Note: Descriptions are shown in the official language in which they were submitted.
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IMMUNE CHECKPOINT INHIBITORS AND CYTOTOXIC T CELLS FOR THE
TREATMENT OF CANCER
RELATED APPLICATIONS
This application claims the benefit of pfiority to U.S. Provisional Patent
Application
serial number 62/341,402, filed May 25, 2016, hereby incorporated by reference
in its
entirety.
BACKGROUND
Nasophatyngeal carcinoma is a type of head and neck cancer that begins in the
nasopharynx. Recently, there is emerging evidence to show that exposure to
Epstein Barr
virus (EBV) can contribute to the pathology of nasopharyngeal carcinoma.
Epstein-Barr
virus associated nasopharyngeal carcinoma (NPC) is endemic in regions of South-
East Asia,
with incidence as high as 25-50 cases per 100,000 people in southern China.
While current
standard therapy is often curative for a subset with stage I or II disease, a
high proportion of
patients relapse and many patients are still initially diagnosed with advanced
stage III or IV
disease, where overall 5-year survival is significantly reduced. Thus, there
exists a need to
develop improved therapies for NPC.
SUMMARY
In certain aspects, provided herein are methods of treating cancer (e.g., NPC)
in a
subject by administering (e.g., conjointly) an immune checkpoint inhibitor and
a
composition comprising cytotoxic T cells (C'TLs) expressing a T cell receptor
specific for a
cancer-associated peptide presented on a class I ME-IC.
In some embodiments, the immune checkpoint inhibitor is a protein or
polypeptide
(e.g., an antibody or antigen-binding fragment thereof) that binds to an
immune checkpoint
protein, such as CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA, KTR, LAG-
3,
TIM-3, IDO, TDO, and VISTA. In some embodiments, the immune checkpoint protein
is
CTLA4, PD-1, PD-L1, TIM-3 or LAG-3. In some embodiments, the immune checkpoint
inhibitor binds to the immune checkpoint protein such that it inhibits its
activity. In some
embodiments, the immune checkpoint inhibitor inhibits the interaction between
the immune
checkpoint protein and an associated receptor/ligand. In some embodiments, the
immune
checkpoint inhibitor is nivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-
514, STI-
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A 1110, TSR-042, RG-7446, BMS-936559, BMS-936558, MK-3475, CT 011, MPDL3280A,
MEDI-4736, MSB-0020718C, AUR-012 and STI-A1010.
In some embodiments, the cytotoxic T cells in the composition can be specific
for
any cancer-associated peptide presented on a class I MI-IC (e.g, a cancer
associated peptide
expressed by a tumor and/or cancer cells in the subject). In some embodiments,
the cancer-
associated peptide is a viral peptide. In some embodiments (e.g, when the
subject has EBV-
associated NPC or another EBV-associated cancer), the viral peptide is an EBV
peptide. In
some embodiments, the EBV peptide comprises a LMP1 peptide, a LMP2A peptide,
and/or
an EBNA1 peptide.
In some embodiments, the CTLs are allogeneic to the subject (e.g, obtained
from a
ecll bank). In some embodiments, the CTLs are autologous to the subject. The
CTLs and the
immune checkpoint inhibitor may be co-administered or administered
sequentially. In some
embodiments, the method further comprises administering to the subject a
chemotherapeutic
agent.
In some aspects, provided herein are methods of treating cancer (e.g.
nasopharyngeal
carcinoma) in a subject, comprising generating peptide-specific CTLs by
incubating a
sample comprising CTLs and antigen-presenting cells (APCs) that present a CMV
peptide,
thereby inducing proliferation peptide-specific CTLs in the sample, and
administering the
peptide-specific CTLs to the subject in combination with an immune checkpoint
inhibitor
described herein. In some embodiments, the APCs are made to present the EBV
peptide by
incubating them with a nucleic acid construct (e.g.. AdEl-LMPpoly) encoding
for the EBV
peptide, thereby inducing the APCs to present the EBV peptide. In some
embodiments, the
APCs may be B cells, antigen-presenting T cells, dendritic cells, or
artificial antigen-
presenting cells (e.g., a cell line expressing CD80, CD83, 41BB-L and/or CD86,
such as
aK562 cells). In some embodiments, the EBV peptide comprises a LMP1 peptide or
a
fragment thereof, a LMP2A peptide or fragment thereof, and/or an EBNA 1
peptide or
fragment thereof. In some embodiments, the EBV peptide comprises a sequence
listed in
Table 1. In some embodiments, one or more immune checkpoint inhibitors are
administered.
The immune checkpoint inhibitors may be administered by any technique known in
the art.
In some embodiments, the immune checkpoint inhibitor is administered
intratumorally. In
some embodiments, the sample comprises one or more cytokines or peripheral
blood
mononuclear cells (PBMCs).
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 has two panels showing the expression of immune checkpoint molecules
(i.e., LAG-3, TIM-3, CTLA4, or PD-1). Panel A depicts percentage of HLA-
multimer
positive CD8-positive lymphocytes that express PD-1, 1IM-3, LAG-3 and CTLA-4.
Panel B
.. depicts the percentage of PD-1 positive, TIM-3 positive, LAG-3 positive and
C'TLA-4
positive lymphocytes in the CU inununotherapy administered in NPC patients
with
no/minimal residual disease (N/MRD) and active-recurrent/metastatic disease
(ARMD) who
either showed stable disease (SD) or progressive disease (PD) following
adoptive T cell
therapy.
DETAILED DESCRIPTION
General
In certain aspects, provided herein are methods to treat cancer in a subject
using a
combination therapy that includes administration, e.g., conjoint
administration, of one or
more immune checkpoint inhibitors combined with cytotoxic T cell (cm)
inununotherapy.
In some embodiments, the cancer is EBV-associated NPC and the CTLs
administered to the
subject express a T cell receptor that has binding specificity for a peptide
expressing and
EBV epitope presented on a class T MHC.
Definitions
For convenience, certain terms employed in the specification, examples, and
appended claims are collected here.
The articles "a" and "an" are used herein to refer to one or to more than one
(i.e.. to at least
one) of the grammatical object of the article. By way of example, "an element"
means one
element or more than one element.
As used herein, the term "administering" means providing a pharmaceutical
agent or
composition to a subject, and includes, but is not limited to, administering
by a medical
professional and self-administering. Such an agent can contain, for example,
peptide
described herein, an antigen presenting cell provided herein and/or a cn,
provided herein.
The term "biological sample," "tissue sample," or simply "sample" each refers
to a
collection of cells obtained from a tissue of a subject. The source of the
tissue sample may be
solid tissue, as from a fresh, frozen and/or preserved organ, tissue sample,
biopsy, or
aspirate; blood or any blood constituents, serum, blood; bodily fluids such as
cerebral spinal
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fluid, amniotic fluid, peritoneal fluid or interstitial fluid, urine, saliva,
stool, tears; or cells
from any time in gestation or development of the subject.
The term "binding" or "interacting" refers to an association, which may be a
stable
association, between two molecules, e.g., between a T cell receptor (TCR) and
a
peptide/ME-IC, due to, for example, electrostatic, hydrophobic, ionic and/or
hydrogen-bond
interactions under physiological conditions.
