U.S. patent application number 17/668746 was filed with the patent office on 2022-07-28 for multiple antigen specific cell therapy methods.
The applicant listed for this patent is HRYZ Biotech Co., SYZ Cell Therapy Co.. Invention is credited to Yanyan HAN, Jin LI, Junyun LIU, Yifan MA, Longqing TANG, Dongyun WU, Xiangjun ZHOU.
Application Number | 20220235324 17/668746 |
Document ID | / |
Family ID | |
Filed Date | 2022-07-28 |
United States Patent
Application |
20220235324 |
Kind Code |
A1 |
ZHOU; Xiangjun ; et
al. |
July 28, 2022 |
MULTIPLE ANTIGEN SPECIFIC CELL THERAPY METHODS
Abstract
The present invention provides methods of preparing a population
of activated T cells by co-culturing T cells with dendritic cells
loaded with a plurality of tumor antigen peptides. Also provided
are methods of treating cancer in an individual using the activated
T cells, pharmaceutical compositions and kits for cell-based cancer
immunotherapy.
Inventors: |
ZHOU; Xiangjun; (Shenzhen,
CN) ; MA; Yifan; (Shenzhen, CN) ; HAN;
Yanyan; (Shenzhen, CN) ; LI; Jin; (Shenzhen,
CN) ; TANG; Longqing; (Shenzhen, CN) ; LIU;
Junyun; (Shenzhen, CN) ; WU; Dongyun;
(Shenzhen, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SYZ Cell Therapy Co.
HRYZ Biotech Co. |
Shenzhen
Shenzhen |
|
CN
CN |
|
|
Appl. No.: |
17/668746 |
Filed: |
February 10, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17043613 |
Sep 29, 2020 |
11248208 |
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PCT/CN2019/080535 |
Mar 29, 2019 |
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17668746 |
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International
Class: |
C12N 5/0783 20060101
C12N005/0783; A61P 35/00 20060101 A61P035/00; A61K 35/17 20060101
A61K035/17 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 30, 2018 |
CN |
PCT/CN2018/081338 |
Claims
1. A method of preparing a population of activated T cells, the
method comprising: a) contacting a population of dendritic cells
with a plurality of tumor antigen peptides to obtain a population
of dendritic cells loaded with the plurality of tumor antigen
peptides; b) co-culturing the population of dendritic cells loaded
with the plurality of tumor antigen peptides and a population of T
cells in an initial co-culture medium comprising a plurality of
cytokines and an immune checkpoint inhibitor to provide a
co-culture; and c) adding an anti-CD3 antibody to the co-culture at
about 3 to 7 days after the co-culturing starts, thereby obtaining
the population of activated T cells.
2. The method of claim 1, wherein step a) further comprises
culturing the population of dendritic cells loaded with the
plurality of tumor antigen peptides in a DC maturation medium
comprising a toll-like receptor (TLR) agonist.
3. The method of claim 2, wherein the TLR agonist is selected from
the group consisting of monophosphoryl lipid (MPLA), Poly I:C,
resquimod, gardiquimod, and CL075.
4. A method of preparing a population of activated T cells, the
method comprising: a) contacting a population of dendritic cells
with a plurality of tumor antigen peptides to obtain a population
of dendritic cells loaded with the plurality of tumor antigen
peptides; b) culturing the population of dendritic cells loaded
with the plurality of tumor antigen peptides in a DC maturation
medium comprising MPLA; and c) co-culturing the population of
dendritic cells loaded with the plurality of tumor antigen peptides
and a population of T cells, thereby obtaining the population of
activated T cells.
5. The method of claim 2, wherein the DC maturation medium
comprises interferon-.gamma. (INF.gamma.), MPLA, and prostaglandin
E2 (PGE2).
6-7. (canceled)
8. The method of claim 5, wherein the MPLA is present in the DC
maturation medium at a concentration of at least about 0.5
.mu.g/mL.
9. (canceled)
10. The method of claim 5, wherein step c) comprises: co-culturing
the population of dendritic cells loaded with the plurality of
tumor antigen peptides and a population of T cells in an initial
co-culture medium comprising a plurality of cytokines and an immune
checkpoint inhibitor to provide a co-culture; and adding an
anti-CD3 antibody to the co-culture, t hereby obtaining the
population of activated T cells.
11. The method of claim 1, wherein the plurality of cytokines
comprises IL-2, IL-7, IL-15 and IL-21.
12. (canceled)
13. The method of claim 1, wherein the immune checkpoint inhibitor
is an anti-PD-1 antibody.
14. (canceled)
15. The method of claim 1, wherein the anti-CD3 antibody is added
to the co-culture at about 3 to 7 days after the co-culturing
starts.
16. (canceled)
17. The method of claim 1, wherein the population of dendritic
cells loaded with the plurality of tumor antigen peptides and the
population of T cells are co-cultured for at least about 10 days in
the presence of the anti-CD3 antibody.
18. (canceled)
19. The method of claim 1, wherein the population of dendritic
cells is obtained by inducing differentiation of a population of
monocytes from PBMCs.
20. The method of claim 1, wherein the population of dendritic
cells and the population of T cells are obtained from the same
individual.
21. The method of claim 1, wherein the plurality of tumor antigen
peptides comprises a neoantigen peptide.
22. An isolated population of activated T cells prepared using the
method of claim 1.
23. A method of treating a cancer in an individual, comprising: a)
contacting a population of dendritic cells with a plurality of
tumor antigen peptides to obtain a population of dendritic cells
loaded with the plurality of tumor antigen peptides; b)
co-culturing the population of dendritic cells loaded with the
plurality of tumor antigen peptides and a population of T cells in
an initial co-culture medium comprising a plurality of cytokines
and an immune checkpoint inhibitor to provide a co-culture; c)
adding an anti-CD3 antibody to the co-culture at about 3 to 7 days
after the co-culturing starts, thereby obtaining activated T cells;
and d) administering to the individual an effective amount of the
activated T cells of claim 22.
24-32. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority benefit of
International Patent Application No. PCT/CN2018/081338, filed Mar.
30, 2018, the disclosure of which is incorporated by reference in
its entirety.
FIELD OF THE INVENTION
[0002] The present application relates to the field of cancer
immunotherapy. More specifically, this application provides
methods, compositions and kits for treating cancer in an individual
using activated T cells.
BACKGROUND OF THE INVENTION
[0003] The human body has an elaborate immune system to defend
itself against diseases. Unleashing the body's own immunity against
cancer has been a long-standing ideal in oncology. Natural immune
response against a tumor is elicited by tumor antigens. Antigen
presenting cells (APCs), notably dendritic cells (DCs), can process
and present the tumor antigens on their cell surface. Upon
maturation, DCs loaded with tumor antigens can trigger T cell
response, which involves cytotoxic T cells, helper T cells, and
functionally distinct effecter and memory T cells against cancer
cells expressing the tumor antigens. A particularly powerful type
of T cell response involves production of cytotoxic T cells that
can kill cancer cells by releasing cytokines, enzymes, and
cytotoxins, or by inducing pro-apoptosis signaling cascades via
cell-cell interactions.
[0004] Cell-based cancer immunotherapy seeks to treat cancer by
administering to patients immunity-mediating cells prepared to
target tumor antigens. FDA-approved PRO VENGE.RTM. (sipuleucel-T)
is a DC-based therapy, comprising exposing a patient's peripheral
blood mononuclear cells (PBMCs) to a fusion protein comprising a
tumor-derived antigen coupled to a cytokine, and then infusing the
PBMCs, presumably containing activated DCs that can present the
tumor-derived antigen to T cells, to the patient. See, U.S. Pat.
No. 6,210,662. Adoptive T-cell therapy involves isolating
tumor-infiltrating lymphocytes (TIL) from a patient's tumor,
expanding the TILs ex vivo, and infusing the TILs back to the
patient after depleting the patient's native non-myeloid
lymphocytes. See, Restifo N P et al. (2012) Nat. Rev. Immunol. 12:
269-81. T cells with engineered T cell receptors (TCR-T) or
chimeric antigen receptors (CAR-T) further expand the capacity of
adoptive T-cell therapy methods by modifying the microenvironment
of T cell-tumor interactions. Recently, a Multiple Antigen Specific
Cell Therapy ("MASCT") method has been designed to harness the
therapeutic capacity of both DCs and activated T cells in order to
provide a safe, durable and customizable treatment to cancer
patients. See, International Patent Application Publication No.
WO2016145578A1.
[0005] The disclosures of all publications, patents, patent
applications and published patent applications referred to herein
are hereby incorporated herein by reference in their entirety.
BRIEF SUMMARY OF THE INVENTION
[0006] The present application provides methods of preparing
activated T cells, and methods of treating cancer in an individual
using the activated T cells.
[0007] One aspect of the present application provides a method of
preparing a population of activated T cells, the method comprising:
a) contacting a population of dendritic cells with a plurality of
tumor antigen peptides to obtain a population of dendritic cells
loaded with the plurality of tumor antigen peptides; b) culturing
the population of dendritic cells loaded with the plurality of
tumor antigen peptides in a DC maturation medium comprising MPLA;
and c) co-culturing the population of dendritic cells loaded with
the plurality of tumor antigen peptides and a population of T
cells, thereby obtaining the population of activated T cells. In
some embodiments, step c) comprises co-culturing the population of
dendritic cells loaded with the plurality of tumor antigen peptides
and a population of T cells in a co-culture medium comprising an
interleukin cocktail, an immune checkpoint inhibitor and an
anti-CD3 antibody. In some embodiments, step c) comprises:
co-culturing the population of dendritic cells loaded with the
plurality of tumor antigen peptides and a population of T cells in
an initial co-culture medium comprising a plurality of cytokines
and an immune checkpoint inhibitor to provide a co-culture; and
adding an anti-CD3 antibody to the co-culture, thereby obtaining
the population of activated T cells. In some embodiments, the
anti-CD3 antibody is added to the co-culture at about 3 to 7 days
(such as about 5 days) after the co-culturing starts. In some
embodiments, the population of dendritic cells loaded with the
plurality of tumor antigen peptides and the population of T cells
are co-cultured for at least about 10 days in the presence of the
anti-CD3 antibody.
[0008] In some embodiments according to any one of the methods
described above, the DC maturation medium comprises INF.gamma. and
MPLA. In some embodiments, the DC maturation medium further
comprises PGE2. In some embodiments, the INF.gamma. is present in
the DC maturation medium at a concentration of at least about 100
IU/mL. In some embodiments, the MPLA is present in the DC
maturation medium at a concentration of at least about 0.5
.mu.g/mL. In some embodiments, the PGE2 is present in the DC
maturation medium at a concentration of at least about 0.1
.mu.g/mL.
[0009] One aspect of the present application provides a method of
preparing a population of activated T cells, the method comprising:
a) contacting a population of dendritic cells with a plurality of
tumor antigen peptides to obtain a population of dendritic cells
loaded with the plurality of tumor antigen peptides; b)
co-culturing the population of dendritic cells loaded with the
plurality of tumor antigen peptides and a population of T cells in
an initial co-culture medium comprising a plurality of cytokines
and an immune checkpoint inhibitor to provide a co-culture; and c)
adding an anti-CD3 antibody to the co-culture at about 3 to 7 days
(such as about 5 days) after the co-culturing starts, thereby
obtaining the population of activated T cells. In some embodiments,
the population of dendritic cells loaded with the plurality of
tumor antigen peptides and the population of T cells are
co-cultured for at least about 10 days in the presence of the
anti-CD3 antibody. In some embodiments, step a) further comprises
culturing the population of dendritic cells loaded with the
plurality of tumor antigen peptides in a DC maturation medium
comprising a toll-like receptor (TLR) agonist. In some embodiments,
the TLR agonist is selected from the group consisting of MPLA, Poly
I:C, resquimod, gardiquimod, and CL075.
[0010] In some embodiments according to any one of the methods
described above, the plurality of cytokines comprises IL-2, IL-7,
IL-15 and IL-21. In some embodiments, the IL-2 is present in the
initial co-culture medium at a concentration of at least about 500
IU/mL.
[0011] In some embodiments according to any one of the methods
described above, the immune checkpoint inhibitor is an anti-PD-1
antibody. In some embodiments, the anti-PD-1 antibody is present in
the initial co-culture medium at a concentration of at least about
10 .mu.g/mL.
[0012] In some embodiments according to any one of the methods
described above, the population of T cells is present in a
population of PBMCs.
[0013] In some embodiments according to any one of the methods
described above, the population of dendritic cells is obtained by
inducing differentiation of a population of monocytes from
PBMCs.
[0014] In some embodiments according to any one of the methods
described above, the population of dendritic cells and the
population of T cells are obtained from the same individual. In
some embodiments, the population of dendritic cells and the
population of T cells are derived from PBMCs of the same
individual.
[0015] In some embodiments according to any one of the methods
described above, the plurality of tumor antigen peptides is a
plurality of synthetic tumor antigen peptides. In some embodiments,
the plurality of tumor antigen peptides is not obtained from a cell
sample.
