NZ794058A - Remnant tumor infiltrating lymphocytes and methods of preparing and using the same - Google Patents
Remnant tumor infiltrating lymphocytes and methods of preparing and using the sameInfo
- Publication number
- NZ794058A NZ794058A NZ794058A NZ79405817A NZ794058A NZ 794058 A NZ794058 A NZ 794058A NZ 794058 A NZ794058 A NZ 794058A NZ 79405817 A NZ79405817 A NZ 79405817A NZ 794058 A NZ794058 A NZ 794058A
- Authority
- NZ
- New Zealand
- Prior art keywords
- tumor
- rtils
- etils
- patient
- cell
- Prior art date
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Abstract
some embodiments, methods of delivering a therapeutically effective amount of an expanded number of tumor infiltrating lymphocytes obtained from tumor remnants to a patient in need thereof, for the treatment of a cancer, are disclosed.
Description
In some embodiments, methods of delivering a therapeutically effective amount of an expanded
number of tumor infiltrating lymphocytes obtained from tumor remnants to a patient in need
f, for the treatment of a cancer, are disclosed.
NZ 794058
REMNANT TUMOR INFILTRATING CYTES AND METHODS OF PREPARING
AND USING THE SAME
CROSS-REFERENCE TO RELATED APPLICATIONS
This international ation claims the benefit of ty to US. Provisional
Application No. 62/423,750, filed November 17, 2016, and US. Provisional Application No.
62/460,441, filed ry 17, 2017, the entirety of which are incorporated herein by reference.
FIELD OF THE INVENTION
Methods and itions for expansion of tumor infiltrating lymphocytes from tumor
ts are disclosed in some embodiments.
BACKGROUND OF THE INVENTION
Treatment of bulky, refractory cancers using adoptive transfer of tumor infiltrating
lymphocytes (TILs) represents a ul approach to therapy for patients with poor prognoses.
Gattinoni, el al., Nat. Rev. Immunol. 2006, 6, 383-393. Adoptive T cell therapy with autologous
TILs provides up to 55% objective response rates and durable regression in >25% of patients
with metastatic melanoma. 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, logistical, and regulatory issues with cell expansion. 1Lbased
TIL expansion ed by a “rapid expansion s” (REP) has become a preferred method
for TIL expansion because of its speed and efficiency. Dudley, el al., Science 2002, 298, 850-
54, Dudley, el al., J. Clin. Oncol. 2005, 23, 7, Dudley, el al., J. Clin. Oncol. 2008, 26,
5233-39, Riddell, el al., Science 1992, 257, 23 8-41, Dudley, el al., J. Immunolher. 2003, 26,
. Processes for generating TILs from resected tumors include the step of morcellating the
tumor into 1-3 mm3 fragments, and expanding TILs in the presence of interleukin 2 (IL-2) in the
pre-rapid expansion protocol (pre-REP or initiation) step. During the pre-REP step, tumor-
resident immune cells emigrate and erate, and these TILs are subjected to a second REP
s, with irradiated peripheral blood mononuclear cell (PBMC) feeders, anti-CD3 antibody
(OKT-3, muromonab), and IL-2, which greatly increases their numbers. To date, all TIL
expansion processes discard residual tumor fragments following the pre-REP process.
Direct enzymatic digestion of resected tumors has been previously explored as an
alternative to pre-REP, but has been reported to yield less TIL cultures, resulting in a decreased
ability to obtain TILs than from pre-REP initiation processes with IL-2. Dudley, el al., J.
lher. 2003, 26, 332-42. For this reason, digestion has not been further explored in the
development of TILs as a therapy for .
TILs obtained from the pre-REP and REP processes have dominated the clinical studies
of TILs to date, which have offered modest clinical responses, and the field remains challenging,
ularly in the extension of TIL therapy from melanoma to other tumor types. Goff, el al., J.
Clin. Oncol. 2016, 34, 2389-97, Dudley, el al., J. Clin. Oncol. 2008, 26, 5233-39, Rosenberg, el
al., Clin. Cancer Res. 2011, 17, 4550-57. Much focus has been placed on selection of TILs
during ion to either select particular subsets (such as CD8+ T cells) or to target driver
mutations such as a mutated ERBBZIP epitope or driver mutations in the KRAS oncogene.
Tran, el al., N. Engl. J. Med. 2016, 375, 2255-62, Tran, el al., Science 2014, 344, 641-45.
However, such ion approaches, even if they can be developed to show efficacy in larger
clinical trials, add significantly to the duration, complexity, and cost of performing TIL therapy
and limit the potential for widespread use of TIL therapy in different types of cancers. Thus,
there is an urgent need to develop ses e of providing TILs with improved properties
for use in cancer therapies.
The invention provides the unexpected finding that TILs with ed properties may
be obtained from ses based on tumor remnant cells, and that such remnant TILs (rTILs)
are phenotypically and functionally ct from normal emigrant TILs (eTILs). The use of
rTILs and combinations of rTILs and eTILs in cancer immunotherapy es cant
advantages over prior eTIL-based therapies.
SUMIVIARY OF THE INVENTION
In an embodiment, the invention includes a method for preparing remnant tumor
infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
infiltrating lymphocytes (TILs),
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and interleukin 2 (IL-2) to e tumor remnants and nt TlLs (eTILs),
(d) removing at least a plurality of the eTILs,
(e) enzymatically digesting the tumor remnants into tumor remnant cells using a digest
mixture; and
(f) expanding the tumor remnant cells with a second cell culture medium sing cell
culture media, ated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an expanded number of remnant tumor infiltrating lymphocytes
(rTILs),
wherein the rTlLs express reduced levels of a T cell tion marker relative to the
eTILs, and wherein the T cell exhaustion marker is selected from the group consisting of
TIM3, LAG3, TIGIT, PD-l, CTLA-4, and combinations thereof.
In an embodiment, the invention includes a method for preparing remnant tumor
infiltrating lymphocytes ) for adoptive T cell therapy, the method comprising:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue ses tumor
infiltrating lymphocytes (TlLs),
(b) nting the tumor ,
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor remnants and emergent TlLs (eTILs),
(d) removing at least a plurality of the eTILs,
(e) enzymatically digesting the tumor remnants into tumor remnant cells using a digest
mixture, and
(f) ing the tumor remnant cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an expanded number of remnant tumor infiltrating lymphocytes
(rTILs),
wherein the rTlLs express reduced levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, CTLA-4, and combinations thereof, and
wherein the tumor tissue is selected from the group consisting of melanoma tumor tissue,
head and neck tumor tissue, breast tumor tissue, renal tumor tissue, pancreatic tumor
tissue, astoma tumor tissue, lung tumor tissue, ctal tumor tissue, sarcoma
tumor tissue, triple negative breast tumor tissue, al tumor tissue, ovarian tumor
tissue, and acute myeloid leukemia bone marrow or tumor tissue.
In an ment, the invention includes a method for preparing remnant tumor
infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
infiltrating lymphocytes (TlLs),
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas ble container with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor ts and emergent TlLs (eTILs),
(d) removing at least a plurality of the eTILs,
(e) enzymatically digesting the tumor remnants into tumor t cells using a digest
mixture, and
(f) expanding the tumor remnant cells with a second cell culture medium comprising cell
e media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an expanded number of remnant tumor infiltrating lymphocytes
(rTILs),
wherein the rTlLs express reduced levels of a T cell exhaustion marker ve to the
eTILs,
wherein the T cell exhaustion marker is ed from the group consisting of TIM3,
LAG3, TIGIT, PD-l, CTLA-4, and combinations thereof, and
wherein the irradiated feeder cells comprise irradiated allogeneic peripheral blood
mononuclear cells.
WO 94167
In an embodiment, the invention includes a method for preparing remnant tumor
infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
infiltrating cytes (TlLs),
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas permeable ner with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor ts and emergent TlLs ),
(d) removing at least a plurality of the eTILs,
(e) enzymatically digesting the tumor remnants into tumor t cells using a digest
mixture, and
(f) expanding the tumor remnant cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an expanded number of remnant tumor infiltrating lymphocytes
(rTILs),
wherein the rTlLs express d levels of a T cell tion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations thereof, and
wherein IL-2 is present in the second cell culture medium at an initial tration of
about 3000 IU/mL and OKT-3 antibody is present in the second cell culture medium at
an initial concentration of about 30 ng/mL.
In an embodiment, the invention includes a method for preparing remnant tumor
infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
infiltrating lymphocytes (TlLs),
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor remnants and emergent TlLs (eTILs),
(d) removing at least a plurality of the eTILs,
(e) enzymatically digesting the tumor ts into tumor remnant cells using a digest
mixture; and
(f) expanding the tumor remnant cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
ner to provide an expanded number of remnant tumor infiltrating lymphocytes
(rTILs),
wherein the rTILs express reduced levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is ed from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations thereof, and
wherein at least one T cell exhaustion marker in CD8+ and CD4+ T cells in the rTILs is
reduced by at least 10% relative to the eTILs.
In an embodiment, the invention includes a method for preparing remnant tumor
infiltrating lymphocytes (rTILs) for adoptive T cell y, the method comprising:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
infiltrating cytes ,
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and eukin 2 (IL-2) to provide tumor remnants and emergent TlLs (eTILs),
(d) removing at least a plurality of the eTILs,
(e) enzymatically digesting the tumor remnants into tumor remnant cells using a digest
mixture, and
(f) ing the tumor remnant cells with a second cell culture medium sing cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an ed number of remnant tumor infiltrating lymphocytes
(rTILs),
wherein the rTlLs express reduced levels of a T cell tion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations thereof, and
wherein the T cell exhaustion marker is a LAG3 marker in CD8+ T cells, and wherein the
LAG3 marker in the rTILs is reduced by at least 2-fold relative to the eTILs.
In an embodiment, the invention includes a method for preparing remnant tumor
ating cytes (rTILs) for ve T cell therapy, the method comprising:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
infiltrating lymphocytes (TlLs),
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor remnants and nt TlLs (eTILs),
(d) removing at least a plurality of the eTILs,
(e) enzymatically digesting the tumor remnants into tumor remnant cells using a digest
mixture, and
(f) ing the tumor remnant cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an expanded number of remnant tumor infiltrating lymphocytes
(rTILs),
wherein the rTlLs express reduced levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations thereof, and
n the T cell tion marker is a TIM3 marker in CD8+ T cells, and wherein the
LAG3 marker in the rTILs is reduced by at least 3-fold relative to the eTILs.
In an embodiment, the invention includes a method for preparing remnant tumor
infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising:
(a) ing tumor tissue from the patient, wherein the tumor tissue comprises tumor
infiltrating lymphocytes (TlLs),
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and interleukin 2 (IL-2) to e tumor remnants and emergent TlLs (eTILs),
(d) removing at least a plurality of the eTILs,
(e) enzymatically digesting the tumor ts into tumor remnant cells using a digest
mixture, and
(f) expanding the tumor remnant cells with a second cell culture medium comprising cell
culture media, ated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an expanded number of remnant tumor infiltrating lymphocytes
wherein the rTILs express reduced levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations thereof, and
wherein the T cell exhaustion marker is a TIM3 marker in CD4+ T cells, and wherein the
LAG3 marker in the rTILs is reduced by at least 2-fold ve to the eTILs.
In an embodiment, the invention includes a method for preparing remnant tumor
infiltrating lymphocytes (rTILs) for adoptive T cell y, the method comprising:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
infiltrating lymphocytes (TlLs),
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas permeable ner with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor remnants and emergent TlLs (eTILs),
(d) removing at least a plurality of the eTILs,
(e) enzymatically digesting the tumor remnants into tumor remnant cells using a digest
mixture; and
(f) expanding the tumor remnant cells with a second cell e medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an expanded number of t tumor infiltrating lymphocytes
(rTILs),
wherein the rTILs express reduced levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations thereof, and
wherein the TIM3 marker and the LAG3 marker in the rTILs are undetectable by flow
cytometry.
In an embodiment, the invention includes a method for ing remnant tumor
infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
ating lymphocytes (TlLs),
(b) nting the tumor tissue,
(c) treating the tumor tissue in a gas ble container with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor remnants and emergent TlLs ),
(d) removing at least a plurality of the eTILs,
(e) tically digesting the tumor remnants into tumor remnant cells using a digest
mixture, and
(f) expanding the tumor remnant cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an expanded number of remnant tumor infiltrating lymphocytes
(rTILs),
wherein the rTlLs express d levels of a T cell exhaustion marker relative to the
eTILs,
n the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and ations f, and
wherein CD56+ expression in the rTILs is reduced by at least 3-fold relative to CD56+
expression in the eTlLs.
In an embodiment, the invention includes a method for preparing remnant tumor
infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising:
(a) obtaining tumor tissue from the patient, n the tumor tissue comprises tumor
infiltrating lymphocytes (TlLs),
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas permeable container with a first cell e medium
and interleukin 2 (IL-2) to provide tumor remnants and emergent TlLs (eTILs),
(d) removing at least a plurality of the eTILs,
(e) enzymatically digesting the tumor remnants into tumor remnant cells using a digest
e, and
(f) expanding the tumor remnant cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to e an expanded number of remnant tumor infiltrating lymphocytes
(rTILs),
wherein the rTlLs express reduced levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations thereof, and
wherein CD69+ expression in the rTILs is increased by at least 2-fold relative to CD69+
expression in the eTlLs.
In an embodiment, the invention includes a method for preparing remnant tumor
infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising:
(a) ing tumor tissue from the patient, wherein the tumor tissue comprises tumor
infiltrating cytes ,
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor remnants and emergent TlLs (eTILs),
(d) removing at least a plurality of the eTILs,
(e) enzymatically digesting the tumor remnants into tumor remnant cells using a digest
mixture, and
(f) expanding the tumor remnant cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an expanded number of remnant tumor infiltrating lymphocytes
wherein the rTlLs express reduced levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations thereof, and
wherein the digest mixture ses ibonuclease, collagenase, and
onidase.
In an embodiment, the invention includes a method for preparing remnant tumor
infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
infiltrating lymphocytes (TlLs),
(b) fragmenting the tumor tissue,
(c) ng the tumor tissue in a gas permeable container with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor remnants and nt TlLs (eTILs),
(d) removing at least a plurality of the eTILs,
(e) enzymatically digesting the tumor remnants into tumor t cells using a digest
mixture; and
(f) expanding the tumor remnant cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
ner to provide an expanded number of remnant tumor infiltrating lymphocytes
(rTILs),
wherein the rTlLs express reduced levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and ations f, and
wherein the first cell culture medium r comprises a cytokine selected from the
group consisting of IL-4, IL-7, IL-15, 1L-21, and combinations thereof.
In an embodiment, the invention includes a method for preparing t tumor
inflltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
inflltrating lymphocytes (TlLs),
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor remnants and emergent TlLs (eTILs),
(d) removing at least a plurality of the eTILs,
(e) enzymatically digesting the tumor remnants into tumor remnant cells using a digest
mixture, and
(f) expanding the tumor remnant cells with a second cell culture medium comprising cell
e media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to e an expanded number of remnant tumor infiltrating lymphocytes
(rTILs),
n the rTlLs express reduced levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations thereof, and
wherein the second cell culture medium r comprises a cytokine selected from the
group consisting of IL-4, IL-7, IL-15, 1L-21, and combinations thereof.
In an embodiment, the invention includes a method of treating a cancer in a patient in
need of such treatment, n the ent comprises delivering a therapeutically effective
amount of rTILs to a patient, wherein the rTILs are prepared according a method sing the
steps of:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
inflltrating lymphocytes (TlLs),
(b) fragmenting the tumor tissue,
(c) ng the tumor tissue in a gas permeable container with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor remnants and emergent TlLs (eTILs),
(d) removing at least a plurality of the eTILs,
(e) enzymatically digesting the tumor remnants into tumor remnant cells using a digest
mixture, and
(f) expanding the tumor remnant cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an expanded number of remnant tumor infiltrating lymphocytes
(rTILs),
wherein the rTlLs express d levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations thereof, and
WO 94167
wherein the second cell culture medium further comprises a cytokine selected from the
group consisting of IL-4, IL-7, IL-15, 1L-21, and combinations thereof.
In an embodiment, the invention includes a method ng a cancer in a patient in
need of such treatment, the method sing:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
infiltrating lymphocytes (TlLs),
(b) nting the tumor tissue,
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor remnants and emergent TlLs (eTILs),
(d) removing at least a plurality of the eTILs,
(e) enzymatically digesting the tumor remnants into tumor remnant cells using a digest
mixture,
(f) expanding the tumor remnant cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an expanded number of remnant tumor infiltrating lymphocytes
wherein the rTILs express reduced levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations thereof, and
wherein the second cell culture medium r ses a ne selected from the
group consisting of IL-4, IL-7, IL-15, 1L-21, and combinations thereof,
(g) treating the patient with a non-myeloablative lymphodepletion regimen prior to
administering the rTILs to the patient,
(h) administering a therapeutically effective amount of rTILs to the patient, and
(i) treating the patient with a high-dose IL-2 regimen starting on the day after
administration of the rTILs to the patient.
In an embodiment, the invention includes a method treating a cancer in a patient in
need of such treatment, the method comprising:
(a) ing tumor tissue from the patient, wherein the tumor tissue comprises tumor
infiltrating lymphocytes (TlLs),
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas permeable ner with a first cell culture medium
and interleukin 2 (IL-2) to e tumor remnants and emergent TlLs (eTILs),
(d) removing at least a plurality of the eTILs,
(e) enzymatically digesting the tumor remnants into tumor remnant cells using a digest
mixture,
(f) expanding the tumor remnant cells with a second cell e medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an ed number of remnant tumor infiltrating lymphocytes
(rTILs),
wherein the rTILs express reduced levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations thereof, and
wherein the second cell e medium further comprises a cytokine selected from the
group consisting of IL-4, IL-7, IL-15, 1L-21, and combinations thereof,
(g) treating the patient with a non-myeloablative lymphodepletion n prior to
administering the rTILs to the patient,
(h) administering a therapeutically effective amount of rTILs to the t, wherein a
therapeutically effective amount of eTILs are simultaneously administered to the patient
in a mixture with the rTILs, and
(i) treating the t with a high-dose IL-2 regimen starting on the day after
administration of the rTILs to the patient.
In an embodiment, the invention es a method treating a cancer in a patient in
need of such treatment, the method comprising:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
infiltrating lymphocytes (TlLs),
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor remnants and emergent TlLs (eTILs),
(d) removing at least a plurality of the eTILs,
(e) enzymatically digesting the tumor ts into tumor remnant cells using a digest
mixture,
(f) expanding the tumor t cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 dy, and IL-2 in a gas permeable
container to provide an expanded number of remnant tumor infiltrating lymphocytes
(rTILs),
wherein the rTILs express reduced levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and ations thereof, and
wherein the second cell e medium r comprises a cytokine selected from the
group consisting of IL-4, IL-7, IL-15, 1L-21, and combinations thereof,
(g) ng the patient with a non-myeloablative lymphodepletion regimen prior to
administering the rTILs to the patient,
(h) administering a therapeutically effective amount of rTILs to the patient, wherein a
therapeutically effective amount of eTILs are aneously administered to the patient
in a mixture with the rTILs, and
(i) treating the patient with a high-dose IL-2 regimen starting on the day after
administration of the rTILs to the patient,
n the cancer is selected from the group consisting of melanoma, double-refractory
melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer,
head and neck , renal cell carcinoma, acute myeloid ia, colorectal cancer,
sarcoma, all cell lung cancer (NSCLC), and triple negative breast cancer.
In an embodiment, the invention includes a method treating a cancer in a patient in
need of such treatment, the method comprising:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
infiltrating lymphocytes (TlLs),
(b) fragmenting the tumor ,
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor remnants and emergent TlLs (eTILs),
(d) removing at least a plurality of the eTILs,
(e) enzymatically digesting the tumor remnants into tumor remnant cells using a digest
mixture,
(f) expanding the tumor remnant cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an expanded number of remnant tumor infiltrating lymphocytes
(rTILs),
wherein the rTlLs s reduced levels of a T cell tion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and ations thereof, and
wherein the second cell culture medium further comprises a cytokine selected from the
group consisting of IL-4, IL-7, IL-15, 1L-21, and combinations f,
(g) ng the patient with a non-myeloablative lymphodepletion regimen prior to
administering the rTlLs to the patient, wherein the non-myeloablative lymphodepletion
regimen comprises the steps of administration of cyclophosphamide at a dose of 60
mg/mZ/day for two days followed by administration of fludarabine at a dose of 25
mg/mZ/day for five days,
(h) administering a therapeutically effective amount of rTILs to the patient, wherein a
therapeutically effective amount of eTILs are simultaneously administered to the patient
in a mixture with the rTILs, and
(i) treating the patient with a high-dose IL-2 regimen ng on the day after
administration of the rTILs to the patient.