As used herein, the term "cancer" includes, but is not limited to, solid
tumors and
blood borne tumors. The term cancer includes diseases of the skin, tissues,
organs, bone,
cartilage, blood and vessels. The term "cancer" further encompasses primary
and metastatic
lo cancers.
The term "epitope" means a protein determinant capable of specific binding to
an
antibody. Epitopes usually consist of chemically active surface groupings of
molecules such
as amino acids or sugar side chains. Certain epitopes can be defined by a
particular sequence
of amino acids to which a T cell receptor or antibody is capable of binding.
As used herein, the phrase "pharmaceutically acceptable" refers to those
agents, compounds,
materials, compositions, and/or dosage forms which are, within the scope of
sound medical
judgment, suitable for use in contact with the tissues of human beings and
animals without
excessive toxicity, irritation, allergic response, or other problem or
complication,
commensurate with a reasonable benefit/risk ratio.
As used herein, the phrase "pharmaceutically-acceptable carrier" means a
pharmaceutically-acceptable material, composition or vehicle, such as a liquid
or solid filler,
diluent, excipient, or solvent encapsulating material, involved in carrying or
transporting an
agent from one organ, or portion of the body, to another organ, or portion of
the body. Each
carrier must be "acceptable" in the sense of being compatible with the other
ingredients of
the formulation and not injurious to the patient. Some examples of materials
which can serve
as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose,
glucose and
sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose,
and its derivatives,
such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
(4) powdered
tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa
butter and suppository
waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame
oil, olive oil, corn oil
and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as
glycerin,
sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate
and ethyl laurate;
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(13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum
hydroxide;
(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)
Ringer's solution; (19)
ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates
and/or
polyanhydrides; and (22) other non-toxic compatible substances employed in
pharmaceutical
formulations.
As used herein, a therapeutic that "prevents" a condition refers to a compound
that,
when administered to a statistical sample prior to the onset of the disorder
or condition,
reduces the occurrence of the disorder or condition in the treated sample
relative to an
untreated control sample, or delays the onset or reduces the severity of one
or more
symptoms of the disorder or condition relative to the untreated control
sample.
As used herein, the tenn "subject" means a human or non-human animal selected
for
treatment or therapy.
The phrases "therapeutically-effective amount" and "effective amount" as used
herein
means the amount of an agent which is effective for producing the desired
therapeutic effect
in at least a sub-population of cells in a subject at a reasonable
benefit/risk ratio applicable to
any medical treatment.
"Treating" a disease in a subject or "treating" a subject having a disease
refers to
subjecting the subject to a pharmaceutical treatment, e.g., the administration
of a drug, such
that at least one symptom of the disease is decreased or prevented from
worsening.
As used herein, the term "conjoint administration" means administration of two
or
more agents to a subject of interest as part of a single therapeutic regimen.
The
administration(s) can be either simultaneous or sequential, i.e.,
administering one agent
followed by administering of a second (and/or a third one, etc.) at a later
time, as long as the
agents administered co-exist in the subject being treated, or at least one
agent will have the
opportunity to act upon the same target tissues of other agents while said
target tissues are
still under the influence of said other agents. In a certain embodiment,
agents to be
administered can be included in a single pharmaceutical composition and
administered
together. In a certain embodiment, the agents are administered simultaneously,
including
through separate routes. In a certain embodiment, one or more agents are
administered
continuously, while other agents are administered only at predetermined
intervals (such as a
single large dosage, or twice a week at smaller dosages, etc.).
Immune Checkpoint Inhibitors
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In certain aspects, provided herein are methods related to treating cancer
(e.g..
nasopharyngeal carcinoma) in a subject by administering to the subject a
combination
therapy, the combination therapy comprising administering to the subject both
an immune
checkpoint inhibitor and a composition comprising cytotoxic T cells (CTLs)
expressing a T
cell receptor specific for a cancer-associated peptide presented on a class I
MHC.
The immune checkpoint inhibitor and CTL composition can be administered
together
or separately. They can be administered simultaneously or sequentially. When
sequentially
administered, in some embodiments the immune checkpoint inhibitor will be
administered
before the CTL composition (e.g.. 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 or 24 hours before, 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 or 30 days
before). When
sequentially administered, in some embodiments the CTL composition will be
administered
before the immune checkpoint inhibitor (e.g.. 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 or 24 hours before, 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 or
30 days before).
Immune checkpoint inhibition broadly refers to inhibiting the biological
pathways
that serve as checkpoints to prevent or downregulate an immune response. Such
pathways
are often used by cancer cells to avoid an anti-tumor immune response. In
certain
embodiments, the method includes administering to the subject one or more
immune
checkpoint inhibitors that target immune checkpoint proteins. Immune
checkpoint proteins
include, but are not limited to, CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-
H4,
BTLA, KIR, LAG-3, TIM-3, MO, TDO, and VISTA. In some embodiments, one or more
immune checkpoint inhibitor may target one or more immune checkpoint proteins.
In some embodiments, the immune checkpoint inhibitor is a protein, such as a
soluble
fusion protein. In some embodiments, such a protein comprises a
receptor/ligand binding
domain (e.g, an extracellular domain) of C'TLA4, PD-1, PD-LI, PD-L2, A2AR, B7-
H3, B7-
H4, BTLA, KIR, LAG-3, TIM-3, IDO, 'TDO, or VISTA. In some embodiments, the
receptor/ligand binding domain is fused to an itruntmoglobulin Fc domain. Such
a fusion
protein can be produced by standard recombinant DNA techniques. For example,
DNA
fragments coding for the different peptide sequences are ligated together in-
frame in
accordance with conventional techniques, for example by employing blunt-ended
or stagger-
ended termini for ligation, restriction enzyme digestion to provide for
appropriate termini,
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filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to
avoid
undesirable joining, and enzymatic ligation. In another embodiment, the fusion
gene can be
synthesized by conventional techniques including automated DNA synthesizers.
Alternatively, PCR amplification of gene fragments can be carried out using
anchor primers
which give rise to complementary overhangs between two consecutive gene
fragments which
can subsequently be annealed and re-amplified to generate a chimeric gene
sequence (see,
for example, Current Protocols in Molecular Biology, Ausubel et al., eds.,
John Wiley &
Sons: 1992). Moreover, many expression vectors are commercially available that
already
encode a fusion moiety.
In certain embodiments, the immune checkpoint inhibitor is an antibody or
antigen-
binding fragment thereof that binds to and inhibits an immune checkpoint
protein (e.g..
CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA, KIR, LAG-3, 1IM-3, WO,
TDO, or VISTA). As used herein, the tertn "antibody" may refer to both an
intact antibody
and an antigen binding fragment thereof. The term "antibody" includes, for
example,
monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized
antibodies,
human antibodies, multispecific antibodies (e.g., bispecific antibodies),
single-chain
antibodies and antigen-binding antibody fragments. An antigen-binding fragment
of an
antibody refers to one or more fragments of an antibody that retain the
ability to bind to an
antigen. Examples of binding fragments include Fab, Fab', F(ab1)2, Fv, scFv,
disulfide linked
Fv, Fd, diabodies, single-chain antibodies, camelid antibodies, isolated
CDRH3, and other
antibody fragments that retain at least a portion of the variable region of an
intact antibody.