[0016] In some embodiments according to any one of the methods
described above, the plurality of tumor antigen peptides comprises
general tumor antigen peptide(s), cancer-type specific antigen
peptide(s), and/or neoantigen peptides. In some embodiments, the
plurality of tumor antigen peptides comprises one or more
neoantigen peptides. In some embodiments, the plurality of tumor
antigen peptides comprises (e.g., consists of) neoantigen peptides.
In some embodiments, the plurality of tumor antigen peptides
comprises at least about 5 (e.g., at least about 10, 20, 30, 40 or
more) different tumor antigen peptides.
[0017] Also provided is an isolated population of activated T cells
prepared using any one of the methods described above.
[0018] Further provided is a method of treating a cancer in an
individual, comprising administering to the individual an effective
amount of the activated T cells prepared using any one of the
methods described above. In some embodiments, the method further
comprises administering to the individual an effective amount of
dendritic cells loaded with the plurality of tumor antigen
peptides. In some embodiments, the population of dendritic cells
and the population of T cells are obtained from the individual
being treated. In some embodiments, the activated T cells are
administered to the individual for at least three times. In some
embodiments, the activated T cells are administered intravenously.
In some embodiments, the dendritic cells loaded with the plurality
of tumor antigen peptides are administered for at least three
times. In some embodiments, the dendritic cells loaded with the
plurality of tumor antigen peptides are administered
subcutaneously, intradermally or intravenously. In some
embodiments, the individual is a human individual. In some
embodiments, the cancer is a solid cancer, such as hepatocellular
carcinoma, gastric cancer, bladder cancer, soft tissue sarcoma,
colorectal cancer, endometrial cancer, or lung cancer.
[0019] Further provided are pharmaceutical compositions, kits, and
articles of manufacture for use in any one of the methods described
above.
[0020] These and other aspects and advantages of the present
invention will become apparent from the subsequent detailed
description and the appended claims. It is to be understood that
one, some, or all of the properties of the various embodiments
described herein may be combined to form other embodiments of the
present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1A shows expression levels of co-stimulatory molecules
on mature DCs after incubation in DC maturation media containing
various Toll-like receptors (TLRs) or combinations. FIG. 1B shows
secretion levels of cytokines by mature DCs. DC3/4: a DC maturation
medium comprising IL6, TNF.alpha., IL1.beta., and Poly I:C; I:
INF.gamma.; M: MPLA; P: Poly I:C; R: resquimod; G: gardiquimod; C:
CL075.
[0022] FIG. 2A shows numbers of mature DCs after incubation in DC
maturation media containing TLRs at different concentrations. FIG.
2B shows expression levels of co-stimulatory molecules on mature
DCs. FIG. 2C shows secretion levels of cytokines by mature DCs.
DC3/4: a DC maturation medium comprising IL6, TNF.alpha.,
IL1.beta., and Poly I:C; I: INF.gamma.; M1: MPLA, low
concentration; M2: MPLA, high concentration; R1: resquimod, low
concentration; R2: resquimod, high concentration; G1: gardiquimod,
low concentration; G2: gardiquimod, high concentration; CL1: CL075,
low concentration; CL2: CL075, high concentration.
[0023] FIG. 3A shows numbers of DCs after induction of maturation
by Toll-like receptor (TLR) compositions having different PGE2
concentrations. FIG. 3B shows secretion levels of co-stimulatory
molecules on induced antigen-loaded mature DCs. FIG. 3C shows
expression levels of cytokines by induced antigen-loaded mature
DCs. DC3/4: a DC maturation medium comprising IL6, TNF.alpha.,
IL1.beta., and Poly I:C; I: INF.gamma.; M: MPLA; P1: PGE2, low
concentration; P2: PGE2, medium concentration; P3: PGE2, high
concentration.
[0024] FIG. 4 shows percentages and numbers of tumor
antigen-specific T cells, which secreted IFN.gamma. after tumor
antigen peptides stimulation, in the co-cultures of antigen-loaded
mature DCs and T cells supplemented with IL-2 or an interleukin
cocktail (IL-2, IL-7, IL-15 and IL-21).
[0025] FIG. 5A shows percentages and numbers of tumor
antigen-specific T cells in cell preparations under various
conditions. CIK: PBMCs cultured with a high concentration of IL-2,
and an anti-CD3 antibody was added after 3 days of co-culturing;
previous MASCT: co-culture of antigen-loaded DCs and PBMCs with a
high concentration of IL-2, and an anti-CD3 antibody was added
after 3 days of co-culturing; 1: co-culture of antigen-loaded DCs
and PBMCs with an anti-PD1 antibody, an interleukin cocktail
(including a high concentration of IL-2), and an anti-CD3 antibody
added after 3 days of co-culturing; 2: co-culture of antigen-loaded
DCs and PBMCs with an anti-PD1 antibody, an interleukin cocktail
(including a high concentration of IL-2), and an anti-CD3 antibody
was added after 5 days of co-culturing.
[0026] FIG. 5B shows percentages and numbers of tumor
antigen-specific T cells in co-cultures of antigen-loaded DCs and
PBMCs with an anti-PD1 antibody, an interleukin cocktail (including
a low concentration or a high concentration of IL-2), and an
anti-CD3 antibody was added after 5 days of co-culturing.
[0027] FIG. 6A shows IL-2 secretion levels by induced
antigen-loaded mature DCs from healthy donors using different DC
preparation methods.
[0028] FIG. 6B shows IL-2 secretion levels by induced
antigen-loaded mature DCs from cancer patients using different DC
preparation methods.
[0029] FIG. 7 shows percentages of IFN-.gamma. producing tumor
antigen-specific T cells in co-cultures prepared using different
methods.
[0030] FIG. 8 shows anti-tumor effects of activated T cells
prepared using a previous MASCT protocol ("P") or an improved MASCT
protocol ("I").
[0031] FIG. 9 shows a schematic workflow of an exemplary neo-MASCT
treatment. The neo-MASCT treatment shown in this figure uses
antigen peptides derived from both neoantigens and tumor-associated
antigens (TAA). In some embodiments, the neo-MASCT treatment uses a
pool of antigen peptides derived only from neoantigens.
[0032] FIG. 10 shows a schematic workflow for designing neoantigen
peptides.
[0033] FIG. 11 shows rates of specific T-cell response against
neoantigen peptides in eight patients who responded to improved
MASCT treatments using the neoantigen peptides ("neo-MASCT").
[0034] FIG. 12 shows ELISPOT results of PBMCs from Patient #2
before and after improved MASCT treatments.
[0035] FIG. 13 shows ELISPOT results of PBMCs from Patient #2
before and after neo-MASCT treatments.
[0036] FIG. 14 shows exemplary ELISPOT results of PBMCs from
Patient #2 and patient #5 before and after neo-MASCT
treatments.
DETAILED DESCRIPTION OF THE INVENTION
[0037] The present application provides improved methods of
preparing activated T cells by co-culturing T cells with dendritic
cells (DCs) loaded with a plurality of tumor antigen peptides. In
some embodiments, the method uses a DC maturation medium comprising
MPLA to induce maturation of antigen-loaded DCs, a co-culture
medium comprising an interleukin cocktail and an immune checkpoint
inhibitor (e.g., anti-PD-1 antibody) for co-culture of T cells with
antigen-loaded DCs, and/or delayed addition of anti-CD3 antibody to
the co-culture to provide activated T cells with an enhanced
proportion of tumor antigen-specific T cells and a reduced
proportion of immunosuppressive regulatory T cells (Tregs). Using
the methods disclosed herein, IL-12 secretion by antigen-loaded
mature DCs derived from cancer patients increased by about 70
times, while the percentage of tumor antigen-specific T cells in
the co-culture increased by about 2-4 times compared to those using
exemplary protocols of a previously reported Multiple Antigen
Specific Cell Therapy ("MASCT" or "previous MASCT") method as
disclosed in WO2016145578A1. Methods for treating cancer using the
activated T cells described herein are referred to as the "improved
MASCT" methods. The improved MASCT methods offer enhanced treatment
efficacy and duration of response in patients having cancer,
including solid cancer.
[0038] Accordingly, one aspect of the present application provides
a method of preparing a population of activated T cells, the method
comprising: a) contacting a population of dendritic cells with a
plurality of tumor antigen peptides to obtain a population of
dendritic cells loaded with the plurality of tumor antigen
peptides; b) co-culturing the population of dendritic cells loaded
with the plurality of tumor antigen peptides and a population of T
cells in an initial co-culture medium comprising a plurality of
cytokines and an immune checkpoint inhibitor to provide a
co-culture, and c) adding an anti-CD3 antibody to the co-culture at
about 3 to 7 days after the co-culturing starts, thereby obtaining
the population of activated T cells.
[0039] Another aspect of the present application provides a method
of preparing a population of activated T cells, the method
comprising: a) contacting a population of dendritic cells with a
plurality of tumor antigen peptides to obtain a population of
dendritic cells loaded with the plurality of tumor antigen
peptides; b) culturing the population of dendritic cells loaded
with the plurality of tumor antigen peptides in a DC maturation
medium comprising MPLA; c) co-culturing the population of dendritic
cells loaded with the plurality of tumor antigen peptides and a
population of T cells, thereby obtaining the population of
activated T cells.
[0040] Activated T cells prepared using the methods described
herein, methods of treating cancer, compositions, kits, and
articles of manufacture are also provided.
I. DEFINITIONS
[0041] Terms are used herein as generally used in the art, unless
otherwise defined as follows.
[0042] As used herein, "treatment" or "treating" is an approach for
obtaining beneficial or desired results including clinical results.
For purposes of this invention, beneficial or desired clinical
results include, but are not limited to, one or more of the
following: decreasing one more symptoms resulting from the disease,
diminishing the extent of the disease, stabilizing the disease
(e.g., preventing or delaying the worsening of the disease),
preventing or delaying the spread (e.g., metastasis) of the
disease, preventing or delaying the occurrence or recurrence of the
disease, delay or slowing the progression of the disease,
ameliorating the disease state, providing a remission (whether
partial or total) of the disease, decreasing the dose of one or
more other medications required to treat the disease, delaying the
progression of the disease, increasing the quality of life, and/or
prolonging survival. Also encompassed by "treatment" is a reduction
of pathological consequence of cancer. The methods of the invention
contemplate any one or more of these aspects of treatment.
[0043] As used herein, "delaying" the development of cancer means
to defer, hinder, slow, retard, stabilize, and/or postpone
development of the disease. This delay can be of varying lengths of
time, depending on the history of the disease and/or individual
being treated. As is evident to one skilled in the art, a
sufficient or significant delay can, in effect, encompass
prevention, in that the individual does not develop the disease. A
method that "delays" development of cancer is a method that reduces
probability of disease development in a given time frame and/or
reduces the extent of the disease in a given time frame, when
compared to not using the method. Such comparisons are typically
based on clinical studies, using a statistically significant number
of individuals. Cancer development can be detectable using standard
methods, including, but not limited to, computerized axial
tomography (CAT Scan), Magnetic Resonance Imaging (MRI), abdominal
ultrasound, clotting tests, arteriography, or biopsy. Development
may also refer to cancer progression that may be initially
undetectable and includes occurrence, recurrence, and onset.
[0044] The terms "individual," "subject" and "patient" are used
interchangeably herein to describe a mammal, including humans. An
individual includes, but is not limited to, human, bovine, horse,
feline, canine, rodent, or primate. In some embodiments, the
individual is human. In some embodiments, an individual suffers
from a disease, such as cancer. In some embodiments, the individual
is in need of treatment.
[0045] As is understood in the art, an "effective amount" refers to
an amount of a composition (e.g. antigen-loaded DCs, or activated T
cells) sufficient to produce a desired therapeutic outcome (e.g.,
reducing the severity or duration of, stabilizing the severity of,
or eliminating one or more symptoms of cancer). For therapeutic
use, beneficial or desired results include, e.g., decreasing one or
more symptoms resulting from the disease (biochemical, histologic
and/or behavioral), including its complications and intermediate
pathological phenotypes presented during development of the
disease, increasing the quality of life of those suffering from the
disease, decreasing the dose of other medications required to treat
the disease, enhancing effect of another medication, delaying the
progression of the disease, and/or prolonging survival of
patients.
[0046] "Adjuvant setting" refers to a clinical setting in which an
individual has had a history of cancer, and generally (but not
necessarily) been responsive to therapy, which includes, but is not
limited to, surgery (e.g., surgery resection), radiotherapy, and
chemotherapy. However, because of their history of cancer, these
individuals are considered at risk of development of the disease.
Treatment or administration in the "adjuvant setting" refers to a
subsequent mode of treatment. The degree of risk (e.g., when an
individual in the adjuvant setting is considered as "high risk" or
"low risk") depends upon several factors, most usually the extent
of disease when first treated.
[0047] "Neoadjuvant setting" refers to a clinical setting in which
the method is carried out before the primary/definitive
therapy.
[0048] As used herein, "combination therapy" means that a first
agent is administered in conjunction with another agent. "In
conjunction with" refers to administration of one treatment
modality in addition to another treatment modality, such as
administration of activated T cells described herein in addition to
administration of another agent (such as an immune checkpoint
inhibitor) to the same individual. As such, "in conjunction with"
refers to administration of one treatment modality before, during,
or after delivery of the other treatment modality to the
individual. Such combinations are considered to be part of a single
treatment regimen or regime.
