell culture plate or tube. When diluting the sample, the dilution was performed
using cell culture medium. 20 ul of cell suspension was added into a well of the ell
cell culture plate or tube. 20ul of 0.2% trypan blue or the AOPI solution was added to the
20ul of cell suspension and the sample mixed thoroughly. 20 ul of the 1:1 solution was
measured and transferred it into one side of the counting chamber. NOTE: ng the clear
area of the slide was avoided. As needed, the samples were repeated on the other side of the
slide. The chamber was inserted into the slot on the front of the eter. For the AOPI
cell counting, “Preview F1” was selected on the main screen to preview the green fluorescent
image (live cell) image. For Trypan blue counting, “Preview Brightfield” was selected. The
focusing wheel was used to bring image into optimal focus. Cells that had a bright center and
a clearly—defined edge. “Count” was selected to begin the counting process. Results were
displayed in a counting results pop-up box on the computer screen that showed the results of
the counting process.
EXAMPLE 3: CELLOMETER IC2 IMAGE CYTOMETER AUTOMATIC CELL
COUNTER
This Example describes the procedure for operation of the eter K2 Image
Cytometer automatic cell counter.
1. Definitions
ul Microliter
AOPI ne Orange Propidium Iodine
BSC Biological Safety Cabinet
DPBS Dulbecco's ate Buffered Saline
ml Milliliter
MNC Mononuclear Blood Cells
NA Not Applicable
PBMC Peripheral Blood Mononuclear Cells
PPE Personal Protective ent
P Initial TIL culture before Rapid Expansion Protocol of culture
REP Rapid Expansion Protocol
TIL Tumor Infiltrating Lymphocytes
Procedure
7.1 Cell suspension preparation
7.1.1 Trypan Blue Preparation
The final Trypan blue concentration was 0.1%. The cturer
recommended preparing a stock solution of 0.2%.
7.1.1.1 When Trypan blue was used on the Cellometer K2, the stock
(0.4 %) was diluted with PBS to 0.2 %.
7.1.1.2 The Trypan blue was filtered with a 0.2-0.4 micron filter and
ted in small volumes into labeled, capped tubes.
7.1.1.3 The cell suspension was mixed at 1:1 with 0.2 % trypan
blue.
7.1.2 AOPI Preparation
7.1.2.1 When AOPI was used on the Cellometer K2, the AOPI
solution was obtained.
7.1.2.2 The cell sample was stained at 1:1 with AOPI solution.
NOTE: When high concentration cultures were counted, the cell samples were
diluted in cell culture medium prior to the final 1:1 dilution with Trypan Blue
or AOPI.
The manufacturer's suggested range of counting was used to determine the
best dilution to use.
7.2 Cellometer K2 Set-Up
7.2.1 The Cellometer K2 equipment was turned on.
7.2.2 The Cellometer Image Cytometer icon was selected on the associated
computer monitor.
7.2.3 On the main screen of the software, one of the Assays listed in the
dropdown box was selected.
7.2.3.1 When the appropriate Assay was selected, the Cell Type and
Image Mode self-populated.
7.2.3.2 Under “Sample” section, Set User/Sample ID was selected
to open another screen to input operator's ation for
specimen.
2.1 The “User ID” was d.
7.2.3.2.2 The “Sample ID” was entered. The sample ID
was derived from incoming specimen
information.
7.2.3.3 Dilution parameters were set up.
7.2.3.3.1 When no other dilution was made besides the
1:1 e, the on factor was 2.
7.2.3.3.2 When a dilution was made prior to the final 1:1
e, the dilution factor was 2 times of the
prior dilution.
7.2.3.3.3 The dilution factor was updated according to the
mixture used in the dilution section of the
screen. The pencil icon was selected to bring up
the dialog screens.
7.2.3.3.4 The F1 Image and F2 Image sections were
verified to be identical to each other.
7.2.3.3.5 The “Save” button was selected after set up was
completed.
7.3 Cell Counting
7.3.1 The plastic backing from both sides of a Cellometer counting chamber
slide (SD100) was removed and placed on top of a clean, lint-free
wipe.
7.3.2 After the cell sion was prepared, a small aliquot of the sample
was removed and transferred into a well of a multiwell cell culture
plate or tube.
7.3.3 When the sample was diluted, the on was performed using cell
e medium.
7.3.4 20 ul of cell suspension was added into a well of the multiwell cell
culture plate or tube.
WO 81473
7.3.5 20 ul of 0.2% trypan blue or the AOPI solution was added to the 20 pl
of cell suspension and mix sample thoroughly.
7.3.6 20 ul of the 1:1 on was measured and erred it into one side
of the counting chamber.
NOTE: Touching the clear area of the slide was avoided.
7.3.7 When necessary, the sample was repeated on the other side of the slide.
7.3 .8. The chamber was inserted into the slot on the front of the
Cellometer.
7.3.8 For the AOPI cell counting, “Preview F1” was selected on the main
screen to preview the green fluorescent image (live cell) image. For
Trypan blue counting, “Preview Brightfield” was selected.
7.3.9 The focusing wheel was used to bring image into optimal focus. Cells
had a bright center and a clearly-defined edge.
7.3.10 “Count” was selected to begin the counting s.
7311 Results were displayed in a counting results pop—up box on the
computer screen that showed the results of the counting process.
EXAMPLE 4: PREPARATION OF IL-2 STOCK SOLUTION (CELLGENIX)
This example describes an ary preparation procedure for an IL-2 stock
solution.
Definitions/Abbreviations
uL: microliter or ul
BSC: Biological Safety Cabinet
BSLZ: Biosafety Level 2
D-PBS: Dulbecco's ate Buffered Saline
G: Gauge
GMP: Good Manufacturing Processing
HAc: Acetic Acid
HSA: Human Serum Albumin
mL: Milliliter
NA: Not applicable
PPE: Personal Protective Equipment
rhlL-Z; IL-Z: Recombinant human eukin-2
COA: Certificate of Analysis
6. Procedure
6.1 Prepared 0.2% Acetic Acid solution (HAc).
6.1.1 Transferred 29 mL sterile water to a 50 mL l tube.
6.1.2 Added 1 mL 1 N acetic acid to the 50 mL conical tube.
6.1.3 Mixed well by inverting tube 2-3 times.
6.1.4 Sterilized the HAc solution by filtration using a Steriflip filter.
6.1.5 Capped, dated and labeled the on “Sterile 0.2% Acetic Acid
Solution.”
6.1.6 Solution expired after 2 months. Stored at room temperature.
6.2 Prepared 1% HSA in PBS.
6.2.1 Added 4mL of 25% HSA stock solution to 96mL PBS in a 150mL
sterile filter unit.
6.2.2 Filtered solution.
6.2.3 Capped, dated and d the solution “1% HSA in PB S.”
6.2.4 Solution expired after 2 months. Stored 4°C.
6.3 For each vial of rhIL-2 prepared, document.
6.4 Prepared rhIL-2 stock solution (6x106 IU/mL final concentration)
6.4.1 Each lot of rh1L-2 was different and required information found in the
manufacturer's Certificate of Analysis (COA), such as:
6.4.1.1 Mass of rh1L-2 per vial (mg)
6.4.1.2 Specific activity of rh1L-2 )
6.4.1.3 Recommended 0.2% HAc reconstitution volume (mL)
6.4.2 Calculated the volume of 1% HSA required for rhlL—2 lot by using the
equation below:
Vial Muss: {mg} 21 Binéogicaé 333331333; {4%}
:94 -— Hm mi {33333 x 1% as3-1 vol {mi}
WWW...
8x335 4.:
6.42.] For example, according to CellGenix's rhIL-2 lot 10200121
COA, the specific activity for the 1 mg vial was 25x106
lU/mg. It ends reconstituting the rh1L-2 in 2mL
0.2% HAc.
a EgrififimH“:
. Ems;
.. : ,m 233-33.. mm. 2.1.6’F-m3i3‘f'fifi
6x305: 3+3 .
$733:
6.4.3 Wiped rubber r of IL-2 vial with alcohol wipe.
6.4.4 Using a 16G needle attached to a 3mL syringe, the recommended
volume of 0.2% HAc was injected into the vial. Care was taken to not
dislodge the stopper as the needle was withdrawn.
6.4.5 Inverted vial 3 times and swirled until all powder was dissolved.
6.4.6 The stopper was carefully removed and set aside on an alcohol wipe.
6.4.7 Added the calculated volume of 1% HSA to the vial.
6.4.8 Capped the vial with the rubber stopper.
6.5 Storage of rhIL-2 solution
6.5.1 For short-term storage (<72hrs), vials were stored at 4°C.
6.5.2 For long-term storage (>72hrs), the vial was ted into smaller
volumes and stored in cryovials at -20°C until ready to use.
Freeze/thaw cycles were avoided, Expired 6 months after date of
preparation.
6.5.3 Rh-IL-2 labels included vendor and catalog number, lot ,
expiration date, operator initials, concentration and volume of t.