Such antibody fragments can be obtained using conventional recombinant and/or
enzymatic
techniques and can be screened for antigen-binding in the same manner as
intact antibodies.
In some embodiments, the immune checkpoint inhibitor is an inhibitory nucleic
acid
(e.g., an siRNA molecule, an shRNA molecule, an antisense RNA) that
specifically binds to
an mRNA that encodes an immune checkpoint inhibitor (e.g, CTLA4, PD-1, PD-L1,
PD-L2,
A2AR, B7-H3, B7-H4, BTLA, KIR, LAG-3, TIM-3, IDO, TDO, or VISTA). Inhibitory
nucleic acid molecules can be prepared by chemical synthesis, in vitro
transcription, or
digestion of long dsRNA by Rnase III or Dicer. Inhibitory nucleic acid
molecules can be
delivered in vitro to cells or in vivo, e.g., to tumors or hypoxic tissues of
a mammal. Typical
delivery means known in the art can be used. For example, an interfering RNA
can be
delivered systemically using, for example, the methods and compositions
described in PCT
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Application No: PCT/US09/036223, PCT/US09/061381 PCT/US09/063927,
PCT/US09/063931 and PCT/US09/063933, each of which is hereby incorporated by
reference in its entirety. In certain embodiments the inhibitory nucleic acid
is delivered
locally. For example, when the inhibitor), nucleic acid described herein is
used to treat
cancer, delivery to a tumor can be accomplished by intratumoral injections, as
described, for
example, in Takahashi et al., Journal of Controlled Release 116:90-95 (2006)
and Kim et al.,
Journal of Controlled Release 129:107-116 (2008), each of which is
incorporated by
reference in its entirety.
In yet other embodiments, the immune checkpoint inhibitor is a small organic
molecule, e.g., a molecule having a molecular weight under about 5 kD,
preferably less than
about 2 kD, and typically exclude oligonucleotides and oligopeptides. Small
molecules
include, for example, peptidomimetics, oligosaccharides, steroids, etc.
Representative small-
molecule checkpoint inhibitors are described in WO 2016/041511, WO
2015/034820, WO
2010/005958, WO 2014/159248, US Published Application 2011/0318373, and
Weinmann,
H., Chem. Med. Chem. 2016, Ii, 450-466 (and in references cited therein).
Various immune checkpoint inhibitors are known in the art. In some
embodiments,
the immune checkpoint inhibitor is nivolumab, pembrolizumab, pidilizumab, AMP-
224,
AMP-514, STI-A1110, TSR-042, RG-7446, BMS-936559, BMS-936558, MK-3475, CT 011,
MPDL3280A, MEDI-4736, MSB-0020718C, AUR-012 and STT-A1010.
Cytotoxic T Lymphocytes
In some embodiments, the CTLs in the CTL composition administered to the
subject
express a T cell receptor that specifically binds to a peptide (e.g., a
peptide comprising a
cancer-associated epitope) presented on a class I MHC. In some embodiments,
the class I
MHC has an a chain polypeptide that is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-
g,
HLA-K or HLA-L. In some embodiments, the peptide is a peptide described
herein. In some
embodiments, the CTLs in the sample express a TCR specific for an Epstein-Barr
Virus
(EBV) peptide (e.g., a LMP1 peptide, a LMP2A peptide or an EBNA1 peptide)
presented on
a class I MHC.
CTLs in the cm compositions described herein may be generated by incubating a
sample comprising CTLs with the antigen-presenting cells (APCs), thereby
inducing the
CTLs to proliferate. In some embodiments, the APCs that present a peptide
described herein
(e.g., a peptide comprising a LMP1, LMP2A, or ERNA1 epitope sequence). In some
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embodiments the APCs are B cells, antigen-presenting T-cells, dendritic cells,
or artificial
antigen-presenting cells (e.g., aK562 cells).
Dendritic cells for use in the process may be prepared by taking peripheral
blood
mononuclear cells (PBMCs) from a patient sample and adhering them to plastic.
Generally
the monocyte population sticks and all other cells can be washed off. The
adherent
population is then differentiated with 1L-4 and GM-CSF to produce monocyte
derived
dendritic cells. These cells may be matured by the addition of IL-113, IL-6,
PGE-1 and TNF-a
(which upregulates the important co-stimulatory molecules on the surface of
the dendritic
cell) and are then transduced with one or more of the peptides provided
herein.
APCs that present one or more peptides described herein may be generated by
contacting an APC with a peptide comprising a CTL epitope and/or with a
nucleic acid
encoding a peptide comprising a cri, epitope. In some embodiments, the APCs
are
irradiated. In some embodiments, the APCs that present a peptide described
herein (e.g., a
peptide comprising a LMP I, LMP2A, or EBNA I epitope sequence). A cell
presenting a
peptide described herein can be produced by standard techniques known in the
art. For
example, a cell may be pulsed to encourage peptide uptake. In some
embodiments, the cells
are transfected with a nucleic acid encoding a peptide provided herein.
Provided herein are
methods of producing antigen-presenting cells (APCs), comprising pulsing a
cell with the
peptides described herein. Exemplary examples of producing antigen-presenting
cells can be
found in W02013088114, hereby incorporated in its entirety.
In some embodiments, the methods provided herein include steps of generating,
activating and/or inducing proliferation of T cells (e.g., C'TLs) that
recognize one or more of
the CTL epitopes described herein. In some embodiments, a sample comprising
CTLs (i.e., a
PBMC sample) is incubated in culture with an APC provided herein (e.g., an APC
that
.. presents a peptide comprising a CTL epitope (e.g., EBV epitope) on a class
I MI-IC
complex). In some embodiments, the APCs are autologous to the subject from
whom the T
cells were obtained. In some embodiments, the APCs are not autologous (i.e.
allogeneic) to
the subject from whom the T cells were obtained. In some embodiments, the
sample
containing T cells is incubated two or more times with APCs provided herein.
In some
embodiments, the T cells are incubated with the APCs in the presence of at
least one
cytokine. In some embodiments, the cytokine is 1L-4, 1L-7 and/or IL-15.
Exemplary methods
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for inducing proliferation of T cells using APCs are provided, for example, in
U.S. Pat. Pub.
No. 2015/0017723, which is hereby incorporated by reference.
In some aspects, provided herein are methods comprising the administration of
samples, comprising immune checkpoint inhibitors and CTLs to a subject in
order to treat
and/or prevent cancer. In some embodiments, the method includes administering
to the
subject an effective amount of a composition comprising CTLs one or more
immune
checkpoint inhibitors, provided herein. In some embodiments, the composition
includes a
combination of multiple (e.g., two or more) CTLs and/ or immune checkpoint
inhibitors
provided herein. In some embodiments, the T cells are autologous to the
subject. In some
embodiments, the T cells are allogeneic to the subject. In some embodiments,
the CTLs are
stored in a cell bank before they are administered to the subject.