[0049] The term "simultaneous administration," as used herein,
means that a first therapy and second therapy in a combination
therapy are administered with a time separation of no more than
about 15 minutes, such as no more than about any of 10, 5, or 1
minutes. When the first and second therapies are administered
simultaneously, the first and second therapies may be contained in
the same composition (e.g., a composition comprising both a first
and second therapy) or in separate compositions (e.g., a first
therapy in one composition and a second therapy is contained in
another composition).
[0050] As used herein, the term "sequential administration" means
that the first therapy and second therapy in a combination therapy
are administered with a time separation of more than about 15
minutes, such as more than about any of 20, 30, 40, 50, 60, or more
minutes. Either the first therapy or the second therapy may be
administered first. The first and second therapies are contained in
separate compositions, which may be contained in the same or
different packages or kits.
[0051] As used herein, the term "concurrent administration" means
that the administration of the first therapy and that of a second
therapy in a combination therapy overlap with each other.
[0052] As used herein, by "pharmaceutically acceptable" or
"pharmacologically compatible" is meant a material that is not
biologically or otherwise undesirable, e.g., the material may be
incorporated into a pharmaceutical composition administered to an
individual without causing any significant undesirable biological
effects or interacting in a deleterious manner with any of the
other components of the composition in which it is contained.
Pharmaceutically acceptable carriers or excipients have preferably
met the required standards of toxicological and manufacturing
testing and/or are included on the Inactive Ingredient Guide
prepared by the U.S. Food and Drug administration.
[0053] As used herein, the terms "cell", "cell line", and "cell
culture" are used interchangeably and all such designations include
progeny. It is understood that all progeny may not be precisely
identical in DNA content, due to deliberate or inadvertent
mutations. Variant progeny that have the same function or
biological activity as the original cells are included.
[0054] The term "peptide" refers to a polymer of amino acids no
more than about 100 amino acids (including fragments of a protein),
which may be linear or branched, comprise modified amino acids,
and/or be interrupted by non-amino acids. The term also encompasses
an amino acid polymer that has been modified naturally or by
intervention, including, for example, disulfide bond formation,
glycosylation, lipidation, acetylation, phosphorylation, or any
other manipulation or modification. Also included within this term
are, for example, peptides containing one or more analogs of an
amino acid (including, for example, unnatural amino acids, etc.),
as well as other modifications known in the art. The peptides
described herein may be naturally-occurring, i.e., obtained or
derived from a natural source (e.g., blood) or synthesized (e.g.,
chemically synthesized or by synthesized by recombinant DNA
techniques).
[0055] As used herein, "a plurality of tumor antigen peptides,"
"multiple tumor antigen peptides," "a pool of tumor antigen
peptides" and "a tumor antigen peptides pool" are used
interchangeably to refer to a combination of two or more tumor
antigen peptides.
[0056] As used herein, "dendritic cells loaded with a plurality of
tumor antigen peptides" and "antigen-loaded dendritic cells" are
used interchangeably to refer to dendritic cells that have enhanced
presentation of one or more tumor antigen peptides among the
plurality of tumor antigen peptides.
[0057] As used herein, "activated T cells" refer to a population of
monoclonal (e.g. encoding the same TCR) or polyclonal (e.g. with
clones encoding different TCRs) T cells that have T cell receptors
that recognize at least one tumor antigen peptide. Activated T
cells may contain one or more subtypes of T cells, including, but
not limited to, cytotoxic T cells, helper T cells, natural killer T
cells, .gamma..delta. T cells, regulatory T cells, and memory T
cells.
[0058] As used herein, "immune checkpoint inhibitor" refers to an
agent (including an antibody) that inhibits or blocks an inhibitory
immune checkpoint molecule on an immune cell (such as T cell) or a
tumor cell. "Immune checkpoint molecules" include molecules that
turn up an immune signal (i.e., "co-stimulatory molecules"), or
molecules that turn down an immune signal (i.e., "inhibitory immune
checkpoint molecules") against a tumor cell.
[0059] The term "antibody" used herein is used in the broadest
sense and specifically covers monoclonal antibodies (including full
length monoclonal antibodies), multispecific antibodies (e.g.,
bispecific antibodies), and antibody fragments so long as they
exhibit the desired biological activity.
[0060] "Antibody fragments" comprise a portion of an intact
antibody, preferably comprising the antigen binding region thereof.
Examples of antibody fragments include Fab, Fab', F(ab').sub.2, and
Fv fragments; diabodies; linear antibodies; single-chain antibody
molecules; and multispecific antibodies formed from antibody
fragments.
[0061] As use herein, the term "specifically binds to,"
"recognizes," "specifically recognizes," "targets," or is "specific
for" refers to measurable and reproducible interactions such as
binding between a target and an antibody, or a receptor and a
ligand, or a receptor and an epitope/MHC complex, which is
determinative of the presence of the target in the presence of a
heterogeneous population of molecules including biological
molecules. For example, an antibody that binds to or specifically
binds to a target (which can be an epitope) is an antibody that
binds this target with greater affinity, avidity, more readily,
and/or with greater duration than it binds to other targets. In one
embodiment, the extent of binding of an antibody to an unrelated
target is less than about 10% of the binding of the antibody to the
target as measured, e.g., by a radioimmunoassay (RIA). In certain
embodiments, an antibody that specifically binds to an antigen
peptide (or an epitope) has a dissociation constant (Kd) of
.ltoreq.1 .mu.M, .ltoreq.100 nM, .ltoreq.10 nM, .ltoreq.1 nM, or
.ltoreq.0.1 nM. In certain embodiments, an antibody specifically
binds to an epitope on a protein that is conserved among the
protein from different species. In another embodiment, specific
binding can include, but does not require exclusive binding.
[0062] It is understood that aspect and embodiments of the
invention described herein include "consisting" and/or "consisting
essentially of" aspects and embodiments.
[0063] Reference to "about" a value or parameter herein includes
(and describes) variations that are directed to that value or
parameter per se. For example, description referring to "about X"
includes description of "X".
[0064] The term "about X-Y" used herein has the same meaning as
"about X to about Y."
[0065] As used herein, reference to "not" a value or parameter
generally means and describes "other than" a value or parameter.
For example, the method is not used to treat cancer of type X means
the method is used to treat cancer of types other than X.
[0066] As used herein and in the appended claims, the singular
forms "a," "an," and "the" include plural referents unless the
context clearly dictates otherwise.
II. METHODS OF PREPARING ACTIVATED T CELLS
[0067] The present application provides methods of preparing
activated T cells with enhanced proportion of tumor
antigen-specific T cells and/or reduced proportion of
immunosuppressive regulatory T cells (Tregs). In some embodiments,
tumor antigen-specific T cells are activated T cells that elicit
specific response to one or more of the tumor antigen peptides used
to load the DCs. Methods of detecting and quantifying such tumor
antigen-specific T cells are known in the art, including, but not
limited to, detection by staining with pentamers and other
multimers (such as dextramers), or detection of
IFN.gamma.-production by T cells after stimulation by tumor antigen
peptides.
[0068] Accordingly, in some embodiments, there is provided a method
of preparing a population of activated T cells, the method
comprising: a) co-culturing a population of dendritic cells loaded
with a plurality of tumor antigen peptides and a population of T
cells in an initial co-culture medium comprising a plurality of
cytokines and an immune checkpoint inhibitor to provide a
co-culture; and c) adding an anti-CD3 antibody to the co-culture at
about 3 to 7 days after the co-culturing starts, thereby obtaining
the population of activated T cells. In some embodiments, the
plurality of cytokines comprises IL-2, IL-7, IL-15 and IL-21. In
some embodiments, the IL-2 is present in the initial co-culture
medium at a concentration of at least about 500 IU/mL. In some
embodiments, the immune checkpoint inhibitor is an anti-PD-1
antibody. In some embodiments, the anti-PD-1 antibody is present in
the initial co-culture medium at a concentration of at least about
10 .mu.g/mL. In some embodiments, the anti-CD3 antibody is added to
the co-culture at about 5 days after the co-culturing starts. In
some embodiments, the population of dendritic cells loaded with the
plurality of tumor antigen peptides and the population of T cells
are co-cultured for at least about 10 days in the presence of the
anti-CD3 antibody. In some embodiments, the population of T cells
is present in a population of PBMCs.
[0069] In some embodiments, there is provided a method of preparing
a population of activated T cells, the method comprising: a)
contacting a population of dendritic cells with a plurality of
tumor antigen peptides to obtain a population of dendritic cells
loaded with the plurality of tumor antigen peptides; b)
co-culturing the population of dendritic cells loaded with the
plurality of tumor antigen peptides and a population of T cells in
an initial co-culture medium comprising a plurality of cytokines
and an immune checkpoint inhibitor to provide a co-culture; and c)
adding an anti-CD3 antibody to the co-culture at about 3 to 7 days
after the co-culturing starts, thereby obtaining the population of
activated T cells. In some embodiments, the plurality of cytokines
comprises IL-2, IL-7, IL-15 and IL-21. In some embodiments, the
IL-2 is present in the initial co-culture medium at a concentration
of at least about 500 IU/mL. In some embodiments, the immune
checkpoint inhibitor is an anti-PD-1 antibody. In some embodiments,
the anti-PD-1 antibody is present in the initial co-culture medium
at a concentration of at least about 10 .mu.g/mL. In some
embodiments, the anti-CD3 antibody is added to the co-culture at
about 5 days after the co-culturing starts. In some embodiments,
the population of dendritic cells loaded with the plurality of
tumor antigen peptides and the population of T cells are
co-cultured for at least about 10 days in the presence of the
anti-CD3 antibody. In some embodiments, the population of T cells
is present in a population of PBMCs. In some embodiments, the
population of dendritic cells and the population of T cells are
obtained from the same individual.
[0070] In some embodiments, there is provided a method of preparing
a population of activated T cells, the method comprising: a)
contacting a population of dendritic cells with a plurality of
tumor antigen peptides to obtain a population of dendritic cells
loaded with the plurality of tumor antigen peptides; b) culturing
the population of dendritic cells loaded with the plurality of
tumor antigen peptides in a DC maturation medium comprising a
toll-like receptor (TLR) agonist; (c) co-culturing the population
of dendritic cells loaded with the plurality of tumor antigen
peptides and a population of T cells in an initial co-culture
medium comprising a plurality of cytokines and an immune checkpoint
inhibitor to provide a co-culture; and d) adding an anti-CD3
antibody to the co-culture at about 3 to 7 days after the
co-culturing starts, thereby obtaining the population of activated
T cells. In some embodiments, the TLR agonist is selected from the
group consisting of MPLA, Poly I:C, resquimod, gardiquimod, and
CL075. In some embodiments, the DC maturation medium comprises
PGE2. In some embodiments, the PGE2 is present in the DC maturation
medium at a concentration of at least about 0.1 .mu.g/mL. In some
embodiments, the plurality of cytokines comprises IL-2, IL-7, IL-15
and IL-21. In some embodiments, the IL-2 is present in the initial
co-culture medium at a concentration of at least about 500 IU/mL.
In some embodiments, the immune checkpoint inhibitor is an
anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is
present in the initial co-culture medium at a concentration of at
least about 10 .mu.g/mL. In some embodiments, the anti-CD3 antibody
is added to the co-culture at about 5 days after the co-culturing
starts. In some embodiments, the population of dendritic cells
loaded with the plurality of tumor antigen peptides and the
population of T cells are co-cultured for at least about 10 days in
the presence of the anti-CD3 antibody. In some embodiments, the
population of T cells is present in a population of PBMCs. In some
embodiments, the population of dendritic cells and the population
of T cells are obtained from the same individual.
[0071] In some embodiments, there is provided a method of preparing
a population of activated T cells, the method comprising: a)
contacting a population of dendritic cells with a plurality of
tumor antigen peptides to obtain a population of dendritic cells
loaded with the plurality of tumor antigen peptides; b) culturing
the population of dendritic cells loaded with the plurality of
tumor antigen peptides in a DC maturation medium comprising MPLA;
and c) co-culturing the population of dendritic cells loaded with
the plurality of tumor antigen peptides and a population of T
cells, thereby obtaining the population of activated T cells. In
some embodiments, step c) comprises co-culturing the population of
dendritic cells loaded with the plurality of tumor antigen peptides
and a population of T cells in a co-culture medium comprising an
interleukin cocktail, an immune checkpoint inhibitor and an
anti-CD3 antibody. In some embodiments, the population of dendritic
cells loaded with the plurality of tumor antigen peptides and the
population of T cells are co-cultured for at least about 10 days in
the presence of the anti-CD3 antibody. In some embodiments, the DC
maturation medium comprises INF.gamma. and MPLA. In some
embodiments, the DC maturation medium further comprises PGE2. In
some embodiments, the MPLA is present in the DC maturation medium
at a concentration of at least about 0.5 .mu.g/mL. In some
embodiments, the INF.gamma. is present in the DC maturation medium
at a concentration of at least about 100 IU/mL. In some
embodiments, the PGE2 is present in the DC maturation medium at a
concentration of at least about 0.1 .mu.g/mL. In some embodiments,
the plurality of cytokines comprises IL-2, IL-7, IL-15 and IL-21.