EXAMPLE 5: PREPARATION OF MEDIA FOR PRE-REP AND REP PROCESSES
This Example describes the procedure for the preparation of tissue culture media for
use in protocols involving the culture of tumor infiltrating cytes (TIL) derived from
various tumor types ing, but not limited to, metastatic melanoma, head and neck
squamous cell carcinoma, ovarian carcinoma, triple-negative breast carcinoma, and lung
adenocarcinoma. In many cases, this media was used for ation of any of the TILs
described in the present application and Examples.
Definition
Hg microgram
um micrometer
uM micromolar
AIM-V® serum-free tissue culture medium (Thermo Fisher Scientific)
BSC Biological Safety Cabinet
CM1 Complete Medium #1
CM2 te Medium #2
CM3 te Medium #3
CM4 Complete Medium #4
1U or U International units
ml milliliter
mM millimolar
NA not applicable
PPE personal protective equipment
Pre-REP pre-Rapid Expansion Process
REP Rapid Expansion Process
rh1L-2, IL-2 recombinant human Interleukin-2
RPM11640 Roswell Park Memorial Institute , formulation 1640
SOP Standard Operating Procedure
TIL tumor infiltrating lymphocytes
Procedure
7.1 All procedures were done using sterile technique in a BSC (Class II, Type
7.1.1 Surface of hood was sprayed with 70% ethanol prior to its use.
7.1.2 All items and reagents were d with 70% ethanol prior to placing
them into tissue culture hood.
7.2 Aliquotting of 200mM L-glutamine
7.2.1 L-glutamine was supplied in larger volumes than needed for the
preparation of serum (e. g., 100ml or 500ml volumes).
7.2.2 Thawed bottle of L-glutamine in 37°C water bath.
7.2.3 Mixed L-glutamine well after thawing, as it precipitates after thaw.
Ensure that all precipitates have returned to solution prior to
aliquotting.
7.2.4 Placed 5-10ml aliquots of L-glutamine into sterile 15ml conical tubes.
7.2.5 Labeled tubes with tration, vendor, lot number, date aliquotted,
and expiration date.
7.2.6 Tubes were stored at —20°C and pulled as needed for media
preparation.
7.3 ation of CMl
7.3.1 Removed the following reagents from cold e and warmed them
in a 37°C water bathe:
7.3.1.1 RPM11640
2 Human AB serum
7.3.1.3 200mM amine
7.3.2 Removed the BME from 4°C storage and place in tissue culture hood.
7.3.3 Placed the gentamycin stock solution from room temperature e
into tissue culture hood.
7.3.4 Prepared CMl medium ing to Table 1 below by adding each of
the ingredients into the top section of a 0.2pm filter unit appropriate to
the volume that was filtered.
Table 11. Preparation of CMl
Ingredient Final concentration Final Volume 500 ml Final Volume IL
RPM11640 NA 450 ml 900 m1
Human AB serum, 50ml 100 ml
heat-inactivated 10%
200mM amine 2mM 5 ml 10 ml
55mM BME SSuM 0.5 ml lml
50mg/ml gentamicin SOug/ml 0.5 ml 1 ml
sulfate
7.3.5 Labeled the CMI media bottle with its name, the initials of the
preparer, the date it was filtered/prepared, the two week expiration date
and stored at 4°C until needed for tissue culture. Media was aliquotted
into smalled volume s as required.
7.3.6 Any remaining RPM11640, Human AB serum, or L-glutamine was
stored at 4°C until next preparation of media.
7.3.7 Stock bottle of BME was returned to 4°C storage.
7.3.8 Stock bottle of icin was returned to its proper RT storage
location.
7.3.9 Because of the limited ing capacity of the , CMl was
discarded no more than two weeks after preparation, or as the phenol
red pH indicator showed an extreme shift in pH (bright red to pink
coloration).
7.3.10 On the day of use, the required amount of CMl was warmed in a 37°C
water bath and 6000 IU/ml IL-2 was added.
7.3.11 Additional supplementation - as was needed
7.3.11.1 CMl was supplemented with GlutaMAX®
7.3.11.1.1 CMl was prepared by substituting 2mM
GlutaMAXTM for 2mM glutamine (final
concentration, see Table 2.) When this was
done, the media bottle was labeled adding
“2mM GlutaMAX” to prevent confusion with
the standard formulation of CM1.
7.3.11.2 CMl was supplemented with extra antibiotic/antimycotic
7.3.11.2.1 Some CMl formulations required additional
antibiotic or antimycotic to prevent
contamination of pre-REP TIL grown from
certain tumor types.
7.3.11.2.2 Antibiotic/antimycotic was added to the final
concentrations shown in Table 2 below.
7.3.11.2.3 When done, the media bottle was d by
adding the name/s of the additional
antibiotic/antimycotic to prevent confusion with
the standard formulation of CM1.
Table 12. Additional supplementation of CM1, as was needed.
Supplement Stock concentration Final concentration
GlutaMAXTm 200mM 1. 100 _
Penicillin/streptomycin 10,000 U/ml : 100 U/ml llin
penicillin 100 ug/ml
,000ug/ml streptomycin
streptomycin
Amphotericin B 250ug/ml 1:100 2.5ug/ml
8.1 Preparation of CM2
8.1.1 Removed prepared CM1 from erator or e fresh CM1 as per
Example above.
8.1.2 Removed AIM-V® from refrigerator.
8.1.3 Prepared the amount of CM2 needed by mixing prepared CM1 with an
equal volume of AIM—V® in a sterile media bottle.
8.1.4 Added 3000 IU/ml IL—2 to CM2 medium on the day of usage.
8.1.5 Made sufficient amount of CM2 with 3000 IU/ml IL-2 on the day of
usage.
8.1.6 Labeled the CM2 media bottle with its name, the initials of the
preparer, the date it was filtered/prepared, the two week expiration date
and stored at 4°C until needed for tissue culture. Media was tted
into smalled volume bottles as required.
8.1.7 Returned any CM2 without IL—2 to the refrigerator where it was stored
for up to two weeks, or until phenol red pH indicator showed an
extreme shift in pH (bright red to pink coloration).
8.2 Preparation of CM3
8.2.1 Prepared CM3 on the day it was ed for use.
8.2.2 CM3 was the same as AIM-V® medium, supplemented with 3000
IU/ml 1L-2 on the day of use.
8.2.3 Prepared an amount of CM3 ient to experimental needs by
adding IL-2 stock solution directly to the bottle or bag of AIM-V.
Mixed well by gentle shaking. Labeled bottle with “3000 IU/ml IL—2”
immediately after adding to the AIM-V. When there was excess CM3,
it was stored in bottles at 4°C labeled with the media name, the initials
of the preparer, the date the media was prepared, and its expiration date
(7 days after preparation).
8.2.4 Discarded media supplemented with IL-2 after 7 days storage at 4°C.
8.3 Preparation of CM4
8.3.1 CM4 was the same as CM3, with the additional supplement of 2mM
GlutaMAXTM (final concentration).
8.3.1.1 For every 1L of CM3, added 10ml of 200mM
GlutaMAXTM.
8.3.2 Prepared an amount of CM4 sufficient to experimental needs by
adding IL—2 stock solution and GlutaMAXTM stock solution directly to
the bottle or bag of AIM-V. Mixed well by gentle shaking.
8.3.3 Labeled bottle with “3000 IL/nil IL—2 and AX” immediately
after adding to the AIM-V.
8.3.4 If there was excess CM4, it was stored in s at 4°C labeled with
the media name, “GlutaMAX”, the initials of the preparer, the date the
media was prepared, and its expiration date (7 days after preparation).
8.3.5 Discarded media supplemented with IL—2 after 7 days storage at 4°C.
EXAMPLE 6: EVALUATION OF IRRADIATED ALLOGENEIC FEEDER CELLS
FOR RAPID EXPANSION PROTOCOL OF LN-144
This Example describes a novel abbreviated ure for qualifying dual lots
of gamma—irradiated peripheral mononuclear cells (PBMCs, also known as MNC) for use as
allogeneic feeder cells in the exemplary methods described .
Each irradiated MNC feeder lot was prepared from an individual donor. Each lot or
donor was ed individually for its ability to expand TIL in the REP in the presence of
purified anti-CD3 (clone OKT3) antibody and interleukin-2 (IL-2). In addition, each lot of
feeder cells was tested without the on of TIL to verify that the ed dose of gamma
radiation was sufficient to render them replication incompetent.