In some embodiments, the methods provided herein include selecting allogeneic
CTLs from a cell bank (e.g. a pre-generated third party donor derived bank of
epitope
specific CTLs) for adoptive immunotherapy by determining the level expression
of a
biomarlcer with in the CU population. In some embodiments, the level of
expression of two
or more biomarkers is determined. In some embodiments, the method further
includes
selecting allogeneic CTLs because they express a TCR restricted to a class I
MI-IC that is
encoded by an HLA allele that is present in the subject. In some embodiments,
the CTLs are
selected if the CTLs and subject share at least 2 (e.g., at least 3, at least
4, at least 5, at least
6) HLA alleles and the CTLs are restricted through a shared HLA allele. In
some
embodiments, the method comprises testing the TCR repertoire of the pre-
generated third-
party-donor-derived epitope-specific T cells (i.e., allogeneic T cells) with
flow cytometry. In
some embodiments epitope-specific T cells are detected using a tetramer assay,
an ELISA
assay, a western blot assay, a fluorescent microscopy assay, an Edman
degradation assay
and/or a mass spectrometry assay (e.g., protein sequencing). In some
embodiments, the TCR
repertoire is analyzed using a nucleic acid probe, a nucleic acid
amplification assay and/or a
sequencing assay.
Peptides
In some embodiments, the methods and compositions provided herein relate to
peptide specific CTLs. In some embodiments, the methods include the generation
of such
CTLs, for example, by incubating a sample comprising CTLs (i.e., a PBMC
sample) with
antigen-presenting cells (APCs) that present one or more of the CTL epitopes
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herein (e.g, APCs that present a peptide described herein comprising a cn,
epitope on a
class I MHC complex).
In some embodiments, the peptides provided herein comprise a sequence of any
EBV
viral protein (e.g., a sequence of at least 5, 6,7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19 or
20 contiguous amino acids of any EBV protein). In some embodiments, the
peptides
provided herein comprise no more than 25, 20, 19, 18, 17, 16, 15, 14, 13, 12,
11 or 10
contiguous amino acids of the EBV viral protein.
In some embodiments, the peptides provided herein comprise a sequence of LMP1
(e.g., a sequence of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19 or 20
.. contiguous amino acids of LMP1). In some embodiments, the peptides provided
herein
comprise no more than 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10
contiguous amino
acids of LMP1. An exemplary LIVIP1 amino acid sequence is provided below (SEQ
ID NO:
1):
1 mdldlergpp gprrpprgpp lssyialall 1111allfwl yiimsnwtgg
allvlyafal
61 mlviiiliif ifrrdllcpl galc1111mi tlllialwnl hgqalylgiv
1fifgcllvl
121 giwvyfleil wrlgatiwql lafflaffld illliialyl qqnwwtllvd
11w111f1ai
181 liwmyyhgqr hsdehhhdds 1phpqqatdd ssnhsdsnsn egrhhllvsg
agdapplcsq
241 nlgapgggpd ngpqdpdntd dngpqdpdnt ddngphdplp qdpdntddng
pqdpdntddn
301 gphdplphnp sdsagndggp pniteevenk ggdrgppsmt dggggdphlp
tlllgtsgsg
361 gddddphgpv qlsyyd
In some embodiments, the peptides provided herein comprise a sequence of LMP2A
(e.g., a sequence of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19 or 20
contiguous amino acids of LMP2A). In some embodiments, the peptides provided
herein
.. comprise no more than 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10
contiguous amino
acids of LMP2A. An exemplary LMP2A amino acid sequence is provided below (SEQ
ID
NO: 2):
1 mgslemvpmg agppspggdp dgddggnnsq ypsasgsdgn tptppndeer
esneeppppy
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61 edldwgngdr hsdyqplognq dpslylglqh dgndglpppp ysprddssqh
iyeeagrgsm
121 npvclpviva pylfwlaaia ascftasyst vvtatglals 1111aavass
yaaaqrkllt
181 pvtvltavvt ffaicltwri edppfnsllf allaaagglq giyvlvm1v1
lilayrrrwr
241 rltvcggimf lacvlvlivd avlqlspllg avtvvsmtll llafv1wlss
pgglgtlgaa
301 lltlaaalal laslilgtln lttmfllmll wtivvllics scsscpltki
llarlflyal
361 allllasali aggsilqtnf kslsstefip nlfcmllliv agilfilail
tewgsgnrty
421 gpvfmclggl ltmvagavwl tvmtntllsa wiltagflif ligfalfgvi
rccryccyyc
481 ltleseerpp tpyrntv
In some embodiments, the peptides provided herein comprise a sequence of EBNA
I
(e.g, a sequence of at least 5, 6, 7, 8, 9, 10, II, 12, 13, 14, 15, 16, 17,
18, 19 or 20
contiguous amino acids of EBNA1). In some embodiments, the peptides provided
herein
comprise no more than 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10
contiguous amino
acids of EBNA I. An exemplary EBNA1 amino acid sequence is provided below (SEQ
ID
NO: 3):
1 pffhpvgead yfeylcieggp dgepdvppga iewpaddpg egpstgprgq
gdggrrkkgg
61 wfgkhrgqgg snpkfeniae glrvllarsh vertteegtw vagvfvyggs
ktslynlrrg
121 talaipqcrl tplsrlpfgm apgpgpqpgp lresivcyfm vflqthifae
vlkdaikdlv
181 mtkpaptcni kvtvcsfddg vdlppwfppm vegaaaegdd gddgdeggdg degeegqe
In some embodiments, the peptide comprises the sequence of an epitope listed
in
Table!.
Table 1. Exemplary EBV viral protein epitopes
Peptide Sequence HLA Restriction SEQ ID No:
PYLFWLAAI A* 2301/A* 2402/03 4
SSCSSCPLSKI A*1101 5
TYGPVFMCL A*2402 6
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RRRWRRLTV B*27/02/04/05/06/09 7
LLSAWILTA A*0203 8
LTAGFLIFL A*0206 9
CLGGLurmv A*0201 10
VMSNTLLSAW A*25/A*26 11
MSNTLLSAW 13*58 12
IEDPPINSL B*4001 13
YLLEMLWRL A*02 14
YLQQNWWTL A*02 15
ALL VLYSFA A*02 16
IALYLQQNW B*57/B*58 17
FLYALALLL A*0201 18
wriNvu..1 A*24 19
CPLSKILL 13*0801 20
HPVGEADYFEY B*35 21
RPQKRPSCI 13*0702 22
IPQCRLTPL 13*0702 23
LSRLPFGMA 13*5701 24
YNLRRGTAL B*0801 25
VLKDAIKDL A*0203 26
FVYGGSKTSL C*0303/C*0304 27
FVYGGSKTay A*26 28
HPVGEADYF B*53 29
LQTHIFAEV A*0206 30
FMVFLQTHI A*0201 31
In some embodiments, the peptides provided herein comprise two or more of the
CU epitopes (e.g., viral epitopes). In some embodiments, the peptides provided
herein
comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19 or 20 CTL
epitopes. For example, in some embodiments, the peptides provided herein
comprise two or
more of the CTL epitopes connected by linkers (e.g., polypeptide linkers).
In some embodiments, the sequence of the peptides comprise a viral protein
sequence
except for 1 or more (e.g.. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)
conservative sequence
modifications. As used herein, the term "conservative sequence modifications"
is intended to
refer to amino acid modifications that do not significantly affect or alter
the interaction
between a T cell receptor (TCR) and a peptide containing the amino acid
sequence presented
on an Such conservative modifications include amino acid substitutions,
additions
(e.g., additions of amino acids to the N or C terminus of the peptide) and
deletions (e.g.,
deletions of amino acids from the N or C terminus of the peptide).