In some embodiments, the IL-2 is present in the co-culture medium
at a concentration of at least about 500 IU/mL. In some
embodiments, the immune checkpoint inhibitor is an anti-PD-1
antibody. In some embodiments, the anti-PD-1 antibody is present in
the co-culture medium at a concentration of at least about 10
.mu.g/mL. In some embodiments, the population of dendritic cells
loaded with the plurality of tumor antigen peptides and the
population of T cells are co-cultured for at least about 10 days in
the presence of the anti-CD3 antibody. In some embodiments, the
population of T cells is present in a population of PBMCs. In some
embodiments, the population of dendritic cells and the population
of T cells are obtained from the same individual.
[0072] In some embodiments, there is provided a method of preparing
a population of activated T cells, the method comprising: a)
contacting a population of dendritic cells with a plurality of
tumor antigen peptides to obtain a population of dendritic cells
loaded with the plurality of tumor antigen peptides; b) culturing
the population of dendritic cells loaded with the plurality of
tumor antigen peptides in a DC maturation medium comprising MPLA;
c) co-culturing the population of dendritic cells loaded with the
plurality of tumor antigen peptides and a population of T cells in
an initial co-culture medium comprising a plurality of cytokines
and an immune checkpoint inhibitor to provide a co-culture; and d)
adding an anti-CD3 antibody to the co-culture, thereby obtaining
the population of activated T cells. In some embodiments, the
anti-CD3 antibody is added to the co-culture when the co-culturing
starts. In some embodiments, the anti-CD3 antibody is added to the
co-culture after the co-culturing starts. In some embodiments, the
DC maturation medium comprises INF.gamma. and MPLA. In some
embodiments, the DC maturation medium further comprises PGE2. In
some embodiments, the MPLA is present in the DC maturation medium
at a concentration of at least about 0.5 .mu.g/mL. In some
embodiments, the INF.gamma. is present in the DC maturation medium
at a concentration of at least about 100 IU/mL. In some
embodiments, the PGE2 is present in the DC maturation medium at a
concentration of at least about 0.1 .mu.g/mL. In some embodiments,
the plurality of cytokines comprises IL-2, IL-7, IL-15 and IL-21.
In some embodiments, the IL-2 is present in the initial co-culture
medium at a concentration of at least about 500 IU/mL. In some
embodiments, the immune checkpoint inhibitor is an anti-PD-1
antibody. In some embodiments, the anti-PD-1 antibody is present in
the initial co-culture medium at a concentration of at least about
10 .mu.g/mL. In some embodiments, the population of dendritic cells
loaded with the plurality of tumor antigen peptides and the
population of T cells are co-cultured for at least about 10 days in
the presence of the anti-CD3 antibody. In some embodiments, the
population of T cells is present in a population of PBMCs. In some
embodiments, the population of dendritic cells and the population
of T cells are obtained from the same individual.
[0073] In some embodiments, there is provided a method of preparing
a population of activated T cells, the method comprising: a)
contacting a population of dendritic cells with a plurality of
tumor antigen peptides to obtain a population of dendritic cells
loaded with the plurality of tumor antigen peptides; b) culturing
the population of dendritic cells loaded with the plurality of
tumor antigen peptides in a DC maturation medium comprising MPLA;
c) co-culturing the population of dendritic cells loaded with the
plurality of tumor antigen peptides and a population of T cells in
an initial co-culture medium comprising a plurality of cytokines
and an immune checkpoint inhibitor to provide a co-culture; and d)
adding an anti-CD3 antibody to the co-culture at about 3 to 7 days
after the co-culturing starts, thereby obtaining the population of
activated T cells. In some embodiments, the DC maturation medium
comprises INF.gamma. and MPLA. In some embodiments, the DC
maturation medium further comprises PGE2. In some embodiments, the
MPLA is present in the DC maturation medium at a concentration of
at least about 0.5 .mu.g/mL. In some embodiments, the INF.gamma. is
present in the DC maturation medium at a concentration of at least
about 100 IU/mL. In some embodiments, the PGE2 is present in the DC
maturation medium at a concentration of at least about 0.1
.mu.g/mL. In some embodiments, the plurality of cytokines comprises
IL-2, IL-7, IL-15 and IL-21. In some embodiments, the IL-2 is
present in the initial co-culture medium at a concentration of at
least about 500 IU/mL. In some embodiments, the immune checkpoint
inhibitor is an anti-PD-1 antibody. In some embodiments, the
anti-PD-1 antibody is present in the initial co-culture medium at a
concentration of at least about 10 .mu.g/mL. In some embodiments,
the anti-CD3 antibody is added to the co-culture at about 5 days
after the co-culturing starts. In some embodiments, the population
of dendritic cells loaded with the plurality of tumor antigen
peptides and the population of T cells are co-cultured for at least
about 10 days in the presence of the anti-CD3 antibody. In some
embodiments, the population of T cells is present in a population
of PBMCs. In some embodiments, the population of dendritic cells
and the population of T cells are obtained from the same
individual.
[0074] In some embodiments, there is provided a method of preparing
a population of activated T cells, the method comprising: a)
contacting a population of dendritic cells with a plurality of
tumor antigen peptides to obtain a population of dendritic cells
loaded with the plurality of tumor antigen peptides; b) culturing
the population of dendritic cells loaded with the plurality of
tumor antigen peptides in a DC maturation medium comprising MPLA,
INF.gamma. and PGE2; c) co-culturing the population of dendritic
cells loaded with the plurality of tumor antigen peptides and a
population of T cells in an initial co-culture medium comprising a
plurality of cytokines comprising IL-2, IL-7, IL-15 and IL-21 and
an anti-PD-1 antibody to provide a co-culture; and d) adding an
anti-CD3 antibody to the co-culture at about 3 to 7 days (e.g.,
about 5 days) after the co-culturing starts, thereby obtaining the
population of activated T cells. In some embodiments, the MPLA is
present in the DC maturation medium at a concentration of at least
about 0.5 .mu.g/mL. In some embodiments, the INF.gamma. is present
in the DC maturation medium at a concentration of at least about
100 IU/mL. In some embodiments, the PGE2 is present in the DC
maturation medium at a concentration of at least about 0.1
.mu.g/mL. In some embodiments, the IL-2 is present in the initial
co-culture medium at a concentration of at least about 500 IU/mL.
In some embodiments, the anti-PD-1 antibody is present in the
initial co-culture medium at a concentration of at least about 10
.mu.g/mL. In some embodiments, the population of dendritic cells
loaded with the plurality of tumor antigen peptides and the
population of T cells are co-cultured for at least about 10 days in
the presence of the anti-CD3 antibody. In some embodiments, the
population of T cells is present in a population of PBMCs. In some
embodiments, the population of dendritic cells and the population
of T cells are obtained from the same individual.
[0075] Also provided are isolated population of activated T cells
and isolated population of dendritic cells loaded with a plurality
of tumor antigen peptides prepared using any of the methods
described herein.
Antigen Loading of Dendritic Cells
[0076] The methods of preparing tumor-antigen specific T cells use
dendritic cells loaded with a plurality of tumor antigen peptides.
In some embodiments, the dendritic cells loaded with a plurality of
tumor antigen peptides are freshly prepared. The improved MASCT
methods described herein may comprise one or more of the following
steps: (1) obtaining PBMCs from an individual; (2) obtaining a
population of monocytes from the PBMCs; (3) inducing
differentiation of the population of monocytes into immature DCs;
(4) contacting the immature DCs with a plurality of tumor antigen
peptides to obtain a population of DCs loaded with the plurality of
tumor antigen peptides; and (5) culturing the population of DCs
loaded with the plurality of tumor antigen peptides in a DC
maturation medium comprising a TLR agonist (such as MPLA).
[0077] In some embodiments, the dendritic cells loaded with a
plurality of tumor antigen peptides are prepared by: (a) contacting
a population of dendritic cells with a plurality of tumor antigen
peptides to obtain a population of dendritic cells loaded with the
plurality of tumor antigen peptides, and (b) culturing the
population of dendritic cells loaded with the plurality of tumor
antigen peptides in a DC maturation medium comprising a toll-like
receptor (TLR) agonist. Exemplary TLR agonists include, but are not
limited to, MPLA (monophosphoryl lipid A), Poly I:C, resquimod,
gardiquimod, and CL075. Cytokines and other appropriate molecules,
such as INF.gamma. and PGE2 (prostaglandin E2) may be further
included in the culturing media in the maturation step.
[0078] In some embodiments, the dendritic cells loaded with a
plurality of tumor antigen peptides are prepared by: (a) contacting
a population of dendritic cells with a plurality of tumor antigen
peptides to obtain a population of dendritic cells loaded with the
plurality of tumor antigen peptides, and (b) culturing the
population of dendritic cells loaded with the plurality of tumor
antigen peptides in a DC maturation medium comprising MPLA,
INF.gamma. and PGE2.
[0079] In some embodiments, the dendritic cells loaded with a
plurality of tumor antigen peptides are prepared by: (a) inducing
differentiation of a population of monocytes into immature DCs; (b)
contacting a population of immature dendritic cells with a
plurality of tumor antigen peptides to obtain a population of
dendritic cells loaded with the plurality of tumor antigen
peptides; and (c) culturing the population of dendritic cells
loaded with the plurality of tumor antigen peptides in a DC
maturation medium comprising MPLA, INF.gamma. and PGE2. In some
embodiments, the population of monocytes is obtained from
PBMCs.
[0080] The DC maturation medium may comprise a suitable
concentration of MPLA, INF.gamma. and/or PGE2. In some embodiments,
the DC maturation medium comprises MPLA at a concentration of at
least about 0.5 .mu.g/mL, such as at least about any one of 1, 2,
3, 4, 5, 6, 7, 8, 9, 10 or more .mu.g/mL. In some embodiments, the
DC maturation medium comprises MPLA at a concentration of any one
of about 0.5-10, 1-5, 5-10, or 2.5-7.5 .mu.g/mL. In some
embodiments, the DC maturation medium comprises INF.gamma. at a
concentration of at least about 100 IU/mL, such as at least about
any one of 150, 200, 250, 300, 400, 500, 600, 800, 1000 or more
IU/mL. In some embodiments, the DC maturation medium comprises
INF.gamma. at a concentration of about any one of 100-1000,
100-250, 250-500, 500-1000, or 250-750 IU/mL. In some embodiments,
the DC maturation medium comprises PGE2 at a concentration of at
least about 0.1 .mu.g/mL, such as at least about any one of 0.15,
0.2, 0.25, 0.3, 0.4, 0.5, or more .mu.g/mL. In some embodiments,
the DC maturation medium comprises PGE2 at a concentration of about
any one of 0.1-0.5, 0.1-0.3, 0.25-0.5 or 0.2-0.4 .mu.g/mL.
[0081] The immature dendritic cells loaded with a plurality of
tumor antigen peptides may be induced by TLR agonists to mature for
at least about any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20
days. In some embodiments, the dendritic cells loaded with a
plurality of tumor antigen peptides are induced to mature for about
8 days.
[0082] In some embodiments, the antigen-loaded dendritic cells are
mature dendritic cells that present one or more tumor antigen
peptides of the plurality of tumor antigen peptides. The mature
dendritic cells prepared by any of the methods described herein may
present at least about any one of 1, 5, 10, 15, 20, 25, 30, 35, 40,
50, 60, 70, 80 or more tumor antigen peptides. Compared to naive
dendritic cells, or dendritic cells that have not been loaded with
a plurality of tumor antigen peptides, the multiple-antigen loaded
dendritic cells may have enhanced level of presentation for at
least about any of 1, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80
or more tumor antigen peptides. In some embodiments, the mature
dendritic cells have enhanced level of presentation for more than
10 tumor antigen peptides. In some embodiments, the mature
dendritic cells have enhanced level of presentation of about any of
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more tumor
antigen peptides derived from proteins selected from the group
consisting of hTERT, p53, Survivin, NY-ESO-1, CEA, CCND1, RGS5,
MMP7, VEGFR1, VEGFR2, MUC1, HER2, MAGE-A1, MAGE-A3, CDCA1, WT1,
KRAS, PARP4, MLL3, MTHFR HBcAg, HBV polymerase, GPC3, SSX, and
AFP.
[0083] In some embodiments, the antigen-loaded dendritic cells are
prepared by pulsing the plurality of tumor antigen peptides into
the population of dendritic cells, such as immature dendritic
cells, or dendritic cells contained in or derived (such as
differentiated) from the PBMCs. As known in the art, pulsing refers
to a process of mixing cells, such as dendritic cells, with a
solution containing antigen peptides, and optionally subsequently
removing the antigen peptides from the mixture. The population of
dendritic cells may be contacted with a plurality of tumor antigen
peptides for seconds, minutes, or hours, such as about at least any
one of 30 seconds, 1 minute, 5 minutes, 10 minutes, 15 minutes, 20
minutes, 30 minutes, 1 hour, 5 hours, 10 hours, 12 hours, 14 hours,
16 hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days, 3 days, 4
days, 5 days, 6 days, one week, 10 days, or more. The concentration
of each tumor antigen peptide used in the contacting step may be at
least about any one of 0.1, 0.5, 1, 2, 3, 5, or 10 .mu.g/mL. In
some embodiments, the concentration of the tumor antigen peptides
is about 0.1-200 .mu.g/mL, including for example about any of
0.1-0.5, 0.5-1, 1-10, 10-50, 50-100, 100-150, or 150-200
.mu.g/mL.