Definitions
AOPI - Acridine Orange/ um Iodide
BSC - Biological Safety Cabinet
CD3 - Cluster of Differentiation 3, surface marker protein for T-lymphocytes
CF - Centrifugal Force
CM1 - Complete Medium for T1L, #1
CM2 - Complete Medium for TIL, #
CMO - Contract Manufacturing Organization
C02 — Carbon Dioxide
EtOH - Ethyl Alcohol
GMP — Good Manufacturing Practices
Gy - Gray
IL-2 — Interleukin 2
IU - International Units
LN2— Liquid Nitrogen
Mini-REP - Mini-Rapid Expansion Protocol
ml - Milliliter
MNC - Mononuclear Cells
NA — Not Applicable
OKT3 - MACS GMP CD3 pure (clone OKT3) antibody
PPE — Personal Protective Equipment
Pre-REP - Before Rapid ion Protocol
QS — Quantum Satis; fill to this quantity
REP - Rapid ion Protocol
TIL - Tumor Infiltrating Lymphocytes
T25 - 25cm2 tissue e flask
ug - Micrograms
pl - Microliter
Equipment, Software, Materials
] Equipment
BSC (Biological Safety Cabinet)
Liquid Nitrogen Freezer
Temperature—controlled water bath
Centrifuge with swinging bucket rotor
Humidified tissue culture incubator
Pipet Aid
2—20ul Pipettor
—20011] Pipettor
lOO—lOOOul Pipettor
Automated Cell Counter
Material
15ml conical centrifuge tubes, sterile
50ml conical centrifuge tubes, sterile
AIM V Medium CTS peutic Grade)
Cell Counter Staining Solution
0 IL—2
0 MACS GMP CD3 pure (clone OKT3) antibody
0 Sterile, disposable gical pipets
o Sterile, disposable transfer pipets
o Sterile, pipet tips
0 24-well tissue culture plate
0 T25 flasks (Greiner #690175)
0 5.3.14. Zipper storage bags
PROCEDURE
Background
Gamma-irradiated, growth-arrested MNC feeder cells were required for REP (Step
D) of TIL expansion. ne receptors on the feeder MNCs bind to anti-CD3 (clone
OKT3) antibody and ink to TIL in the REP (Step D) flask, stimulating the TIL to
expand. Feeder lots were prepared from the leukapheresis of whole blood taken from
individual donors. The leukapheresis product was subjected to centrifugation over Ficoll-
e, , irradiated, and cryopreserved under GMP conditions.
It was important that patients who received TIL y not be infused with viable
feeder cells as this can result in Graft-Versus-Host Disease (GVHD). Feeder cells were
ore growth—arrested by dosing the cells with gamma—irradiation, which resulted in
double strand DNA breaks and the loss of cell viability of the MNC cells upon reculture.
Evaluation Criteria and Experimental Set-Up
Feeder lots were ted on two criteria: 1) their ability to expand TIL in ure
>100-fold and 2) their replication incompetency.
Feeder lots were tested in mini-REP format utilizing two primary pre-REP TIL lines
grown in upright T25 tissue culture flasks. Feeder lots were tested against two distinct TIL
lines, as each TIL line was unique in its ability to proliferate in response to activation in a
REP. As a control, a lot of irradiated MNC feeder cells which was historically been shown to
meet the criteria of 1) and 2): (1) their ability to expand TIL in co-culture old and (2)
their replication incompetency was run alongside the test lots.
To ensure that all lots tested in a single experiment receive equivalent testing,
sufficient stocks of the same pre—REP TIL lines were used to test all conditions and all feeder
lots. For each lot of feeder cells tested, there was a total of six T25 flasks:
o Pre-REP TIL line #1 (2 flasks)
o Pre-REP Tll line #2 (2 flasks)
o Feeder control (2 flasks)
0 NOTE: Flasks containing TIL lines #1 and #2 evaluated the ability of the feeder
lot to expand TIL. The feeder control flasks ted the replication
etence of the feeder lot.
mental Protocol
Day -2/3, Thaw of TIL lines
Prepared CM2 medium as per Example 5, Pre-REP and REP Media Preparation.
Warmed CM2 in 37°C water bath. Prepared 40 ml of CM2 supplemented with 3000IU/ml IL-
2. Kept warm until use. Placed 20ml of pre-warmed CM2 t lL-2 into each of two 50ml
conical tubes labeled with names of the TIL lines used. Removed the two designated pre-REP
TIL lines from LN2 storage and transfer the vials to the tissue culture room. Recorded TIL
line fication form. Thawed vials by placing them inside a sealed zipper storage bag in a
37°C water bath until a small amount of ice remains. Sprayed or wiped thawed vials with
70% ethanol and transferred vials to BSC. Used a sterile transfer pipet to ately
transfer the contents of vial into the 20ml of CM2 in the prepared, labeled 50ml conical tube.
QS (filled to this quantity) to 40ml using CM2 without IL-2 to wash cells. Centrifuged at 400
x CF for 5 minutes. Aspirated the atant and ended in 5ml warm CM2
mented with 3000 IU/ml IL-2. Removed small aliquot (20 ul) in duplicate for cell
counting using an automated cell counter. Recorded the counts. While counting, placed the
50ml conical tube with TIL cells into a humidified 370C, 5% C02 incubator, with the cap
loosened to allow for gas exchange. Determined cell concentration and dilute TIL to 1 x 106
cells/ml in CM2 supplemented with IL-2 at 3000 IU/ml. Cultured in 2ml/well of a 24-well
tissue culture plate in as many wells as needed in a humidified 370C incubator until Day 0 of
the mini-REP. Cultured the different TIL lines in separate 24-well tissue culture plates to
avoid confusion and potential cross-contamination.
Day 0, initiate Mini-REP
Prepared enough CM2 medium for the number of feeder lots to be tested. (e.g, for
testing 4 feeder lots at one time, prepare 800ml of CM2 medium). Aliquoted a portion of the
CM2 prepared in Example 5 and supplemented it with 3000 IU/ml IL-2 for the culturing of
the cells. (e.g., for g 4 feeder lots at one time, prepare 500ml of CM2 medium with 3000
IU/ml IL-2). The remainder of the CM2 with no IL-2 was used for washing of cells as
described below.
Prepared TIL
7.3.2.4.Working with each TIL line separately to prevent cross-contamination, the 24-
well plate with TIL e was removed from the incubator and erred to
the B SC.
7.3.2.5. Using a sterile transfer pipet or 100-1000u1 Pipettor and tip, d about 1ml
of medium from each well of TIL to be used and placed in an unused well of the
24-well tissue culture plate. This was used for washing wells.
6. Using a fresh sterile transfer pipet or 100-1000ul Pipettor and tip, mixed
remaining medium with TIL in wells to resuspend the cells and then transferred
the cell suspension to a 50ml l tube labeled with the TIL name and
recorded the volume.
7.3.2.7. Washed the wells with the reserved media and transferred that volume to the
same 50ml conical tube.
7.3.2.8. Spun the cells at 400 x CF to collect the cell pellet.
7.3.2.9. Aspirated off the media supernatant and resuspended the cell pellet in 2-5ml of
CM2 medium containing 3000 IU/ml IL-2, volume used was based on the
number of wells harvested and the size of the pellet — volume was sufficient to
ensure a concentration of >1 .3 x 106 cells/ml.
7.3.2.10. Using a serological pipet, mixed the cell suspension thoroughly and recorded the
volume.
7.3.2.11. Removed 200 pl for a cell count using an automated cell counter.
7.3.2.12. While counting, the 50ml conical tube with TIL cells was placed into a
humidified, 5% C02, 37°C tor, with the cap loosened to allow gas
7.3.2.13. Recorded the counts.
7.3.2.14. Removed the 50ml conical tube containing the TIL cells from the incubator and
resuspended them cells at a concentration of 1.3 x106 cells/ml in warm CM2
supplemented with 30001U/ml IL-2. Returned the 50ml conical tube to the
incubator with a loosened cap.
7.3.2.15 When needed, the original 24—well plate was kept to reculture any residual TIL.
7.3.2.16. Repeated steps 7.3.2.4 - 7.3.2.15 for the second TIL line.
7.3.2.17. Just prior to plating the TIL into the T25 flasks for the experiment, TIL were
diluted 1:10 for a final tration of 1.3 x 105 ml as per step 7.3.2.35
below.
Prepare MACS GMP CD3 pure {OKT3 2 working solution
7.3.2.18. Took out stock on of OKT3 (1mg/ml) from 4°C refrigerator and placed in
7.3.2.19. A final concentration of 30ng/ml OKT3 was used in the media of the mini-REP.
7.3.2.20. 600ng of OKT3 were needed for 20ml in each T25 flask of the experiment; this
is the equivalent of 60111 of a 10pg/ml solution for each 20ml, or 360pl for all 6
flasks tested for each feeder lot.
7.3.2.21. For each feeder lot tested, 400ul of a 1:100 dilution of 1mg/ml OKT3 was made
for a working tration of 10ug/ml (e.g., for testing 4 feeder lots at one
time, made 1600ul ofa 1:100 dilution of 1mg/ml OKT3: l6ul of 1mg/ml OKT3
+ 1.584ml of CM2 medium with 3000IU/ml IL-2.)
Prepare T25 flasks
7.3.2.22. Labeled each flask with the name of the TIL line tested, flask replicate ,
feeder lot number, date, and ls of analyst.
7.3.2.23. Filled flask with the CM2 medium prior to preparing the feeder cells.
7.3.2.24. Placed flasks into 37°C humidified 5% CO2 incubator to keep media warm
while waiting to add the remaining components.