Conservative amino acid
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substitutions are ones in which the amino acid residue is replaced with an
amino acid residue
having a similar side chain. Families of amino acid residues having similar
side chains have
been defined in the art. These families include amino acids with basic side
chains (e.g,
lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,
glutamic acid), uncharged
.. polar side chains (e.g.. glycine, asparagine, glutamine, serine, threonine,
tyrosine, cysteine,
hyptophan), nonpolar side chains (e.g, alanine, valine, leucine, isoleucine,
proline,
phenylalanine, methionine), beta-branched side chains (e.g., threonine,
valine, isoleucine)
and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,
histidine). Thus, one or
more amino acid residues of the peptides described herein can be replaced with
other amino
acid residues from the same side chain family and the altered peptide can be
tested for
retention of TCR binding using methods known in the art. Modifications can be
introduced
into an antibody by standard techniques known in the art, such as site-
directed mutagenesis
and PCR-mediated mutagenesis.
In some embodiments, the peptides provided herein comprise a sequence that is
at
least 80%, 85%, 90%, 95% or 100% identical to a protein sequence (e.g., the
sequence of a
fragment of a viral protein). To determine the percent identity of two amino
acid sequences,
the sequences are aligned for optimal comparison purposes (e.g, gaps can be
introduced in
one or both of a first and a second amino acid sequence for optimal alignment
and non-
identical sequences can be disregarded for comparison purposes). The amino
acid residues at
.. corresponding amino acid positions are then compared. When a position in
the first sequence
is occupied by the same amino acid residue as the corresponding position in
the second
sequence, then the molecules are identical at that position. The percent
identity between the
two sequences is a function of the number of identical positions shared by the
sequences,
taking into account the number of gaps, and the length of each gap, which need
to be
introduced for optimal alignment of the two sequences.
The peptides provided herein can be isolated from cells or tissue sources by
an
appropriate purification scheme using standard protein purification
techniques, and can be
produced by recombinant DNA techniques, and/or can be chemically synthesized
using
standard peptide synthesis techniques. The peptides described herein can be
produced in
prokaryotic or eukaryotic host cells by expression of nucleotides encoding a
peptide(s) of the
present invention. Alternatively, such peptides can be synthesized by chemical
methods.
Methods for expression of heterologous peptides in recombinant hosts, chemical
synthesis of
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peptides, and in vitro translation are well known in the art and are described
further in
Maniatis et al., Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold
Spring
Harbor, N. Y.; Berger and Kimmel, Methods in Enzymology, Volume 152, Guide to
Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, Calif.;
Merrifield,
J. (1969) J. Am. Chem. Soc. 91:501; Chaiken I. M. (1981) CRC Crit. Rev.
Biochem. 11:255;
Kaiser et al. (1989) Science 243:187; Merrifield, B. (1986) Science 232:342;
Kent, S. B. H.
(1988) Annu. Rev. Biochem. 57:957; and Offord, R. E. (1980) Semisynthetic
Proteins,
Wiley Publishing, which are incorporated herein by reference.
In certain aspects, provided herein are nucleic acid molecules encoding the
peptides
described herein. In some embodiments, the nucleic acid molecule is a vector.
In some
embodiments, the nucleic acid molecule is a viral vector, such as an
adenovirus based
expression vector, that comprises the nucleic acid molecules described herein.
In some
embodiments, the vector provided herein encodes a plurality of epitopes
provided herein
(e.g., as a polyepitope). In some embodiments, the vector provided herein
encodes at least 2,
.. 3,4. 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 epitopes
provided herein (e.g.,
epitopes provided in Table 1).
In some embodiments, the vector is AdEl-LMPpoly. The AdEl-LMPpoly vector
encodes a polyepitope of defined CTL epitopes from LMP1 and LMP2 fused to a
Gly-Ala
repeat-depleted EBNA I sequence. The AdEl-LMPpoly vector is described, for
example, in
Smith et al., Cancer Research 72:1116 (2012); Duraiswamy etal., Cancer
Research
64:1483-9 (2004); and Smith etal., J Immunol 117:4897-906, each of which is
hereby
incorporated by reference.
As used herein, the term "vector," refers to a nucleic acid molecule capable
of
transporting another nucleic acid to which it has been linked. One type of
vector is a
"plasmid", which refers to a circular double-stranded DNA loop into which
additional DNA
segments may be ligated. Another type of vector is a viral vector, wherein
additional DNA
segments may be ligated into the viral genome. Certain vectors are capable of
autonomous
replication in a host cell into which they are introduced (e.g, bacterial
vectors having a
bacterial origin of replication, episomal mammalian vectors). Other vectors
(e.g., non-
episomal mammalian vectors) can be integrated into the genome of a host cell
upon
introduction into the host cell, and thereby be replicated along with the host
genome.
Moreover, certain vectors are capable of directing the expression of genes.
Such vectors are
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referred to herein as "recombinant expression vectors- (or simply, "expression
vectors"). In
some embodiments, provided herein are nucleic acids operably linked to one or
more
regulatory sequences (e.g, a promotor) in an expression vector. In some
embodiments, the
cell transcribes the nucleic acid provided herein and thereby expresses a
peptide described
herein. The nucleic acid molecule can be integrated into the genome of the
cell or it can be
extrachromasomal.
In some embodiments, provided herein are cells that contain a nucleic acid
described
herein (e.g., a nucleic acid encoding a peptide described herein). The cell
can be, for
example, prokaryotic, eukaryotic, mammalian, avian, murine and/or human. In
some
embodiments, the cell is a mammalian cell. In some embodiments the cell is an
APC (e.g. an
antigen-presenting T cell, a dendritic cell, a B cell, or an aK562 cell). In
the present methods,
a nucleic acid described herein can be administered to the cell, for example,
as nucleic acid
without delivery vehicle, in combination with a delivery reagent. In some
embodiments, any
nucleic acid delivery method known in the art can be used in the methods
described herein.
Suitable delivery reagents include, but are not limited to, e.g., the Mints
Transit TKO
lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g.,
polylysine),
atelocollagen, nanoplexes, and liposomes. In some embodiments of the methods
described
herein, liposomes are used to deliver a nucleic acid to a cell or subject.
Liposomes suitable
for use in the methods described herein can be formed from standard vesicle-
forming lipids,
which generally include neutral or negatively charged phospholipids and a
sterol, such as
cholesterol. The selection of lipids is generally guided by consideration of
factors such as the
desired liposome size and half-life of the liposomes in the blood stream. A
variety of
methods are known for preparing liposomes, for example, as described in Szoka
et al.
(1980), Ann. Rev. Biophys. Bioeng. 9:467; and U.S. Pat. Nos. 4,235,871,
4,501,728,
.. 4,837,028, and 5,019,369, the entire disclosures of which are herein
incorporated by
reference.
Therapeutic Methods
In some embodiments, the provided herein are methods of treating a cancer in a
subject by administering to the subject a combination therapy described
herein.
In some embodiments, the methods provided herein can be used to treat any
cancer.