[0084] In some embodiments, the population of dendritic cells is
contacted with the plurality of tumor antigen peptides in the
presence of a composition that facilitates the uptake of the
plurality of tumor antigen peptides by the dendritic cells. In some
embodiments, compounds, materials or compositions may be included
in a solution of the plurality of tumor antigen peptides to
facilitate peptide uptake by the dendritic cells. Compounds,
materials or compositions that facilitate the uptake of the
plurality of tumor antigen peptides by the dendritic cells include,
but are not limited to, lipid molecules and peptides with multiple
positively charged amino acids. In some embodiments, more than
about any of 50%, 60%, 70%, 80%, 90%, or 95% of the tumor antigen
peptides are uptaken by the population of dendritic cells. In some
embodiments, more than about any of 50%, 60%, 70%, 80%, 90%, or 95%
of the dendritic cells in the population uptake at least one tumor
antigen peptide.
[0085] Dendritic cells (such as immature dendritic cells) may be
obtained from various sources, including autologous sources, i.e.
from the individual receiving the treatment. A convenient source of
dendritic cells is the PBMCs from the peripheral blood. For
example, monocytes, a type of white blood cells, are abundant in
PBMCs, comprising about 5-30% of total PBMCs. Monocytes can be
induced to differentiate into dendritic cells, such as immature
dendritic cells, using cytokines. In some embodiments, the immature
dendritic cells are prepared by obtaining a population of PBMCs,
obtaining a population of monocytes from the population of PBMCs,
and contacting the population of monocytes with a plurality of
cytokines to obtain a population of immature dendritic cells.
Exemplary cytokines that may be used to induce differentiation of
monocytes include, but are not limited to, GM-C SF and IL-4, with
conditions (such as concentrations, temperature, CO.sub.2 level
etc.) known in the art.
[0086] The adherent fraction of PBMCs contains the majority of
monocytes in PBMCs. In some embodiments, the monocytes from the
adherent fraction of PBMCs are contacted with cytokines to obtain a
population of immature dendritic cells. PBMCs can be conveniently
obtained by centrifugation of a sample of peripheral blood, or
using apheresis methods to collect from an individual. In some
embodiments, the population of PBMCs is obtained by density
gradient centrifugation of a sample of human peripheral blood. In
some embodiments, the sample is from the individual that receives
the multiple-antigen loaded dendritic cells, activated T cells, or
other immunotherapeutic compositions prepared using the
multiple-antigen loaded dendritic cells.
[0087] Further provided by the present application is an isolated
population of dendritic cells prepared by any of the embodiments of
the methods described herein. In some embodiments, the isolated
population of dendritic cells is capable of eliciting
MHC-restricted T cell response in vivo or ex vivo. In some
embodiments, the MHC-restricted T cell response is mediated by both
MHC class I and MHC class II molecules. In some embodiments, the
isolated population of dendritic cells is capable of inducing
differentiation and proliferation of tumor antigen-specific T
cells.
Preparation of Activated T Cells
[0088] The methods described herein for preparing activated T cells
comprise co-culturing a population of T cells with a population of
dendritic cells loaded with a plurality of tumor antigen peptides.
In some embodiments, the population of dendritic cells loaded with
the plurality of tumor antigen peptides and the population of T
cells are cultured in a co-culture medium comprising a plurality of
cytokines, an immune checkpoint inhibitor, and an anti-CD3
antibody. In some embodiments, the activated T cells are prepared
by: (a) co-culturing the population of dendritic cells loaded with
a plurality of tumor antigen peptides and a population of T cells
in an initial co-culture medium comprising a plurality of cytokines
and an immune checkpoint inhibitor to provide a co-culture; and (b)
adding an anti-CD3 antibody to the co-culture.
[0089] In some embodiments, the co-culture medium (including the
initial co-culture medium) comprises a plurality of cytokines (also
referred herein as "cytokine cocktail"). Exemplary cytokines
include, but are not limited to, IL-2, IL-7, IL-15, IL-21 and the
like. In some embodiments, the co-culture medium (including the
initial co-culture medium) comprises IL-2, IL-7, IL-15 and IL-21.
In some embodiments, each cytokine in the cytokine cocktail is
present at a concentration of at least about any one of 0.1, 0.5,
1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50 or higher ng/mL. In some
embodiments, each cytokine in the cytokine cocktail is present at a
concentration of about any one of 0.1-1, 1-5, 5-10, 10-20, 20-30,
1-30, 1-10, 30-50 or 1-50 ng/mL. In some embodiments, the IL-2 is
present at least about any of 100, 200, 300, 400, 500, 600, 700,
800, 900, 1000, 1500, or 2000 IU/ml in the co-culture medium
(including the initial co-culture medium). In some embodiments, the
IL-2 is present at a concentration of about any one of 100-500,
500-1000, 1000-1500, 1500-2000, 100-2000, 500-1500, 100-1000 or
1000-2000 IU/ml. The cytokines may facilitate activation,
maturation, and/or proliferation of the T cells, to prime T cells
for later differentiation into effector T cells and memory T cells,
and/or suppress the percentage of T.sub.REG in the population of
activated T cells in the co-culture.
[0090] In some embodiments, the co-culture medium (including the
initial co-culture medium) comprises one or more (such as any of 1,
2, 3, or more) immune checkpoint inhibitors. Any known immune
checkpoint inhibitors may be used. In some embodiments, the immune
checkpoint inhibitor is a natural or engineered ligand of an
inhibitory immune checkpoint molecule, including, for example,
ligands of CTLA-4 (e.g., B7.1, B7.2), ligands of TIM-3 (e.g.,
Galectin-9), ligands of A2a Receptor (e.g., adenosine,
Regadenoson), ligands of LAG-3 (e.g., MHC class I or MHC class II
molecules), ligands of BTLA (e.g., HVEM, B7-H4), ligands of KIR
(e.g., MHC class I or MHC class II molecules), ligands of PD-1
(e.g., PD-L1, PD-L2), ligands of IDO (e.g., NKTR-218, Indoximod,
NLG919), and ligands of CD47 (e.g., SIRP-alpha receptor).
[0091] The immune checkpoint inhibitors may be of any suitable
molecular modality, including, but not limited to, small molecules,
nucleic acids (such as DNA, RNAi, or aptamer), peptides, or
proteins (such as antibodies).
[0092] In some embodiments, the immune checkpoint inhibitor is an
antibody (such as antagonist antibody) that targets an inhibitory
immune checkpoint protein selected from the group consisting of
anti-CTLA-4 (e.g., Ipilimumab, Tremelimumab, KAHR-102), anti-TIM-3
(e.g., F38-2E2, ENUM005), anti-LAG-3 (e.g., BMS-986016, IMP701,
IMP321, C9B7W), anti-KIR (e.g., Lirilumab and IPH2101), anti-PD-1
(e.g., Nivolumab, Pidilizumab, Pembrolizumab, BMS-936559,
atezolizumab, Pembrolizumab, MK-3475, AMP-224, AMP-514, STI-A1110,
TSR-042), anti-PD-L1 (e.g., KY-1003 (EP20120194977), MCLA-145,
RG7446, BMS-936559, MEDI-4736, MSB0010718C, AUR-012, STI-A1010,
PCT/US2001/020964, MPDL3280A, AMP-224, Dapirolizumab pegol
(CDP-7657), MEDI-4920), anti-CD73 (e.g., AR-42
(OSU-HDAC42,HDAC-42,AR42,AR 42,OSU-HDAC 42,OSU-HDAC-42,NSC
D736012,HDAC-42,HDAC 42,HDAC42,NSCD736012,NSC-D736012), MEDI-9447),
anti-B7-H3 (e.g., MGA271, DS-5573a, 8H9), anti-CD47 (e.g.,
CC-90002, TTI-621, VLST-007), anti-BTLA, anti-VISTA, anti-A2aR,
anti-B7-1, anti-B7-H4, anti-CD52 (such as alemtuzumab), anti-IL-10,
anti-IL-35, and anti-TGF-.beta. (such as Fresolumimab). In some
embodiments, the antibody is a monoclonal antibody. In some
embodiments, the antibody is a full-length antibody. In some
embodiments, the antibody is an antigen-binding fragment selected
from the group consisting of Fab, Fab', F(ab').sub.2, Fv, scFv,
BiTE, nanobody, and other antigen-binding subsequences of the full
length antibody or engineered combinations thereof. In some
embodiments, the antibody is a human antibody, a humanized
antibody, or a chimeric antibody. In some embodiments, the antibody
is a bispecific or multispecific antibody.
[0093] In some embodiments, the immune checkpoint inhibitor is an
inhibitor of PD-1. In some embodiments, the immune checkpoint
inhibitor is an anti-PD-1 antibody. Exemplary anti-PD-1 antibodies
include, but are not limited to, Nivolumab, pembrolizumab,
pidilizumab, BMS-936559, and atezolizumab, Pembrolizumab, MK-3475,
AMP-224, AMP-514, STI-A1110, and TSR-042. In some embodiments, the
immune checkpoint inhibitor is nivolumab (for example,
OPDIVO.RTM.). In some embodiments, the immune checkpoint inhibitor
is Pembrolizumab (for example, KEYTRUDA.RTM.). In some embodiments,
the immune checkpoint inhibitor is SHR-1210. In some embodiments,
the initial co-culture medium comprises IL-2, IL-7, IL-15, IL-21
and an anti-PD-1 antibody (e.g., SHR-1210).
[0094] A suitable concentration of the immune checkpoint inhibitor
(e.g., anti-PD-1 antibody) in the co-culture medium (including the
initial co-culture medium) include, but are not limited to, at
least about any of 1, 2, 5, 10, 15, 20, 25 or more .mu.g/mL. In
some embodiments, the immune checkpoint inhibitor (e.g., anti-PD-1
antibody) is present in the co-culture medium (including the
initial co-culture medium) is any one of about 1 .mu.g/mL to about
10 .mu.g/mL, about 10 .mu.g/mL to about 20 .mu.g/mL, about 1
.mu.g/mL to about 25 .mu.g/mL, or about 5 .mu.g/mL to about 20
.mu.g/mL.
[0095] The anti-CD3 antibody may be present in the co-culture at
the time the co-culturing starts, or added to the co-culture after
the co-culturing of the antigen-loaded dendritic cells and the T
cells starts. In some embodiments, the anti-CD3 antibody is
included in the co-culture medium (including the initial co-culture
medium). In some embodiments, the initial co-culture medium does
not comprise the anti-CD3 antibody. In some embodiments, the
anti-CD3 antibody is added to the co-culture at about any one of 1,
2, 3, 4, 5, 6, 7, or more days after the co-culturing starts. In
some embodiments, the anti-CD3 antibody is added to the co-culture
at about any one of 1-7, 1-3, 3-5, 5-7 or 3-7 days after the
co-culturing starts. In some embodiments, the anti-CD3 antibody is
added to the co-culture at about 5 days after the co-culturing
starts. Any suitable anti-CD3 antibody may be used, including, but
not limited to OKT3.
[0096] In some embodiments, the population of T cells and the
population of dendritic cells are derived from the same individual,
such as an individual with a cancer (e.g., low to moderate grade
cancer). In some embodiments, the population of T cells, the
population of dendritic cells, or both is derived from autologous
sources, i.e., from the individual that receives the activated T
cells, the multiple-antigen loaded dendritic cells, or both.
[0097] In some embodiments, the population of T cells and the
population of dendritic cells loaded with the plurality of tumor
antigen peptides are co-cultured for at least about any one of 2,
4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 days. In
some embodiments, the population of T cells is co-cultured with the
population of dendritic cells loaded with the plurality of tumor
antigen peptides for about 7 days to about 21 days, such as about
any one of 7-14, 14-21, 12-18, 10-16, 10, 14 , 16, 18 , or 21 days.
In some embodiments, the population of T cells is co-cultured with
the population of dendritic cells loaded with the plurality of
tumor antigen peptides for about 10 days. In some embodiments, the
population of T cells is co-cultured with the population of
dendritic cells loaded with the plurality of tumor antigen peptides
for about 14 days.
[0098] In some embodiments, the population of T cells and the
population of dendritic cells loaded with the plurality of tumor
antigen peptides are co-cultured in the presence of the anti-CD3
antibody for at least about any one of 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 12, 15, 20, or more days. In some embodiments, the population
of T cells and the population of dendritic cells loaded with the
plurality of tumor antigen peptides are co-cultured in the presence
of the anti-CD3 antibody for about any one of 1-20, 5-15, 1-10, or
10-20 days.
[0099] The population of T cells used in any embodiment of the
methods described herein may be derived from a variety of sources.
A convenient source of T cells is from the PBMCs of the human
peripheral blood. The population of T cells may be isolated from
the PBMCs, or alternatively, a population of PBMCs enriched with T
cells (such as by addition of T cell specific antibodies and
cytokines) can be used in the co-culture. In some embodiments, the
PBMCs are obtained by density gradient centrifugation of a sample
of peripheral blood. In some embodiments, the population of T cells
is present in the PBMCs. In some embodiments, PBMCs are used in the
co-culture.