In an embodiment, the invention includes a method treating a cancer in a patient in
need of such treatment, the method comprising:
(a) ing tumor tissue from the patient, wherein the tumor tissue comprises tumor
inflltrating lymphocytes (TlLs),
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas ble container with a first cell culture medium
and eukin 2 (IL-2) to provide tumor remnants and emergent TlLs (eTILs),
(d) removing at least a ity of the eTILs,
(e) enzymatically digesting the tumor remnants into tumor remnant cells using a digest
mixture,
(f) expanding the tumor remnant cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an expanded number of remnant tumor ating lymphocytes
wherein the rTILs s reduced levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations thereof, and
wherein the second cell culture medium further comprises a cytokine selected from the
group consisting of IL-4, IL-7, IL-15, 1L-21, and ations thereof,
(g) treating the patient with a non-myeloablative lymphodepletion regimen prior to
WO 94167
administering the rTlLs to the patient;
(h) administering a therapeutically effective amount of rTILs to the patient, wherein a
therapeutically effective amount of eTILs are simultaneously administered to the patient
in a mixture with the rTlLs, and
(i) treating the t with a high-dose IL-2 regimen ng on the day after
administration of the rTlLs to the patient,
wherein the high-dose IL-2 regimen comprises 600,000 or 0 IU/kg of aldesleukin,
or a biosimilar or variant thereof, stered as a 15-minute bolus intravenous infusion
every eight hours until tolerance.
In an embodiment, the invention includes a method for preparing remnant tumor
inf11trating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
inf11trating lymphocytes (TlLs),
(b) nting the tumor tissue,
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor remnants and emergent TlLs (eTILs),
(d) removing the eTILs,
(e) enzymatically digesting the tumor remnants into tumor remnant cells using a digest
mixture, and
(f) ing the tumor remnant cells with a second cell culture medium comprising cell
culture media, ated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an expanded number of t tumor infiltrating lymphocytes
(rTILs),
wherein the rTlLs express reduced levels of a T cell exhaustion marker relative to the
eTILs, and wherein the T cell tion marker is selected from the group consisting of
TIM3, LAG3, TIGIT, PD-l, CTLA-4, and combinations thereof.
In an embodiment, the invention includes a method for preparing remnant tumor
rating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
ating lymphocytes (TlLs),
(b) fragmenting the tumor tissue;
(c) ng the tumor tissue in a gas permeable container with a first cell e medium
and interleukin 2 (IL-2) to provide tumor remnants and emergent TlLs (eTILs),
(d) removing the eTILs,
(e) enzymatically digesting the tumor remnants into tumor t cells using a digest
mixture; and
(f) expanding the tumor remnant cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
ner to provide an expanded number of remnant tumor infiltrating lymphocytes
(rTILs),
wherein the rTlLs express reduced levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, , and combinations thereof, and
wherein the tumor tissue is selected from the group consisting of melanoma tumor tissue,
head and neck tumor , breast tumor tissue, renal tumor tissue, pancreatic tumor
tissue, glioblastoma tumor tissue, lung tumor tissue, colorectal tumor tissue, a
tumor tissue, triple negative breast tumor tissue, cervical tumor tissue, ovarian tumor
tissue, and acute myeloid leukemia bone marrow or tumor tissue.
In an embodiment, the invention includes a method for preparing remnant tumor
inflltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
inflltrating lymphocytes (TlLs),
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor remnants and emergent TlLs (eTILs),
(d) removing the eTILs,
(e) enzymatically digesting the tumor remnants into tumor remnant cells using a digest
mixture; and
(f) expanding the tumor remnant cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an expanded number of remnant tumor infiltrating lymphocytes
(rTILs),
wherein the rTlLs express d levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, CTLA-4, and combinations thereof, and
n the irradiated feeder cells se irradiated allogeneic peripheral blood
mononuclear cells.
In an ment, the invention includes a method for preparing remnant tumor
inf11trating lymphocytes (rTILs) for adoptive T cell therapy, the method sing:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
inf11trating lymphocytes (TlLs),
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas permeable ner with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor remnants and emergent TlLs (eTILs),
(d) removing the eTILs,
(e) enzymatically digesting the tumor remnants into tumor remnant cells using a digest
e, and
(f) expanding the tumor remnant cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 dy, and IL-2 in a gas permeable
container to provide an expanded number of remnant tumor ating lymphocytes
(rTILs),
wherein the rTlLs express reduced levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group ting of TIM3,
LAG3, TIGIT, PD-l, and combinations thereof, and
wherein IL-2 is present in the second cell culture medium at an initial concentration of
about 3000 IU/mL and OKT-3 antibody is present in the second cell culture medium at
an initial concentration of about 30 ng/mL.
In an embodiment, the invention includes a method for preparing remnant tumor
infiltrating lymphocytes (rTILs) for ve T cell therapy, the method comprising:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
infiltrating lymphocytes (TlLs),
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor remnants and nt TlLs (eTILs),
(d) removing the eTILs,
(e) enzymatically ing the tumor remnants into tumor remnant cells using a digest
mixture, and
(f) expanding the tumor remnant cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an ed number of t tumor infiltrating lymphocytes
(rTILs),
wherein the rTlLs express reduced levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations thereof, and
wherein at least one T cell exhaustion marker in CD8+ and CD4+ T cells in the rTILs is
reduced by at least 10% relative to the eTILs.
In an embodiment, the invention es a method for preparing remnant tumor
infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
infiltrating lymphocytes (TlLs),
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor remnants and emergent TlLs (eTILs),
(d) removing the eTILs,
(e) enzymatically digesting the tumor remnants into tumor t cells using a digest
e, and
(f) expanding the tumor remnant cells with a second cell e medium comprising cell
culture media, ated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an expanded number of remnant tumor infiltrating lymphocytes
(rTILs),
wherein the rTILs express d levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations thereof, and
wherein the T cell exhaustion marker is a LAG3 marker in CD8+ T cells, and wherein the
LAG3 marker in the rTILs is reduced by at least 2-fold relative to the eTILs.
In an ment, the invention includes a method for preparing remnant tumor
infiltrating cytes (rTILs) for adoptive T cell therapy, the method comprising:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
infiltrating lymphocytes (TlLs),
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and interleukin 2 (IL-2) to e tumor remnants and emergent TlLs (eTILs),
(d) removing the eTILs,
(e) enzymatically digesting the tumor remnants into tumor remnant cells using a digest
mixture; and
(f) expanding the tumor remnant cells with a second cell e medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an expanded number of remnant tumor infiltrating lymphocytes
wherein the rTILs express reduced levels of a T cell tion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations thereof, and
wherein the T cell exhaustion marker is a TIM3 marker in CD8+ T cells, and wherein the
LAG3 marker in the rTILs is reduced by at least 3-fold relative to the eTILs.
In an embodiment, the invention includes a method for preparing remnant tumor
infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
infiltrating lymphocytes (TlLs),
(b) nting the tumor tissue,
(c) treating the tumor tissue in a gas permeable container with a first cell e medium
and interleukin 2 (IL-2) to provide tumor remnants and emergent TlLs (eTILs),
(d) removing the eTILs,
(e) enzymatically ing the tumor remnants into tumor remnant cells using a digest
mixture, and
(f) expanding the tumor remnant cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an expanded number of remnant tumor infiltrating cytes
(rTILs),
wherein the rTlLs express reduced levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and ations thereof, and
wherein the T cell exhaustion marker is a TIM3 marker in CD4+ T cells, and wherein the
LAG3 marker in the rTILs is reduced by at least 2-fold relative to the eTILs.
In an embodiment, the invention includes a method for preparing remnant tumor
infiltrating cytes (rTILs) for adoptive T cell therapy, the method comprising:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue ses tumor
infiltrating lymphocytes (TlLs),
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor ts and emergent TlLs (eTILs),
(d) removing the eTILs,
(e) enzymatically digesting the tumor remnants into tumor remnant cells using a digest
mixture, and
(f) ing the tumor t cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an expanded number of t tumor infiltrating lymphocytes
(rTILs),
wherein the rTlLs express reduced levels of a T cell tion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations thereof, and
wherein the TIM3 marker and the LAG3 marker in the rTILs are undetectable by flow
cytometry.
In an embodiment, the invention includes a method for preparing remnant tumor
infiltrating lymphocytes (rTILs) for adoptive T cell y, the method comprising:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
infiltrating lymphocytes (TlLs),
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor remnants and emergent TlLs (eTILs),
(d) ng the eTILs,
(e) enzymatically digesting the tumor remnants into tumor remnant cells using a digest
mixture, and
(f) expanding the tumor t cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 dy, and IL-2 in a gas permeable
container to provide an expanded number of remnant tumor infiltrating lymphocytes
wherein the rTILs express reduced levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations thereof, and
wherein CD56+ expression in the rTILs is reduced by at least 3-fold relative to CD56+
expression in the eTlLs.
In an embodiment, the invention es a method for preparing remnant tumor
infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising:
(a) obtaining tumor tissue from the patient, n the tumor tissue comprises tumor
infiltrating cytes (TlLs),
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and eukin 2 (IL-2) to provide tumor remnants and emergent TlLs (eTILs),
(d) removing the eTILs,
(e) enzymatically ing the tumor remnants into tumor remnant cells using a digest
mixture; and
(f) expanding the tumor remnant cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas ble
container to provide an expanded number of remnant tumor infiltrating cytes
wherein the rTILs express reduced levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations thereof, and
wherein CD69+ expression in the rTILs is increased by at least 2-fold relative to CD69+
expression in the eTlLs.
In an embodiment, the invention includes a method for preparing remnant tumor
infiltrating cytes (rTILs) for ve T cell therapy, the method comprising:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
infiltrating lymphocytes (TlLs),
(b) nting the tumor tissue,
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor remnants and emergent TlLs ),
(d) removing the eTILs,
(e) enzymatically digesting the tumor remnants into tumor remnant cells using a digest
mixture, and
(f) expanding the tumor remnant cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an expanded number of remnant tumor infiltrating lymphocytes
(rTILs),
wherein the rTlLs express reduced levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations thereof, and
wherein the digest mixture comprises deoxyribonuclease, collagenase, and
onidase.
In an embodiment, the invention es a method for preparing remnant tumor
ating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
infiltrating lymphocytes (TlLs),
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas permeable ner with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor remnants and emergent TlLs (eTILs),
(d) removing the eTILs,
(e) enzymatically digesting the tumor remnants into tumor remnant cells using a digest
mixture, and
(f) expanding the tumor remnant cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
ner to provide an expanded number of remnant tumor infiltrating lymphocytes
(rTILs),
n the rTlLs express reduced levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group ting of TIM3,
LAG3, TIGIT, PD-l, and combinations f, and
wherein the first cell culture medium further comprises a cytokine selected from the
group consisting of IL-4, IL-7, IL-15, 1L-21, and combinations thereof.
In an embodiment, the invention includes a method for preparing remnant tumor
infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising:
(a) obtaining tumor tissue from the t, wherein the tumor tissue comprises tumor
infiltrating cytes (TlLs),
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor remnants and emergent TlLs (eTILs),
(d) removing the eTILs,
(e) enzymatically digesting the tumor remnants into tumor remnant cells using a digest
mixture, and
(f) expanding the tumor remnant cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas ble
container to provide an expanded number of remnant tumor infiltrating lymphocytes
(rTILs),
wherein the rTILs express reduced levels of a T cell exhaustion marker relative to the
eTILs,
n the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations f, and
n the second cell culture medium further comprises a cytokine selected from the
group consisting of IL-4, IL-7, IL-15, 1L-21, and ations thereof.
In an embodiment, the invention includes a method of ng a cancer in a patient in
need of such treatment, wherein the treatment comprises delivering a therapeutically effective
amount of rTILs to a patient, wherein the rTILs are prepared according a method comprising the
steps of:
(a) obtaining tumor tissue from the patient, n the tumor tissue comprises tumor
infiltrating lymphocytes (TlLs),
(b) fragmenting the tumor tissue,
WO 94167
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor remnants and emergent TlLs (eTILs),
(d) removing the eTILs,
(e) enzymatically digesting the tumor remnants into tumor remnant cells using a digest
mixture; and
(f) expanding the tumor remnant cells with a second cell e medium comprising cell
culture media, ated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an expanded number of remnant tumor infiltrating lymphocytes
(rTILs),
wherein the rTlLs express reduced levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations thereof, and
n the second cell culture medium further comprises a cytokine selected from the
group consisting of IL-4, IL-7, IL-15, 1L-21, and combinations thereof.
In an embodiment, the ion includes a method treating a cancer in a patient in
need of such treatment, the method comprising:
(a) obtaining tumor tissue from the t, wherein the tumor tissue comprises tumor
inflltrating lymphocytes (TlLs),
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor remnants and emergent TlLs (eTILs),
(d) removing the eTILs,
(e) tically digesting the tumor remnants into tumor remnant cells using a digest
mixture,
(f) expanding the tumor t cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to e an expanded number of remnant tumor infiltrating lymphocytes
(rTILs),
wherein the rTlLs express reduced levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and ations thereof, and
wherein the second cell culture medium further comprises a cytokine selected from the
group consisting of IL-4, IL-7, IL-15, 1L-21, and combinations thereof,
(g) treating the patient with a non-myeloablative lymphodepletion regimen prior to
administering the rTlLs to the patient;
(h) administering a eutically effective amount of rTILs to the patient; and
(i) treating the patient with a high-dose IL-2 regimen starting on the day after
administration of the rTlLs to the patient.
In an embodiment, the invention es a method treating a cancer in a patient in
need of such treatment, the method comprising:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
inflltrating lymphocytes (TlLs),
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor ts and emergent TlLs (eTILs),
(d) ng the eTILs,
(e) enzymatically digesting the tumor remnants into tumor remnant cells using a digest
mixture,
(f) expanding the tumor remnant cells with a second cell culture medium comprising cell
e media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an ed number of remnant tumor infiltrating lymphocytes
(rTILs),
wherein the rTlLs s reduced levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations thereof, and
wherein the second cell culture medium r ses a cytokine selected from the
group consisting of IL-4, IL-7, IL-15, 1L-21, and combinations thereof,
(g) treating the patient with a non-myeloablative lymphodepletion regimen prior to
stering the rTlLs to the patient;
(h) administering a therapeutically effective amount of rTILs to the patient, wherein a
eutically effective amount of eTILs are simultaneously administered to the patient
in a e with the rTlLs, and
(i) treating the patient with a ose IL-2 regimen starting on the day after
administration of the rTlLs to the patient.
In an embodiment, the invention includes a method treating a cancer in a patient in
need of such treatment, the method comprising:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
inflltrating lymphocytes (TlLs),
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and eukin 2 (IL-2) to provide tumor remnants and emergent TlLs (eTILs),
(d) removing the eTILs,
(e) tically digesting the tumor remnants into tumor remnant cells using a digest
mixture,
(f) expanding the tumor remnant cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an expanded number of remnant tumor infiltrating lymphocytes
(rTILs),
wherein the rTlLs express reduced levels of a T cell tion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations thereof, and
wherein the second cell culture medium r comprises a cytokine selected from the
group consisting of IL-4, IL-7, IL-15, 1L-21, and combinations thereof,
(g) treating the patient with a non-myeloablative lymphodepletion regimen prior to
administering the rTlLs to the t;
(h) administering a therapeutically effective amount of rTILs to the patient, wherein a
therapeutically effective amount of eTILs are aneously administered to the patient
in a mixture with the rTlLs, and
(i) treating the patient with a high-dose IL-2 regimen starting on the day after
administration of the rTlLs to the patient,
wherein the cancer is selected from the group consisting of melanoma, double-refractory
melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer,
head and neck cancer, renal cell carcinoma, acute myeloid leukemia, ctal cancer,
sarcoma, non-small cell lung cancer (NSCLC), and triple negative breast cancer.
In an embodiment, the invention includes a method ng a cancer in a t in
need of such treatment, the method comprising:
(a) obtaining tumor tissue from the t, wherein the tumor tissue comprises tumor
inflltrating lymphocytes (TlLs),
(b) fragmenting the tumor ,
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor remnants and emergent TlLs (eTILs),
(d) removing the eTILs,
(e) enzymatically digesting the tumor remnants into tumor remnant cells using a digest
mixture,
(f) expanding the tumor remnant cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an expanded number of remnant tumor infiltrating lymphocytes
(rTILs),
wherein the rTILs s reduced levels of a T cell exhaustion marker ve to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations thereof, and
wherein the second cell culture medium r ses a cytokine selected from the
group consisting of IL-4, IL-7, IL-15, 1L-21, and combinations thereof,
(g) treating the patient with a non-myeloablative lymphodepletion regimen prior to
administering the rTILs to the patient, wherein the non-myeloablative lymphodepletion
n comprises the steps of administration of cyclophosphamide at a dose of 60
mg/mZ/day for two days followed by administration of fludarabine at a dose of 25
mg/mZ/day for five days,
(h) stering a therapeutically ive amount of rTILs to the patient, wherein a
therapeutically effective amount of eTILs are simultaneously administered to the patient
in a mixture with the rTILs, and
(i) treating the t with a high-dose IL-2 regimen starting on the day after
administration of the rTILs to the patient.
In an embodiment, the ion includes a method treating a cancer in a patient in
need of such treatment, the method comprising:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
inflltrating lymphocytes (TlLs),
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas permeable ner with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor remnants and emergent TlLs (eTILs),
(d) removing the eTILs,
(e) enzymatically digesting the tumor remnants into tumor remnant cells using a digest
(f) expanding the tumor remnant cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an expanded number of remnant tumor infiltrating cytes
(rTILs),
n the rTILs express reduced levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations f, and
wherein the second cell culture medium further comprises a cytokine ed from the
group consisting of IL-4, IL-7, IL-15, IL-21, and combinations f,
(g) treating the patient with a non-myeloablative lymphodepletion regimen prior to
administering the rTILs to the patient,
(h) stering a therapeutically effective amount of rTILs to the patient, wherein a
eutically effective amount of eTILs are aneously administered to the patient
in a e with the rTILs, and
(i) treating the patient with a high-dose IL-2 regimen starting on the day after
administration of the rTILs to the patient,
wherein the high-dose IL-2 regimen comprises 600,000 or 720,000 IU/kg of aldesleukin,
or a biosimilar or variant thereof, administered as a 15-minute bolus intravenous infusion
every eight hours until tolerance.
In an embodiment, the invention includes a process for generating an expanded number
of tumor remnant cells that include tumor infiltrating lymphocytes (TILs) from a patient for
adoptive T cell therapy. In some embodiments, the process of the invention may include the step
of obtaining tumor tissue from the patient, wherein the tumor tissue comprises TILs. In some
embodiments, the process of the invention may include the step of fragmenting the tumor .
In some embodiments, the process of the invention may include the step of treating the tumor
tissue in a gas ble container with cell culture media and interleukin 2 (IL-2) and other T
cell growth factors or agonistic antibodies to e tumor remnants and an expanded number
of TILs. In some embodiments, the process of the invention may include the step of ng
the expanded number of TILs. In some embodiments, the process of the invention may include
the step of tically digesting the tumor remnants into tumor remnant cells. In some
embodiments, the process of the invention may include the step of treating the tumor remnant
cells with cell culture media, irradiated feeder cells, anti-CD3 monoclonal antibody (muromonab
or OKT-3), and IL-2 to provide the ed number of tumor remnant cells. In some
embodiments, the tumor remnant cells prepared according to the processes of the invention may
include TILs that express reduced levels of at least one marker selected from the group
consisting of TIM3, LAG3, PD-l, and combinations thereof. In some embodiments, the tumor
tissue may be selected from the group consisting of melanoma tumor tissue, head and neck tumor
tissue, breast tumor tissue, renal tumor tissue, pancreatic tumor , lung tumor tissue, and
colorectal tumor tissue.
In an embodiment, the invention may include method of treating a tumor in a patient in
need of such treatment. In some embodiments, the treatment may include delivering a
therapeutically effective amount of an expanded number of tumor remnant cells to the patient,
wherein the expanded number of tumor remnant cells may be prepared according to any s
described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing summary, as well as the following detailed description of the invention,
will be better tood when read in conjunction with the appended drawings.
illustrates an exemplary diagram of the tumor digestion solution preparation.
illustrates an ary flow-through diagram of the tumor digestion ure.
In this example, two digestion methods are performed aneously and are seeded separately
as a part of two distinct pre-REPs ed to compare the efficacy of each digestion method.
illustrates ential phenotypic expression of key markers in eTILs and rTILs.