For example, in some embodiments, the methods and CTLs described herein may be
used to
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treat any cancerous or pre-cancerous tumor. In some embodiments, the cancer
includes a
solid tumor. In some embodiments, cancers that may be treated by methods and
compositions provided herein include, but are not limited to, cancer cells
from the bladder,
blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine,
gum, head,
kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach,
testis, tongue, or
uterus. In addition, the cancer may specifically be of the following
histological type; though
it is not limited to these: neoplasm, malignant; carcinoma; carcinoma,
undifferentiated; giant
and spindle cell carcinoma; small cell carcinoma; papillary carcinoma;
squamous cell
carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix
carcinoma;
.. transitional cell carcinoma; papillary transitional cell carcinoma;
adenocarcinoma;
gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined
hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma;
adenoid
cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma,
familial
polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-
alveolar
adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil
carcinoma;
oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma;
granular cell
carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma;
nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma;
endometrioid
carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous
adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma;
cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous
cystadenocarcinoma;
mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell
carcinoma;
infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma;
inflammatory
carcinoma; mammary paget's disease; acinar cell carcinoma; adenosquamous
carcinoma;
.. adenocarcinoma w/squamous metaplasia; malignant thy-moma; malignant ovarian
stromal
tumor; malignant thecoma; malignant granulosa cell tumor; and malignant
roblastoma;
sertoli cell carcinoma; malignant leydig cell tumor; malignant lipid cell
tumor; malignant
paraganglioma; malignant extra-mammary paraganglioma; pheochromocytoma;
glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial
spreading
melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell
melanoma;
malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma;
myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embiyonal
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rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed
tumor;
mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma;
malignant
mesenchymoma; malignant brenner tumor; malignant phyllodes tumor; synovial
sarcoma;
malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma;
malignant struma ovarii; choriocarcinoma; malignant mesonephroma;
hemangiosarcoma;
malignant hemangioendothelioma; kaposi's sarcoma; malignant
hemangiopericytoma;
lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma;
malignant
chondroblastoma; mesenchymal cbondrosarcoma; giant cell tumor of bone; ewing's
sarcoma;
malignant odontogenic tumor; ameloblastic odontosarcoma; malignant
ameloblastoma;
ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma;
ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary, astrocytoma;
astroblastoma;
glioblastoma; oligodendroglioma; oligodendroblastoma; primitive
neuroectodermal;
cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma;
olfactory
neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant
neurilemmoma;
malignant granular cell tumor; malignant lymphoma; Hodgkin's disease;
Hodgkin's
lymphoma; paragranuloma; small lymphocytic malignant lymphoma; diffuse large
cell
malignant lymphoma; follicular malignant lymphoma; mycosis fungoides; other
specified
non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell
sarcoma;
immunoproliferative small intestinal disease; leukemia; lymphoid leukemia;
plasma cell
leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia;
basophilic
leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia;
megakwyoblastic
leukemia; myeloid sarcoma; and hairy cell leukemia.
In some embodiments, the methods provided herein are used to treat EBV
associated
cancer. In some embodiments, the EBV-associated cancer is EBV-associated NPC.
In some
embodiments, the EBV associated cancer is post-transplant ly-mphoproliferative
disorder (PTLD). NKJT cell lymphoma, EBV+ gastric cancer, or EBV+
leiomyosarcoma.
In some embodiments, the combination therapy further comprises a
chemotherapeutic agent (e.g, alkylating agents or agents with an alkylating
action, such as
cyclophosphamide (CTX; e.g., CYTOXAN9), chlorambucil (CHL; e.g., LEUKERANC),
cisplatin (Cis P; e.g., PLATINOLS) busulfan (e.g., MYLERANO), melphalan,
carmustine
(BCNU), streptozotocin, triethylenemelamine (TEM), mitomycin C, and the like;
anti-
metabolites, such as methotrexate (MTX), etoposide (VP16; e.g., VEPESIDO), 6-
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mercaptopurine (6MP), 6-thioguanine (6TG), cytarabine (Ara-C), 5-fluorouracil
(5-FU),
capecitabine (e.g. XELODA1)), dacarbazine (DTIC), and the like; antibiotics,
such as
actinomycin D, doxombicin (DXR; e.g., ADRIAMYCINS), daunombicin (daunomycin),
bleomycin, mithramycin and the like; alkaloids, such as vinca alkaloids such
as vincristine
(VCR), vinblastine, and the like; and other antitumor agents, such as
paclitaxel (e.g.,
TAXOLC) and pactitaxel derivatives, the cytostatic agents, glucocorticoids
such as
dexamethasone (DEX; e.g., DECADRONS)) and corticosteroids such as prednisone,
nucleoside enzyme inhibitors such as hydroxyurea, amino acid depleting enzymes
such as
asparaginase, leucovorin and other folic acid derivatives, and similar,
diverse antitumor
agents. The following agents may also be used as additional agents: amifostine
(e.g.,
ETHYOL01), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin,
cyclophosphamide, lomustine (CCNU), doxorubicin lipo (e.g., DOXILe),
gemcitabine (e.g.,
GEMZARO)õ daunorubicin lipo (e.g., DAUNOXOME0), procarbazine, mitomycin,
docetaxel (e.g., TAXOTERE0), aldesleukin, carboplatin, oxaliplatin,
cladribine,
camptothecin, CPT 11 (irinotecan), 10-hydroxy 7-ethyl-camptothecin (SN38),
floxuridine,
fludarabine, ifosfamide, idarubicin, mesna, interferon beta, interferon alpha,
mitoxantrone,
topotecan, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin,
mitotane,
pegaspargase, pentostatin, pipobroman, plicamycin, tamoxifen, teniposide,
testolactone,
thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil).
Actual dosage levels of the active ingredients in the pharmaceutical
compositions
provided herein may be varied so as to obtain an amount of the active
ingredient which is
effective to achieve the desired therapeutic response for a particular
patient, composition,
and mode of administration, without being toxic to the patient.
The selected dosage level will depend upon a variety of factors including the
activity
of the particular agent employed, the route of administration, the time of
administration, the
rate of excretion or metabolism of the particular compound being employed, the
duration of
the treatment, other drugs, compounds and/or materials used in combination
with the
particular compound employed, the age, sex, weight, condition, general health
and prior
medical history of the patient being treated, and like factors well known in
the medical arts.
The CTLs and the immune checkpoint inhibitors described herein may be co-
administered or
administered sequentially. The immune checkpoint inhibitors may be
administered by any
technique known in the art. In some embodiments, the immune checkpoint
inhibitor is
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administered intratumorally. In some embodiments, the immune checkpoint
inhibitor is
administered intravenously. In some embodiments, the immune checkpoint
inhibitor is
administered parenterally.
In some embodiments, the subject has been exposed to a virus (e.g. EBV) such
that
virus particles are detectable in the subject's blood. In some embodiments,
the method
further comprises measuring viral load in the subject (e.g, before or after
administering the
peptide specific CTLs to the subject). Determining viral load in a subject may
be a good
prognostic marker for immunotherapy effectiveness. In some embodiments,
selecting CTLs
further comprises determining the number of viral DNA copies in the subject
(e.g. in a tissue
or blood sample). In some embodiments, viral load is measured two or more
times.