[0100] Further provided by the present application is an isolated
population of activated T cells prepared by any embodiment of the
methods described herein. Also provided herein is a co-culture
useful for treating cancer in an individual, comprising a
population of T cells and a population of dendritic cells loaded
with a plurality of tumor antigen peptides. In some embodiments,
the population of T cells and the population of dendritic cells
loaded with the plurality of tumor antigen peptides are derived
from the same individual, such as the individual being treated.
[0101] The isolated population of activated T cells and the
co-culture described in this section may be used for treating
cancer, such as solid caner. Immunotherapeutic compositions
comprising the isolated population of activated T cells or the
co-culture are useful for treating cancer, preventing tumor
progression or metastasis, or reducing cancer immune escape are
provided herein. The isolated population of activated T cells and
the co-culture may also be used in the manufacture of a medicament
for treating cancer, preventing tumor progression or metastasis, or
reducing cancer immune escape.
[0102] It is intended that any of the steps and parameters
described herein for preparing a population of dendritic cells
loaded with a plurality of tumor antigen peptides or for preparing
a population of activated T cells can be combined with any of the
steps and parameters described herein for the improved MASCT
method, as if each and every combination is individually
described.
Plurality of Tumor Antigen Peptides
[0103] The methods described herein use a plurality of tumor
antigen peptides to prepare dendritic cells and activated T cells
that can trigger specific T cell response ex vivo and in vivo. In
some embodiments, the plurality of tumor antigen peptides is a
plurality of synthetic tumor antigen peptides. In some embodiments,
the plurality of tumor antigen peptides is not obtained from a cell
sample, such as a lysed cell composition.
[0104] In some embodiments, each tumor antigen peptide comprises at
least about any one of 1, 2, 3, 4, 5, or 10 epitopes from a single
protein antigen (including a neoantigen). In some embodiments, each
tumor antigen peptide in the plurality of tumor antigen peptides
comprises at least one epitope recognizable by a T cell receptor.
In some embodiments, the plurality of tumor antigen peptides
comprises at least one tumor antigen peptide that comprises at
least 2 epitopes from a single protein antigen. The tumor antigen
peptide can be a naturally derived peptide fragment from a protein
antigen containing one or more epitopes, or an artificially
designed peptide with one or more natural epitope sequences,
wherein a linker peptide can optionally be placed in between
adjacent epitope sequences. In some preferred embodiments, the
epitopes contained in the same tumor antigen peptide are derived
from the same protein antigen.
[0105] The tumor antigen peptide may contain at least one MHC-I
epitope, at least one MHC-II epitope, or both MHC-I epitope(s) and
MHC-II epitope(s). In some embodiments, the plurality of tumor
antigen peptides comprises at least one peptide comprising an MHC-I
epitope. In some embodiments, the plurality of tumor antigen
peptides comprises at least one peptide comprising an MHC-II
epitope. In some embodiments, at least one tumor antigen peptide in
the plurality of tumor antigen peptides comprises both MHC-I and
MHC-II epitopes.
[0106] Special design strategies can be applied to the sequence of
the tumor antigen peptides (including neoantigen peptides) in order
to optimize the immune response to dendritic cells loaded with the
tumor antigen peptides. Typically, a peptide longer than the exact
epitope peptide can increase uptake of the peptide into dendritic
cells. In some embodiments, an MHC-I or MHC-II epitope sequence is
extended at the N terminus or the C terminus or both termini
according to the natural sequence of the protein harboring the
epitope to obtain an extended sequence, wherein the extended
sequence is amenable for presentation by both class I and class II
HLA molecules, and by different subtypes of HLA molecules in
different individuals. In some embodiments, the epitope sequence is
extended at one or both termini by at least about any one of 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20 amino acid residues to
generate the extended epitope. In some embodiments, the peptides
comprising an MHC-I or MHC-II epitope further comprise additional
amino acids flanking the epitope at the N-terminus, the C-terminus,
or both. In some embodiments, each tumor antigen peptide in the
plurality of tumor antigen peptides is at least about any one of 7,
8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100
amino acids long. Different tumor antigen peptides in the plurality
of tumor antigen peptides may have the same length, or different
lengths. In some embodiments, the plurality of tumor antigen
peptides is each about 20-40 amino acids long.
[0107] In some embodiments, the amino acid sequences of one or more
epitope peptides used to design a tumor antigen peptide in the
present application are based on sequences known in the art or
available in public databases, such as the Peptide Database
(Vigneron N. et al. Cancer Immunity, 13:15 (2013)).
[0108] In some embodiments, the amino acid sequences of one or more
epitope peptides are predicted based on the sequence of the antigen
protein using a bioinformatics tool for T cell epitope prediction.
Exemplary bioinformatics tools for T cell epitope prediction are
known in the art, for example, see Yang X. and Yu X. (2009) "An
introduction to epitope prediction methods and software" Rev. Med.
Viral. 19(2): 77-96. In some embodiments, the sequence of the
antigen protein is known in the art or available in public
databases. In some embodiments, the sequence of the antigen protein
is determined by sequencing a sample (such as a tumor sample) of
the individual being treated.
[0109] The present application contemplates tumor antigen peptides
derived from any tumor antigens and epitopes known in the art,
including neoantigens and neoepitopes, or specially developed or
predicted using bioinformatics tools by the inventors.
[0110] In some embodiments, the plurality of tumor antigen peptides
comprises a first core group of general tumor antigen peptides. In
some embodiments, the plurality of tumor antigen peptides further
comprises a second group of cancer-type specific antigen peptides.
In some embodiments, the plurality of tumor antigen peptides
comprises one or more neoantigen peptides. In some embodiments,
neoantigen peptides are cancer-type specific antigen peptides. In
some embodiments, the plurality of tumor antigen peptides consists
of the first core group of general tumor antigen peptides. In some
embodiments, the plurality of tumor antigen peptides consists of
the first core group of general tumor antigen peptides and the
second group of cancer-type specific antigen peptides. In some
embodiments, the plurality of tumor antigen peptides consists of
neoantigen peptides. In some embodiments, the plurality of tumor
antigen peptides comprises a first core group of general tumor
antigen peptides and one or more neoantigen peptides. In some
embodiments, the plurality of tumor antigen peptides comprises a
first core group of general tumor antigen peptides, a second group
of cancer-type specific antigen peptides, and one or more
neoantigen peptides.
[0111] The first core group of general tumor antigen peptides is
derived from tumor antigens commonly overexpressed by a variety of
cancers of different types. Therefore, the first core group of
general tumor antigen peptides is useful to prepare dendritic cells
and/or activated T cells for treating individuals with different
cancer types. For example, in some embodiments, the first core
group of general tumor antigen peptides is useful for methods
described herein for treating a variety of cancers, such as lung
cancer, colon cancer, gastric cancer, prostate cancer, melanoma,
lymphoma, pancreatic cancer, ovarian cancer, breast cancer, glioma,
esophageal cancer, nasopharyngeal carcinoma, cervical cancer, renal
carcinoma, or hepatocellular carcinoma. Exemplary tumor antigen
peptides of the first core group include, but are not limited to,
peptides derived from hTERT, p53, Survivin, NY-ESO-1, CEA, CCND1,
MET, MUC1, Her2, MAGEA1, MAGEA3, WT-1, RGS5, MMP7, VEGFR (such as
VEGFR1 and VEGFR2), and CDCA1. The first core group may comprise
peptides derived from at least about any one of 1, 2, 5, 10, 15,
20, 25, 30, 40, 50, 60, 70, 80 or more tumor antigens. The first
core group may comprise at least about any one of 1, 2, 5, 10, 15,
20, 25, 30, 40, 50, 60, 70, 80 or more general tumor antigen
peptides. In some embodiments, the first core group comprises more
than one general tumor antigen peptides. In some embodiments, the
first core group comprises about 10 to about 20 general tumor
antigen peptides.
[0112] The second group of cancer-type specific antigen peptides is
derived from tumor antigens that are overexpressed only in one or a
limited number of cancer types. Therefore, the second group of
cancer-type specific antigen peptides is useful to prepare
dendritic cells and/or activated T cells for treating individuals
with a particular type of cancer. Exemplary cancer-type specific
antigen peptides for treating hepatocellular carcinoma (HCC)
include, but are not limited to, peptides derived from SSX, AFP,
and GPC3. In some embodiments, one or more cancer-specific antigen
peptide is a virus-specific antigen peptide derived from a virus
that can induce cancer, or relates to cancer development in the
individual when infecting the individual. In some embodiments, the
virus-specific antigen peptide is specific to the subtype of the
virus infecting the individual. Exemplary virus-specific antigen
peptides for treating an HCC patient with concurrent infection of
HBV include, but are not limited to, peptides derived from HBV core
antigen (HBcAg), and HBV DNA polymerase. In some embodiments, the
second group comprises virus-specific antigen peptides derived from
HBV antigens, wherein the method is to treat hepatocellular
carcinoma in an individual. In some embodiments, the second group
comprises virus-specific antigen peptides derived from HPV
antigens, wherein the method is to treat cervical cancer in an
individual. In some embodiments, the second group comprises
virus-specific antigen peptides derived from EBV antigens, wherein
the method is to treat nasopharyngeal carcinoma in an individual.
The second group of cancer-type specific antigen peptides may
comprise peptides derived from at least about any one of 1, 2, 5,
10, 15, 20, 25, 30, 40, 50 or more cancer-type specific antigens.
The second group of cancer-type specific antigen peptides may
comprise at least about any one of 1, 2, 5, 10, 15, 20, 25, 30, 40,
50 or more cancer-type specific antigen peptides. In some
embodiments, the second group comprises more than one cancer-type
specific antigen peptides. In some embodiments, the second group
comprises about 1 to about 10 cancer-type specific antigen
peptides. In some embodiments, the type of cancer targeted by the
cancer-type specific antigen peptides is selected from the group
consisting essentially of hepatocellular carcinoma, cervical
cancer, nasopharyngeal carcinoma, endometrial cancer, colorectal
cancer, breast cancer, and lymphoma.
[0113] In some embodiments, the plurality of tumor antigen peptides
comprises one or more (such as any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10
or more) neoantigen peptides. In some embodiments, the plurality of
tumor antigen peptides consists of neoantigen peptides. In some
embodiments, the plurality of tumor antigen peptides comprises
neoantigen peptides and no general tumor antigen peptides. In some
embodiments, the plurality of tumor antigen peptides comprises one
or more general tumor antigen peptides and one or more neoantigen
peptides. In some embodiments, the plurality of tumor antigen
peptides comprises one or more general tumor antigen peptides, one
or more cancer-type specific antigen peptides, and one or more
neoantigen peptides. The neoantigen peptides are derived from
neoantigens. Neoantigens are newly acquired and expressed antigens
present in tumor cells of the individual, such as the individual
being treated for cancer. In some embodiments, neoantigens are
derived from mutant protein antigens that are only present in
cancer cells, but absent in normal cells. Neoantigens may be
uniquely present in the tumor cells (such as all tumor cells or a
portion of tumor cells) of the individual being treated for cancer,
or present in individuals having similar types of cancer as the
individual being treated. In some embodiments, the neoantigen is a
clonal neoantigen. In some embodiments, the neoantigen is a
subclonal neoantigen. In some embodiments, the neoantigen is
present in at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 95% or more tumor cells in the individual. In some
embodiments, the neoantigen peptide comprises an MHC-I restricted
neoepitope. In some embodiments, the neoantigen peptide comprises
an MHC-II restricted neoepitope. In some embodiments, the
neoantigen peptide is designed to facilitate presentation of the
neoepitope by both class I and class II MHC molecules, for example,
by extending the neoepitope at both the N- and the C-termini.
Exemplary neoantigen peptides include, but are not limited to,
neoepitope derived from mutant KRAS (e.g., KRAS.sup.G12A).sup.,
PARP4 (e.g., PARP4.sup.T1170T), MLL3 (e.g.,MLL3.sup.C988F), and
MTHFR (e.g., MTHFR.sup.A222V).
[0114] Neoantigen peptides can be selected based on the genetic
profile of one or more tumor sites of the individual being treated,
and neoantigens are not be expressed in normal tissues. In some
embodiments, the genetic profile of the tumor sample comprises
sequence information of the full genome. In some embodiments, the
genetic profile of the tumor sample comprises sequence information
of the exome. In some embodiments, the genetic profile of the tumor
sample comprises sequence information of cancer-associated
genes.
[0115] Neoantigen peptides suitable for use in the present
application may be derived from any mutant proteins, such as those
encoded by mutant cancer-associated genes, in the tumor cells. In
some embodiments, the neoantigen peptide comprises a single
neoepitope derived from a cancer-associated gene. In some
embodiments, the neoantigen peptide comprises more than one (such
as 2, 3, or more) neoepitope derived from a cancer-associated gene.