7.3.2.25. Once feeder cells were prepared, the components were added to the CM2 in
each flask as shown in Table 14, Flask Set-up, below.
CM2 + 3000 IU/ml IL-2
VINC: 1.3 X 107/1111 in CM2 + 3000IU IL-Z (final
concentration 1.3 X 107/flask
OKT3: lOug/ml in CM2 + 3000IU IL-Z
TIL: 1.3 X lOS/ml in CM2 with 30001U of IL-2
(final concentration 1 _3 X l 05/flask)
ed Feeder Cells
7.3.2.26. A minimum of 78 X 106 feeder cells were needed per lot tested for this protocol.
Each lml vial frozen by SDBB had 100 X 106 viable cells upon freezing.
Assuming a 50% recovery upon thaw from LN2 storage, it was recommended to
thaw at least two lml vials of feeder cells per lot giving an estimated 100 X 106
viable cells for each REP. Alternately, if supplied in 1.8ml vials, only one vial
would provide enough feeder cells
27. Before thawing feeder cells, pre-warmed approximately 50ml of CM2 without
1L-2 for each feeder lot to be tested.
7.3.2.28. Removed the designated feeder lot vials from LN2 storage, placed in zipper
storage bag, and place on ice. erred vials to tissue culture room.
7.3.2.29. Thawed vials inside closed zipper storage bag by ing in a 37°C water
bath.
7.3.2.30. Removed vials from zipper bag, spray or wipe with 70% EtOH and transferred
vials to BSC.
7.3.2.31. Using a er pipet, the contents of feeder vials were immediately transferred
into 30ml of warm CM2 in a 50ml conical tube. Washed vial with a small
volume of CM2 to remove any residual cells in the vial.
7.3.2.32. Centrifuged at 400 X CF for 5 minutes.
7.3.2.33. Aspirated the supernatant and resuspended in 4ml warm CM2 plus 3000 IU/ml
1L-2.
7.3.2.34. Removed 200 pl for cell counting using the Automated Cell Counter. Record
the counts.
7.3.2.35. ended cells at 1.3 X ls/ml in warm CM2 plus 3000 IU/ml lL-2.
Setup Co-Culture
7.3.2.36. Diluted TIL cells from 1.3 X 106 cells/ml to 1.3 X 105 cells/ml. Worked with
each TIL line independently to prevent cross-contamination.
732.36.]. Added 45ml of CM2 medium to a 15ml conical tube.
7.3.2.36.2. Removed TIL cells from incubator and resuspended well using a 10ml
serological pipet.
7.3.2.363. Removed 0.5ml of cells from the 1.3 X 106 cells/ml TIL suspension and
add to the 4.5ml of medium in the 15ml conical tube. Returned TIL stock
vial to tor.
7.3.2.364. Mixed well.
7.3.2.365. Repeated steps 7.3.2.361— 364 for the second TIL line.
7.3 2.36.6. When testing more than one feeder lot at one time, diluted the TIL to the
lower concentration for each feeder lot just prior to plating the TIL.
7.3.2.37. Transferred flasks with pre-warmed media for a single feeder lot from the
incubator to the BSC.
7.3.2.38. Mixed feeder cells by pipetting up and down l times with a 1ml pipet tip
and transfer 1 ml (1.3 X 107 cells) to each flask for that feeder lot.
7.3.2.39. Added 60ul of OKT3 working stock ml) to each flask. 7.3.2.40. Returned
the two control flasks to the incubator.
7.3.2.41. Transferred 1 ml (1.3 X 105) of each TIL lot to the correspondingly labeled T25
flask.
7.3.2.42. Returned flasks to the incubator and incubated t. Did not disturb until Day
7.3.2.43. Repeated 36 — 7.3.2.42 for all feeder lots tested.
7.3.3. Day 5: Media changed
7.3.3.1. Prepared CM2 with 3000 IU/ml IL-2. 10ml is needed for each flask
7.3.3.2. To prevent cross—contamination, handled the flasks for a single feeder lot at a
time. Removed flasks from the incubator and transferred to the BSC, and care
was taken not to disturb the cell layer on the bottom of the flask.
7.3.3.3. Gently removed 10ml of the media from flask and ded.
7.3.3.4. Repeated for all flasks ing control flask.
7.3.3.5. With a 10ml pipette, transferred 10ml warm CM2 with 3000 IU/ml IL-2 to each
flask.
7.3.3.6. Returned flasks to the incubator and incubate upright until Day 7. 7.3.3.7.
Repeat 1 - 7.3.3.6 for all feeder lots tested.
7.3.4. Day 7: Harvest
7.3.4.1. To prevent cross-contamination, d the flasks for a single feeder lot at a
t1me.
7.3.4.2. Removed flasks from the incubator and transferred to the BSC, and care was
taken not to disturb the cell layer on the bottom of the flask.
7.3.4.3. Without disturbing the cells growing on the bottom of the flasks, removed 10ml
of medium from each test flask and 15ml of medium from each of the control
flasks.
7.3.4.4. Using a 10ml serological pipet, resuspended the cells in the remaining medium
and mixed well to break up any clumps of cells.
7.3.4.5. Recorded the volumes for each flask in Day 7.
6. After thoroughly mixing cell suspension by ing, removed 200 pl for cell
counting.
7.3.4.7. Counted the TIL using the appropriate standard operating procedure in
conjunction with the automatic cell counter equipment.
7.3.4.8. Recorded counts for Day 7.
9. Repeated 7.3.4.1 — 7.3.4.8 for all feeder lots tested.
7.3.4.10. Feeder control flasks were evaluated for replication incompetence and flasks
containing TIL were evaluated for fold expansion from Day 0 according to the
criteria listed in Figure 2.
7.3.5. Day 7 : Continuation of Feeder Control Flasks to Day 14
1. After completing the Day 7 counts of the feeder control flasks, added 15ml of
fresh CM2 medium containing 3000 IU/ml IL-2 to each of the l flasks.
7.3.5.2. Returned the control flasks to the incubator and incubated in an upright on
until Day 14.
7.3.6. Day 14 Extended Non-proliferation of Feeder Control Flasks
7.3.6.1 To prevent contamination, handled the flasks for a single feeder lot at a
time.
73.6.2 Removed flasks from the incubator and erred to the BSC, and care was
taken not to disturb the cell layer on the bottom of the flask.
7.3.6.3. Without disturbing the cells growing on the bottom of the flasks, removed
approximately 17ml of medium from each control flasks.
7.3.6.4. Using a 5ml serological pipet, resuspended the cells in the remaining medium
and mixed well to break up any clumps of cells.
7.3.6.5. Recorded the volumes for each flask.
7.3.6.6. After thoroughly mixing cell suspension by ing, removed 200ul for cell
counting.
7.3.6.7. d the TIL using the appropriate standard operating procedure in
conjunction with the automatic cell counter equipment
8. Recorded counts for Day 14.
7.3.6.9. Repeated 7.3.4.1 — 7.3.4.8 for all feeder lots tested.
Expected Results and Acceptance Criteria
Expected Results
The dose of gamma irradiation was sufficient to render the feeder cells replication
incompetent. All lots were expected to meet the evaluation criteria and also demonstrated a
reduction in the total viable number of feeder cells remaining on Day 7 of the REP culture
compared to Day 0.
] All feeder lots were ed to meet the evaluation criteria of lOO-fold ion
of TIL growth by Day 7 of the REP culture.
Day 14 counts of Feeder Control flasks were expected to continue the non—
proliferative trend seen on Day 7.
Acceptance criteria
The following acceptance criteria had to be met for each replicate TIL line tested for
each lot of feeder cells.
Acceptance was two-fold: as follows [outlined in Figure 2: Acceptance Criteria):
Whether the dose of radiation was sufficient to render the MNC feeder cells
replication incompetent when cultured in the ce of 30ng/ml OKT3 antibody and 3000
IU/ml [L-Z was evaluated,
ation incompetence was evaluated by total viable cell count (TVC) as
determined by ted cell counting on Day 7 and Day 14 of the REP.
Acceptance criteria is “No Growth,” meaning the total viable cell number had not
increased on Day 7 and Day 14 from the initial viable cell number put into culture on Day 0
of the REP.
Evaluate the ability of the feeder cells to support TIL expansion.
TIL growth was measured in terms of fold expansion of viable cells from the onset
of culture on Day 0 of the REP to Day 7 of the REP.
On Day 7, TIL cultures achieved a minimum of lOO-fold expansion, (i.e., r
than 100 times the number of total viable TIL cells put into culture on REP Day 0), as
evaluated by automated cell counting.
MNC feeder lots that did not meet these two criteria above were typically ed.
Any MNC feeder lots that meet ance criteria but are judged to have poor
mance in regard to the ability to expand TIL relative to other previous feeder lots tested
in parallel with the same pre-REP TIL lines, as judged by those of skill in the art could have
been excluded. See Table 15 below for ance criteria used.