WO 94167
illustrates studies of eTIL and rTIL by 2-(N—(7-nitrobenzoxa-l,3-diazol
yl)amino)deoxyglucose (2-NBDG) (A) and Mitotracker (B) to assess metabolic capacity prior
to rapid expansion.
shows the results of experiments wherein eTIL and rTIL were stimulated with
CD3/CD28/4-1BB beads with brefeldin A overnight for CD4+ and CD8+ T cells. PMA and
cin was added for 4-5 hours. Interferon-y was assessed by intracellular flow cytometric
analysis (n=3).
illustrates results showing that (A) rTIL expand and (B) remain phenotypically
distinct from eTIL during rapid expansion.
illustrates an exemplary process for treating a patient using rTILs of the
illustrates an ary timeline of the process for treating a patient using rTILs
of the ion.
rates the diversity of the TCRvB repetoire (i.e., the diversity score) in eTIL
and rTIL.
illustrates the percent of shared CDR3s in eTIL and rTIL.
illustrates cell proliferation analyses in triple negative breast carcinoma,
colorectal carcinoma, lung carcinoma, renal carcinoma, and melanoma where eTIL from either
the CD4+ or CD8+ population in all five tumors demonstrated an enhancement in the
proliferative capacity upon co-culture with rTIL with anti-CD3 antibody as demonstrated by a
shift (or dye dilution) in the Cell Trace dye, when compared to eTIL alone. The red ents
the eTIL and the blue represents the eTIL when co-cultured with the rTIL.
illustrates a heat map prepared from a Nanostring analysis, which shows that
the gene expression profile for eTIL and rTIL is significantly different.
illustrates a graph prepared from a Nanostring analysis, which shows that
several genes are significantly upregulated or gulated in the rTIL as compared to the
eTIL.
illustrates a clonotype graph showing the top 50 shared CDR3s between eTIL
and rTIL (for three TIL pairs) from n carcinoma.
illustrates a ype graph showing the top 50 shared CDR3s n eTIL
and rTIL (for three eTIL/rTIL pairs) from renal carcinoma.
illustrates a clonotype graph showing the top 50 shared CDR3s between eTIL
and rTIL (for three TIL pairs) from triple ve breast carcinoma.
BRIEF DESCRIPTION OF THE SEQUENCE G
SEQ ID \O:l is the amino acid sequence of the heavy chain of muromonab.
SEQ ID \O:2 is the amino acid sequence of the light chain of muromonab.
SEQ ID \O:3 is the amino acid sequence of a recombinant human IL-2 protein.
SEQ ID \O:4 is the amino acid sequence of aldesleukin.
SEQ ID \O:5 is the amino acid sequence of a inant human IL-4 protein.
SEQ ID \O:6 is the amino acid sequence of a recombinant human IL-7 protein.
SEQ ID \O:7 is the amino acid sequence of a recombinant human IL-15 protein.
SEQ ID \O:8 is the amino acid sequence of a recombinant human IL-21 protein.
DETAILED DESCRIPTION OF THE INVENTION
Unless defined otherwise, all technical and scientific terms used herein have the same
meaning as is commonly understood by one of skill in the art to which this invention belongs.
All patents and ations referred to herein are incorporated by reference in their entireties.
Definitions
The terms “exhausted phenotype” and “exhaustion marker” refer to cell surface
markers characteristic of T cell exhaustion in response to chronic T cell receptor (TCR)
stimulation by antigen. T cells exhibiting an exhausted phenotype express inhibitory receptors,
such as T cell immunoglobulin and mucin-domain containing-3 (TIM3 or THVI-3), lymphocyte-
activation gene 3 (LAG3 or LAG-3), T cell immunoreceptor with immunoglobulin and ITIM
domains (TIGIT), and programmed cell death protein 1 (PD-l), and lack effector cytokine
production and the ability to mount an effective immune response. Exhaustion in T cells is
described in Yi, er al., Immunology 2010, 129, 474-81, the disclosure of which is incorporated by
reference herein.
The terms “co-administration, 77 (L co-administering,77 (L administered in combination
with,” “administering in combination with,” “simultaneous,” and “concurrent,” as used ,
encompass administration of two or more active pharmaceutical ingredients to a human subject
so that both active pharmaceutical ingredients and/or their metabolites are present in the human
t at the same time. Co-administration includes aneous administration in separate
compositions, administration at different times in separate compositions, or administration in a
ition in which two or more active pharmaceutical ingredients are present. Simultaneous
administration in separate compositions and stration in a composition in which both
agents are present is also encompassed in the s of the invention.
The term “in viva” refers to an event that takes place in a subject’s body.
The term “in vilro” 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 ed.
The term “antigen” refers to a substance that induces an immune response. In some
embodiments, an antigen is a molecule capable of being bound by an dy or a TCR if
presented by major histocompatibility compleX (MHC) molecules. The term “antigen”, as used
herein, also encompasses T cell epitopes. An antigen is additionally capable of being recognized
by the immune system. In some embodiments, an antigen is capable of inducing a humoral
immune response or a cellular immune response leading to the activation of B lymphocytes
and/or T lymphocytes. In some cases, this may require that the antigen contains or is linked to a
Th cell epitope. An n can also have one or more epitopes (e.g., B- and T-epitopes). In
some embodiments, an antigen will ably react, typically in a highly specific and selective
manner, with its corresponding antibody or TCR and not with the multitude of other antibodies
or TCRs which may be induced by other antigens.
The term “effective amount” or “therapeutically effective amount” refers to that amount
of a nd or combination of compounds as bed herein that is sufficient to effect the
intended application including, but not d to, disease treatment. A therapeutically effective
amount may vary depending upon the intended application (in vitro or in vivo), or the human
subject and disease condition being treated (e.g., the weight, age and gender of the subject), the
severity of the e ion, the manner of administration, etc. which can readily be
determined by one of ordinary skill in the art. The term also applies to a dose that will induce a
particular response in target cells (e.g., the reduction of platelet adhesion and/or cell migration).
The specific dose will vary depending on the particular compounds chosen, the dosing n
to be followed, whether the compound is stered in combination with other compounds,
timing of administration, the tissue to which it is administered, and the physical delivery system
in which the compound is carried. A therapeutically effective amount may be “an anti-tumor
effective amount” and/or a “tumor-inhibiting effective amount,” which may be 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 ct). It can generally be stated that a
pharmaceutical composition comprising the cytotoxic lymphocytes or rTILs 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 weight), ing all integer values within those ranges. Cytotoxic
lymphocyte or rTIL itions may also be administered multiple times at these dosages. The
cytotoxic cytes or rTILs can be administered by using infusion techniques that are
commonly known in immunotherapy (see, e.g., Rosenberg et al., N. Eng. J. Med. 319: 1676,
1988). The optimal dosage and treatment regime for a particular patient can readily be
determined by one skilled in the art of medicine by monitoring the patient for signs of disease
and adjusting the treatment ingly.
A “therapeutic effect” as that term is used herein, encompasses a therapeutic benefit
and/or a lactic benefit in a human subject. A prophylactic effect includes delaying or
eliminating the appearance of a disease or condition, delaying or eliminating the onset of
ms of a disease or condition, slowing, g, or reversing the progression of a disease or
condition, or any combination thereof.
“Pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” is
intended to include any and all solvents, dispersion media, coatings, antibacterial and ngal
agents, isotonic and absorption delaying agents, and inert ingredients. The use of such
pharmaceutically acceptable rs or pharmaceutically acceptable excipients for active
pharmaceutical ingredients is well known in the art. Except insofar as any conventional
pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible
with the active pharmaceutical ingredient, its use in the therapeutic compositions of the invention
is plated. Additional active pharmaceutical ients, such as other drugs, can also be
incorporated into the described compositions and methods.
The terms “treatment”, “treating”, “treat”, and the like, refer to obtaining a desired
cologic and/or physiologic effect. The effect may be prophylactic in terms of completely
or partially preventing a disease or symptom f and/or may be therapeutic in terms of a
partial or complete cure for a disease and/or adverse effect attributable to the disease.
ment”, as used herein, covers any treatment of a disease in a mammal, particularly in a
human, and es: (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 disease 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 condition. 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 vaccine.
The term “heterologous” when used with reference to ns of a nucleic acid or
protein indicates that the nucleic acid or protein comprises two or more subsequences that are not
found in the same relationship to each other in nature. For ce, the nucleic acid is typically
recombinantly produced, having two or more sequences from unrelated genes arranged to make a
new functional nucleic acid, e.g., a promoter from one source and a coding region from another
, or coding regions from different sources. Similarly, a heterologous protein indicates that
the n comprises two or more subsequences that are not found in the same relationship to
each other in nature (e.g., a fusion n).
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
ols are outlined herein.
By “tumor infiltrating lymphocytes” or “TILs” herein is meant a population of cells
originally obtained as white blood cells that have left the bloodstream of a t 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 macrophages. TILs include
both primary and secondary TILs. ry TILs” are those that are obtained from patient tissue
samples as outlined herein imes referred to as “freshly harvested”), and “secondary TILs”
are any TIL cell populations that have been expanded or proliferated as discussed herein,
ing, but not limited to bulk TILs and expanded TILs (“REP TILs” or REP TILs”).
In certain embodiments, the term “Primary TILs” may e rTILs and mixtures of eTILs and
rTILs.
By “population of cells” (including TILs) herein is meant a number of cells that share
common traits. In general, populations lly range from l X 106 to l X 1010 in number, with
different TIL populations comprising different numbers. For example, initial growth of primary
TILs in the presence of IL-2 results in a population of bulk TILs of roughly l X 108 cells. REP
expansion is lly done to provide populations of 1.5 X 109 to 1.5 X 1010 cells for on.
The terms “peripheral blood mononuclear cells” and “PBMCs” refers to a peripheral
blood cell haVing a round nucleus, including lymphocytes (such as T cells, B cells, and NK cells)
and monocytes. Preferably, the peripheral blood mononuclear cells are irradiated allogeneic
peripheral blood mononuclear cells. PBMCs are a type of antigen-presenting cell.
By “cryopreserved TILs” herein is meant that TILs, either primary, bulk, or expanded
(REP TILs), are d and stored in the range of about -lSO°C to -60°C. General methods for
cryopreservation are also described elsewhere herein. For clarity, “cryopreserved TILs” are
distinguishable from frozen tissue samples which may be used as a source of primary TILs
including rTILs.
By “thawed cryopreserved TILs” herein is meant a population of TILs (such as rTILs)
that was preViously cryopreserved and then treated to return to room temperature or higher,
ing but not limited to cell culture temperatures or temperatures wherein TILs may be
administered to a patient.
The terms “sequence ty,77 (Lpercent identity,” and “sequence percent identity” 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 aligned (introducing gaps, if necessary) for
maximum correspondence, not considering any conservative amino acid substitutions as part of
the sequence identity. The percent identity can be measured using sequence comparison
re 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 ty include for example the BLAST suite of
programs available from the US. Government’ s National Center for Biotechnology Information
BLAST web site. Comparisons between two sequences can be carried using either the BLASTN
or BLASTP thm. BLASTN is used to compare nucleic acid sequences, while BLASTP is
used to compare amino acid sequences. ALIGN, ALIGN—2 tech, South San Francisco,
rnia) 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 re are used.
The term “conservative amino acid tutions” means amino acid sequence
modifications which do not abrogate the binding of the antibody to the antigen. Conservative
amino acid substitutions e the substitution of an amino acid in one class by an amino acid
of the same class, where a class is defined by common physicochemical amino acid side chain
properties and high substitution frequencies in homologous proteins found in nature, as
determined, for example, by a standard Dayhoff frequency exchange matrix or BLOSUIVI .
Six general classes of amino acid side chains have been categorized and include: Class I (Cys),
Class II (Ser, Thr, Pro, Ala, Gly), Class III (Asn, Asp, Gln, Glu), Class IV (His, Arg, Lys), Class
V (Ile, Leu, Val, Met), and Class VI (Phe, Tyr, Trp). For example, substitution of an Asp for
another class III residue such as Asn, Gln, or Glu, is a conservative substitution. Thus, a
predicted ential amino acid residue in a protein is preferably replaced with another amino
acid residue from the same class. Methods of identifying amino acid conservative tutions
which do not eliminate antigen g are well-known in the art (see, e.g., Brummell, er al.,
Biochemistry 1993, 32, 1180-1187, Kobayashi, et al., Protein Eng. 1999, 12, 879-884 (1999),
and Burks, el al., Proc. Natl. Acad. Sci. USA 1997, 94, 412-417).
“Pegylation” refers to a modified antibody or fusion protein, or a fragment thereof, that
typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde
derivative of PEG, under conditions in which one or more PEG groups become attached to the
antibody or antibody fragment. Pegylation may, for example, increase the biological (e.g.,
serum) half life of the antibody. Preferably, the pegylation is carried out via an acylation
reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-
soluble r). As used herein, the term “polyethylene glycol” is intended to encompass any
of the forms of PEG that have been used to derivatize other proteins, such as mono (Cl-Clo)
alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. The protein or
dy to be ted may be an aglycosylated protein or antibody. Methods for pegylation
are known in the art and can be applied to the antibodies of the invention, as described for
example in European Patent Nos. EP 0154316 and EP 0401384 and US. Patent No. 778,
the disclosures of each of which are incorporated by reference herein.
The term “OKT-3” (also referred to herein as “OKT3”) refers to a monoclonal antibody
or ilar or variant thereof, ing human, humanized, chimeric, or murine antibodies,
directed t the CD3 receptor in the T cell antigen receptor of mature T cells, and includes
cially-available forms such as OKT-3 (30 ng/mL, MACS GMP CD3 pure, Miltenyi
Biotech, Inc., San Diego, CA, USA) and muromonab or variants, vative amino acid
substitutions, orms, or biosimilars thereof. The amino acid sequences of the heavy and
light chains of muromonab are given in Table l (SEQ ID N011 and SEQ ID N012). 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. Anti-CD3 antibodies also include the UHCTl clone
(commercially available from BioLegend, San Diego, CA, USA), also known as T3 and CD38.
TABLE 1. Amino acid sequences of muromonab.
Sequence (One-Letter Amino Acid Symbols)
SEQ ID NO:1 QVQLQQSGAE LARPGASVKM SCKASGYTFT RYTMHWVKQR PGQGLEWIGY INPSRGYTNY 60
Muromonab heavy KATL STAY MQLSSLTSED SAVYYCARYY DDHYCLDYWG QGTTLTVSSA 120
chain KTTAPSVYPL APVCGGTTGS SVTLGCLVKG TLTW NSGSLSSGVH TFPAVLQSDL 180
VTVT SSTWPSQSIT CNVAHPASST KVDKKIEPRP KSCDKTHTCP PCPAPELLGG 240
PSVFLFPPKP KDTLMISRTP VDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN
STYRVVSVLT VLHQDWLNGK EYKCKVSNKA KTIS KAKGQPREPQ VYTLPPSRDE
LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW
QQGNVFSCSV MHEALHNHYT QKSLSLSPGK
SEQ ID NO:2 SPAI MSASPGEKVT MTCSASSSVS YMNWYQQKSG TSPKRWIYDT SKLASGVPAH
Muromonab light FRGSGSGTSY SLTISGMEAE DAATYYCQQW SSNPFTFGSG RADT APTVSIFPPS
chain SEQLTSGGAS VVCFLNNFYP KDINVKWKID GVLN 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 eukin-2, and es all forms of IL-2 including human and mammalian forms,
vative amino acid substitutions, glycoforms, biosimilars, and variants f. 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 1L-2 suitable for use in the invention is given in Table 2 (SEQ
ID NO:3). For example, the term IL-2 asses human, recombinant forms of 1L-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 1L-2 commercially supplied by CellGeniX,
Inc., Portsmouth, NH, USA RO GMP) or ProSpec-Tany TechnoGene Ltd., East
Brunswick, NJ, USA (Cat. No. CYTb) and other commercial equivalents from other
s. Aldesleukin (des-alanyl-l, serine-125 human IL-2) is a nonglycosylated human
recombinant form of IL-2 with a molecular weight of approximately 15 kDa. The amino acid
sequence of eukin suitable for use in the invention is given in Table 2 (SEQ ID N04).
The term IL-2 also encompasses ted forms of IL-2, as described herein, including the
pegylated 1L2 prodrug NKTR-214, available from Nektar Therapeutics, South San Francisco,
CA, USA. NKTR—214 and pegylated 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.
herein. Alternative forms of conjugated IL-2 suitable for use in the invention are described in
US. Patent Nos. 4,766,106, 5,206,344, 5,089,261 and 4902,502, the disclosures of which are
incorporated by reference herein. Formulations of 1L-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 ces of interleukins.
Identifier Sequence (One-Letter Amino Acid Symbols)
SEQ ID NO:3 MAPTSSSTKK TQLQLEHLLJ DLQMILNGIN NYKNPKLTRM MPKK LQCL 60
recombinant EEELKPLEEV LNLAQSKNFl LRPRDLISNI NVIVLELKGS ETTFMCEYAD ETATIVEFLN I20
human ILi2 RWITFCQSII STLT I34
(rhILe2)
SEQ ID NO:4 PTSSSTKKTQ LQLEHLLLDJ QMILNGINNY KNPKLTRMLT FKFYMPKKAT ELKlLQCLEE 60
AIdesIeukin ELKPLEEVLN LAQSKNFHLR PRDLISNINV IVLELKGSET TFMCEYADET ATIVEFLNRW I20
ITFSQSIIST LT I32
SEQ ID NO:5 MHKCDITLQE IIKTLNSLTE QKTLCTELTV TDIFAASKNT TEKETECRAA TVLRQFYSHH 60
recombinant LGAT AQQFHRHKQJ IRFLKRLDRN LWGLAGLNSC PVKEANQSTL LKTI I20
human ILi4 MREKYSKCSS I30
(rhILe4)
SEQ ID NO:6 MDCDIEGKDG KQYESVLMVS IDQLLDSMKE IGSNCLNNEF NEFKRHICDA NKEGMFLFRA 60
recombinant ARKLRQFLKM NSTGDFDLHJ LKVSEGTTIL LNCTGQVKGR EAQP TKSJEENKSL I20
human ILi7 KEQKKLNDLC FLKRLLQEI{ TCWNKILMGT KEH I53
(rhILe7)
SEQ ID NO:7 MNWVNVISDL KKIEDLIQSM YTES DVHPSCKVTA MKCFLLELQV ISLESGDASI 60
recombinant HDTVENLIIL ANNSLSSNGN VTESGCKECE ELEEKNIKEF LQSFVHIVQM FINTS II5
human IL7I5
(rhILeIS)
SEQ ID NO:8 MQDRHMIRMR QLIDIVDQLK NYVNDLVPEF LPAPEDVETN CEWSAFSCFQ KAQJKSANTG 60
inant NNERIINVSI KKLKRKPPST NAGRRQKHRL TCPSCDSYEK KPPKEFLERF KSLJQKMIHQ I20
human ILi2I HLSSRTHGSE DS I32
The term “IL-4” (also referred to herein as “IL4”) refers to the cytokine known as
eukin 4, which is produced by Th2 T cells and by eosinophils, basophils, 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 1L-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 expression from B cells.
Recombinant human IL-4 suitable for use in the invention is cially 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-4 recombinant
protein, Cat. No. Gibco CTPOO43). The amino acid ce of inant human IL-4
suitable for use in the invention is given in Table 2 (SEQ ID N05).
The term “IL-7” (also referred to herein as “IL7”) refers to a glycosylated tissuederived
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 receptor, a heterodimer consisting of
IlL-7 receptor alpha and common gamma chain receptor, which in a series of s important
for T cell development within the thymus and survival within the periphery. 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. 4) and
Fisher Scientific, Inc., Waltham, MA, USA (human IL-7 recombinant protein, Cat. No.
Gibco PHCOO7l). The amino acid sequence of recombinant human IL-7 suitable for use in the
invention is given in Table 2 (SEQ ID N06).
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 mammalian forms,
vative amino acid substitutions, glycoforms, biosimilars, and variants f. 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-15 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.,
m, MA, USA (human IL-15 recombinant protein, Cat. No. 9-82). The amino acid
sequence of recombinant human 1L-15 suitable for use in the invention is given in Table 2 (SEQ
ID N07).
The term “IL-21” (also referred to herein as “IL21”) refers to the pleiotropic cytokine
protein known as interleukin-21, and includes all forms of 1L-21 including human and
mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants
thereof. 1L-21 is described, e.g., in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-
95, the disclosure of which is incorporated by nce herein. IL-21 is primarily produced by
l killer T cells and activated human CD4+ T cells. Recombinant human IL-21 is a single,
ycosylated polypeptide chain containing 132 amino acids with a molecular mass of 15.4
kDa. Recombinant human IL-21 is commercially available from multiple ers, including
ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYTb) and
ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-21 inant protein, Cat. No.
1480). The amino acid sequence of recombinant human lL-21 suitable for use in the
invention is given in Table 2 (SEQ ID NO:8).