In some embodiments, the neoantigen peptide comprises more than one
(such as 2, 3, or more) neoepitope derived from more than one (such
as 2, 3, or more) cancer-associated genes. In some embodiments, the
plurality of tumor antigens comprises a plurality of neoantigen
peptides derived from a single cancer-associated gene. In some
embodiments, the plurality of tumor antigens comprises a plurality
of neoantigen peptides derived from more than one (such as any of
2, 3, 4, 5, or more) cancer-associated genes.
[0116] Cancer-associated genes are genes that are overexpressed in
cancer cells, but expressed at low levels in normal cells.
Exemplary cancer-associated genes include, but are not limited to,
ABL1, AKT1, AKT2, AKT3, ALK, ALOX12B, APC, AR, ARAF, ARID1A,
ARID1B, ARID2, ASXL1, ATM, ATRX, AURKA, AURKB, AXL, B2M, BAP1,
BCL2, BCL2L1, BCL2L12, BCL6, BCOR, BCORL1, BLM, BMPR1A, BRAF,
BRCA1, BRCA2, BRD4, BRIP1, BUB1B, CADM2, CARD11, CBL, CBLB, CCND1,
CCND2, CCND3, CCNE1, CD274, CD58, CD79B, CDC73, CDH1, CDK1, CDK2,
CDK4, CDK5, CDK6, CDK9, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B,
CDKN2C, CEBPA, CHEK2, CIITA, CREBBP, CRKL, CRLF2, CRTC1, CRTC2,
CSF1R, CSF3R, CTNNB1, CUX1, CYLD, DDB2, DDR2, DEPDC5, DICER1, DIS3,
DMD, DNMT3A, EED, EGFR, EP300, EPHA3, EPHA5, EPHA7, ERBB2, ERBB3,
ERBB4, ERCC2, ERCC3, ERCC4, ERCC5, ESR1, ETV1, ETV4, ETV5, ETV6,
EWSR1, EXT1, EXT2, EZH2, FAM46C, FANCA, FANCC, FANCD2, FANCE,
FANCF, FANCG, FAS, FBXW7, FGFR1, FGFR2, FGFR3, FGFR4, FH, FKBP9,
FLCN, FLT1, FLT3, FLT4, FUS, GATA3, GATA4, GATA6, GLI1, GLI2, GLI3,
GNA11, GNAQ, GNAS, GNB2L1, GPC3, GSTM5, H3F3A, HNF1A, HRAS, ID3,
IDH1, IDH2, IGF1R, IKZF1, IKZF3, INSIG1, JAK2, JAK3, KCNIP1, KDM5C,
KDM6A, KDM6B, KDR, KEAP1, KIT, KRAS, LINC00894, LMO1, LMO2, LMO3,
MAP2K1, MAP2K4, MAP3K1, MAPK1, MCL1, MDM2, MDM4, MECOM, MEF2B,
MEN1, MET, MITF, MLH1, MLL (KMT2A), MLL2 (KTM2D), MPL, MSH2, MSH6,
MTOR, MUTYH, MYB, MYBL1, MYC, MYCL1 (MYCL), MYCN, MYD88, NBN,
NEGR1, NF1, NF2, NFE2L2, NFKBIA, NFKBIZ, NKX2-1, NOTCH1, NOTCH2,
NPM1, NPRL2, NPRL3, NRAS, NTRK1, NTRK2, NTRK3, PALB2, PARK2, PAX5,
PBRM1, PDCD1LG2, PDGFRA, PDGFRB, PHF6, PHOX2B, PIK3C2B, PIK3CA,
PIK3R1, PIM1, PMS1, PMS2, PNRC1, PRAME, PRDM1, PRF1, PRKAR1A,
PRKCI, PRKCZ, PRKDC, PRPF40B, PRPF8, PSMD13, PTCH1, PTEN, PTK2,
PTPN11, PTPRD, QKI, RAD21, RAF1, RARA, RB1, RBL2, RECQL4, REL, RET,
RFWD2, RHEB, RHPN2, ROS1, RPL26, RUNX1, SBDS, SDHA, SDHAF2, SDHB,
SDHC, SDHD, SETBP1, SETD2, SF1, SF3B1, SH2B3, SLITRK6, SMAD2,
SMAD4, SMARCA4, SMARCB1, SMC1A, SMC3, SMO, SOCS1, SOX2, SOX9,
SQSTM1, SRC, SRSF2, STAG1, STAG2, STAT3, STAT6, STK11, SUFU, SUZ12,
SYK, TCF3, TCF7L1, TCF7L2, TERC, TERT, TET2, TLR4, TNFAIP3, TP53,
TSC1, TSC2, U2AF1, VHL, WRN, WT1, XPA, XPC, XPO1, ZNF217, ZNF708,
and ZRSR2.
[0117] In some embodiments, the plurality of tumor antigen peptides
comprises at least one (such as at least about any of 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) tumor antigen
peptide each comprising one or more epitopes encoded by a
cancer-associated gene selected from the group consisting of hTERT,
p53, Survivin, NY-ESO-1, CEA, CCND1, RGS5, MMP7, VEGFR1, VEGFR2,
MUC1, HER2, MAGE-A1, MAGE-A3, CDCA1, WT1, KRAS, PARP4, MLL3, MTHFR,
HBcAg, HBV polymerase, GPC3, SSX, and AFP. In some embodiments, the
plurality of tumor antigen peptides comprises at least 10 tumor
antigen peptides. In some embodiments, the plurality of tumor
antigen peptides comprises tumor antigen peptides derived from
hTERT, p53, Survivin, NY-ESO-1, CEA, CCND1, MUC1, Her2, MAGEA1,
MAGEA3, WT-1, RGS5, VEGFR1, VEGFR2, and CDCA1.
[0118] In some embodiments, the plurality of tumor antigen peptides
is present in a composition having at least about any one of 95%,
96%, 97%, 98%, 99%, 99.9% or higher percentage of tumor antigen
peptides. In some embodiments, the purity of the plurality of tumor
antigen peptides is at least about 98%. In some embodiments, the
solubility of the plurality of tumor antigen peptides in the medium
for pulsing the tumor antigen peptides into the dendritic cells is
at least about any one of 80%, 85%, 90%, 95%, 98%, 99%, 99.9% or
higher. In some embodiments, the plurality of tumor antigen
peptides is about 100% soluble in the medium for pulsing the tumor
antigen peptides into the dendritic cells.
III. METHODS OF TREATMENT
[0119] The present application provides cell-based immunotherapy
methods of treating cancer in an individual, comprising
administering to the individual an effective amount of activated T
cells prepared using any one of the methods described in Section
II. In some embodiments, the method further comprises administering
to the individual an effective amount of dendritic cells loaded
with a plurality of tumor antigen peptides.
[0120] In some embodiments, there is provided a method of treating
a cancer (e.g., solid cancer) in an individual, comprising
administering to the individual an effective amount of activated T
cells, wherein the activated T cells are prepared by: (a)
co-culturing a population of dendritic cells loaded with a
plurality of tumor antigen peptides and a population of T cells in
an initial co-culture medium comprising a plurality of cytokines
and an immune checkpoint inhibitor to provide a co-culture; and b)
adding an anti-CD3 antibody to the co-culture at about 3 to 7 days
after the co-culturing starts, thereby obtaining the population of
activated T cells. In some embodiments, the plurality of cytokines
comprises IL-2, IL-7, IL-15 and IL-21. In some embodiments, the
IL-2 is present in the initial co-culture medium at a concentration
of at least about 500 IU/mL. In some embodiments, the immune
checkpoint inhibitor is an anti-PD-1 antibody. In some embodiments,
the anti-PD-1 antibody is present in the initial co-culture medium
at a concentration of at least about 10 .mu.g/mL. In some
embodiments, the anti-CD3 antibody is added to the co-culture at
about 5 days after the co-culturing starts. In some embodiments,
the population of dendritic cells loaded with the plurality of
tumor antigen peptides and the population of T cells are
co-cultured for at least about 10 days in the presence of the
anti-CD3 antibody. In some embodiments, the population of T cells
is present in a population of PBMCs. In some embodiments, the
population of dendritic cells and the population of T cells are
obtained from the individual being treated. In some embodiments,
the activated T cells are administered to the individual for at
least three times. In some embodiments, the activated T cells are
administered intravenously. In some embodiments, the method further
comprises administering to the individual an effective amount of
dendritic cells loaded with the plurality of tumor antigen
peptides. In some embodiments, the dendritic cells loaded with the
plurality of tumor antigen peptides are administered for at least
three times. In some embodiments, the dendritic cells loaded with
the plurality of tumor antigen peptides are administered
subcutaneously, intradermally or intravenously. In some
embodiments, the plurality of tumor antigen peptides comprises
tumor antigen peptides derived from hTERT, p53, Survivin, NY-ESO-1,
CEA, CCND1, MUC1, Her2, MAGEA1, MAGEA3, WT-1, RGS5, VEGFR1, VEGFR2,
and CDCA1. In some embodiments, the cancer is a solid cancer
selected from the group consisting of hepatocellular carcinoma,
gastric cancer, bladder cancer, soft tissue sarcoma, colorectal
cancer, endometrial cancer, and lung cancer.
[0121] In some embodiments, there is provided a method of treating
a cancer (e.g., solid cancer) in an individual, comprising
administering to the individual an effective amount of activated T
cells, wherein the activated T cells are prepared by: a) contacting
a population of dendritic cells with a plurality of tumor antigen
peptides to obtain a population of dendritic cells loaded with the
plurality of tumor antigen peptides; b) culturing the population of
dendritic cells loaded with the plurality of tumor antigen peptides
in a DC maturation medium comprising MPLA; and c) co-culturing the
population of dendritic cells loaded with the plurality of tumor
antigen peptides and a population of T cells, thereby obtaining the
population of activated T cells. In some embodiments, step c)
comprises co-culturing the population of dendritic cells loaded
with the plurality of tumor antigen peptides and a population of T
cells in a co-culture medium comprising an interleukin cocktail, an
immune checkpoint inhibitor and an anti-CD3 antibody. In some
embodiments, the population of dendritic cells loaded with the
plurality of tumor antigen peptides and the population of T cells
are co-cultured for at least about 10 days in the presence of the
anti-CD3 antibody. In some embodiments, the DC maturation medium
comprises INF.gamma. and MPLA. In some embodiments, the DC
maturation medium further comprises PGE2. In some embodiments, the
MPLA is present in the DC maturation medium at a concentration of
at least about 0.5 .mu.g/mL. In some embodiments, the INF.gamma. is
present in the DC maturation medium at a concentration of at least
about 100 IU/mL. In some embodiments, the PGE2 is present in the DC
maturation medium at a concentration of at least about 0.1
.mu.g/mL. In some embodiments, the plurality of cytokines comprises
IL-2, IL-7, IL-15 and IL-21. In some embodiments, the IL-2 is
present in the co-culture medium at a concentration of at least
about 500 IU/mL. In some embodiments, the immune checkpoint
inhibitor is an anti-PD-1 antibody. In some embodiments, the
anti-PD-1 antibody is present in the co-culture medium at a
concentration of at least about 10 .mu.g/mL. In some embodiments,
the population of dendritic cells loaded with the plurality of
tumor antigen peptides and the population of T cells are
co-cultured for at least about 10 days in the presence of the
anti-CD3 antibody. In some embodiments, the population of T cells
is present in a population of PBMCs. n some embodiments, the
population of dendritic cells and the population of T cells are
obtained from the individual being treated. In some embodiments,
the activated T cells are administered to the individual for at
least three times. In some embodiments, the activated T cells are
administered intravenously. In some embodiments, the method further
comprises administering to the individual an effective amount of
dendritic cells loaded with the plurality of tumor antigen
peptides. In some embodiments, the dendritic cells loaded with the
plurality of tumor antigen peptides are administered for at least
three times. In some embodiments, the dendritic cells loaded with
the plurality of tumor antigen peptides are administered
subcutaneously, intradermally or intravenously. In some
embodiments, the plurality of tumor antigen peptides comprises
tumor antigen peptides derived from hTERT, p53, Survivin, NY-ESO-1,
CEA, CCND1, MUC1, Her2, MAGEA1, MAGEA3, WT-1, RGS5, VEGFR1, VEGFR2,
and CDCA1. In some embodiments, the cancer is a solid cancer
selected from the group consisting of hepatocellular carcinoma,
gastric cancer, bladder cancer, soft tissue sarcoma, colorectal
cancer, endometrial cancer, and lung cancer.