Table 14 ce ntance Criteria
0 growth observed at 7 and 14 days
At least a lOO-fold expansion of each TIL
TIL eXpanswn. (minimum of 1.3 x 107 viable cells)
Whether the dose of radiation was sufficient to render the MNC feeder cells
replication incompetent when cultured in the presence of 30ng/ml OKT3 antibody and 3000
IU/ml lL—2 was evaluated.
.2.2.1.1 Replication incompetence was evaluated by total viable cell count (TVC)
as determined by automated cell counting on Day 7 and Day 14 of the
.2.2.1.2 Acceptance criteria was “No Growth,” meaning the total viable cell
number was not increased on Day 7 and Day 14 from the initial viable cell
number put into culture on Day 0 of the REP.
.2 The ability of the feeder cells to support TIL expansion was evaluated.
.2.2.2.1 TIL growth was measured in terms of fold expansion of viable cells from
the onset of e on Day 0 of the REP to Day 7 of the REP.
.2.2.2.1 On Day 7, TIL cultures achieved a m of 100-fold expansion, (i.e.,
greater than 100 times the number of total viable TIL cells put into culture
on REP Day 0), as evaluated by automated cell ng.
.2.2.3 When a lot failed to meet the two criteria above, the lot was retested
according to the contingency plan outlined in Section 10.3 below.
.2.2.4 Following retesting of a failed lot, any MNC feeder lot that did not meet
the two acceptance criteria in both the original evaluation and the
contingency testing was ed.
.2.2.5 Any MNC feeder lots that met acceptance criteria but were judged to have
poor performance in regard to the ability to expand TIL relative to other
previous feeder lots tested in parallel with the same pre-REP TIL lines
were excluded as appropriate.
Contingency Testing ofMNC Feeder Lots that do not meet acceptance criteria
.3.1 In the event that an MNC feeder lot met either of the acceptance ia
ed in Section 10.2 above, the ing steps were taken to retest the
lot to rule out simple experimenter error as its cause.
.3.2 If there were two or more remaining satellite testing vials of the lot, then
the lot could be retested. If there were one or no remaining satellite testing
vials of the lot, then the lot was failed according to the acceptance criteria
listed in Section 10.2 above.
.3.3 Two trained personnel, include the original person who evaluated the lot in
question, had to both test the lot at the same time.
.3.4 Repeating Section 7.2 — 7.3 was done to re-evaluate the lot in question.
.3.5 Each person would test the lot in question as well as a control lot (as
defined in Section 7.2.4 above).
.3.6 In order to be qualified, the lot in question and the control lot had to
achieve the ance criteria of Section 10.2 for both of the nel
doing the contingency testing.
.3.7 Upon g these criteria, the lot could then be released for CMO use as
outlined in Section 10.2 above.
EXAMPLE 7: PROCEDURE FOR QUALIFYING DUAL LOTS OF GAMMA-
IRRADIATED PERIPHERAL BLOOD MONONUCLEAR CELLS
This Example describes a novel abbreviated procedure for qualifying individual lots
of gamma-irradiated peripheral blood clear cells (PBMC) for use as allogeneic feeder
cells in the exemplary methods described . This example provides a protocol for the
evaluation of irradiated PBMC cell lots for use in the production of clinical lots of TIL. Each
irradiated PBMC lot was ed from an dual donor. Over the course of more than
100 qualification protocols, it has been shown that, in all cases, irradiated PBMC lots from
SDBB (San Diego Blood Bank) can expand TILs >100—fold on Day 7 of a REP. This
modified qualification protocol is intended to apply to irradiated donor PBMC lots from
SDBB which must still be tested to verify that the received dose of gamma radiation was
sufficient to render them replication incompetent. Once demonstrated that they maintain
replication incompetence over the course of 14 days, donor PBMC lots were ered
“qualified” for usage to produce clinical lots of TIL.
Key Terms and ions
pg — Microgram
ul — Microliter
AIM-V — commercially available cell e medium Biological Safety Cabinet
BSC — Cluster of entiation
CD — Complete Medium for TIL #2
CMZ — CM2 supplemented with 3000 IU/ml 1L—2
CMZILZ — Contract Manufacturing Organization
C02 — Carbon Dioxide
EtOH — Ethanol
GMP — Good Manufacturing Practices
Gy — Gray
IL — Interleukin
IU — International Units
LN2 7 Liquid Nitrogen
MI — Milliliter
NA — Not Applicable
OKT3 — anti-CD3 monoclonal antibody designation
P20 — 2-20pl pipettor
P200 — 20—20mm pipettor
PBMC — peripheral blood mononuclear cells
P1000 — lOO-lOOOul pipettor
PPE — Personal Protective Equipment
REP 7 Rapid Expansion Protocol
SDBB — San Diego Blood Bank
TIL — Tumor Infiltrating Lymphocytes
T25 — 25cm2 tissue culture flask
WO 81473
x g — “times gravity” — measure of relative centrifugal force
Specimens included Irradiated donor PBMC (SDBB).
Procedure
Background
7.1.1 Gamma-irradiated, growth-arrested PBMC were required for current standard
REP of TIL. Membrane receptors on the PBMCs bind to anti-CD3 (clone
OKT3) antibody and crosslink to TIL in culture, stimulating the TIL to
expand. PBMC lots were prepared from the leukapheresis of whole blood
taken from individual donors. The heresis product was subjected to
centrifugation over Ficoll-Hypaque, , irradiated, and cryopreserved
under GMP conditions.
It is important that patients who e TIL therapy not be infused with viable
PBMCs as this can result in Graft—Versus—Host e . Donor
PBMCs were therefore growth-arrested by dosing the cells with gamma-
irradiation, resulting in double strand DNA breaks and the loss of cell viability
of the PBMCs upon reculture.
Evaluation Criteria
7.2.1 Evaluation criterion for irradiated PBMC lots was their replication
incompetency.
Experimental Set-up
7.3.1 Feeder lots were tested in mini—REP format as if they were to be co—cultured
with TIL, using upright T25 tissue culture flasks.
7.3.1.1 Control lot: One lot of irradiated PBMCs, which had historically
been shown to meet the criterion of 7.2. 1, was run alongside the
experimental lots as a control.
7.3 .2 For each lot of irradiated donor PBMC tested, duplicate flasks were run.
Experimental Protocol
All tissue culture work in this ol was done using sterile technique in a
Day 0
7.4.1 ed ~90ml of CM2 medium for each lot of donor PBMC to be tested.
Kept CM2 warm in 37°C water bath.
7.4.2 Thawed an aliquot of6 x 106 IU/ml IL-2.
7.4.3 Returned the CM2 medium to the BSC, wiping with 70% EtOH prior to
g in hood. For each lot ofPBMC tested, about 60ml of CM2 was
removed to a separate sterile bottle. Added IL—2 from the thawed 6 x 106
IU/ml stock solution to this medium for a final concentration of 3000 IU/ml.
Labeled this bottle as “CM2/ILZ” (or similar) to distinguish it from the
unsupplemented CMZ.
7.4.4 Labeled two T25 flasks for each lot of PBMC to be tested. Minimal label
included:
7.4.4.1 Lot number
7.4.4.2 Flask number (1 or 2)
7.4.4.3 Date of initiation of culture (Day 0)
ed OKT3
7.4.5 Took out the stock solution of anti-CD3 (OKT3) from the 4°C refrigerator and
placed in the BSC.
7.4.6 A final concentration of 30ng/ml OKT3 was used in the media of the mini—
7.4.7 Prepared a lOug/ml g solution of anti-CD3 (OKT3) from the lmg/ml
stock solution. Placed in refrigerator until needed.
7.4.7.1 For each PBMC lot tested, prepared 150 pl of a 1:100 dilution of
the anti-CD3 (OKT3) stock.
E. g., for testing 4 PBMC lots at one time, prepared 600111 of
10ug/ml D3 (OKT3) by adding 6111 of the 1mg/ml stock
solution to 594 pl of CM2 supplemented with 3000 IU/ml IL-2.
ed Flasks
7.4.8 Added 19ml per flask of CM2/IL-2 to the labeled T25 flasks and place flasks
into 37°C, humidified, 5% C02 incubator while preparing cells.
Prepared Irradiated PBMC
7.4.9 Worked with each donor PBMC lot individually to avoid the potential cross-
contamination of the lots.
7.4.10 Retrieved vials of PBMC lots to be tested from LN2 storage. These were
placed at —80°C or kept on dry ice prior to thawing.
7.4.11 Placed 30ml of CMZ (without IL-Z supplement) into 50ml conical tubes for
each lot to be thawed. Labeled each tube with the different lot numbers of the
PBMC to be thawed. Capped tubes tightly and place in 37°C water bath prior
to use. As needed, returned 50ml conical tubes to the BSC, wiping with 70%
EtOH prior to placing in the hood.
7.4.12 d a vial PBMC from cold e and place in a floating tube rack in a
37°C water bath to thaw. Allowed thaw to proceed until a small amount of ice
remains in the vial.