The term “biosimilar” means a biological product, including a monoclonal antibody or
fusion protein, that is highly similar to a US. licensed reference biological product
notwithstanding minor differences in clinically inactive components, and for which there are no
ally gful differences n the biological product and the reference product in
terms of the safety, purity, and potency of the product. Furthermore, a similar biological or
“biosimilar” medicine is a biological medicine that is similar to another biological medicine that
has already been authorized for use by the European Medicines Agency. The term milar”
is also used synonymously by other national and regional regulatory es. Biological
products or biological medicines are medicines that are made by or derived from a biological
source, such as a bacterium or yeast. They can consist of relatively small molecules such as
human insulin or erythropoietin, or compleX molecules such as monoclonal antibodies. For
example, if the nce IL-2 protein is aldesleukin (PROLEUKIN), a protein ed by drug
regulatory authorities with reference to aldesleukin is a “biosimilar to” aldesleukin or is a
“biosimilar thereof’ of aldesleukin. In Europe, a similar biological or milar” medicine is a
biological medicine that is similar to another biological medicine that has already been
authorized for use by the European Medicines Agency (EMA). The relevant legal basis for
similar biological applications in Europe is Article 6 of Regulation (EC) No 726/2004 and
e 10(4) of Directive 2001/83/EC, as amended and therefore in Europe, the biosimilar may
be authorized, approved for authorization or subject of an application for authorization under
Article 6 of Regulation (EC) No 726/2004 and Article 10(4) of ive 2001/83/EC. The
already authorized original biological medicinal product may be referred to as a ence
medicinal product” in Europe. Some of the requirements for a product to be considered a
biosimilar are outlined in the CHMP Guideline on r Biological Medicinal Products. In
on, product specific guidelines, including guidelines relating to monoclonal antibody
biosimilars, are provided on a product-by-product basis by the EMA and published on its
website. A biosimilar as bed herein may be similar to the reference medicinal product by
way of quality characteristics, biological activity, mechanism of action, safety profiles and/or
cy. In addition, the biosimilar may be used or be intended for use to treat the same
ions as the reference medicinal product. Thus, a biosimilar as described herein may be
deemed to have similar or highly similar quality characteristics to a reference medicinal product.
Alternatively, or in on, a biosimilar as described herein may be deemed to have similar or
highly similar ical activity to a nce medicinal product. Alternatively, or in addition,
a biosimilar as described herein may be deemed to have a similar or highly similar safety profile
to a nce medicinal product. Alternatively, or in addition, a biosimilar as described herein
may be deemed to have similar or highly similar efficacy to a reference medicinal t. As
described herein, a biosimilar in Europe is compared to a reference medicinal product which has
been authorized by the EMA. However, in some instances, the biosimilar may be compared to a
biological medicinal t which has been authorized outside the European Economic Area (a
A authorized “comparator”) in certain studies. Such studies include for e certain
clinical and in vivo inical studies. As used herein, the term “biosimilar” also relates to a
biological medicinal product which has been or may be compared to a non-EEA authorized
comparator. n biosimilars are proteins such as antibodies, antibody fragments (for
example, antigen binding ns) and fusion proteins. A protein biosimilar may have an amino
acid sequence that has minor modifications in the amino acid structure (including for example
deletions, ons, and/or substitutions of amino acids) which do not significantly affect the
function of the polypeptide. The ilar may comprise an amino acid sequence having a
sequence identity of 97% or greater to the amino acid sequence of its reference medicinal
product, e.g., 97%, 98%, 99% or 100%. The biosimilar may comprise one or more post-
translational modifications, for example, although not limited to, glycosylation, oxidation,
deamidation, and/or tion which is/are different to the post-translational modifications of
the reference medicinal product, provided that the differences do not result in a change in safety
and/or efficacy of the medicinal product. The biosimilar may have an identical or different
glycosylation pattern to the reference medicinal product. ularly, although not exclusively,
the biosimilar may have a different glycosylation pattern if the differences address or are
intended to address safety ns associated with the reference medicinal product.
Additionally, the biosimilar may deviate from the reference medicinal product in for example its
th, pharmaceutical form, formulation, excipients and/or presentation, providing safety and
efficacy of the medicinal product is not compromised. The biosimilar may comprise ences
in for e pharmacokinetic (PK) and/or pharmacodynamic (PD) profiles as compared to the
reference nal product but is still deemed sufficiently similar to the reference medicinal
t as to be authorized or considered suitable for authorization. In certain circumstances, the
biosimilar exhibits different binding characteristics as compared to the reference medicinal
product, wherein the different binding characteristics are considered by a Regulatory Authority
such as the EMA not to be a barrier for authorization as a similar biological product. The term
“biosimilar” is also used mously by other national and regional regulatory agencies.
] 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 substitutions, 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 nce antibody. The term variant also
es pegylated antibodies or proteins.
“Pegylation” refers to a modified antibody, or a fragment thereof, that typically is
reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde tive of PEG,
under conditions in which one or more PEG groups become attached to the antibody, antibody
nt, or protein. Pegylation may, for e, increase the biological (e.g., serum) half life
of the antibody or protein. Preferably, the pegylation is d out via an acylation reaction or
an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble
polymer). As used herein, the term “polyethylene glycol” is intended to ass any of the
forms of PEG that have been used to derivatize other proteins, such as mono (Cl-Clo) alkoxy- or
aryloxy-polyethylene glycol or polyethylene glycol-maleimide. The antibody or protein to be
pegylated may be an aglycosylated antibody. Methods for pegylation are known in the art and
can be applied to the antibodies of the invention, as described for example in European Patent
Nos. EP 6 and EP 0401384.
] The term “hematological malignancy” refers to mammalian cancers and tumors of the
hematopoietic and lymphoid tissues, including but not limited to tissues of the blood, bone
, lymph nodes, and lymphatic system. Hematological malignancies are also referred to as
“liquid tumors.” Hematological malignancies include, but are not limited to, acute
blastic leukemia (ALL), chronic cytic lymphoma (CLL), small lymphocytic
lymphoma (SLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML),
acute monocytic leukemia , 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 y 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 d 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
microenvironment.
] 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, myelomas, and lymphomas, as
well as other hematological malignancies. TILs obtained from liquid tumors may also be referred
to herein as marrow infiltrating lymphocytes (MILs).
The term “microenvironment,” as used , may refer to the solid or hematological
tumor microenvironment as a whole or to an individual subset of cells within the
nvironment. The tumor microenvironment, as used herein, refers to a x mixture of
“cells, soluble factors, ing molecules, extracellular matrices, and mechanical cues that
promote stic transformation, support tumor growth and on, protect the tumor from
host ty, foster therapeutic resistance, and provide niches for dominant ases to
thrive,” as described in Swartz, el al., Cancer Res, 2012, 72, 2473. Although tumors express
antigens that should be ized by T cells, tumor clearance by the immune system is rare
because of immune suppression by the microenvironment.
The terms “fragmenting,” “fragment,” and “fragmented,” as used herein to describe
processes for disrupting a tumor, includes mechanical fragmentation methods such as crushing,
slicing, dividing, and morcellating tumor tissue as well as any other method for disrupting the
physical structure of tumor tissue.
The terms “about” and “approximately” mean within a statistically meaningful range of
a value. Such a range can be within an order of magnitude, preferably within 50%, more
preferably within 20%, more preferably still within 10%, and even more preferably within 5% of
WO 94167
a given value or range. The ble variation encompassed by the terms ” or
“approximately” s on the particular system under study, and can be readily appreciated by
one of ordinary skill in the art. Moreover, as used herein, the terms ” and “approximately”
mean that dimensions, sizes, formulations, parameters, shapes and other quantities and
characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as
desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like,
and other factors known to those of skill in the art. In general, a dimension, size, formulation,
parameter, shape or other quantity or characteristic is “about” or “approximate” whether or not
expressly stated to be such. It is noted that embodiments of very different sizes, shapes and
dimensions may employ the described arrangements.
The transitional terms “comprising,77 (L consisting essentially of,” and “consisting of,”
when used in the appended claims, in original and amended form, define the claim scope with
respect to what unrecited additional claim elements or steps, if any, are excluded from the scope
of the claim(s). The term “comprising” is intended to be inclusive or open-ended and does not
e any additional, unrecited t, method, step or material. The term “consisting of’
excludes any element, step or material other than those specified in the claim and, in the latter
instance, impurities ordinary associated with the specified material(s). The term “consisting
essentially of’ limits the scope of a claim to the ed elements, steps or material(s) and those
that do not materially affect the basic and novel characteristic(s) of the claimed invention. All
itions, methods, and kits described herein that embody the invention can, in alternate
embodiments, be more specifically defined by any of the transitional terms “comprising,”
“consisting essentially of,” and “consisting of.”
For the avoidance of doubt, it is intended herein that ular features (for example
rs, characteristics, values, uses, diseases, formulae, compounds or groups) bed in
conjunction with a particular aspect, embodiment or example of the invention are to be
understood as applicable to any other , embodiment or example bed herein unless
incompatible therewith. Thus such features may be used where appropriate in conjunction with
any of the definition, claims or embodiments defined herein. All of the features disclosed in this
specification (including any accompanying claims, abstract and drawings), and/or all of the steps
of any method or process so disclosed, may be combined in any combination, except
combinations where at least some of the features and/or steps are mutually exclusive. The
invention is not restricted to any details of any disclosed embodiments. The invention extends to
any novel one, or novel combination, of the features disclosed in this specification (including any
accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of
the steps of any method or process so disclosed.
s of Expanding Remnant Tumor Infiltrating Lymphocytes
In an ment, the invention es a method of expanding remnant tumor
infiltrating lymphocytes (rTILs) after digestion of a tumor as described herein.
In an ment, the invention includes a method of expanding rTILs, the method
comprising contacting a population of rTILs comprising at least one rTIL with IL-2, thereby
expanding rTILs.
In an embodiment, the ion provides a method of expanding a population of rTILs,
the method comprising the steps as described in Jin, er al., J. Immunolherapy 2012, 35, 2,
the disclosure of which is orated by reference . For example, the tumor may be
placed in enzyme media and mechanically fragmented for approximately 1 minute. The mixture
may then be incubated for 30 minutes at 37 0C in 5% C02 and then mechanically fragmented
again for approximately 1 minute. After incubation for 30 s at 37 0C in 5% C02, the
tumor may be mechanically fragmented a third time for approximately 1 minute. If after the
third mechanical disruption, large pieces of tissue are present, 1 or 2 additional mechanical
iations may be applied to the sample, with or without 30 additional minutes of tion
at 37 0C in 5% C02. At the end of the final incubation, if the cell suspension contains a large
number of red blood cells or dead cells, a density gradient separation using Ficoll may be
performed to remove these cells. TIL cultures were initiated in 24-well plates (Costar 24-well
cell culture cluster, flat bottom, Corning Incorporated, Corning, NY), each well may be seeded
with l>< 106 tumor digest cells or one tumor fragment approximately l—8 mm3 in size in 2 mL of
complete medium (CM) with IL-2 (6000 IU/mL, Chiron Corp., Emeryville, CA). CM consists
of RPMI 1640 with GlutaMAX, mented with 10% human AB serum, 25mM Hepes, and
mg/mL gentamicin. Cultures may be initiated in gas-permeable flasks with a 40 mL capacity
and a 10 cm2 gas-permeable silicon bottom (G—Rex 10, Wilson Wolf Manufacturing, New
Brighton), each flask may be loaded with lO-40>< lO6 viable tumor digest cells or 5—30 tumor
fragments in 10—40 mL of CM with IL-2. G—Rex 10 and 24-well plates may be incubated in a
humidified tor at 37 0C in 5% C02 and 5 days after culture initiation, half the media may
be removed and replaced with fresh CM and IL-2 and after day 5, half the media may be
changed every 2—3 days. A rapid expansion protocol (REP) for TILs may be med using T-
175 flasks and gas-permeable bags or gas-permeable G—Rex flasks, as described elsewhere
herein. For REP in T-l75 flasks, l><106 rTILs may be suspended in 150 mL of media in each
flask. The rTIL may be cultured in a l to 1 mixture of CM and AIM-V medium (50/50
), supplemented with 3000 IU/mL of IL-2 and 30 ng/mL of anti-CD3 antibody (OKT-3).
The T-l75 flasks may be incubated at 37 0C in 5% C02. Half the media may be changed on day
using 50/50 medium with 3000 IU/mL of IL-2. On day 7, cells from 2 T-l75 flasks may be
combined in a 3 L bag and 300 mL of AIM-V with 5% human AB serum and 3000 IU/mL of IL-
2 may be added to the 300 mL of TIL suspension. The number of cells in each bag may be
counted every day or two days, and fresh media may be added to keep the cell count between 0.5
and 2.0><106 cells/mL. For REP in 500 mL capacity flasks with 100 cm2 rmeable n
bottoms (e.g., G—Rex 100, Wilson Wolf cturing, as described ere herein), 5><106 or
><106 TILs may be cultured in 400 mL of 50/50 medium, supplemented with 3000 IU/mL of
IL-2 and 30 ng/mL of anti-CD3 antibody (OKT-3). The G—Rex100 flasks may be incubated at
37 0C in 5% C02. On day five, 250 mL of supernatant may be removed and placed into
fuge bottles and centrifuged at 1500 rpm (491 g) for 10 minutes. The obtained TIL pellets
may be resuspended with 150 mL of fresh 50/50 medium with 3000 IU/mL of IL-2 and added
back to the G—Rex 100 flasks. When TIL are expanded serially in G—Rex 100 flasks, on day
seven the TIL in each G-Rex100 are suspended in the 300 mL of media t in each flask and
the cell suspension may be divided into three 100 mL aliquots that may be used to seed 3 G-
Rex100 flasks. About 150 mL of AIM-V with 5% human AB serum and 3000 IU/mL of IL-2
may then be added to each flask. G—Rex100 flasks may then be incubated at 37 0C in 5% C02,
and after four days, 150 mL of AIM-V with 3000 IU/mL of IL-2 may be added to each G-
Rex100 flask. After this, the REP may be completed by harvesting cells on day 14 of culture.
In an embodiment, a method of expanding or treating a cancer includes a step wherein
TILs are obtained from a patient tumor sample. A patient tumor sample may be obtained using
methods known in the art. For example, TILs may be cultured from enzymatic tumor digests and
tumor fragments (about 1 to about 8 mm3 in size) from sharp dissection. Such tumor digests may
be produced by incubation in enzymatic media (e.g., Roswell Park Memorial Institute (RPMI)
1640 buffer, 2 mM glutamate, 10 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 or fragmenter).
Tumor digests may be ed by placing the tumor in enzymatic media and mechanically
fragmenting the tumor for approximately 1 minute, followed by incubation for 30 minutes at 37
0C in 5% C02, followed by ed cycles of mechanical dissociation and incubation 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 performed 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 disclosure of which is incorporated by
reference herein. Any of the foregoing methods may be used in any of the embodiments
described herein for methods of expanding TILs or methods ng a cancer.
In an embodiment, REP of rTILs can be performed in a gas permeable container using
any suitable method. For example, rTILs can be rapidly expanded using non-specif1c T cell
receptor stimulation in the presence of interleukin-2 (IL-2), interleukin-15 (IL-15), and/or
interleukin-21 (IL-21), as described, e.g., in International Patent Application ation Nos.
incorporated by reference herein. The non-specific T cell receptor stimulus can include, for
example, about 30 ng/mL of OKT-3, a monoclonal anti-CD3 antibody (commercially available
from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Inc., San Diego, CA, USA). TILs can be
y ed by further stimulation of the TILs in vitro with one or more ns, 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-A2) binding peptide, e.g., 0.3 uM
MART-1:26-35 (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 antigens may e, e.g., -l,
TRP-l, TRP-2, nase cancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic
portions thereof. TIL may also be rapidly ed by re-stimulation with the same antigen(s) of
the cancer pulsed onto HLA-A2-expressing antigen-presenting cells. Alternatively, the TILs can
be further re-stimulated with, e.g., example, irradiated, autologous lymphocytes or with
ated HLA-A2+ neic lymphocytes and IL-2.
In an embodiment, a method for expanding TILs may include using about 5000 mL to
about 25000 mL of cell medium, about 5000 mL to about 10000 mL of cell culture medium, or
about 5800 mL to about 8700 mL of cell culture medium. In an embodiment, a method for
ing TILs may include using about 1000 mL to about 2000 mL of cell medium, about 2000
mL to about 3000 mL of cell culture medium, about 3000 mL to about 4000 mL of cell culture
medium, about 4000 mL to about 5000 mL of cell culture medium, about 5000 mL to about 6000
mL of cell culture medium, about 6000 mL to about 7000 mL of cell culture medium, about
7000 mL to about 8000 mL of cell culture medium, about 8000 mL to about 9000 mL of cell
culture medium, about 9000 mL to about 10000 mL of cell culture medium, about 10000 mL to
about 15000 mL of cell culture medium, about 15000 mL to about 20000 mL of cell culture
medium, or about 20000 mL to about 25000 mL of cell culture medium. In an ment,
expanding the number of TILs uses no more than one type of cell culture . Any suitable
cell culture medium may be used, e.g., AHVI-V cell medium tamine, 50 M streptomycin
sulfate, and 10 M gentamicin sulfate) cell e medium (Invitrogen, Carlsbad CA). In this
, 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, ing the
number of TIL may comprise feeding the cells no more frequently than every third or fourth day.
Expanding the number of cells in a gas permeable container simplifies the procedures necessary
to expand the number of cells by reducing the feeding frequency necessary to expand the cells.
In an ment, the rapid expansion is performed using a gas permeable container.
Such embodiments allow for cell populations to expand from about 5 X 105 cells/cm2 to between
X 106 and 30 X 106 cells/cmz. In an embodiment, this expansion occurs without feeding. In
an embodiment, this expansion occurs without feeding so long as medium resides at a height of
about 10 cm in a rmeable flask. In an embodiment this is without 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 cytokine with the medium. Such containers, s, and methods
are known in the art and have been used to expand TILs, and include those described in US.
Patent ation Publication No. US 3 77739 A1, International Patent Application
Publication No.
2013/0115617 A1, International Publication No.
Publication No. US 2011/0136228 A1, US. Patent No. 8,809,050, International Patent
WO 94167
Application Publication No.
2016/0208216 A1, US. Patent Application ation No. US 2012/0244133 A1, International
Patent Application Publication No.
No. US 2013/0102075 A1, US. Patent No. 8,956,860, International Patent Application
Publication No.
2015/0175966 Al, the disclosures of which are incorporated herein by reference. Such
processes are also bed in Jin, el al., J. Immunolherapy 2012, 35, 283-292, the disclosure of
which is incorporated by reference herein.
In an embodiment, the gas permeable container is a G—ReX 10 flask (Wilson Wolf
Manufacturing Corporation, New Brighton, MN, USA). In an embodiment, the gas permeable
container includes a 10 cm2 gas ble culture surface. In an embodiment, the gas permeable
container includes a 40 mL cell culture medium capacity. In an embodiment, the gas permeable
container provides 100 to 300 million TILs after 2 medium exchanges.
In an embodiment, the gas permeable container is a G—ReX 100 flask (Wilson Wolf
Manufacturing Corporation, New Brighton, MN, USA). In an embodiment, the gas permeable
container includes a 100 cm2 gas ble culture surface. In an embodiment, the gas
permeable container includes a 450 mL cell e medium capacity. In an embodiment, the gas
permeable container provides 1 to 3 billion TILs after 2 medium exchanges.
In an embodiment, the gas ble container is a G—ReX 100M flask (Wilson Wolf
cturing Corporation, New Brighton, MN, USA). In an embodiment, the gas permeable
container includes a 100 cm2 gas permeable culture surface. In an embodiment, the gas
ble container includes a 1000 mL cell culture medium capacity. In an embodiment, the
gas permeable container provides 1 to 3 billion TlLs without medium ge.
In an embodiment, the gas permeable container is a G—ReX lOOL flask (Wilson Wolf
Manufacturing Corporation, New Brighton, MN, USA). In an embodiment, the gas ble
container includes a 100 cm2 gas permeable culture surface. In an embodiment, the gas
permeable ner includes a 2000 mL cell culture medium capacity. In an embodiment, the
gas permeable container provides 1 to 3 billion TlLs t medium exchange.
In an embodiment, the gas permeable container is a G—ReX 24 well plate (Wilson Wolf
Manufacturing Corporation, New Brighton, MN, USA). In an embodiment, the gas permeable
container includes a plate with wells, n each well includes a 2 cm2 gas permeable culture
e. In an embodiment, the gas permeable container includes a plate with wells, wherein
each well includes a 8 mL cell culture medium capacity. In an embodiment, the gas permeable
container provides 20 to 60 million cells per well after 2 medium exchanges.
In an embodiment, the gas permeable container is a G—Rex 6 well plate (Wilson Wolf
Manufacturing Corporation, New Brighton, MN, USA). In an embodiment, the gas permeable
container includes a plate with wells, wherein each well es a 10 cm2 gas permeable culture
surface. In an embodiment, the gas permeable container includes a plate with wells, wherein
each well includes a 40 mL cell e medium capacity. In an embodiment, the gas permeable
container provides 100 to 300 million cells per well after 2 medium exchanges.
In an embodiment, the cell medium in the f1rstand/or second gas permeable container
is unf11tered. The use of red cell medium may simplify the procedures necessary 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 ing a tumor tissue
sample from the mammal, culturing the tumor tissue sample in a first gas permeable container
containing cell medium therein, obtaining TILs from the tumor tissue , expanding the
number of TILs in a second gas permeable container containing cell medium therein for a
duration of about 14 to about 42 days, e.g., about 28 days.
In an embodiment, the ratio of rTILs to PBMCs in the rapid 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 ment, the ratio of rTILs to
PBMCs in the rapid expansion is between 1 to 50 and 1 to 300. In an ment, the ratio of
rTILs to PBMCs in the rapid expansion is between 1 to 100 and 1 to 200.