In some embodiments, the method further includes selecting allogeneic CTLs for
combination therapy because they express a TCR restricted to a class I MHC
that is encoded
by an HLA allele that is present in the subject. In some embodiments, the CTLs
are selected
if the CTLs and subject share at least 2 (e.g.. at least 3, at least 4, at
least 5, at least 6) HLA
alleles and the CTLs are restricted through a shared HLA allele. In some
embodiments, the
method comprises testing the TCR repertoire of the pre-generated third-party-
donor-derived
epitope-specific T cells (i.e., allogeneic T cells) with flow cytometr3,7. in
some embodiments
epitope-specific T cells are detected using a tetramer assay, an ELISA assay,
a western blot
assay, a fluorescent microscopy assay, an Edman degradation assay and/or a
mass
spectrometry assay (e.g., protein sequencing). In some embodiments, the TCR
repertoire is
analyzed using a nucleic acid probe, a nucleic acid amplification assay and/or
a sequencing
assay. In some embodiments, the allogeneic CTLs are obtained from a cell bank.
Exemplification:
Example 1: immune checkpoint protein expression in NPC patients
Fifty-two patients were enrolled in a study applying LMP1&2 and EBNA 1 -
specific CTL immunotherapy for the treatment of EBV-associated NPC, including
41 with
active progressive disease following pallatative chemotherapy and 11 patients
with minimal
or no residual disease (N/MRD) following standard radio/chemotherapeutic
treatment.
Twenty active disease patients and 9 N/MRD patients received the minimum 2
doses
(range 2-8 doses) and a median total of 1.1x108 cells (range: 5.7x107 to
2.4x108). The
clinical characteristics of the patients who received adoptive T cell therapy
are provided in
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Table 1 and 2. Of the remaining 23 patients, 1 patient died after the
administration of a single
dose, T cell therapy was manufactured for 5 patients but not administered due
to illness,
12 failed to meet release criteria due to low specificity or cell yield and 5
were withdrawn
prior to the commencement of T cell manufacture.
To generate LMP/EBNA I specific T cells 100-300mI, of peripheral blood was
harvested and used to generate peripheral blood mononuclear cells (PBMC). The
.AdEl -
12v1Ppoly vector was then used to infect 30% of the PBMC (MOT of 10:1) that
were then
irradiated and co-cultured with the remaining PBMC for two weeks. Cultures
were
supplemented with fresh erowth medium and I 201.UIna, of recombinant every
3-4 days
(Komtur Pharmaceuticals, Frieburg, Germany). Cultured T cells were tested for
antigen
specificity using intracellular cytokine analysis and microbial contamination
prior to release
for infusion.
FACS profiling was performed to characterize the expression of immune
checkpoint
proteins by the T cells administered to the subjects. MHC tetramers were
generated in house.
T cells were incubated for 20 minutes at 4 C with APC-labelled NfFIC class I
tetramers
specific for the HLA A 11-restricted epitope SSCSSCPLSKI (LMP2A), the HLA A24
restricted epitope TYGPVFMCL (LMP2A) and the HLA Cw03 restricted epitope
FVYGGSKTSL (EBNA1). Cells were then incubated for a further 30 minutes with
one or
more of the following antibodies: PE-conjugated anti-TIM-3, F1TC conjugated
anti-LAG-
3. BV786-conjugated anti-PD-1 and BV421-conjugated anti-CTLA4. Cells were
acquired
using a BD LSR Fortessa with FACSDiva software (BD Biosciences) and post-
acquisition
analysis was performed using Flowio software (TreeStar).
Figure 1 shows the expression of immune checkpoint molecules (i.e., LAG-3, TIM-
3,
CTLA4, or PD-1). Panel A depicts percentage of HLA-multimer positive CDR-
positive
lymphocytes that express PD-1, TIM-3, LAG-3 and CTLA-4. Panel B depicts the
percentage
of PD-1 positive, TIM-3 positive, LAG-3 positive and CTLA-4 positive
lymphocytes in the
CTL immunotherapy administered in NPC patients with no/minimal residual
disease
(N/MRD) and active-recurrent/metastatic disease (ARMD) who either showed
stable disease
(SD) or progressive disease (PD) following adoptive T cell therapy.
Example 2: Adoptive transfer of ERV-Cas and Checkpoint Inhibitor Therapy in
NPC
Patients
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Patients with platinum resistant or recurrent EBV-associated nasophary, ngeal
carcinoma (NPC) are treated with adoptive transfer of allogeneic Epstein-Barr
virus
cytotoxic T lymphocytes (EBV-CTLs) in combination with a checkpoint inhibitor
(Pembrolizumab). Up to a total of 48 subjects with metastatic, platinum-
resistant or recurrent
EBV-associated NPC are enrolled in the study.
Study Protocol and Dosing
The study has two parts: Cohort! is enrolled as the Phase 1B portion of the
study to
determine the Phase 2 dose; Cohort 2 is enrolled as the Phase 2 portion of the
study to
examine the clinical benefits of combined adoptive cellular and checkpoint
inhibitor
therapies for NPC. The protocol will enroll 48 subjects in total. Phase 1B
(Cohort 1) will
enroll 12 subjects whose disease progressed despite prior PD1 inhibitor
therapy, and Phase 2
(Cohort 2) will enroll 36 subjects naïve to PD1 inhibitor therapy.
Allogeneic third-party EBV-CTLs are selected for each subject from the bank of
available EBV-CTLs based on matching > 2 HLA alleles, at least one of which is
a
restricting HLA allele, shared between the EBV-CTLS source material (donor)
and the
subject. High resolution HLA typing will be performed during screening to
facilitate
selection of the EBV-CTLS cell product. Historical HLA typing will be
acceptable if
performed at high resolution (DNA based versus serologic assessment).
In the Phase 1B (Cohort 1), EBV-CTLs are administered at doses ranging from
500,000 to 200,000,000 T-cells per infusion intravenously on Day 1, Day 8, and
Day 15 of a
21 Day Cycle to six subjects with advanced NPC. The subject population is
selected based
on previous Phase 1 safety and efficacy data which showed adequate EBV cm,
expansion
and antitumor activity in patients with advanced NPC. Likewise, pembrolizumab
is
administered to Cohort 1 subjects at a dose of 200 mg IV Q3 weeks to adults
(adults are
greater than or equal to 18 years old) and at 2mg/kg IV Q3 weeks to pediatric
subjects (less
than 18 years old).
If less than 2 of the initial 6 Phase 1B Cohort 1 subjects experience dose
limiting
toxicity in the first 21 days, dose reduction of EBV-CTLS occurs, and the
subsequent 6
subjects are treated with the combination of EBV-CTLS and pembroliztunab at
the
recommended dose level.
22
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Screening will begin up to 28 days prior to dosing (Cycle 1 Day 1). Subjects
will be
treated with EBV-CTLS in combination with pembrolizumab until disease
progression or an
unacceptable toxicity is observed.