7.4.13 Sprayed or wiped thawed vial with 70% EtOH and er to BSC.
7.4.14 Using a sterile transfer pipet, the contents of the vial were immediately
transferred into the 30ml of CM2 in the 50ml conical tube. Removed about
1ml of medium from the tube to rinse the vial; returned rinse to the 50ml
conical tube. Capped tightly and swirl gently to wash cells.
7.4.15 Centrifuged at 400 x g for 5min at room temperature.
7.4.16 Aspirated the supernatant and resuspended the cell pellet in 1ml of warm
CM2/IL-2 using a 1000ul pipet tip. atively, prior to adding ,
resuspended cell pellet by dragging capped tube along an empty tube rack.
After resuspending the cell pellet, bring volume to 4ml using CM2/IL-2
medium. Recorded volume.
7.4.17 Removed a small aliquot (e.g., 100ul) for cell counting using an automated
cell counter.
7.4.17.1 Performed counts in ate according to the particular
automated cell counter SOP. It was often necessary to perform a
dilution of the PBMC prior to performing the cell counts. A
recommended starting dilution was 1:10, but this could vary
depending on the type of cell counter used.
7.4.17.2 Recorded the counts.
7418 Adjusted concentration of PBMC to 1.3 x 107 ml as per step 7.4.15.2
using CM2/lL-2 medium. Mixed well by gentle swirling or by gently
ting up-and-down using a serological pipet.
Set Up Culture Flasks
7.4.19 ed two labeled T25 flasks to the BSC from the tissue culture incubator.
7.4.20 Returned the lOug/ml vial of anti-CD3/OKT3 to the BSC.
7.4.21 Added 1ml of the 1.3 x 107 PBMC cell suspension to each flask.
7.4.22 Added 60ul ofthe lOug/ml anti-CD3/OKT3 to each flask.
7.4.23 ed capped flasks to the tissue culture incubators for 14 days of growth
without disturbance.
7.4.24 The anti-CD3/OKT3 vial was placed back into the refrigerator until needed for
the next lot.
7.4.25 Repeated steps 7.4.9 — 7.4.24 for each lot of PBMC to be evaluated.
Day 14, Measurement of Non-proliferation of PBMC
7.4.26 Working with each lot independently, carefully returned the duplicate T25
flasks to the BSC.
7.4.27 For each flask, using a fresh 10ml serological pipet, removed ~l7ml from each
of the flasks, then lly pulled up the remaining media to measure the
volume remaining in the flasks. ed volume.
7.4.28 Mixed sample well by ing up and down using the same serological pipet.
7.4.29 Removed a 200ul sample from each flask for counting.
7.4.30 Counted cells using an automated cell counter.
7.4.31 Repeated steps 7.4.26 — 7.4.31 for each lot of PBMC being evaluated.
RESULTS AND ACCEPTANCE CRITERION
Results
.1.1 The dose of gamma irradiation was ent to render the feeder cells
replication incompetent. All lots were expected to meet the evaluation
criterion and demonstrated a reduction in the total viable number of feeder
cells remaining on Day 14 of the REP culture compared to Day 0.
Acceptance ion
.2.1 The following acceptance criterion was met for each irradiated donor PBMC
lot tested:
.2.2 “No growth” — meaning that the total number of viable cells on Day 14 was
less than the initial viable cell number put into culture on Day 0 of the REP.
.2.3 Should a lot fail to meet the ion above, the lot was retested per the
Contingency Testing Procedure outlined in the section 10.4.
.2.4 ing retesting of a failed lot, any MNC feeder lot that did not meet the
acceptance criterion in both the original evaluation and the contingency testing
was excluded.
Contingency Testing ofPBMC lots which did not meet acceptance ion.
.4.1 In the event than an irradiated donor PBMC lot did not meet the acceptance
criterion above, the following steps were taken to retest the lot to rule out
simple experimenter error as the cause of its failure.
.4.2 If there were two or more ing satellite vials of the lot, then the lot was
retested. If there were one or no remaining satellite vials of the lot, then the lot
was failed according to the acceptance ion of section 10.2 above.
.4.3 Whenever possible, two trained personnel rably including the original
person who evaluated the lot in question) did the testing of the two separate
vials independently. This was the preferred method of contingency testing.
Aside from the separate vials of PBMC, the same reagents can be used by both
personnel.
.4.3.1. If two personnel were not available, one person did the testing of
the two PBMC vials for the failed lot, working with each vial
independently.
.4.4 Repeating of section 7.4 “Experimental Protocol” was done to re-evaluated the
lot in question.
.4.5 In addition to the lot in question, a control lot was tested by each person
carrying out the contingency testing.
.4.5.1 If two personnel m gency testing, both personnel tested
the control lot ndently.
.4.5.2 If only one person was available to perform contingency testing, it
was not necessary for the control lot to be run in duplicate.
.4.5.3 To be qualified, a PBMC lot going through contingency testing
must have had both the control lot and both replicates of the lot in
question achieve the acceptance criterion of Section 10.2 to pass.
.4 Upon meeting this criterion, the lot was then be released for CMO usage as
outlined in section 10.2.
EXAMPLE 8: COMPARISON OF PRE- AND RYOPRESERVED TILS
Antibody cocktails for the samples and the FMO controls were made before starting
the sample preparation and staining procedure. The cocktails were stored at 4°C in the dark
for up to 60 days. See Cocktail Preparation section below.
Table 15: Stainin Procedure:
Removed Aqua dye aliquot from the freezerptdark.
Added 3mL lXPBS to each sample tube
Spun tubes at 300g for 5 minutes.
Prepared Aqua Live/Dead stain. Dilute 1:200 in PBS. 25 uL per sample and
FMO control tube is .
1:200 = LAuua+ mL PBS
Aspirated or decanted atant from step 3
Added 25uL of Aqua L/D to each sample tube. Resuspended cells by ng
along rack Incubated 15min, dark, room temperature.
Without washing, added SOpL of appropnate Ab cocktail to each tube
Incubated tubes for 15 minutes at room temperature
Added 3mLs ofFACS Wash buffer
Spun at 330g for 5 min at 4°C.
Resuspended tubes by dragging along an empty tube rack.
Added 100“L 1% PFA/PBS solution at 4°C.
Stored samples at 4°C in dark for up to 72 hours.
Ran s on Flow Cytometer
Table 16: Differentiation Panel 1 DF1 :
PE/Cy7 IP26 BioLegend 306720
Pcergl: HNK-l BioLegend 359622
PE CD282 BioLegend 302908
CD4 FITC OKT4 eBioscience 11 042 2
Coulter
Table 17: Differentiation Panel 2 DF2
CD45RA* PE-Cy7 H1100 560675
CD8a PerCP/Cy5.5 RPA-T8 BioLegend 301032
150503 BD Biosciences 560765
110048-
OKT4 eBioscience
ATP/CW BioLegend 300318
CD3 8* 113-7 BioLegend 356606
HLA-DR L243 BioLegend 3 0763 3
>“Denotes FMO (Fluorescence Minus One) control should be made.
Table 18: T cell Activation Panel 1 Tactl
CD137* PE/C7 4B4-1 309818 -
PerCP/C5.5 RPA-T8 301032 -
Lag3"< PE 3DS223H eBioscience 1242
CD4 FITC OKT4 BioLeend 317408
CD3 APC/C 7 HIT3a 300318 -
EH122H7 329908 -
BV421 F38—2E2 345008 -
Table 19: T cell Activation Panel 2 (Tath)
CD69* PE-C 7 FNSO ilznces
CD8a PerCP/C 5.5 RPA-T8 BioLe_end
TIGIT* PE MBSA43 eBioscience
CD4 FITC OKT4 BioLe_end
CD3 APC/C 7 HIT3a BioLe_end
KLRG1* Ax647 SA231A2 BioLe_end
CD154* BV421 TRAP1 B' ' 563886
>“Denotes FMO (Fluorescence M1nus One) control should be made.
Compensation Controls
1. Added one drop ofBD Comp beads to 11 tubes.
2. Labeled tubes 1 through 7 with the chromophores from DF1
3. Labeled tubes 8 through ten with APCy7, BV421, and AX647.
4 Tube 11 was for unlabeled beads.
. Added 5 uL of Antibody to each tube.
6. Incubated 10 to 30 minutes in dark, room temperature.
7. Washed with 3mLs FACS Buffer
8. Resuspended with SOOuL 1% PFA.
9. Added one drop ofBD Comp negative bead to each tube.
. Stored at 4°C in dark. Could be used for one week.
Aqua l:
1. Added one drop of Arc positive control to tube labelled Aqua.
2. Added 3 uL of thawed aqua on to tube.
3. Repeated steps 6 — 10 as above. Except used the negative Arc bead for step 9.
Table 20: Setup.