In an embodiment, the ratio of rTILs to PBMCs (rTIL:PBMC) is selected from the
group consisting of1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65,
1:70,1:75,1:80,1:85,1:90,1:95,1:100,1:105,1:110,1:115,1:120,1:125,1:130,1:135,1:140,
1:145,1:150,1:155,1:160,1:165,1:170,1:175,1:180,1:185,1:190,1:195,1:200,1:225,1:250,
1:275, 1:300, 1:350, 1:400, 1:450, and 1:500. In a preferred embodiment, the ratio of rTILs to
PBMCs (rTILzPBMC) is about 1:90. In a preferred embodiment, the ratio of TILs to PBMCs
(rTILzPBMC) is about 1:95. In a preferred embodiment, the ratio of rTlLs to PBMCs
(TIL:PBMC) is about 1:100. In a preferred embodiment, the ratio of rTlLs to PBMCs
(TIL:PBMC) is about 1:105. In a preferred embodiment, the ratio of rTlLs to PBMCs
(TIL:PBMC) is about 1:110.
In an embodiment, the cell e medium further comprises IL-2. In a preferred
embodiment, 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 ses 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 an embodiment, the cell e medium comprises OKT-3 antibody. In a preferred
embodiment, the cell e medium comprises about 30 ng/mL of OKT-3 dy. In an
embodiment, the cell culture medium comprises 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 OKT-3 antibody. In an embodiment,
the cell culture medium comprises n 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 OKT-3 antibody.
] In an embodiment, a rapid expansion process for TILs may be performed using T-175
flasks and gas permeable bags as preViously described (Tran, el al., J. Immunolher. 2008, 31,
742-51, Dudley, er al., J. Immunolher. 2003, 26, 332-42) or gas ble cultureware (G—ReX
flasks, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton,
MN, USA). For TIL rapid expansion in T-175 flasks, 1 X 106 TILs suspended in 150 mL of
WO 94167
media may be added to each T-l75 flask. The TlLs may be cultured in a l to 1 mixture of CM
and AIM-V medium, supplemented with 3000 IU (injection units) per mL of IL-2 and 30 ng per
mL of anti-CD3 antibody (e.g., OKT-3). The T-l75 flasks may be incubated at 37° C in 5%
C02. Half the media may be exchanged on day 5 using 50/50 medium with 3000 IU per mL of
IL-2. On day 7 cells from two T-l75 flasks may be combined in a 3 liter bag and 300 mL of
AIM V with 5% human AB serum and 3000 IU per mL of IL-2 was added to the 300 ml of TIL
suspension. The number of cells in each bag was d every day or two and fresh media was
added to keep the cell count between 0.5 and 2.0 x 106 cells/mL.
In an embodiment, for TIL rapid expansions in 500 mL capacity gas permeable flasks
with 100 cm gas-permeable silicon s (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 in 50/50 medium, supplemented with 5% human AB serum, 3000 IU per mL of IL-2
and 30 ng per mL of anti-CD3 (OKT-3). The G-Rex 100 flasks may be incubated at 37°C in 5%
C02. On day 5, 250 mL of atant may be removed and placed into centrifuge bottles and
centrifuged at 1500 rpm utions per minute, 491 X g) for 10 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
t 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 AHVI-V with 5% human AB serum and 3000
IU per mL of 1L-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—Rex 100 flask. The cells may be harvested on day 14 of culture.
In an embodiment, TILs may be prepared as follows. 2 mm3 tumor fragments are
cultured in complete media (CM) comprised of AIM-V medium (Invitrogen Life Technologies,
Carlsbad, CA) supplemented with 2 mM glutamine (Mediatech, Inc. Manassas, VA), 100 U/mL
penicillin (Invitrogen Life Technologies), 100 ug/mL streptomycin (Invitrogen Life
Technologies), 5% heat-inactivated human AB serum (Valley Biomedical, Inc. Winchester, VA)
and 600 IU/mL rhIL-2 (Chiron, ille, CA). For enzymatic digestion of solid tumors, tumor
ens was diced into RPMI—l640, washed and centrifuged at 800 rpm for 5 minutes at 15-
22°C, and resuspended in enzymatic ion buffer (0.2 mg/mL Collagenase and 30 units/ml of
DNase in RPMI-1640) followed by overnight rotation at room temperature. TILs established
from fragments may be grown for 3-4 weeks in CM and ed fresh or cryopreserved in
heat-inactivated HAB serum with 10% dimethylsulfoxide (DMSO) and stored at -180°C until the
time of study. Tumor associated lymphocytes (TAL) obtained from ascites collections were
seeded at 3 X 106 cells/well of a 24 well plate in CM. TIL growth was inspected about every
other day using a low-power ed cope.
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.
US. Patent Application Publication No. 2005/0106717 Al, the sures 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 expansion 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 Expansion
System W5 (GE Healthcare). In an embodiment, the cell expansion system includes a gas
permeable cell bag with a volume ed 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, about 10 L, about 11 L, about 12 L, about 13 L, about 14 L, about 15 L, about 16
L, about 17 L, about 18 L, about 19 L, about 20 L, about 25 L, and about 30 L. In an
embodiment, the cell expansion system includes a gas permeable cell bag with a volume range
selected from the group consisting of between 50 and 150 mL, between 150 and 250 mL,
between 250 and 350 mL, between 350 and 450 mL, between 450 and 550 mL, between 550 and
650 mL, between 650 and 750 mL, n 750 and 850 mL, between 850 and 950 mL, and
between 950 and 1050 mL. In an embodiment, the cell expansion system es a gas
permeable cell bag with a volume range selected from the group ting of between 1 L and 2
L, between 2 L and 3 L, between 3 L and 4 L, between 4 L and 5 L, between 5 L and 6 L,
between 6 L and 7 L, between 7 L and 8 L, between 8 L and 9 L, n 9 L and 10 L, between
L and 11 L, between 11 L and 12 L, between 12 L and 13 L, between 13 L and 14 L, between
14 L and 15 L, between 15 L and 16 L, between 16 L and 17 L, between 17 L and 18 L, between
18 L and 19 L, and between 19 L and 20 L. In an embodiment, the cell expansion system
includes a gas permeable cell bag with a volume range selected from the group consisting of
between 0.5 L and 5 L, between 5 L and 10 L, between 10 L and 15 L, between 15 L and 20 L,
between 20 L and 25 L, and between 25 L and 30 L. In an embodiment, the cell expansion
system utilizes a g time of about 30 minutes, about 1 hour, about 2 hours, about 3 hours,
about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about
hours, about 11 hours, about 12 hours, about 24 hours, about 2 days, about 3 days, about 4
days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about
11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17
days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days,
about 24 days, about 25 days, about 26 days, about 27 days, and about 28 days. In an
embodiment, the cell expansion system utilizes a rocking time of between 30 minutes and 1
hour, n 1 hour and 12 hours, between 12 hours and 1 day, between 1 day and 7 days,
between 7 days and 14 days, between 14 days and 21 days, and between 21 days and 28 days. In
an embodiment, the cell expansion system utilizes a rocking rate of about 2 rocks/minute, about
rocks/minute, about 10 rocks/minute, about 20 rocks/minute, about 30 minute, and about
40 rocks/minute. In an embodiment, the cell expansion system utilizes a rocking rate of
between 2 rocks/minute and 5 rocks/minute, 5 rocks/minute and 10 minute, 10
rocks/minute and 20 rocks/minute, 20 rocks/minute and 30 rocks/minute, and 30 minute
and 40 rocks/minute. In an embodiment, the cell expansion system es a rocking angle of
about 2°, about 3°, about 4°, about 5°, about 6°, about 7°, about 8°, about 9°, about 10°, about
11°, and about 12°. In an embodiment, the cell ion system utilizes a rocking angle of
between 2° and 3°, between 3° and 4°, n 4° and 5°, between 5° and 6°, between 6° and 7°,
between 7° and 8°, between 8° and 9°, between 9° and 10°, between 10° and 11°, and between
11° and 12°.
In an embodiment, a method of expanding rTILs further comprises a step n
rTILs 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 ion of TILs for superior tumor reactiVity.
Characteristics of rTILs
In an embodiment, the rTILs of the invention exhibit an exhausted T cell phenotype
characterized by one or more T cell exhaustion s. In an embodiment, the rTILs of the
invention exhibit an exhausted T cell phenotype characterized by one or more T cell exhaustion
markers using flow cytometry analysis. In an embodiment, the T cell exhaustion marker is PD-l.
In an embodiment, the T cell exhaustion marker is LAG3. In an ment, the T cell
exhaustion marker is TIM3.
In an embodiment, PD-l expression in rTILs is reduced by at least 5%, at least 10%, at
least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at
least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least
150% relative to the eTILs. In an embodiment, PD-l expression in rTILs is reduced by at least
2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold,
at least 9-fold, or at least 10-fold ve to the eTILs.
] In an embodiment, LAG3 expression in rTILs is d by at least 5%, at least 10%, at
least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at
least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least
150% relative to the eTILs. In an embodiment, LAG3 expression in rTILs is reduced by at least
2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold,
at least 9-fold, or at least 10-fold relative to the eTILs. In an embodiment, LAG3 sion in
rTILs is undetectable by flow cytometry.
In an embodiment, TIM3 expression in rTILs is reduced by at least 5%, at least 10%, at
least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at
least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least
150% relative to the eTILs. In an embodiment, T11VI3 expression in rTILs is reduced by at least
2-fold, at least 3-fold, at least , at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold,
at least 9-fold, or at least 10-fold relative to the eTILs. In an ment, TIM3 sion in
rTILs is undetectable by flow cytometry.
In an embodiment, TIGIT expression in rTILs is reduced by at least 5%, at least 10%,
at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at
least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least
150% relative to the eTILs. In an embodiment, TIGIT expression in rTILs is reduced by at least
2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold,
at least 9-fold, or at least 10-fold relative to the eTILs. In an embodiment, TIGIT expression in
rTILs is undetectable by flow cytometry.
In an embodiment, CTLA-4 expression in rTILs is reduced by at least 5%, at least 10%,
at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at
least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least
150% relative to the eTILs. In an embodiment, CTLA-4 expression in rTILs is reduced by at
least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-
fold, at least 9-fold, or at least 10-fold relative to the eTILs. In an embodiment, CTLA-4
expression in rTILs is undetectable by flow cytometry.
In an embodiment, CD69 expression in rTILs is increased by at least 10%, at least 20%,
at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at
least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% ve
to the eTILs. In an embodiment, CD69 expression in rTILs is increased by at least 2-fold, at
least , at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least , at least 9-
fold, or at least 10-fold relative to the eTILs.
In an embodiment, SlPRl (sphingosine-l-phosphate or 1) expression in rTILs is
decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at
least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%,
at least 140%, or at least 150% relative to the eTILs. In an embodiment, SlPRl sion in
rTILs is sed by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold,
at least 7-fold, at least 8-fold, at least 9-fold, or at least d relative to the eTILs.
] In an embodiment, telomere length in rTILs is increased by at least 10%, at least 20%,
at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at
least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% relative
to the eTILs. In an ment, telomere length in rTILs is increased by at least 2-fold, at least
3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold,
or at least 10-fold relative to the eTILs.
In an embodiment, CD28 expression in rTILs is increased by at least 10%, at least 20%,
at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at
least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% relative
to the eTILs. In an embodiment, CD28 expression in rTILs is increased by at least 2-fold, at
least , at least 4-fold, at least , at least 6-fold, at least 7-fold, at least 8-fold, at least 9-
fold, or at least 10-fold relative to the eTILs.
In an embodiment, CD27 expression in rTILs is increased by at least 10%, at least 20%,
at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at
least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% relative
to the eTILs. In an embodiment, CD27 expression in rTILs is increased by at least , at
least 3-fold, at least 4-fold, at least 5-fold, at least , at least 7-fold, at least 8-fold, at least 9-
fold, or at least d relative to the eTILs.
] In some embodiments, the methods described herein may include an optional
eservation of eTIL and/or rTIL in a storage media (for example, media containing 5%
DMSO) prior to performing an additional step desribed herein or after completion of a REP step
described herein, prior to transport, g, and/or administration to a patient. In some
embodiments, the methods described herein may include a step of thawing cryopreserved TILs
(e. g. cryopreserved eTIL, cryopreserved rTIL, or a combination or mixture thereof) prior to
performing an additional step described herein. In some embodiments, the additional step may
be an additional or repeated expansion of the eTIL and/or rTIL (e.g., a reREP), which may be
performed on the thawed cells, using, for example, a supplemented cell culture medium
sing IL-2, OKT-3, and/or feeder cells (e.g., antigen presenting cells), generally
comprising peripheral blood mononuclear cells , or, atively, using antigen
ting cells), wherein the additional expansion step may be performed for at least 14 days.
In some embodiments, such media may also contain combinations of IL-2, IL-15, and/or IL-23
rather than IL-2 alone.
As discussed herein, cryopreservation can occur at numerous points throughout the TIL
expansion process. In some embodiments, a bulk TIL population (e.g., eTILs, rTILs, or a
combination or mixture thereof) after expansion can be cryopreserved. Cryopreservation can be
generally accomplished by placing the TIL population into a freezing solution, e.g., 85%
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complement inactivated AB serum and 15% dimethyl sulfoxide . The cells in solution
are placed into cryogenic vials and stored for 24 hours at -80 0C, with optional transfer to
s nitrogen freezers for cryopreservation. See, Sadeghi, el al., Acla Oncologica 2013, 52,
978-986. In some embodiments, the TILs described herein may be eserved in 5% DMSO.
In some embodiments, the TILs described herein may be cryopreserved in cell culture media
plus 5% DMSO.
When riate, the cryopreserved cells described herein, such as cryopreserved
rTILs, are removed from the freezer and thawed in a 37 oC water bath until approximately 4/5 of
the solution is thawed. The cells are generally resuspended in complete media and ally
washed one or more times. In some embodiments, the thawed TILs can be d and assessed
for viability as is known in the art.
Methods of Digesting Tumors to Obtain rTILs
In an ment, a method of obtaining rTILs includes a step wherein a tumor is
digested using one or more enzymes. Enzymes le for ion of tumors are described in
Volvitz, el al., BMC Neuroscience 2016, 17, 30, the disclosure of which is incorporated by
reference herein.
In some embodiments, the invention may include methods of obtaining rTILs that
include a step wherein a tumor, which may include tumor tissue or a portion thereof, is digested
using an deoxyribonuclease, a collagenase, a hyaluronidase, or a combination thereof.
In an embodiment, a method of obtaining rTILs includes a step wherein a tumor is
digested using any enzyme that catalyzes the hydrolytic cleavage of odiester linkages in
the DNA backbone, thus degrading DNA. In an ment, a method of obtaining rTILs
includes a step wherein a tumor is digested using a deoxyribonuclease (DNase). In an
embodiment, a method of obtaining rTILs includes a step wherein a tumor is digested using a
deoxyribonuclease and at least one other enzyme. In an embodiment, the deoxyribonuclease is
deoxyribonuclease I. In an embodiment, the deoxyribonuclease is deoxyribonuclease II. In an
embodiment, the deoxyribonuclease is deoxyribonuclease I from bovine pancreas (Sigma D5025
or equivalent). In an embodiment, the deoxyribonuclease is recombinant deoxyribonuclease I
from bovine expressed in Pichiapastoris (Sigma D2821 or equivalent). In an embodiment, the
deoxyribonuclease is inant human deoxyribonuclease I (thNAase I, also known as
dornase alfa, commercially ble as PULMOZYME from Genentech, Inc.). In an
embodiment, the ibonuclease is deoxyribonuclease II from bovine spleen (Sigma D8764
or equivalent). In an embodiment, the deoxyribonuclease is deoxyribonuclease II from porcine
spleen (Sigma D4138 or equivalent). In an embodiment, any of the foregoing
deoxyribonucleases is present in the tumor digest. The preparation and properties of
deoxyribonucleases suitable for use in the invention are described in US. Patent Nos. 433,
6,391,607 , 7,407,785, and 7,297,526, and International Patent Application Publication No. WC
2016/108244 Al, the disclosures of each of which are incorporated by reference herein.
In an ment, a method of obtaining rTILs es a step wherein a tumor is
digested using any enzyme that catalyzes the cleavage of peptide linkages in collagen, thus
degrading collagen. In an embodiment, a method of obtaining rTILs includes a step n a
tumor is digested using a collagenase. In an embodiment, a method of obtaining rTILs includes
a step wherein a tumor is digested using a collagenase and at least one other enzyme. In an
embodiment, the collagenase is collagenase from Clostridium histolylicum. In an embodiment,
the collagenase is Clostridiopeptidase A. In an embodiment, the collagenase is collagenase I. In
an embodiment, the collagenase is collagenase II. In an embodiment, the collagenase is
collagenase from Clostridium ylicum (Sigma C5138 or equivalent). The preparation and
properties of collagenases suitable for use in the ion are described in US. Patent Nos.
3,201,325, 3,705,083, 3,821,364, 5,177,017, 5,422,261, 5,989,888, 9,211,316, the disclosures of
each of which are incorporated by nce herein.
In an ment, a method of obtaining rTILs includes a step wherein a tumor is
digested using any enzyme that catalyzes the degradation of hyaluronic acid. In an embodiment,
a method of obtaining rTILs includes a step wherein a tumor is digested using a hyaluronidase.
In an ment, a method of ing rTILs includes a step wherein a tumor is ed using
a hyaluronoglucosidase. In an embodiment, the onidase is hyaluronidase Type I from
bovine testes (Sigma H3506 or equivalent). In an embodiment, the hyaluronidase is
hyaluronidase Type II from sheep testes (Sigma H2126 or equivalent). In an embodiment, the
hyaluronidase is hyaluronidase Type III. In an embodiment, the hyaluronidase is hyaluronidase
Type IV (Type IV-S) from bovine testes (Sigma H3884 or equivalent). In an embodiment, the
hyaluronidase is hyaluronidase Type V from sheep testes (Sigma H6254 or equivalent). In an
embodiment, the hyaluronidase is hyaluronidase Type VIII from bovine testes (Sigma H3757 or
equivalent). In an embodiment, the hyaluronidase is recombinant human onidase
rcially available as X from Halozyme, Inc). The ation and properties of
hyaluronidases suitable for use in the invention are described in US. Patent Nos. 4,820,516,
,593,877, 6,057,110, 6,123,938, 7,767,429, 8,202,517, 8,431,124, and 8,431,380, the
disclosures of each of which are incorporated by reference herein.
In an embodiment, a method of obtaining rTILs includes a step wherein a tumor is
digested using a deoxyribonuclease and a hyaluronidase. In an embodiment, a method of
obtaining rTILs includes a step wherein a tumor is digested using a deoxyribonuclease and a
collagenase. In an embodiment, a method of obtaining rTILs includes a step wherein a tumor is
digested using a hyaluronidase and a collagenase. In an embodiment, a method of obtaining
rTILs includes a step wherein a tumor is digested using a deoxyribonuclease, a hyaluronidase,
and a collagenase.
] In an embodiment, a method of obtaining rTILs includes a step wherein a tumor is
digested using a deoxyribonuclease and a hyaluronidase and at least one additional enzyme. In
an embodiment, a method of obtaining rTILs includes a step wherein a tumor is digested using a
deoxyribonuclease and a collagenase and at least one additional enzyme. In an embodiment, a
method of obtaining rTILs includes a step wherein a tumor is digested using a hyaluronidase and
a collagenase and at least one onal enzyme. In an embodiment, a method of obtaining
rTILs includes a step wherein a tumor is digested using a ibonuclease, a onidase,
and a collagenase and at least one additional enzyme. In any of the ing embodiments, the
additional enzyme is selected from the group consisting of caseinase, clostripain, trypsin, and
combinations thereof.
In an embodiment, a method of ing rTILs includes a step wherein a tumor is
digested using any enzyme described above, and further comprises the step of mechanically
disrupting or fragmenting the tumor before, during, or after ion.
In an embodiment, a method of obtaining rTILs includes a step wherein a tumor is
digested using any enzyme described above, wherein the digestion is performed over a period
selected from the group consisting of 15 minutes, 30 s, 45 minutes, 1 hour, 90 minutes, 2
hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours,
18 hours, 24 hours, 36 hours, and 48 hours.
In an embodiment, a method of obtaining rTILs includes a step wherein a tumor is
digested using any enzyme described above, wherein the digestion is performed over a period
selected from the group consisting of about 15 minutes, about 30 minutes, about 45 minutes,
about 1 hour, about 90 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about
6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12
hours, about 18 hours, about 24 hours, about 36 hours, and about 48 hours.
In an ment, a method of obtaining rTILs includes a step wherein a tumor is
digested using any enzyme described above, wherein the digestion is performed over a period
selected from the group consisting of less than 15 minutes, less than 30 s, less than 45
s, less than 1 hour, less than 90 minutes, less than 2 hours, less than 3 hours, less than 4
hours, less than 5 hours, less than 6 hours, less than 7 hours, less than 8 hours, less than 9 hours,
less than 10 hours, less than 11 hours, less than 12 hours, less than 18 hours, less than 24 hours,
less than 36 hours, and less than 48 hours.