Study Participants
Summary of Subject Enrollment Criteria is as follows:
Inclusion criteria: A patient will be considered eligible to participate in
this study if all of the
following inclusion criteria are satisfied:
1. Males and females 2 years of age
2. Patients with advanced or metastatic NPC who are considered platinum
refractory/resistant, defined as having at least one prior platinum-based
chemotherapeutic regimen with a subsequent platinum-free interval of < 12
months,
having progression during platinum-based therapy, or having persistent disease
after
a platinum-based therapy, are eligible
3. Patients with NPC in whom the EBV-genome or antigens have been demonstrated
in
tissue biopsy samples
4. Histologically or cytologically-confirmed EBV-associated locally recurrent,
metastatic or persistent NPC (WHO type II/III) and meeting the following
corresponding requirements for the cohort of the study they will enroll into:
a. Phase IB (Cohort 1): patients who have received prior treatment with
pembrolizumab anti-PD!, who have not received prior treatment with anti-
PD-L1, anti-PD-L2, anti-CD137, anti-OX-40 or anti-CTLA-4 antibodies
b. Phase 2 (Cohort 2): patients who have not received prior treatment with
pembrolizumab or other anti-PD!, anti-PD-Li, anti-PD-L2, anti-CD137, anti-
0X40 or anti-CTLA 4 antibodies
5. Life expectancy 4 months at time of screening
6. Measurable disease using RECIST 1.1. Tumor lesions situated in a previously
irradiated area are considered measurable if progression has been demonstrated
in
such lesions
7. Patients in Phase 1B must have a biopsy at baseline and on treatment of a
metastatic
lesion that can be biopsied with acceptable clinical risk (as judged by the
investigator), and must agree to undergo biopsy.
8. Patients must agree to submit prior biopsy material for biomarker
assessment.
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WO 2017/203362 PCT/IB2017/000740
9. Eastern Cooperative Oncology Group (ECOG) performance status of 1 for
patients
aged > 16 years; Lansky score? 70 for patients aged < 16 years.
10. Adequate organ function per the following (unless deemed to be caused by
the
underlying EBV -driven
process which EBV-CTLs are intended to treat, or its prior therapy):
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11. Willing and able to provide written informed consent
Exclusion criteria: A patient will not be eligible to participate in the study
if any of the
following criteria are met:
1. Has disease that is suitable for local therapy administered with curative
intent.
2. Need for methotrexate or extracorporeal photopheresis
3. Need for vasopressor or ventilator support
4. Antithymocyte globulin or similar anti-I cell antibody therapy < 4 weeks
prior to
Cycle I Day 1
5. Has a diagnosis of immunodeficiency or is receiving systemic steroid
therapy or any
other form of irumunosuppressive therapy within 7 days prior to the first dose
of trial
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CA 03023845 2018-11-09
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treatment. The use of physiologic doses of corticosteroids may be approved
after
consultation with the Sponsor.
6. Patients with history or evidence of interstitial lung disease
7. Patients with an active infection requiring systemic therapy
8. Patients with history of (non-infectious) pneumonitis that required
steroids or current
pneumonitis
9. Patients who have received transfusion of blood products (including
platelets or red
blood cells) or administration of colony stimulating factors (including G-CSF,
GM-
CSF or recombinant erythropoetin) within 4 weeks prior to study Day 1.
10. Pregnancy or breastfeeding; females of childbearing potential must have a
negative
urine or serum pregnancy test. If the urine test is positive or cannot be
confirmed as
negative, a serum pregnancy test will be required. The serum pregnancy must be
confirmed negative within 72 hours of first dose for the patient to be
eligible.
11. Full resolution of immunotherapy related adverse effects and no treatment
for these
adverse events (AEs) for at least 4 weeks prior to enrollment
12. No history of severe inununotherapy related adverse effects (CTCAE Grade
4;
CTCAE Grade 3 requiring treatment > 4 weeks)
13. Patients who have received any non-oncology vaccine therapy used for
prevention of
infectious diseases for up to 30 days prior to enrollment. Examples include,
but are
not limited to: measures, mumps, rubella, chicken pox, yellow fever, rabies,
BCG,
and typhoid vaccine. Seasonal flu vaccines that do not contain live virus are
acceptable.
14. Has a known additional malignancy that is progressing or requires active
treatment.
Exceptions include basal cell carcinoma of the skin, squamous cell carcinoma
of the
skin that has undergone potentially curative therapy or in situ cervical
cancer.
15. Female of childbearing potential or male with a female partner of
childbearing
potential unwilling to use a highly effective method of contraception
(abstinence is
acceptable) for the course of the study through 120 days after the last study
dose.
16. Inability to comply with study procedures
17. Chemotherapy, targeted small molecule therapy, hormonal therapy, or
radiation
therapy within 2 weeks of Cycle 1 Day 1 or who has not recovered (i.e., <
Grade 1 or
at baseline) from adverse events due to a previously administered agent.
Subjects
CA 03023845 2018-11-09
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PCT/IB2017/000740
with S Grade 2 neuropathy or < Grade 2 alopecia are an exception to this
criterion
and may qualify for the study.
18. Antibody/biologic therapy within 5 half-lives or 4 weeks (whichever is
longer) of
Cycle 1 Day 1 or who has not recovered (i.e., S Grade 1 or at baseline) from
adverse
events due to agents administered more than 4 weeks earlier.
19. Patient with carcinomatous meningitis and/or active CNS metastases, unless
metastases are treated and stable and the patient does not require systemic
steroids
NOTE: Subjects with previously treated brain metastases may participate
provided
they are stable (without evidence of progression by imaging (using the
identical
imaging modality for each assessment, either MR1 or CT scan) for at least four
weeks
prior to the first dose of trial treatment and any neurologic symptoms have
returned
to baseline), have no evidence of new or enlarging brain metastases, and are
not
using steroids for at least 7 days prior to trial treatment. This exception
does not
include carcinomatous meningitis which is excluded regardless of clinical
stability.
20. Patients with a history or current evidence of any condition, therapy, or
laboratory
abnormality that might confound the results of the trial, interfere with the
subject's
participation for the full duration of the trial, or is not in the best
interest of the
subject to participate, in the opinion of the treating investigator.
21. Has known psychiatric or substance abuse disorders that would interfere
with
cooperation with the requirements of the trial.
22. Known history of HIV, known active Hepatitis B (e.g. HBsAg reactive),
Hepatitis C
(e.g. HCV RNA is detected.
23. Prior treatment with any investigational product within 4 weeks of Cycle 1
Day 1
24. Has had a prior anti-cancer monoclonal antibody (mAb) within 4 weeks prior
to
study Day 1 or who has not recovered (i.e., < Grade 1 or at baseline) from
adverse
events due to agents administered more than 4 weeks earlier.
25. Prior treatment with EBV T-cells
The following are indications of efficacy of treatment:
1) The change in NPC disease progression and other clinically relevant
outcomes, as
measured by complete response (CR) rate, duration of response (DOR),
progression-free
survival (PFS) and overall survival (OS).
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2) Increases in immune response rate (irRR = irCR + irPR) and/or duration of
response (DOirR).
All publications, patents, patent applications and sequence accession numbers
.. mentioned herein are hereby incorporated by reference in their entirety as
if each individual
publication, patent or patent application was specifically and individually
indicated to be
incorporated by reference. In case of conflict, the present application,
including any
definitions herein, will control.
Those skilled in the art will recognize, or be able to ascertain using no more
than
.. routine experimentation, many equivalents to the specific embodiments of
the invention
described herein. Such equivalents are intended to be encompassed by the
following claims.
27