2 PerCP-CyS .5
———3 U‘IUILI‘I
“——kl‘lkl‘l
———kl‘lkl‘lU‘IUI
EXAMPLE 9: REMARKABLY STABLE TUMOR-INFILTRATING
LYMPHOCYTES (TIL) FOR INFUSION PHENOTYPE FOLLOWING
CRYOPRESERVATION
Abstract Background:
This Example discusses the development of cancer immunotherapies based on
infiltrating lymphocytes (TIL) with the ultimate goal of developing therapeutic
populations of TILs. Cryopreservation of TILs allows the final cell product to be shipped in a
safe manner with fewer temporal constraints (Axelsson S, Faresjo M, Hedman M, Ludvigsson
J, Casas R: Cryopreserved peripheral blood mononuclear cells are suitable for the assessment
of immunological markers in type 1 diabetic children. Cryobiology 2008, 57:201—8.)
Here, fresh versus frozen/thawed TIL samples were evaluated for the expression of
individual phenotypic markers to assess whether ypic changes occur with
cryopreserved TILs. (See, for example, i A, ag G, Wagenius G, man TH,
Eriksson F: Rapid ion of T cells: Effects of e and cryopreservation and
importance of short-term cell recovery. Acta Oncol. 2013,52z978-86.)
Results:
No significant ences in CD4, CD8, NK, TCROLB expression, or memory
markers comparing fresh versus thawed TIL were observed. The activation status of TIL as
defined by HLA-DR, CD38, and CD69 expression was maintained while regulatory
molecules LAG-3 and TIM-3 demonstrated a slight decrease in expression. In addition, the
viability of both the fresh and thawed product was greater than 86%.
Methods:
PreREP TILs were obtained by culturing melanoma tumor fragments in IL-2 (6000
IU/ml).
Rapid Expansion ol (REP) cells were initiated using irradiated allogeneic
PBMC feeder cells with OKT3 and IL-2 in a GREX-lOO flask for 11-14 days.
Cultured cells were cryopreserved in 5% DMSO.
Flow tric evaluation of fresh and thawed TIL following rest for 1 to 2 hours
in IL-2 was performed using four panels consisting of lineage, differentiation, activation, and
regulatory markers.
Conclusion:
Cryopreservation did not affect the measured ypic characteristics of TIL,
with the exception of modest changes in some regulatory molecules. We are investigating the
possibility of using cryopreserved TIL in a clinical setting.
E 10: MEMORY CELL SUBSETS IN FRESH VERSUS REREP TIL
POPULATIONS
In previous experiments, no central memory subset was seen with fresh TIL
populations (see, Figure 8). However, after the ReREP nearly 60% central memory cells, as
provided in Table 22 below.
Based on the raw numbers, the rested cells had a slightly higher CD4 population
than the not rested. l the CD8 percentage was high as expected. It’s roughly a 60/40
split for CM al memory—Q3)/EM (effector memory—Q4) among the CD8s. The
CD8+CD28+ expression looks interesting. The rested cells have a higher amount. See also,
Figure 9 and lOA—lOB. See, also Figure 15.
EXAMPLE 11: ADMINISTRATION OF AUTOLOGOUS TUMOR INFILTRATING
LYMPHOCYTES (TILS) IN MELANOMA PATIENTS
Administration of autologous tumor infiltrating lymphocytes (TILS) in
melanoma patients has shown an overall response of 55% at NCI, 38% at Moffitt Cancer
Center, 48% at MD Anderson Cancer Center, and 40% in Sheba at the Ella Cancer Institute,
Israel. The durable responses observed in melanoma patients using ACT may permit broader
ation to other solid tumors. As shown herein, the ility of growing TILs and
ping TIL therapies for other solid tumors is demonstrated. The example es data
showing “Successful expansion and characterization of tumor infiltrating lymphocytes (TILs)
from non-melanoma tumors”, see, Figures 12-14.
Phenotypic terization of TILs from bladder, cervical, and lung cancer
were greater than 60-70% CD8+ T-cells whereas TILs from head and trated variable
distribution of CD8+ and CD4+ T-cells. TILs propagated from TNBC were greater than
80% CD4+ T-cells. Regardless of the tumors, most cultures had less than 20% CD56+
NK cells.
TILs were prepared by:
a. Washing an obtained tumor in HBSS;
b. Dicing the tumor into fragments (e.g., 2-3 mm3 fragments);
c. g the tumor fragments in G-REX 10 cell e flasks with
medium containing serum and IL-2,
d. Exchanging media on day 7 and every 4-5 days from day 11 until day 21,
e. Assessing cell count, viability, and phenotyping followed by
cryopreservation for future purposes including, but not limited to, future
delivery to patients for the treatment of , as described herein.
As demonstrated herein, TILs were grown from lung, bladder, head and
neck, cervical, and TNBC patient tumors.
Moreover, as demonstrated herein, lung, bladder, and cervical tumors
showed greater proportion of CD8+ TILs. Head and neck and TNBC tumors were mostly
CD4+ TILs. In addition, further terization of CD4+ and CD8+ TILs demonstrated
effector memory phenotypic cells that were also CD27+ and CD28+.
WO 81473
The examples set forth above are provided to give those of ordinary skill in the
art a complete disclosure and description of how to make and use the embodiments of the
compositions, systems and methods of the invention, and are not intended to limit the scope
of what the inventors regard as their invention. Modifications of the above-described modes
for carrying out the invention that are s to persons of skill in the art are intended to be
within the scope of the following claims. All patents and publications mentioned in the
specification are indicative of the levels of skill of those skilled in the art to which the
ion ns. All references cited in this disclosure are incorporated by reference to the
same extent as if each reference had been incorporated by nce in its entirety
individually.
All headings and n ations are used for clarity and reference
purposes only and are not to be considered limiting in any way. For example, those of skill in
the art will appreciate the usefulness of combining various aspects from different headings
and sections as appropriate according to the spirit and scope of the invention described
herein.
All references cited herein are hereby incorporated by reference herein in their
ties and for all purposes to the same extent as if each individual publication or patent or
patent application was specifically and individually ted to be incorporated by reference
in its ty for all purposes.
Many modifications and variations of this application can be made without
departing from its spirit and scope, as will be apparent to those skilled in the art. The specific
embodiments and examples described herein are offered by way of example only, and the
application is to be limited only by the terms of the appended claims, along with the full
scope of equivalents to which the claims are entitled.
Claims (54)
1. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic tion of TILs comprising: (i) ming a first expansion by culturing a first population of TILs obtained from a tumor sample from a patient in a cell e medium comprising IL-2 to produce a second population of TILs; and (ii) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, and wherein the second expansion is performed for about 7 to 11 days in order to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs.
2. The method according to claim 1, wherein the tumor sample comprises one or more tumor fragments.
3. The method according to claim 1, wherein the tumor sample comprises a tumor digest.
4. The method ing to claim 3, wherein the tumor digest was prepared by ting a sample of the tumor that was resected from the subject in an enzymatic media.
5. The method ing to claim 4, further comprising disrupting the tumor sample mechanically so as to dissociate the tumor sample.
6. The method according to claim 5, further comprising purifying the disassociated tumor sample using a density gradient tion.
7. The method according to any one of claims 4 to 6, wherein the enzymatic media comprises DNase.
8. The method according to any one of claims 4 to 7, wherein the enzymatic media comprises 30 units/mL of DNase.
9. The method according to any one of claims 4 to 8, wherein the enzymatic media comprises collagenase.
10. The method according to any one of claims 4 to 9, wherein the enzymatic media comprises 1.0 mg/mL of collagenase.
11. The method according to any one of claims 1 to 10, wherein the tumor sample comprising the first population of TILs is cryopreserved and thawed prior to step (i).
12. The method according to any one of claims 1 to 11, wherein the method further comprises: (iii) performing an onal second expansion by supplementing the cell culture medium of the third population of TILs with additional IL-2, additional OKT-3, and additional APCs, wherein the additional second expansion is med for about 7 to 11 days to obtain a larger therapeutic population of TILs than ed in step (ii).
13. The method according to claim 12, wherein after step (ii), the cells are removed from the cell e and cryopreserved in a e medium prior to performing step (iii).
14. The method according to claim 12 or 13, wherein the cells are thawed prior to performing step (iii).
15. The method according to any one of claims 1 to 14, wherein step (ii) is repeated one to four times in order to obtain sufficient TILs in the therapeutic population of TILs for a therapeutically effective dosage of the TILs.
16. The method according to any one of claims 1 to 15, wherein the first expansion is performed from about 11 to 21 days.
17. The method according to any one of claims 1 to 15, wherein the first expansion is performed within about 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or 21 days.
18. The method according to any one of claims 1 to 15, wherein the first expansion is med within about 11 days, 12 days, 13 days or 14 days.
19. The method according to any one of claims 1 to 15, wherein the first ion is performed for about 11 to 12 days.
20. The method according to any one of claims 1 to 19, wherein the second expansion is performed for about 7 days, 8 days, 9 days, 10 days, or 11 days.
21. The method according to any one of claims 1 to 19, wherein the second expansion is performed for at least 14 days in order to obtain the third population of TILs, wherein the third population of TILs is a therapeutic tion of TILs.