In an embodiment, a method of obtaining rTILs includes a step wherein a tumor is
digested using any enzyme described above, wherein the digestion is performed over a period
selected from the group consisting of greater than 15 s, greater than 30 minutes, r
than 45 minutes, greater than 1 hour, greater than 90 minutes, greater than 2 hours, greater than 3
hours, greater than 4 hours, greater than 5 hours, greater than 6 hours, greater than 7 hours,
greater than 8 hours, greater than 9 hours, greater than 10 hours, greater than 11 hours, greater
than 12 hours, greater than 18 hours, greater than 24 hours, greater than 36 hours, and greater
than 48 hours.
In an embodiment, a method of obtaining rTILs includes a step wherein a tumor is
digested using any enzyme described above, wherein the digestion is performed over a period
selected from the group consisting of n 30 minutes and 1 hour, between 1 hours and 2
hours, between 2 hours and 3 hours, n 3 hours and 4 hours, between 4 hours and 5 hours,
n 5 hours and 6 hours, between 6 hours and 12 hours, between 12 hours and 18 hours,
between 18 hours and 24 hours, and between 24 hours and 48 hours.
In an embodiment, a method of obtaining rTILs includes a step wherein a tumor is
digested using any enzyme bed above, wherein the digestion is performed at a temperature
selected from the group consisting of about 20 oC, about 25 oC, about 30 oC, about 35 oC, about
40 oC, about 45 oC, about 50 oC, about 55 oC, about 60 oC, about 65 oC, about 70 oC, about 75
oC, and about 80 0C.
In an embodiment, a method of obtaining rTILs includes a step n a tumor is
digested using any enzyme described above, wherein the digestion is med at a temperature
selected from the group consisting of between 20 oC and 25 oC, between 25 oC and 30 oC,
between 30 oC and 35 oC, between 35 oC and 40 oC, between 40 oC and 45 oC, between 45 oC
and 50 oC, between 50 oC and about 55 oC, between 55 oC and 60 oC, n 60 oC and 65 oC,
between 65 oC and 70 oC, between 70 oC and 75 oC, and between 75 oC and 80 0C.
In an embodiment, a method of obtaining rTILs includes a step wherein a tumor is
digested using any enzyme described above, wherein the time and temperature of the digestion
are each decreased if tumor remnants (after pre-REP) are digested. In an embodiment, a method
of obtaining rTILs es a step wherein a tumor is digested using any enzyme described
above, wherein the time and temperature of the digestion are each se if whole tumor
fragments (without pre-REP) are ed.
s of Modulating rTIL to eTIL Ratio
In an embodiment, the concentration of rTILs relative to eTILs may be modulated or
controlled by use of any expansion and digestion steps as described herein (including pre-REP),
such that a therapeutic TIL product for use in the treatment of cancers described herein may
contain a desirable rTIL to eTIL ratio. In an embodiment, the invention provides a method of
removing eTILs from a mixture of eTILs and rTILs. In an embodiment, the invention provides a
method of removing rTILs from a mixture of eTILs and rTILs.
In some embodiments of the methods of the ion, eTILs and/or rTILs may be
added to a culture before an initial expansion step, at a first expansion step (e.g., pre-REP),
and/or at a second expansion step (e.g., REP). In some embodiments of the s of the
invention, eTILS may be separately ed according to the culture or expansion steps
described herein through one, two, three, or more expansions, and added to a population of
rTILS and eTILS at a selected rTIL to eTIL ratio. In some embodiments of the methods of the
invention, rTILS may be tely cultured according to the culture or expansion steps
bed herein through one, two, three, or more expansions, and added to a population of
eTlLS to provide a mixture of rTILS and eTILS at a selected rTIL to eTIL ratio.
In an embodiment, eTILs prepared according to the methods described herein may be
added to a population of rTILs to provide a selected rTIL to eTIL ratio in the resulting rTIL/eTIL
mixture. In an ment, rTILs prepared according to the methods described herein may be
added to a tion of eTILs to provide a selected rTIL to eTIL ratio in the resulting
rTIL/eTIL mixture.
In an embodiment, the invention provides a method of treating a cancer wherein the
treatment comprises delivering a therapeutically ive amount of TILs to a patient, n
the ratio of rTILs to eTILs in the TILs (e.g., a selected rTIL to eTIL ratio) is selected from the
group consisting of about 0:100, about 1:99, about 5:95, about 10:90, about 15:85, about 20:80,
about 25:75, about 30:70, about 35:65, about 40:60, about 45:55, about 50:50, about 55:45, about
60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85:15, about 90: 10, about 95:5,
about 99:1, and about 100:0 rTIL to eTIL.
In an embodiment, the rTIL to eTIL ratio is adjusted using a selection method, which
may be used to enrich or reduce rTILs relative to eTILs as required by the d artisan. In an
embodiment, the selection method is based on the lack of exhaustion markers, including TIM3,
LAG3, TIGIT, PD-l, and . In an embodiment, the selection method is based on
enhanced CD69 expression. In an embodiment, the selection method is based on superior
mitochondrial mass. In an embodiment, the selection method is based on a subset of cell surface
proteins. In an embodiment, the selection method is based on phenotype. In an embodiment, the
ion method is based on function.
In an embodiment, the rTIL to eTIL ratio is adjusted by co-culturing rTILs and eTILs
in the same cell culture medium until a desirable ratio is obtained. In an embodiment, the rTIL
to eTIL ratio is adjusted by co-culturing rTILs and eTILs in the same cell e medium,
including the addition of rTILs or eTILs to the cell culture medium at different timepoints during
expansion, until a desirable ratio is obtained. In an embodiment, rTIL growth is preferentially
expanded in the cell culture medium by addition of cytokines other than IL-2, including IL-4, IL-
7, IL-l5, and/or IL-21.
In an embodiment, the ratio of rTlLs to eTlLs (e.g., a selected rTIL to eTIL ratio)
provided by the methods described herein may be at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,
9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,
26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,
42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,
58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,
74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% rTIL to eTIL.
In an embodiment, the ratio of rTlLs to eTlLs (e.g., a selected rTIL to eTIL ratio)
provided by the methods described herein may be at most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,
9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,
26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,
42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,
58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,
74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% rTIL to eTIL.
In an embodiment, the ratio of rTlLs to eTlLs (e.g., a selected rTIL to eTIL ratio)
provided by the methods bed herein may be about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,
%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,
26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,
42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,
58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,
74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% rTIL to eTIL.
s of Treating Cancers and Other Diseases
The rTILs and combinations of rTILs and eTlLs described herein may be used in a
method for treating diseases in a human. In an embodiment, they are for use in treating a
roliferative disorder. In some embodiments, the hyperproliferative disorder is cancer. In
some ments, the hyperproliferative disorder is a solid tumor cancer. In some
embodiments, the solid tumor cancer is selected from the group consisting of melanoma, double-
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refractory melanoma (1'.e., melanoma tory to at least two prior treatments including
chemotherapy and checkpoint de), ovarian cancer, cervical cancer, non-small-cell lung
cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human oma
virus, head and neck , renal cancer, renal cell carcinoma, and sarcoma. In some
embodiments, the hyperproliferative disorder is a hematological malignancy (or liquid tumor
cancer). In some embodiments, the hematological malignancy is selected from the group
consisting of acute d leukemia, chronic lymphocytic leukemia, acute lymphoblastic
leukemia, diffuse large B cell lymphoma, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma,
follicular lymphoma, and mantle cell lymphoma. The rTILs and ations of rTILs and
eTILs described herein may also be used in treating other disorders as bed herein and in the
following paragraphs.
In an embodiment, the invention includes a method of treating a cancer in a patient in
need of such treatment, wherein the treatment comprises delivering a eutically effective
amount of rTILs to a patient, wherein the rTILs are prepared according a method comprising the
steps of:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
inflltrating lymphocytes (TILs),
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas ble container with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor remnants and emergent TILs (eTILs),
(d) removing at least a plurality of the eTILs,
(e) enzymatically digesting the tumor remnants into tumor remnant cells using a digest
mixture, and
(f) expanding the tumor remnant cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an expanded number of t tumor infiltrating cytes
(rTILs),
wherein the rTILs express reduced levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell tion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations thereof, and
wherein the second cell culture medium further comprises a cytokine ed from the
group consisting of IL-4, IL-7, IL-15, 1L-21, and combinations f.
In an embodiment, the invention includes a method treating a cancer in a patient in
need of such treatment, the method sing:
(a) obtaining tumor tissue from the t, wherein the tumor tissue comprises tumor
infiltrating lymphocytes (TlLs),
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor remnants and emergent TlLs (eTILs),
(d) removing at least a plurality of the eTILs,
(e) tically digesting the tumor remnants into tumor remnant cells using a digest
mixture,
(f) expanding the tumor remnant cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an expanded number of remnant tumor infiltrating lymphocytes
(rTILs),
wherein the rTILs express reduced levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations thereof, and
wherein the second cell culture medium further comprises a ne selected from the
group consisting of IL-4, IL-7, IL-15, 1L-21, and ations thereof,
(g) treating the patient with a non-myeloablative lymphodepletion regimen prior to
administering the rTILs to the patient,
(h) administering a therapeutically ive amount of rTILs to the patient, and
(i) treating the patient with a high-dose IL-2 n starting on the day after
administration of the rTlLs to the patient.
In an embodiment, the invention includes a method treating a cancer in a t in
need of such treatment, the method comprising:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
infiltrating lymphocytes (TlLs),
(b) fragmenting the tumor ,
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor remnants and nt TlLs (eTILs),
(d) ng at least a ity of the eTILs,
(e) enzymatically digesting the tumor remnants into tumor remnant cells using a digest
mixture,
(f) expanding the tumor remnant cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an expanded number of remnant tumor infiltrating lymphocytes
(rTILs),
wherein the rTlLs express reduced levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and ations thereof, and
wherein the second cell culture medium further comprises a cytokine selected from the
group consisting of IL-4, IL-7, IL-15, 1L-21, and ations thereof,
(g) treating the patient with a non-myeloablative lymphodepletion regimen prior to
administering the rTlLs to the patient,
(h) administering a therapeutically effective amount of rTILs to the patient, wherein a
therapeutically effective amount of eTILs are simultaneously stered to the patient
in a mixture with the rTlLs, and
(i) treating the patient with a high-dose IL-2 regimen starting on the day after
administration of the rTlLs to the patient.
In an embodiment, the invention includes a method treating a cancer in a patient in
need of such treatment, the method comprising:
(a) obtaining tumor tissue from the t, wherein the tumor tissue comprises tumor
infiltrating lymphocytes (TlLs),
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor remnants and emergent TlLs (eTILs),
(d) removing at least a plurality of the eTILs,
(e) enzymatically digesting the tumor remnants into tumor t cells using a digest
mixture,
(f) ing the tumor remnant cells with a second cell culture medium sing cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
ner to provide an expanded number of remnant tumor infiltrating lymphocytes
(rTILs),
wherein the rTlLs express reduced levels of a T cell tion marker relative to the
eTILs,
wherein the T cell exhaustion marker is ed from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations thereof, and
wherein the second cell culture medium further comprises a cytokine selected from the
group consisting of IL-4, IL-7, IL-15, 1L-21, and combinations thereof,
(g) treating the patient with a eloablative lymphodepletion regimen prior to
administering the rTlLs to the patient,
(h) administering a therapeutically effective amount of rTILs to the t, wherein a
therapeutically effective amount of eTILs are simultaneously administered to the patient
in a mixture with the rTlLs, and
(i) treating the patient with a high-dose IL-2 regimen ng on the day after
administration of the rTlLs to the patient,
wherein the cancer is selected from the group consisting of melanoma, double-refractory
melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer,
head and neck cancer, renal cell oma, acute myeloid leukemia, colorectal ,
sarcoma, non-small cell lung cancer (NSCLC), and triple ve breast cancer.
In an embodiment, the ion includes a method treating a cancer in a patient in
need of such treatment, the method comprising:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
infiltrating lymphocytes (TlLs),
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor remnants and emergent TlLs (eTILs),
(d) ng at least a plurality of the eTILs,
(e) tically digesting the tumor remnants into tumor remnant cells using a digest
mixture,
(f) expanding the tumor remnant cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas ble
container to provide an expanded number of remnant tumor infiltrating lymphocytes
(rTILs),
wherein the rTlLs express reduced levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations thereof, and
wherein the second cell culture medium further comprises a cytokine selected from the
group consisting of IL-4, IL-7, IL-15, 1L-21, and ations thereof,
(g) treating the patient with a non-myeloablative lymphodepletion regimen prior to
stering the rTlLs to the t, wherein the non-myeloablative lymphodepletion
regimen comprises the steps of administration of cyclophosphamide at a dose of 60
day for two days followed by administration of fiudarabine at a dose of 25
mg/mZ/day for five days,
(h) administering a therapeutically effective amount of rTILs to the patient, wherein a
therapeutically effective amount of eTILs are simultaneously administered to the patient
in a mixture with the rTILs, and
(i) treating the patient with a high-dose IL-2 regimen starting on the day after
administration of the rTILs to the patient.
In some embodiments of the methods described herein, the step of removing at least a
plurality of the eTILs includes removing at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,
11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%,
27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%,
43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,
59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,
75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% ofthe eTILs.
Efficacy of the compounds and ations of compounds described herein in
treating, preventing and/or managing the indicated diseases or ers can be tested using
various models known in the art, which provide guidance for treatment of human disease. For
example, models for determining efficacy of ents for ovarian cancer are bed, e.g., in
Mullany, et al., Endocrinology 2012, 153, 1585-92, and Fong, et al., J. Ovarian Res. 2009, 2, 12.
Models for determining efficacy of treatments for atic cancer are described in Herreros-
Villanueva, et al., World J. Gastroenterol. 2012, 18, 1286-1294. Models for determining
efficacy of treatments for breast cancer are described, e.g., in Fantozzi, Breast Cancer Res. 2006,
8, 212. Models for determining efficacy of treatments for melanoma are bed, e.g., in
Damsky, et al., Pigment Cell & ma Res. 2010, 23, 853—859. Models for determining
efficacy of treatments for lung cancer are described, e.g., in sen, et al., Genes &
Development, 2005, 19, 643-664. Models for determining efficacy of treatments for lung cancer
are described, e.g., in Kim, Clin. Exp. Otorhinolaryngol. 2009, 2, 55-60, and Sano, Head Neck
Oncol. 2009, I, 32.
inistration of IL-2
In an embodiment, the invention provides a method of treating a cancer in a patient in
need of such treatment, comprising the steps of:
(a) obtaining rTILs from a tumor resected from a patient according to a method described
herein;
(b) treating the patient with a non-myeloablative depletion regimen prior to
administering the rTILs to the patient;
(c) administering a therapeutically effective amount of rTILs to the patient; and
(d) treating the patient with an IL-2 regimen starting on the day after administration of the
rTILs to the patient.
In an embodiment; the IL-2 regimen comprises a high-dose IL-2 regimen; wherein the
high-dose IL-2 regimen comprises aldesleukin; or a ilar or variant thereof; administered
intravenously starting on the day after administering a eutically effective portion of the
third population of TILs; wherein the aldesleukin or a biosimilar or variant thereof is
administered at a dose of 600,000 or 0 IU/kg (patient body mass) using 15-minute bolus
intravenous ons every eight hours until tolerance; for a maXimum of 14 doses. Following 9
days of rest; this schedule may be ed for another 14 doses; for a maXimum of 28 doses in
total.
In an embodiment; the IL-2 regimen comprises a high-dose IL-2 regimen; wherein the
high-dose IL-2 regimen comprises aldesleukin; or a biosimilar or variant f; administered
intravenously starting on the day after administering a eutically effective n of the
third population of TILs; wherein the aldesleukin or a biosimilar or variant f is
administered at a dose of 0.037 mg/kg or 0.044 mg/kg IU/kg (patient body mass) using 15-
minute bolus intravenous infusions every eight hours until tolerance; for a maXimum of 14 doses.
Following 9 days of rest; this schedule may be repeated for another 14 doses; for a maXimum of
28 doses in total.
In an embodiment; the invention includes a method treating a cancer in a patient in
need of such treatment; the method comprising:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
infiltrating lymphocytes (TlLs),
(b) fragmenting the tumor tissue;
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor remnants and emergent TlLs (eTILs),
(d) ng at least a plurality of the eTILs,
(e) enzymatically digesting the tumor remnants into tumor remnant cells using a digest
(f) ing the tumor remnant cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an expanded number of remnant tumor infiltrating lymphocytes
(rTILs),
wherein the rTILs express d levels of a T cell exhaustion marker relative to the
eTILs,
n the T cell exhaustion marker is ed from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations thereof, and
wherein the second cell culture medium further comprises a cytokine ed from the
group consisting of IL-4, IL-7, IL-15, 1L-21, and combinations thereof,
(g) treating the t with a non-myeloablative lymphodepletion regimen prior to
administering the rTILs to the patient,
(h) administering a therapeutically effective amount of rTILs to the patient, wherein a
eutically effective amount of eTILs are simultaneously administered to the patient
in a mixture with the rTILs, and
(i) treating the patient with a high-dose IL-2 regimen starting on the day after
administration of the rTILs to the patient,
wherein the high-dose IL-2 regimen comprises 600,000 or 720,000 IU/kg of aldesleukin,
or a biosimilar or variant thereof, stered as a 15-minute bolus intravenous infusion
every eight hours until tolerance.
] In an embodiment, the IL-2 regimen comprises a decrescendo IL-2 regimen.
Decrescendo IL-2 regimens have been described in O’Day, el al., J. Clin. Oncol. 1999, 17, 2752-
61 and Eton, er al., Cancer 2000, 88, 1703-9, the disclosures of which are incorporated herein by
nce. In an embodiment, a decrescendo IL-2 regimen comprises 18 X 106 IU/m2
administered intravenously over 6 hours, followed by 18 X 106 IU/m2 administered intravenously
over 12 hours, followed by 18 X 106 IU/m2 stered intravenously over 24 hours, followed
by 4.5 X 106 IU/m2 administered enously over 72 hours. This treatment cycle may be
repeated every 28 days for a maximum of four cycles. In an embodiment, a decrescendo IL-2
regimen comprises 18,000,000 IU/m2 on day 1, 9,000,000 IU/m2 on day 2, and 000
IU/m2 on days 3 and 4.
] In an embodiment, the invention includes a method treating a cancer in a patient in
need of such treatment, the method comprising:
(a) obtaining tumor tissue from the patient, wherein the tumor tissue comprises tumor
rating lymphocytes (TILs),
(b) nting the tumor tissue,
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor remnants and emergent TILs (eTILs),
(d) removing at least a plurality of the eTILs,
(e) enzymatically digesting the tumor remnants into tumor remnant cells using a digest
mixture,
(f) expanding the tumor remnant cells with a second cell culture medium comprising cell
e media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable
container to provide an expanded number of remnant tumor infiltrating lymphocytes
(rTILs),
wherein the rTILs express reduced levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations thereof, and
wherein the second cell culture medium further comprises a cytokine ed from the
group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof,
(g) ng the patient with a non-myeloablative lymphodepletion regimen prior to
administering the rTILs to the patient;
(h) administering a therapeutically effective amount of rTILs to the patient, wherein a
therapeutically effective amount of eTILs are simultaneously administered to the patient
in a mixture with the rTILs, and
(i) treating the patient with a decrescendo IL-2 regimen starting on the day after
administration of the rTILs to the patient,
n the decrescendo IL-2 n ses 18 X 106 IU/m2 administered
intravenously over 6 hours, followed by 18 X 106 IU/m2 administered intravenously over
12 hours, followed by 18 X 106 IU/m2 administered intravenously over 24 hours,
followed by 4.5 X 106 IU/m2 administered intravenously over 72 hours, repeated every
28 days for a maXimum of four cycles.
] In an embodiment, the IL-2 regimen comprises administration of pegylated IL-2,
including pegylated eukin. In an embodiment, the IL-2 regimen comprises administration
of pegylated IL-2 every 1, 2, 4, 6, 7, 14 or 21 days at a dose of O. 10 mg/day to 50 mg/day.
In an embodiment, the invention includes a method treating a cancer in a patient in
need of such ent, the method comprising:
(a) obtaining tumor tissue from the patient, n the tumor tissue comprises tumor
infiltrating lymphocytes (TILs),
(b) fragmenting the tumor tissue,
(c) treating the tumor tissue in a gas permeable container with a first cell culture medium
and interleukin 2 (IL-2) to provide tumor ts and emergent TILs (eTILs),
(d) removing at least a plurality of the eTILs,
(e) enzymatically digesting the tumor remnants into tumor remnant cells using a digest
mixture,
(f) expanding the tumor t cells with a second cell culture medium comprising cell
culture media, irradiated feeder cells, OKT-3 dy, and IL-2 in a gas permeable
container to provide an expanded number of remnant tumor infiltrating lymphocytes
(rTILs),
wherein the rTILs express reduced levels of a T cell exhaustion marker relative to the
eTILs,
wherein the T cell exhaustion marker is selected from the group consisting of TIM3,
LAG3, TIGIT, PD-l, and combinations thereof, and
wherein the second cell culture medium further comprises a cytokine selected from the
group ting of IL-4, IL-7, IL-15, IL-21, and combinations thereof,
(g) treating the patient with a non-myeloablative depletion regimen prior to
administering the rTILs to the patient,
(h) administering a therapeutically effective amount of rTILs to the patient, wherein a
therapeutically effective amount of eTILs are simultaneously administered to the patient
in a mixture with the rTILs, and
(i) treating the patient with a pegylated IL-2 regimen starting on the day after
administration of the rTILs to the patient,
wherein the pegylated IL-2 regimen comprises administration of pegylated IL-2 every 1,
2, 4, 6, 7, 14 or 21 days at a dose of 0.10 mg/day to 50 mg/day.
Non-Myeloablative Lymphodepletion with Chemotherapy
In an embodiment, the invention includes a method of treating a cancer with a
population of rTILs, wherein a patient is pre-treated with non-myeloablative herapy prior
to an on of rTILs according to the ion. In some embodiments, the population of
rTILs may be provided with a population of eTils, wherein a patient is eated with non-
myeloablative chemotherapy prior to an infusion of rTILs and eTils according to the invention.
In an embodiment, the eloablative chemotherapy is cyclophosphamide 6O mg/kg/d for 2
days (days 27 and 26 prior to rTIL infusion) and fludarabine 25 mg/mZ/d for 5 days (days 27 to
23 prior to rTIL infusion). In an embodiment, after non-myeloablative chemotherapy and rTIL
infusion (at day 0) ing to the invention, the patient receives an intravenous infusion of IL-
2 enously at 720,000 IU/kg every 8 hours to physiologic tolerance.
Experimental findings indicate that lymphodepletion prior to adoptive transfer of
tumor-specific T lymphocytes plays a key role in ing treatment efficacy by eliminating
regulatory T cells and competing elements of the immune system (“cytokine sinks”).
Accordingly, some embodiments of the ion utilize a lymphodepletion step (sometimes also
referred to as osuppressive ioning”) on the patient prior to the introduction of the
rTILs of the invention.
In l, lymphodepletion is achieved using administration of fludarabine or
cyclophosphamide (the active form being ed to as mafosfamide) and combinations thereof.
Such methods are described in Gassner, el al., Cancer Immunol. Immunolher. 2011, 60, 75—85,
Muranski, el al., Nat. Clin. Pract. 011001., 2006, 3, 668—681, Dudley, el al., J. Clin. Oncol. 2008,
26, 5233-5239, and Dudley, el al., J. Clin. Oncol. 2005, 23, 2346—2357, all ofwhich are
incorporated by reference herein in their entireties.
In some embodiments, the fludarabine is administered at a concentration of 0.5 ug/mL -
ug/mL fludarabine. In some embodiments, the fludarabine is administered at a concentration
of l ug/mL fludarabine. In some embodiments, the fludarabine treatment is administered for 1
day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more. In some embodiments, the
fludarabine is stered at a dosage of 10 mg/kg/day, 15 mg/kg/day, 20 day,
mg/kg/day, 30 mg/kg/day, 35 mg/kg/day, 40 day, or 45 mg/kg/day. In some
embodiments, the fludarabine treatment is administered for 2-7 days at 35 mg/kg/day. In some
embodiments, the fludarabine treatment is administered for 4-5 days at 35 day. In some
embodiments, the fludarabine treatment is administered for 4-5 days at 25 mg/kg/day.
In some embodiments, the mafosfamide, the active form of cyclophosphamide, is
obtained at a concentration of 0.5 ug/mL -10 ug/mL by administration of cyclophosphamide. In
some embodiments, mafosfamide, the active form of cyclophosphamide, is obtained at a
concentration of l ug/mL by administration of hosphamide. In some embodiments, the
cyclophosphamide treatment is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or
7 days or more. In some embodiments, the cyclophosphamide is administered at a dosage of
100 mg/mZ/day, l50 mg/mZ/day, l75 mg/mZ/day, 200 mg/mZ/day, 225 mg/mZ/day, 250
mg/mZ/day, 275 mg/mZ/day, or 300 mg/mZ/day. In some embodiments, the cyclophosphamide is
administered intravenously (i.v.) In some embodiments, the cyclophosphamide ent is
administered for 2-7 days at 35 mg/kg/day. In some embodiments, the cyclophosphamide
ent is administered for 4-5 days at 250 mg/mZ/day i.v. In some embodiments, the
cyclophosphamide treatment is administered for 4 days at 250 day i.v.
In some embodiments, depletion is performed by administering the bine
and the cyclophosphamide are together to a patient. In some embodiments, fludarabine is
administered at 25 mg/mZ/day iv. and cyclophosphamide is administered at 250 mg/mZ/day i.v.
over 4 days.
In an embodiment, the lymphodepletion is performed by administration of
cyclophosphamide at a dose of 60 mg/mZ/day for two days ed by administration of
fludarabine at a dose of 25 mg/mZ/day for five days.
Pharmaceutical Compositions, s, and Dosing Regimens
In an embodiment, rTILs expanded using methods of the invention are administered to
a patient as a pharmaceutical composition. In an embodiment, the pharmaceutical composition is
a suspension of rTILs in a e buffer. rTILs expanded using methods of the invention may be
administered by any suitable route as known in the art. Preferably, the rTILs are administered as
a single intra-arterial or intravenous infusion, which preferably lasts approximately 30 to 60
minutes. Other suitable routes of administration include eritoneal, intrathecal, and
intralymphatic administration.
In an embodiment, rTILs and eTILs ed using methods of the invention are
stered to a patient as a pharmaceutical composition. In an embodiment, the
pharmaceutical composition is a suspension of rTILs and eTILs in a sterile buffer. rTILs and
eTILs expanded using s of the invention may be administered by any suitable route as
known in the art. Preferably, the rTILs and eTILs are administered as a single intra-arterial or
enous infusion, which preferably lasts approximately 30 to 60 minutes. Other suitable
routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.
Any suitable dose of rTILs can be administered. Preferably, from about 2.3 X 1010 to
about 13 .7>< lO10 rTILs are administered, with an average of around 7. 8 X 1010 rTILs, particularly if
the cancer is melanoma. In an embodiment, about 1.2>< 1010 to about 4.3 X1010 of rTILs are
administered.
Any suitable dose of rTILs and eTILs can be stered. Preferably, from about
2.3 X 1010 to about l3.7>< lO10 rTILs and eTILs are administered, with an average of around
7 .8>< lO10 rTILs and eTILs, particularly if the cancer is ma. In an embodiment, about
1.2>< 1010 to about 4.3><1010 of rTILs and eTILs are administered.
In some ments, the number of the rTILs provided in the pharmaceutical
itions ofthe invention is about 1x106, 2x106, 3x106, 4x106, 5x106, 6><106, 7x106, 8x1067
9><106,1><107,2><107,3><107,4><107,5><107,6><107,7><107,8><107,9><107,1><108,2><108,3><108,
4><108,5><108,6><108,7><108,8><108,9><108,1><109,2><109,3><109,4><109,5><109,6><109,7><109,
8><109,9><109,1><1010,2><1010,3><1010,4><1010,5><1010,6><1010,7><1010,8><1010,9><1010,1X10”,
2><1011,3><1011,4><1OH,5><1011,6><1011,7><1011,8><1011,9><1OH,1><1012,2><1012,3><1012,4><10127
><1012,6><1012,7><1012,8><1012,9><1012,1><1013,2><1013,3><1013,4><1013,5><1013,6><1013,7><10137
8><1013, and 9><1013. In an embodiment, the number of the rTILs provided in the pharmaceutical
compositions ofthe invention is in the range of 1x106 to 5x106, 5x106 to 1x107, 1x107 to 5x1077
5x107 to 1x10", 1x108 to 5x10", 5x108 to 1x109, 1x109 to 5x109, 5x109 to 1x10“), 1x1010 to
5x10“), 5x1010 to 1x10“, 5x1011 to 1x10”, 1x1012 to 5x10”, and 5x1012 to 1x10”.
In some embodiments, the number of the rTILs and eTILs provided in the
pharmaceutical itions of the invention is about l><lO6, 2><lO6, 3><106, 4><lO6, 5><106,
6><106,7><106,8><106,9><106,1><107,2><107,3><107,4><107,5><107,6><107,7><107,8><107,9><107,
1x108,2x108,3x108,4x108,5x108,6x108,7x108,8x108,9x108,1x109,2x109,3x109,4x109,
><109,6><109,7><109,8><109,9><109,1><1010,2><1010,3><1010,4><1010,5><1010,6><1010,7><1010,
8><1010,9><1010,1><1OH,2><1011,3><1011,4><1011,5><1011,6><1OH,7><10”,8><1011,9><1011,1><10127
2><1012,3><1012,4><1012,5><1012,6><1012,7><1012,8><1012,9><1012,1><1013,2><1013,3><1013,4><10137
5x10”, 6><1013, 7x10”, 8x10”, and 9x10”. In an embodiment, the number ofthe rTILs and
eTILs provided in the pharmaceutical compositions of the invention is in the range of l>< 106 to
5x106, 5x106 to 1x107, 1x107 to 5x107, 5x107 to 1x10", 1x108 to 5x10", 5x108 to 1x109, 1x109
to 5x109, 5x109 to 1x10”, 1x1010 to 5><1010,5><1010tol><1011,5><1011tol><1012, l><1012to
5x10”, and 5x1012 to 1x10”.
] In some embodiments, the concentration of the rTILs provided in the pharmaceutical
compositions of the invention is less than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%,
%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 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%, , 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%,
0.001%, 0.0009%, 0.0008%, 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 rTILs and eTILs provided in the
ceutical 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%, 5%, 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%, 0.0008%, 0.0007%, %, 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 rTILs provided in the ceutical
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%, 16.50%, 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%, , 12.25% 12%, 11.75%, , 11.25% 11%,
.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%, 5.25% 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%, , 0.005%, 0.004%, 0.003%, 0.002%, , 0.0009%,
0.0008%, 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 ments, the concentration of the rTILs and eTILs provided in the
ceutical compositions of the invention is greater than 90%, 80%, 70%, 60%, 50%, 40%,
%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%,
17.25% 17%, 16.75%, 16.50%, 16.25% 16%, 15.75%, 15.50%, 15.25% 15%, , 14.50%,
14.25% 14%, 13.75%, 13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 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%, 5.25% 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%, 0.0005%, 0.0004%, %, 0.0002% or 0.0001%
w/w, w/v, or v/v of the pharmaceutical composition.
In some embodiments, the concentration of the rTILs provided in the pharmaceutical
compositions of the invention is in the range from about 0.0001% 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 25%, 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 rTILs and eTILs ed in the
pharmaceutical compositions of the invention is in the range from about 0.0001% 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 25%,
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
ition.
In some embodiments, the tration of the rTILs 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 concentration of the rTlLs and eTILs 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 ition.
In some embodiments, the amount of the rTILs ed 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,
0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 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 rTILs and eTILs provided in the
pharmaceutical compositions of the ion 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, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 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 rTILs provided in the pharmaceutical
compositions ofthe 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, 0.2 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, 1
g, 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
In some embodiments, the amount of the rTILs and eTILs provided in the
pharmaceutical itions 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,
0.2 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, 1 g, 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 rTILs provided in the pharmaceutical compositions of the invention are effective
over a wide dosage range. The exact dosage will depend upon the route of stration, the
form in which the compound is administered, the gender and age of the subject to be treated, the
body weight of the t to be treated, and the preference and experience of the attending
physician. The clinically-established dosages of the rTILs may also be used if appropriate. The
amounts of the pharmaceutical compositions administered using the methods , such as the
dosages of rTILs, 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 treating physician.
The rTILs and eTILs provided in the pharmaceutical compositions of the invention are
effective over a wide dosage range. The exact dosage will depend upon the route of
stration, the form in which the compound is administered, the gender and age of the
subject to be treated, the body weight of the subject to be treated, and the preference and
experience of the attending physician. The clinically-established dosages of the rTILs and eTILs
may also be used if appropriate. The amounts of the pharmaceutical compositions stered
using the methods herein, such as the s of rTILs and eTILs, 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
treating physician.
In some embodiments, rTILs may be administered in a single dose. Such
administration may be by injection, e.g., intravenous injection. In some embodiments, rTILs
may be administered in le 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 rTILs may continue as long as
necessary.
In some embodiments, rTILs and eTILs may be administered in a single dose. Such
administration may be by ion, e.g., intravenous injection. In some ments, rTILs
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 rTILs and eTILs may continue
as lOIlg as necessary.
In some embodiments, an effective dosage of rTILs is about l><106, 2><106, 3><106,
,5><106,6><106,7><106,8><106,9><106,1><107,2><107,3><107,4><107,5><107,6><107,7><107,
8><107,9><107,1><108,2><108,3><108,4><108,5><108,6><108,7><108,8><108,9><108,1><109,2><109,
3><1O9,4><109,5><1O9,6><1O9,7><109,8><109,9><109,1><1010,2><1010,3><1010,4><1010,5><1010,
6><1010,7><1010,8><1010,9><1010,1><1011,2><1011,3><1011,4><1011,5><10H,6><1011,7><1011,8X10”,
9X10”,1><1012,2><1012,3><1012,4><1012,5><1012,6><1012,7><1012,8><1012,9><1012,1><1013,2><1013,
,4x1013, 3,6><1013,7><1013, 8><1013, and 9x10”. In some embodiments, an effective
dosage ofrTILs is in the range of 1x106 to 5x106, 5x106 to 1x107, 1x107 to 5x107, 5x107 to
1x10", 1x108 to 5x10", 5x108 to 1x109, 1x109 to 5x109, 5x109 to 1x10”, 1x1010 to 5x10“),
5x1010 to 1x10“, 5x1011 to 1x10”, 1x1012 to 5x10”, and 5x1012 to 1x10”.
In some embodiments, an effective dosage of rTILs and eTILs is about l>< 106, 2>< 106,
3><106,4><106,5><106,6><106,7><106,8><106,9><106,1><107,2><107,3><107,4><107,5><107,6><107,
7><107,8><107,9><107,1><108,2><108,3><108,4><108,5><108,6><108,7><108,8><108,9><108,1><109,
2><109,3><109,4><109,5><109,6><109,7><109,8><109,9><109,1><1010,2><1010,3><1010,4><1010,
><1010,6><1010,7><1010,8><1010,9><1010,1><1011,2><1011,3><1011,4><10H,5><1OH,6><1011,7><1OH,
8x10“,9x10“, 1x1012,2x1012,3x1012,4x1012, 5><1012,6><1012,7><1012, 8><1012,9><1012, 1x10”,
2x1013,3x1013,4x1013, 5><1013,6><1013,7><1013, 8><1013,and 9x10”. In some embodiments, an
effective dosage of rTILs and eTILs is in the range of l>< 106 to 5><106, 5>< 106 to l>< 107, l>< 107 to
5x107, 5x107 to 1x10", 1x108 to 5x10", 5x108 to 1x109, 1x109 to 5x109, 5x109 to 1x10”, 1x1010
to 5x10“), 5x1010 to 1x10“, 5x1011 to 1x10”, 1x1012 to 5x10”, and 5x1012 to 1x10”.
In some embodiments, an ive dosage of rTlLs 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 rTlLs and eTILs 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 rTlLs 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,
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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.
In some embodiments, an ive dosage of rTlLs and eTILs 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 rTlLs and/or eTILs may be administered in either single or
multiple doses by any of the accepted modes of administration of agents having similar utilities,
including by infusion into the bloodstream, infusion into a tumor, intra-arterial ion,
intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, topically, by
intranasal administration, by transplantation, or by inhalation.
EXAMPLES
The embodiments encompassed herein are now described with reference to the
following examples. These examples are provided for the purpose of illustration only and the
disclosure encompassed herein should in no way be construed as being limited to these
examples, but rather should be construed to encompass any and all variations which become
t as a result of the teachings provided herein.
Example 1 — Expansion of rTILs from Tumor Digests
Tumor ts were digested according to the following exemplary procedure. This
procedure describes the digestion of a fresh human tumor sample into a , single-cell
suspension, to obtain and isolate tumor-infiltrating lymphocytes, and may use DNase-
Collagenase-Hyaluronidase (DCH) methods (as described herein) or MACS human tumor
WO 94167
dissociation kit (TDK) (Miltenyi Biotech, Inc., San Diego, CA, USA) digestion protocols for the
dissociation of human tumors.
Preparation of the CMl + IL-2 working culture medium is as follows. Place 500 mL
RPMI 1640, 200 mM L-glutamine, and 100 mL human AB serum in a water bath at 37°C to
equilibrate for at least 30 s. Transfer the contents of this mixture from the water bath to a
biosafety cabinet along with 1000X B-ME stock and 50 mg/mL gentamicin stock solution from
the refrigerator. Remove 50 mL from the RPMI 1640, add: 50 mL human AB serum, 5mL 200
mM L-glutamine, 500 uL 1000X B-ME, and 500 uL 50 mg/mL gentamicin. To complete
medium 1, add 500 uL of 6000 U/mL reconstituted human rh1L-2 (CellGeniX, Inc., outh,
NH, USA).
The 10>< DCH stock solution is prepared using the following procedure, which is
depicted in First, the volume ed to reconstitute each enzyme to obtain the desired
working solution concentrations is calculated. For example, titute 150,000 U
national units) of deoxyribonuclease in 15 mL to obtain a 10,000 U/mL g solution.
Aliquot leftover working solution. Reconstitute the lyophilized enzymes in an amount of sterile
Hanks’ balanced salt solution (HBSS, Sigma H6648, Sigma-Aldrich Co., St. Louis, MO, USA,
or lent) previously calculated above at room temperature. Remove any residual powder
from the sides of the bottles and from the protective foil. Pipette up and down several times and
swirl to ensure complete reconstitution. Add 100,000 U of DNase (deoxyribonuclease I from
bovine pancreas, Sigma D5025 or equivalent), 1 g of collagenase (Sigma C5138 from
Closlridium histolylicum or equivalent), and 100 mg of hyaluronidase (Type V from sheep testes,
Sigma H6254 or lent) to a final volume of 100 mL sterile HBSS to obtain a 10>< triple
enzyme ion stock solution for human tumors. Aliquot the remaining enzyme working
solutions into 10,000 U/mL DNase, 10 mg/mL collagenase and 1 mg/mL hyaluronidase. The
>< DCH stock solution at 100 mL final volume has the following concentrations: DNase I 1000
U/mL, collagenase 10 mg/mL, and hyaluronidase 1 mg/mL. The 10>< DCH stock solution is
d to 1>< DCH in HBSS for tumor digestion.
Concurrently, for comparison of the DCH digest with MACS TDK, prepare the
reagents included in the MACS TDK to manufacturer specifications, if desired. Thaw aliquots
that have been stored at -20 0C at room temperature.
The tumor may be prepared for digestion as follows. Remove the tumor from its
primary and secondary packaging and weigh the vial, record the mass, and transfer to a biosafety
cabinet. Cut the tumor into fragments, or morcellate the tumor. Several fragments are selected
to be used in the digestion protocol, and additional fragments are retained for histology and DNA
extractions if desired.
An exemplary DCH-based tumor digestion procedure is depicted in and includes
the following steps. The lO>< DCH stock solution must be diluted to a l>< working tration
for digestions. Calculate the total volume needed for the digestion of the tumor, which is about 5
mL of solution per cm2 of tumor. Dilute the DCH working solution to l>< by adding 1 part DCH
to 9 parts HBSS. Transfer tumor fragments to a 50 mL Flacon conical tube in the volume of
HBSS calculated above. Add the amount of lO>< DCH ated above, cap the tube, and
optionally seal. Transfer to the MACS tube rotator (Miltenyi h, Inc., San Diego, CA,
USA) in a 37 oC, 5% C02 humidified incubator on constant rotation for l to 2 hours.
atively, the tumor fragments can be digested at room temperature overnight, also with
constant rotation. Attach a 0.70 um strainer to sterile Falcon l tube. Obtain the digestion
from the incubator and using a pipette, and add all contents of the digestion to the strainer. Use
the butt of a sterile syringe r to push any solid h the strainer. The tube is capped
and contains DCH-digested rTILs. The cells may be washed to remove the digest cocktail,
counted, and resuspended in media for REP expansion as described ere herein.
If a pre-REP step is desired to provide eTlLs for comparison with rTILs (as in the
following Examples), seed G—REX flasks for pre-REP using the DCH-digested rTILs. Label the
necessary number of G—REX lO flasks and add the digest. Add CMl + IL-2 to obtain 40 mL
final volume. Place the flasks in a 5% C02 incubator at 37 0C with humidity. Cell counting and
viability may be performed using a Nexcelom eter K2 using 40 uL of sample to 40 uL of
acrydine orange and propidium iodide dual staining solution (AOPI) on, and count in
ate for each digest or condition, diluting as needed. Mix samples well to avoid clumping,
and pipette AOPI promptly before running each sample to ensure viability isn’t obfuscated by
the cytotoxic effect of propidium iodide.