22. The method according to any one of claims 1 to 20, wherein steps (i) h (ii) or (iii) are performed within a period of about 40 days to about 50 days.
23. The method according to any one of claims 1 to 20, wherein steps (i) through (ii) or (iii) are performed within a period of about 42 days to about 48 days.
24. The method according to any one of claims 1 to 20, wherein steps (i) through (ii) or (iii) are performed within a period of about 42 days to about 45 days.
25. The method according to any one of claims 1 to 20, wherein steps (i) through (ii) or (iii) are performed within about 44 days.
26. The method according to any one of claims 1 to 20, wherein steps (i) through (ii) or (iii) are performed within about 24 days.
27. The method according to any one of claims 1 to 20, wherein steps (i) through (ii) or (iii) are performed within about 26 days.
28. The method according to any one of claims 1 to 27, wherein the cells from steps (ii) or (iii) express CD4, CD8, and TCR αβ at levels similar to freshly harvested cells.
29. The method according to any one of claims 1 to 28, wherein the third population of TILs is at least 100-fold greater in number than the second population of TILs.
30. The method according to any one of claims 1 to 29, wherein the third population of TILs is a therapeutic population of TILs which comprises an increased subpopulation of effector T cells and/or l memory T cells ve to the second population of TILs.
31. The method according to claim 30, wherein the effector T cells and/or central memory T cells in the therapeutic population of TILs exhibit one or more characteristics selected from the group consisting of expression of CD27, expression of CD28, longer telomeres, increased CD57 expression, and decreased CD56 expression, relative to effector T cells and/or central memory T cells in the third population of cells.
32. The method according to any one of claims 1 to 31, wherein the antigen presenting cells are eral blood mononuclear cells (PBMCs).
33. The method according to claim 32, wherein the PBMCs are added to the cell culture on any of days 9 h 11 in step (ii).
34. The method ing to any one of claims 1 to 31, n the APCs are artificial APCs (aAPCs).
35. The method ing to any one of claims 1 to 34, further comprising the step of transducing the first population of TILs with an expression vector comprising a nucleic acid encoding a high-affinity T cell receptor.
36. The method according to any one of claims 1 to 34, further comprising the step of transducing the first population of TILs with an expression vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising a single chain variable nt antibody fused with at least one endodomain of a T-cell signaling molecule.
37. The method according to any one of claims 1 to 36, wherein the therapeutic tion of TILs are formulated for infusion into a patient.
38. The method according to any one of claims 1 to 37, where the number of TILs sufficient for a therapeutically effective dosage is from about 2.3×1010 to about 13.7×1010.
39. The method according to any one of claims 1 to 38, the method further comprising: (a) performing an additional expansion of a portion of the third population of TILs by supplementing the cell culture medium of the third population with IL-2, OKT-3, and APCs for a reREP period of at least 3 days, wherein the portion of the third population of TILs is previously cryopreserved and thawed prior to the additional expansion, wherein the third expansion is performed to obtain a fourth population of TILs, wherein the number of TILs in the fourth population of TILs is ed to the number of TILs in the third population of TILs to obtain a ratio; (b) determining based on the ratio in step (a) whether the thawed tion of TILs is suitable for administration to a patient; and (c) formulating a eutically effective dosage of the thawed third population of TILs for administration to the patient when the ratio of the number of TILs in the fourth population of TILs to the number of TILs in the third population of TILs is determined to be greater than 5:1 in step (b).
40. A therapeutic tion of tumor infiltrating lymphocytes (TILs) obtainable by the method comprising: (i) performing a first ion by culturing a first population of TILs obtained from a tumor sample from a patient in a cell culture medium comprising IL-2 to produce a second population of TILs; (ii) ming a second expansion by supplementing the cell culture medium of the second population of TILs with onal IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, and wherein the second expansion is performed for about 7 to 11 days in order to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs; (iii) harvesting the third population of TILs obtained from step (ii); (iv) transferring the harvested third population of TILs from step (iii) into an on bag; (v) cryopreserving the infusion bag comprising the harvested TIL population from step (iv) using a cryopreservation process.
41. A eutic population of tumor infiltrating lymphocytes (TILs) obtainable by the method according to any of claims 1 to 38.
42. Use of expanded tumor infiltrating lymphocytes (TILs) in the manufacture of a medicament for the treatment of a cancer in a subject, wherein treatment comprises administration of the TILs to the subject, n the expanded TILs are obtained as follows: (i) performing a first expansion by culturing a first population of TILs obtained from a tumor sample from a patient in a cell e medium comprising IL-2 to produce a second population of TILs; (ii) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and antigen presenting cells , to e a third population of TILs, and wherein the second expansion is performed for about 7 to 11 days in order to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs; (iii) harvesting the third population of TILs obtained from step (ii); (iv) transferring the harvested third population of TILs from step (iii) into an infusion bag; (v) cryopreserving the infusion bag comprising the harvested TIL population from step (iv) using a cryopreservation s; and (vi) formulating for administration a therapeutically effective dosage of the third population of TILs to the subject.
43. The use according to claim 42, r comprising: (a) performing an additional ion of a portion of the third population of TILs by menting the cell culture medium of the third population with IL-2, OKT-3, and APCs for a reREP period of at least 3 days, wherein the portion of the third population of TILs is previously cryopreserved and thawed prior to the additional expansion, wherein the third expansion is performed to obtain a fourth population of TILs, wherein the number of TILs in the fourth population of TILs is compared to the number of TILs in the third population of TILs to obtain a ratio; (b) determining based on the ratio in step (a) whether the thawed tion of TILs is suitable for administration to a t; and (c) formulating a therapeutically effective dosage of the thawed third tion of TILs for administration to the patient when the ratio of the number of TILs in the fourth population of TILs to the number of TILs in the third population of TILs is determined to be greater than 5:1 in step (b).
44. Use of expanded tumor infiltrating lymphocytes (TILs) in the manufacture of a medicament for the treatment of cancer in a subject, wherein treatment comprises administration of the TILs to the subject, wherein the TILs are obtained by the method according to any one of claims 1 to 38.
45. The use according to claim 43 or 44, wherein prior to the stration of a therapeutically effective dosage of TIL cells, a non-myeloablative lymphodepletion regimen has been administered to the subject, n the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg/m2/day for two days followed by administration of fludarabine at a dose of 25 mg/m2/day for five days.
46. The use according to any of claims 43 to 45, n the subject is further treated with a high-dose IL-2 regimen starting on the day after stration of the TIL cells to the subject, wherein optionally the high-dose IL-2 regimen comprises 600,000 or 720,000 IU/kg administered as a 15-minute bolus intravenous on every eight hours until tolerance.
47. The use according to any of claims 43 to 46, n the cancer is selected from the group consisting of melanoma (including metastatic melanoma), ovarian cancer, cervical cancer, non-small-cell lung cancer (NSCLC), lung , bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, and renal cell carcinoma.
48. A method for ing the metabolic activity of a TIL cell population obtained by the method according to any one of claims 1 to 38, sing measuring the basal glycolysis of the cells, ing the basal respiration of the cells, measuring the spare respiratory capacity (SRC) of the cells, and/or ing the ytic reserve of the cells.
49. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs according to the drawings and/or the specification.
50. A therapeutic population of tumor infiltrating lymphocytes (TILs) obtainable by the method according to the drawings and/or the specification.
51. Use of expanded tumor infiltrating lymphocytes (TILs) in the manufacture of a medicament for the treatment of cancer in a subject according to the drawings and/or the specification.
52. A cryopreserved population of tumor infiltrating lymphocytes (TILs) obtainable by the method according to the drawings and/or the specification.
53. A cryopreserved therapeutic population of tumor infiltrating lymphocytes (TILs) obtainable by the method according to the drawings and/or the specification.
54. A method for assessing the metabolic activity of a TIL cell population ing to the drawings and/or the specification. WO 81473 mN mN mHH i E Eaten. co=m_:E_Hmm._-Hmon H H H H m.NN wda DVD imHHmOH 3H mm: umiwmmaoru auspice. m? HNH 153 NH N.~ mm H H H md HmNN mdwm Hm.- EN 0de N unseen. Hmwimzhmon #4» 06 wam :HHHm 3 w: m§ME H H H H H 5.2”. mNH mdfi mdfim in 32 AEEVEEE EE “5 c0385m9 :38 E E m_m>_8>
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US62/413,283 | 2016-10-26 | ||
US62/413,387 | 2016-10-26 | ||
US62/415,452 | 2016-10-31 |
Publications (1)
Publication Number | Publication Date |
---|---|
NZ793724A true NZ793724A (en) | 2022-10-28 |
Family
ID=
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11304980B2 (en) | Restimulation of cryopreserved tumor infiltrating lymphocytes | |
NZ793724A (en) | Restimulation of cryopreserved tumor infiltrating lymphocytes | |
NZ793726A (en) | Restimulation of cryopreserved tumor infiltrating lymphocytes | |
NZ793725A (en) | Restimulation of cryopreserved tumor infiltrating lymphocytes |