The DCH procedure described above was found to be surprisingly superior to the
MACS TDK enzymatic digest mixture and procedure for several reasons. Three independent
experiments using the MACS TDK mixture were performed. The first experiment was
performed using a melanoma tumor, where a significant downregulation of the CD4+/CD8+
population in rTILs was observed by flow cytometry using the MACS system. Such an effect
was not observed in DCH-digested rTILs, indicating that the MACS digest procedure may
adversely affect the expression of surface s. In a second experiment, an estrogen
receptor-positive (ER+)/progesterone receptor-positive (PR+) breast tumor was used, and the
MACS digest let to the appearance of debris in the 24-well G—REX plate, whereas the DCH
digest did led to clear material, ting poor digestion for the MACS enzyme cocktail.
Finally, a second digest of a different ER+/PR+ breast tumor using the MACS TDK enzymatic
digest mixture and procedure led to both poor yield and viability of rTILs.
Example 2 — Phenotyp_ic Characterization of rTILs from Tumor Digests
] During the pre-REP, tumor-resident TILs emigrate as eTILs and erate. The length
of the pre-REP used to prepare eTILs for comparison with rTILs may vary between 11-21 days,
ing on cell growth. Residual tumor fragments (remnants) are normally discarded and the
expanded eTIL are subjected to a REP with irradiated PBMC feeders, anti-CD3 and IL-2.
Viable TILs remaining in the tumor ts (rTILs) ing the P were investigated
after digestion according to Example 1 as described above to assess their function and phenotype
in comparison to eTILs.
Cell populations from the tumor remnants and P suspension (1'.e., the ed
cell population) in melanoma, head and neck, breast, renal, pancreatic, lung and colorectal
tumors (n=l7) were evaluated and compared. Interestingly, rTILs are consistently phenotypically
distinct from eTILs, as determined herein and shown by differential expression of s
markers ing LAG3, TIM-3, PD-l, CD69, CD45RO, CD27, CD56, CD57 and HLA-DR. A
REP of the tumor remnant and pre-REP populations resulted in comparable expansion, but
similar to the pre-REP results, the phenotypic signature varied between the two populations with
respect to LAG3, THVI-3, HLA-DR and CD28.
The rTIL and eTIL obtained from melanoma, breast, renal, pancreatic, lung and
colorectal tumors (11 = 9) were evaluated and compared. Tumor rTIL are consistently
phenotypically distinct from eTIL, as determined by differential expression of s markers
(Table 3 and .
TABLE 3. Summary of phenotypic characterization results for nine tumors.
Marker - CD56
Expression J“ + + + + + +
+ +
eTIL 507/ 2832/ 36.95/ 1320/ 1498/ 1163/ 18.76/ 5. 6/15
144 1756 47 1543 3751 5036 19.6
rTIL 209/ 877/ 42.88/ 3437/ 1034/ 458.3/ 1.027
106 742 223.4 1167 2795 9.156/
*P-Values 0.05/ 0.05/ 0. 38/ 0. 11/ 0. 55/ 0.05/ 0.05/
(CD8/ 0.21 0.01 0.89 0.001 0.01 0.11 0.06
>“P-values represent the difference between rTlL and eTlL using students unpaired T test.
The fundamental differences in rTILs as compared to eTILs were increased CD69
expression (7-fold median fluorescence intensity (MFI) in CD4+) (p<.OOOl), diminished LAG3
expression d MFI in CD8+ T cells) (p < 0.05) and TIM3 sion (3- and 2-fold MFI in
CD8+ and CD4+ T cells, respectively) (p < 0.05/0.0l), diminished CD154 expression (3-fold
MFI in CD4+ T cells) (p < 0.01), and diminished CD56 expression (5%) (p < 0.05).
Surprisingly, a REP of rTILs and eTILs resulted in comparable expansion, similar to the pre-
REP results, as described in Example 4. The phenotypic ure of rTILs was sustained after
REP with fidelity with of expression to the dual levels of LAG3, TIM3, and CD28, as
described in Example 4. Furthermore, since CD57 is a receptor associated with terminal
differentiation, the results suggest that rTILs are less terminally differentiated than eTILs (i.e.,
less likely to die).
The results reported in and Table 3 show that eTIL and rTIL t ct yet
consistent differences in phenotypic expression in various tumor histologies. Most notably, there
was a reduction in the expression of the so called “exhaustion markers” (LAG3 and TIM3) in the
rTIL. Interestingly, PD-l was similarly expressed in the eTIL and rTIL. Additionally, there was
an enhancement in CD69 expression in the rTIL, compared to the eTIL, yet KLRGl was similar
between the two tions (data not shown). This provides r evidence that the rTlL do
not appear to be terminally differentiated, but phenotypically resemble tissue-resident effector
memory T cells.
Collectively, these results have identified significant ences in the y of cell
populations that remain in the tumor or expand and progress out of the tumor, and the signals
associated with emigration and retention.
Example 3 — onal Characterization of rTILs from Tumor Digests
T cell dysfunction is directly associated with a loss of mitochondrial function.
Sharping, el al., Immunity 2016, 45, 374-88. Moreover, the reprogramming of T cells to favor
mitochondrial esis can increase intratumoral T cell persistence and function. Therefore,
more lically active T cells are pivotal in mounting an efficient immune response to tumor.
In an effort to assess the eTIL and rTIL onally, eTIL and rTIL were compared in terms of
metabolic capacity via Mitotracker and 2-(N-(7-nitrobenzoxa-l,3-diazolyl)amino)
lucose (2-NBDG). 2-NBDG may be used to measure e uptake, but does not
specify the primary metabolic process, 1'.e., full ion in the mitochondria or only glycolysis
and generation of lactate. acker dye (ThermoFisher Scientific, Inc., Waltham, MA, USA)
may be used to measure mitochondrial mass. Comparison of the eTIL and rTIL by this approach
demonstrated an enhancement in glucose update in the rTIL, as shown in This result is
surprising because rTILs, being directly liberated from the tumor, are expected to be more
glycolytic, however, when the mitochondrial mass the rTILs was assessed, they exhibited a
slightly enhanced level of Mitotracker compared to the eTIL. These results demonstrate that
rTIL were more metabolically active than eTIL, when expected to be less active, and suggest that
the rTIL may have a greater capacity to amount an immune response to tumor than eTIL.
To further assess their functional capabilities, rTILs were stimulated overnight with
brefeldin A and beads coated with anti-CD3, anti-CD28, and anti-CD137 antibodies
(DYNABEADS, catalog no. lll62D, cially available from Fisher Scientific,
Inc., Waltham, MA, USA) and IFN—y was measured. Cells were harvested and d
intracellularly for IFN-y following permeabilization and assessed by flow cytometry. The results
are shown in
Cells were also stimulated with phorbol 12-myristate l3-acetate (PMA) and ionomycin
to evaluate the lity of TILs to produce cytokine. The results are also shown in
Granzyme B, TNF-oc and IL-l7A levels were also assessed. There was negligible IL-l7A, and
no differences in TNFoc or granzyme B between rTILs and eTILs (data not shown).
Surprisingly, a slightly elevated level of IFN—y in the CD4+ subset (but not in CD8+ T cells) was
observed (11 = 3) in the anti-CD3/anti-CD28/anti-CDl37 bead and PMA/ ionomycin conditions
in rTILs compared to eTILs. This data suggests that rTILs are functionally competent cells, and
show evidence of greater functional competence that eTILs.
Example 4 — Comparison of REP of rTILs and eTILs from Tumor Digests
eTILs and rTILs were subjected to a rapid expansion protocol (REP) with irradiated
PBMC feeders, D3 antibody (OKT3), and IL-2 for 14 days. Viability and cells counts
were assessed in duplicate in 3 ndent tumors (11 = 3). ypic expression was assessed
by flow try. Successful initiation in mini-REP experiments was observed for both rTILs
and eTILs. The REP performance of the TILs with anti-CD3 antibody and feeders was similar
(), although the rTILs surprisingly exhibited a slightly enhanced number of cells (p <
0.08), compared to the eTIL. As observed prior to REP (see Example 2), the eTILs and rTILs
obtained post-REP were phenotypically distinct. Many of the phenotypic differences observed
in the pre-REP were preserved during the REP, such as a reduction in LAG3 and TIM3
expression in the rTIL ().
Additional properties of eTILs and rTILs may be compared based on the results of (1)
deep TCR sequencing, (2) co-culture proliferation (rTIL/eTIL co-culture with ne mixtures)
and additional functional assays, (3) assays for transcriptional ng (e.g., using a
NanoString Technologies NCOUNTER system). TCR cing may assess the ity
and/or diversity of the TCR repertoire, including Vb repertoire. Telomere length may also be
assessed to compare rTILs to eTILs.
Example 5 — Treatment of Human Disease with rTILs and ations of rTILs and eTILs
] The rTILs of the invention may be used in the treatment of cancers as described herein.
An overall process flow diagram for the expansion of rTILs from a patient tumor and treatment
of a patient is depicted in The process allows for tailoring of the rTIL to eTIL ratio in the
TIL product infused to the patient as shown. The ratio of rTIL to eTIL may be selected by way
of an affinity assay or other cell sorting assay known by persons having ordinary skill in the art
based on the differential expression of CD69 and/or T-cell tive markers in rTILs and
eTILs.
In a timeline showing an exemplary process of obtaining rTILs from a patient
tumor, expanding the rTILs from tumor remnants after pre-REP using a REP stage, performing
lymphodepletion, and infusion of rTILs into a patient is shown in conjunction with a parallel
eTIL process.
Example 6 — Study to Assess the Vlfi Repertoire in eTIL and rTIL
The eTIL and rTIL were assessed for differences in the VB T cell receptor repertoire,
with respect to diversity and frequency.
In the study, 6 pre-REP eTIL/rTIL pairs were ted from the following histologies:
n cancer, renal cancer (n=2), and breast cancer (TNBC n=2, ER+PR+ n=l). The cell
pellets were shipped on dry ice to iRepertoire (Huntsville, AL, USA) for RNA tion and VB
cing.
The results of this study are illustrated in FIGS. 9-10 and 14-16. In particular, FIGS. 9
and 10 illustrate the diversity score and the % of shared CDR3 s, respectively. rmore, three
clonotype graphs g the top shared 50 CDR3s are shown in FIGS. 14, 15, and 16 for
ovarian carcinoma, renal carcinoma, and triple negative breast carcinoma, respectively.
Surprisingly, the diversity of the TCRvB repertoire is greater in the rTIL than in the
eTIL (. Approximately 30-50% of the total CDR3 in the eTIL and rTIL are shared (), trating that a large percentage of the total CDR3’s are differentially expressed in the
two populations. However, of the shared CDR3s the top 50 clones were mostly shared between
the two populations, suggesting that eTIL and rTIL have clones with similar antigen city
(see FIGS. 14-16). Moreover, the frequency of the top 50 clones varied, suggesting again that
the eTIL and rTIL are surprisingly distinct T cell populations.
Example 7 — Study of Co-Culture Proliferation Assays
The eTIL and rTIL were ed determine whether the rTIL can alter the proliferation
status of the eTIL, upon co-culture (or vice versa).
In the study, 5 pre-REP eTIL/rTIL pairs were harvested from the following histologies:
renal cancer, triple-negative breast cancer (TNBC), melanoma, lung , and colorectal
. The rTIL were isolated from the tumor remnants by a 60-min enzymatic digestion at
37°C. eTIL were stained with Cell Trace Yellow and rTIL with Cell Trace Red to independently
track the two distinct tions. 166 of 6TIL, 565 6TIL + 565 rTIL, and 166 rTIL were cultured
for 4 days at 37°C with IL-2 +/- and OKT3 (anti-CD3 antibody) and ed for proliferation by
flow cytometry.
The results of this study are illustrated in . In , the 6TIL from either the
CD4+ or CD8+ population in all five tumors trated an ement in the erative
capacity upon co-culture with rTIL with anti-CD3 antibody as demonstrated by a shift (or dye
on) in the Cell Trace dye, when compared to 6TIL alone. The red represents the 6TIL and
the blue represents the 6TIL when co-cultured with the rTIL.
Example 8 — Study of Co-Culture Proliferation Assays
] The 6TIL and rTIL were assessed identify similarities and/or differences in the gene
expression profile of rTIL and 6TIL.
In the study, Nanostring’s nCounter technology was utilized, which employs a color-
coded barcode multiplexed to mRNA to deliver a digital readout of gene expression. Purified
RNA (RNeasy, Qiagen) from six matched 6TIL and rTIL samples were hybridized with an
nCounter Immunology V2 panel codeset for 16 hours on a thermocycler. Codesets consist of a
mixture of capture and er probes that are multiplexed with the target RNA through 22bp
interactions during thermocycling. Samples were loaded into a 12-well SPRINT cartridge and
ran on an nCounter SPRINT device. Count data are exported in a custom RCC format and
matched to an RLF file which matches gene names to probe IDs. Normalization and analysis
were done on nSolver 3.0 (NanoString Technologies, Inc.).
The results of this study are illustrated in FIGS. 12 and 13. As shown in FIGS. 12 and
13, the gene expression profile is significantly different when ing the 6TIL and rTIL (see
the heat map in ). There are several genes that are significantly upregulated or
downregulated in the rTIL compared to the 6TIL ().
Claims (18)
1. Use of remnant tumor rating lymphocytes (rTILs) in the manufacture of a medicament for treating a cancer in a patient in need of such treatment, wherein the rTILs are obtained by the method comprising: obtaining a therapeutically effective amount of remnant tumor infiltrating lymphocytes (rTILs), by enzymatically digesting tumor remnants separated from emergent tumor infiltrating lymphocytes ) using a digest e comprising a deoxyribonuclease, a collagenase, a hyaluronidase, or a combination thereof, and ing the rTILs with a second cell culture medium comprising cell culture media, irradiated feeder cells, OKT-3 antibody, and interleukin 2 (IL-2), n the tumor remnants and the eTILs are derived from tumor tissue that was previously obtained from the patient, fragmented and treated with a first cell culture medium comprising IL-2, and wherein treatment comprises administration of a therapeutically effective amount of the rTILs to the patient.
2. The use of claim 1, wherein the tumor tissue is selected from the group consisting of melanoma tumor tissue, head and neck tumor tissue, breast tumor tissue, renal tumor tissue, pancreatic tumor tissue, glioblastoma tumor tissue, lung tumor tissue, colorectal tumor tissue, sarcoma tumor tissue, triple negative breast tumor tissue, cervical tumor tissue, ovarian tumor tissue, and acute myeloid leukemia bone marrow or tumor .
3. The use of claim 1, n the irradiated feeder cells comprise irradiated allogeneic peripheral blood mononuclear cells.
4. The use of claim 1, wherein IL-2 is present in the second cell culture medium at an l concentration of about 3000 IU/mL and OKT-3 antibody is present in the second cell culture medium at an initial concentration of about 30 ng/mL.
5. The use of claim 1, n CD56+ sion in the rTILs is reduced by at least 3-fold relative to CD56+ expression in the eTILs.
6. The use of claim 1, wherein CD69+ expression in the rTILs is increased by at least 2-fold relative to CD69+ expression in the eTILs.
7. The use of claim 1, wherein the first cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and ations thereof.
8. The use of claim 1, wherein the second cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.
9. The use of claim 1, wherein the rTILs are cryopreserved prior to obtaining the rTILs.
10. The use of claim 9, further comprising the step of thawing the cryopreserved rTILs prior to ing the rTILs.
11. The use of claim 1, further comprising the steps of: administration of a eloablative lymphodepletion regimen to the patient prior to administration of the therapeutically ive amount of rTILs to the patient; administration of a high-dose IL-2 regimen to the patient starting on the day after administration of the eutically effective amount of rTILs to the patient.
12. The use of claim 11, wherein a therapeutically effective amount of eTILs is simultaneously obtained in a mixture with the therapeutically effective amount of rTILs.
13. The use of claim 11, wherein the non-myeloablative lymphodepletion regimen comprises the steps of stration of cyclophosphamide to the patient at a dose of 60 mg/m2/day for two days ed by administration of fludarabine to the patient at a dose of 25 mg/m2/day for five days.
14. The use of claim 11, wherein the high-dose IL–2 regimen comprises 600,000 or 720,000 IU / kg of aldesleukin, or a biosimilar or variant thereof, stered to the patient as a 15-minute bolus intravenous infusion every eight hours until tolerance.
15. The use of claim 1, wherein the cancer is selected from the group ting of melanoma, double–refractory melanoma, uveal melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, renal cell carcinoma, acute myeloid leukemia, colorectal cancer, and sarcoma.
16. The use of claim 15, wherein the cancer is breast cancer, and wherein the breast cancer is selected from the group consisting of triple negative breast cancer , estrogen receptor-positive breast cancer, progesterone receptor-positive breast cancer, and estrogen receptor-positive/progesterone receptor-positive breast cancer.
17. The use of claim 15, n the cancer is lung cancer, and the lung cancer is selected from the group ting of non-small cell lung cancer and small cell lung cancer.
18. The use of claim 1, wherein the therapeutically effective amount of rTILs is provided in a pharmaceutical composition, wherein the concentration of the rTILs in the pharmaceutical composition is greater than 50 %. auuuuuuuu'uuu, I,I,II,I,II,”Innnnnnnnn,”\\\\\\\\\\\\\\\\\ \kn“ \\\\\\\\\\\\\\\\\\\\\\\\\\\\\ ,u'uuuuuuuuuuu, . ,u'uuuuuuuuuuu, Ill/II/ ,III,I,II,I,II,”Innnnnnnnn,’ mm““WNW“ \\\\\\\\\\\\\\\\\\\\\\\\\v \xxxxxxxx\xxxxxxxx-nx-nxxxx-nx-m‘xx xxxx{\xxxxxxxx\xxxxxxxxxxxxxxxxxxxkxx-n uuuuuuuuu'uuu, : 3 ~ \ . \ . \ . \ . \ . \ x: \ a“: S . \ . \ . \ . \ . \ . \ . \ . \ ksss \\\\\\\\\\\\\\\\\\\\\\ “\ \\\\\\\\\\\\\\\\\\\\\\\\\‘ L\\\\\\ sss\s\s\s\ss\s\s\\\\\\\\\\\\\\\\\\\\\\\\ \s‘s‘é rnurnrnnnnnnnn rIrrrrrrrrrrrrrrrrrrrrrrr munuuu‘“ ““‘u‘u‘u‘a ‘xxx xxxxxxxxxxxxxxxxxxxxxx xxx , ‘ xxx \xxxxxxxx \\\\\\\\\\\\ w. x\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\;M 4Idummy, 5/aaaaaaa5 .2 l.w "4. runny” o \ “ ‘ xxxv\\\\\\\\:\{{\““\\“‘\“ zunnannannunuu~« ««««««««««« 55‘ {3055 m xQt: “£3 u Q a Q oN E: , £3 ‘ \ w i “‘ ,N E E: % “ x m__v c W; g .23 E: . e o‘ . I x E: 33m 3 {cm *} ‘T. i mA3 (xcaafl. sR mw v‘ m . w! Q m§vuv .§xx $5.,“ \\\\\\\ mw i n i :a\v 3:“: \\\\\\\ v .\\» ‘Q m \“ GS me a x9. \\\\\\\ «Mia» “was." t i o . xx a .,\xxxxxxm x \\ 3 {cmfi m .x 3 . a A.‘m "32.3,. m . 3 Hu u \\\\\\\\\\ a ..\.w, a ~.mw.~.m&m \\\\\\\\\\ I a §§I§§§Ra \\\\\\\\\\ I fix» m ¢ p mm, , a P 333‘! {634+} .{ Ca, M.“ C w“\n h. i # ml t wy.“ ..u « v .. «335. u. ‘ ‘ $3 h~ ,~‘‘m‘ m AN. I! 3‘“ 6%1 A mg:“a x, §»« 33$ a,. h \\~ a , \ gig c {153:6mm a Bs8 .35c a Maxxxxxxx*xxxxxxx‘xxxxxxxaxxxxxxx ‘iiiuiufliiuiiioiuiiii Q «uR my@ m “$3“ 3 fan. .Mw 1 mg_. .. xw Svax . sw § w Lm . . «.3» D g I mfl“ a whmmhmh x gnnnnnmmh xxmnmw a.»m ‘9‘ c as “s {8 am ”.1 , {éC,a 8 s, . wK as. t nR a R a n 3 FE». x u‘ .9 we“, .. .323 «“39 wa m. o 3 \\\\\\\\\\\\\\\\\\\\\\\\\\\\\ MakmsmmmSWNW(“mean r‘tifi amam; wuss swam 8:13:33“ ‘f‘tast: we»: «Mm 913.86%. a mam: asset aw smsszimg simzt‘snzsmna me dam‘iy ‘Ezwnsm steam J ‘9 A 1 it 3 mm: 3w: A g ‘ a: mm j 293 - 9 éM. is: 777777777777777777777711‘ ,,,,,,,, ‘ z7>>>>>>>>>>>>>>>>>>>>>zz’ iwis paaaaaaaaay ;aaaaaaaaaaaaaaaaaaaaaav 444444444444444444444444’ ML (3‘?! L m3. N35» *5? 3” a, m*5» 3‘9““ ‘ seam: 1 may E x.
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