[0122] In some embodiments, there is provided a method of treating
a cancer (e.g., solid cancer) in an individual, comprising
administering to the individual an effective amount of activated T
cells, wherein the activated T cells are prepared by: a) contacting
a population of dendritic cells with a plurality of tumor antigen
peptides to obtain a population of dendritic cells loaded with the
plurality of tumor antigen peptides; b) co-culturing the population
of dendritic cells loaded with the plurality of tumor antigen
peptides and a population of T cells in an initial co-culture
medium comprising a plurality of cytokines and an immune checkpoint
inhibitor to provide a co-culture; and c) adding an anti-CD3
antibody to the co-culture at about 3 to 7 days after the
co-culturing starts, thereby obtaining the population of activated
T cells. In some embodiments, step (a) further comprises culturing
the population of dendritic cells loaded with the plurality of
tumor antigen peptides in a DC maturation medium comprising a
toll-like receptor (TLR) agonist. In some embodiments, the TLR
agonist is selected from the group consisting of MPLA, Poly I:C,
resquimod, gardiquimod, and CL075. In some embodiments, the DC
maturation medium comprises PGE2. In some embodiments, the
plurality of cytokines comprises IL-2, IL-7, IL-15 and IL-21. In
some embodiments, the IL-2 is present in the initial co-culture
medium at a concentration of at least about 500 IU/mL. In some
embodiments, the immune checkpoint inhibitor is an anti-PD-1
antibody. In some embodiments, the anti-PD-1 antibody is present in
the initial co-culture medium at a concentration of at least about
10 .mu.g/mL. In some embodiments, the anti-CD3 antibody is added to
the co-culture at about 5 days after the co-culturing starts. In
some embodiments, the population of dendritic cells loaded with the
plurality of tumor antigen peptides and the population of T cells
are co-cultured for at least about 10 days in the presence of the
anti-CD3 antibody. In some embodiments, the population of dendritic
cells and the population of T cells are obtained from the
individual being treated. In some embodiments, the activated T
cells are administered to the individual for at least three times.
In some embodiments, the activated T cells are administered
intravenously. In some embodiments, the method further comprises
administering to the individual an effective amount of dendritic
cells loaded with the plurality of tumor antigen peptides. In some
embodiments, the dendritic cells loaded with the plurality of tumor
antigen peptides are administered for at least three times. In some
embodiments, the dendritic cells loaded with the plurality of tumor
antigen peptides are administered subcutaneously, intradermally or
intravenously. In some embodiments, the plurality of tumor antigen
peptides comprises tumor antigen peptides derived from hTERT, p53,
Survivin, NY-ESO-1, CEA, CCND1, MUC1, Her2, MAGEA1, MAGEA3, WT-1,
RGS5, VEGFR1, VEGFR2, and CDCA1. In some embodiments, the cancer is
a solid cancer selected from the group consisting of hepatocellular
carcinoma, gastric cancer, bladder cancer, soft tissue sarcoma,
colorectal cancer, endometrial cancer, and lung cancer.
[0123] In some embodiments, there is provided a method of treating
a cancer (e.g., solid cancer) in an individual, comprising
administering to the individual an effective amount of activated T
cells, wherein the activated T cells are prepared by: a) contacting
a population of dendritic cells with a plurality of tumor antigen
peptides to obtain a population of dendritic cells loaded with the
plurality of tumor antigen peptides; b) culturing the population of
dendritic cells loaded with the plurality of tumor antigen peptides
in a DC maturation medium comprising MPLA; c) co-culturing the
population of dendritic cells loaded with the plurality of tumor
antigen peptides and a population of T cells in an initial
co-culture medium comprising a plurality of cytokines and an immune
checkpoint inhibitor to provide a co-culture; and d) adding an
anti-CD3 antibody to the co-culture, thereby obtaining the
population of activated T cells. In some embodiments, the anti-CD3
antibody is added to the co-culture when the co-culturing starts.
In some embodiments, the anti-CD3 antibody is added to the
co-culture after the co-culturing starts. In some embodiments, the
DC maturation medium comprises INF.gamma. and MPLA. In some
embodiments, the DC maturation medium further comprises PGE2. In
some embodiments, the MPLA is present in the DC maturation medium
at a concentration of at least about 0.5 .mu.g/mL. In some
embodiments, the INF.gamma. is present in the DC maturation medium
at a concentration of at least about 100 IU/mL. In some
embodiments, the PGE2 is present in the DC maturation medium at a
concentration of at least about 0.1 .mu.g/mL. In some embodiments,
the plurality of cytokines comprises IL-2, IL-7, IL-15 and IL-21.
In some embodiments, the IL-2 is present in the initial co-culture
medium at a concentration of at least about 500 IU/mL. In some
embodiments, the immune checkpoint inhibitor is an anti-PD-1
antibody. In some embodiments, the anti-PD-1 antibody is present in
the initial co-culture medium at a concentration of at least about
10 .mu.g/mL. In some embodiments, the population of dendritic cells
loaded with the plurality of tumor antigen peptides and the
population of T cells are co-cultured for at least about 10 days in
the presence of the anti-CD3 antibody. In some embodiments, the
population of dendritic cells and the population of T cells are
obtained from the individual being treated. In some embodiments,
the activated T cells are administered to the individual for at
least three times. In some embodiments, the activated T cells are
administered intravenously. In some embodiments, the method further
comprises administering to the individual an effective amount of
dendritic cells loaded with the plurality of tumor antigen
peptides. In some embodiments, the dendritic cells loaded with the
plurality of tumor antigen peptides are administered for at least
three times. In some embodiments, the dendritic cells loaded with
the plurality of tumor antigen peptides are administered
subcutaneously, intradermally or intravenously. In some
embodiments, the plurality of tumor antigen peptides comprises
tumor antigen peptides derived from hTERT, p53, Survivin, NY-ESO-1,
CEA, CCND1, MUC1, Her2, MAGEA1, MAGEA3, WT-1, RGS5, VEGFR1, VEGFR2,
and CDCA1. In some embodiments, the cancer is a solid cancer
selected from the group consisting of hepatocellular carcinoma,
gastric cancer, bladder cancer, soft tissue sarcoma, colorectal
cancer, endometrial cancer, and lung cancer.
[0124] In some embodiments, there is provided a method of treating
a cancer (e.g., solid cancer) in an individual, comprising
administering to the individual an effective amount of activated T
cells, wherein the activated T cells are prepared by: a) contacting
a population of dendritic cells with a plurality of tumor antigen
peptides to obtain a population of dendritic cells loaded with the
plurality of tumor antigen peptides; b) culturing the population of
dendritic cells loaded with the plurality of tumor antigen peptides
in a DC maturation medium comprising MPLA; c) co-culturing the
population of dendritic cells loaded with the plurality of tumor
antigen peptides and a population of T cells in an initial
co-culture medium comprising a plurality of cytokines and an immune
checkpoint inhibitor to provide a co-culture; and d) adding an
anti-CD3 antibody to the co-culture at about 3 to 7 days after the
co-culturing starts, thereby obtaining the population of activated
T cells. In some embodiments, the DC maturation medium comprises
INF.gamma. and MPLA. In some embodiments, the DC maturation medium
further comprises PGE2. In some embodiments, the MPLA is present in
the DC maturation medium at a concentration of at least about 0.5
.mu.g/mL. In some embodiments, the INF.gamma. is present in the DC
maturation medium at a concentration of at least about 100 IU/mL.
In some embodiments, the PGE2 is present in the DC maturation
medium at a concentration of at least about 0.1 .mu.g/mL. In some
embodiments, the plurality of cytokines comprises IL-2, IL-7, IL-15
and IL-21. In some embodiments, the IL-2 is present in the initial
co-culture medium at a concentration of at least about 500 IU/mL.
In some embodiments, the immune checkpoint inhibitor is an
anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is
present in the initial co-culture medium at a concentration of at
least about 10 .mu.g/mL. In some embodiments, the anti-CD3 antibody
is added to the co-culture at about 5 days after the co-culturing
starts. In some embodiments, the population of dendritic cells
loaded with the plurality of tumor antigen peptides and the
population of T cells are co-cultured for at least about 10 days in
the presence of the anti-CD3 antibody. In some embodiments, the
population of dendritic cells and the population of T cells are
obtained from the individual being treated. In some embodiments,
the activated T cells are administered to the individual for at
least three times. In some embodiments, the activated T cells are
administered intravenously. In some embodiments, the method further
comprises administering to the individual an effective amount of
dendritic cells loaded with the plurality of tumor antigen
peptides. In some embodiments, the dendritic cells loaded with the
plurality of tumor antigen peptides are administered for at least
three times. In some embodiments, the dendritic cells loaded with
the plurality of tumor antigen peptides are administered
subcutaneously, intradermally or intravenously. In some
embodiments, the plurality of tumor antigen peptides comprises
tumor antigen peptides derived from hTERT, p53, Survivin, NY-ESO-1,
CEA, CCND1, MUC1, Her2, MAGEA1, MAGEA3, WT-1, RGS5, VEGFR1, VEGFR2,
and CDCA1. In some embodiments, the cancer is a solid cancer
selected from the group consisting of hepatocellular carcinoma,
gastric cancer, bladder cancer, soft tissue sarcoma, colorectal
cancer, endometrial cancer, and lung cancer.
[0125] In some embodiments, there is provided a method of treating
a cancer (e.g., solid cancer) in an individual, comprising
administering to the individual an effective amount of activated T
cells, wherein the activated T cells are prepared by: a) contacting
a population of dendritic cells with a plurality of tumor antigen
peptides to obtain a population of dendritic cells loaded with the
plurality of tumor antigen peptides; b) culturing the population of
dendritic cells loaded with the plurality of tumor antigen peptides
in a DC maturation medium comprising MPLA, INF.gamma. and PGE2; c)
co-culturing the population of dendritic cells loaded with the
plurality of tumor antigen peptides and a population of T cells in
an initial co-culture medium comprising a plurality of cytokines
comprising IL-2, IL-7, IL-15 and IL-21 and an anti-PD-1 antibody to
provide a co-culture; and d) adding an anti-CD3 antibody to the
co-culture at about 3 to 7 days (e.g., about 5 days) after the
co-culturing starts, thereby obtaining the population of activated
T cells. In some embodiments, the MPLA is present in the DC
maturation medium at a concentration of at least about 0.5
.mu.g/mL. In some embodiments, the INF.gamma. is present in the DC
maturation medium at a concentration of at least about 100 IU/mL.
In some embodiments, the PGE2 is present in the DC maturation
medium at a concentration of at least about 0.1 .mu.g/mL. In some
embodiments, the IL-2 is present in the initial co-culture medium
at a concentration of at least about 500 IU/mL. In some
embodiments, the anti-PD-1 antibody is present in the initial
co-culture medium at a concentration of at least about 10 .mu.g/mL.
In some embodiments, the population of dendritic cells loaded with
the plurality of tumor antigen peptides and the population of T
cells are co-cultured for at least about 10 days in the presence of
the anti-CD3 antibody. In some embodiments, the population of
dendritic cells and the population of T cells are obtained from the
individual being treated. In some embodiments, the activated T
cells are administered to the individual for at least three times.
In some embodiments, the activated T cells are administered
intravenously. In some embodiments, the method further comprises
administering to the individual an effective amount of dendritic
cells loaded with the plurality of tumor antigen peptides. In some
embodiments, the dendritic cells loaded with the plurality of tumor
antigen peptides are administered for at least three times. In some
embodiments, the dendritic cells loaded with the plurality of tumor
antigen peptides are administered subcutaneously, intradermally or
intravenously. In some embodiments, the plurality of tumor antigen
peptides comprises tumor antigen peptides derived from hTERT, p53,
Survivin, NY-ESO-1, CEA, CCND1, MUC1, Her2, MAGEA1, MAGEA3, WT-1,
RGS5, VEGFR1, VEGFR2, and CDCA1. In some embodiments, the cancer is
a solid cancer selected from the group consisting of hepatocellular
carcinoma, gastric cancer, bladder cancer, soft tissue sarcoma,
colorectal cancer, endometrial cancer, and lung cancer.
[0126] In addition to the administration step(s), some embodiments
of the improved MASCT method further comprise one or more of the
following cell preparation steps: 1) obtaining PBMCs from the
individual; 2) obtaining a population of DCs from the PBMCs (e.g.,
by inducing differentiation of a population of monocytes from the
PBMCs); 3) obtaining a population of T cells from the PBMCs; 4)
preparing a population of dendritic cells loaded with the plurality
of tumor antigen peptides; 5) inducing maturation of the population
of dendritic cells loaded with the plurality of tumor antigen
peptides in a DC maturation medium; 6) co-culturing the population
of dendritic cells loaded with the plurality of tumor antigen
peptides and a population of T cells (including, for example,
co-culturing the population of dendritic cells loaded with the
plurality of tumor antigen peptides and a population of T cells in
an initial co-culture medium; and adding an anti-CD3 antibody to
the co-culture); and 8) co-culturing the population of dendritic
cells loaded with the plurality of tumor antigen peptides and the
population of T cells in the presence of the anti-CD3 antibody for
at least about 10 days.
[0127] Thus, in some embodiments, there is provided a method of
treating a cancer (e.g., solid cancer) in an individual,
comprising: (a) obtaining a population of PBMCs from the
individual; (b) obtaining a population of dendritic cells from the
population of PBMCs; (c) contacting the population of dendritic
cells with a plurality of tumor antigen peptides to obtain a
population of dendritic cells loaded with the plurality of tumor
antigen peptides; (d) culturing the population of dendritic cells
loaded with the plurality of tumor antigen peptides in a DC
maturation medium comprising MPLA, INF.gamma. and PGE2; (e)
optionally administering an effective amount of the dendritic cells
loaded with the plurality of tumor antigen peptides to the
individual; (f) obtaining a population of T cells from the PBMCs;
(g) co-culturing the population of dendritic cells loaded with the
plurality of tumor antigen peptides and the population of T cells
in an initial co-culture medium comprising a plurality of cytokines
comprising IL-2, IL-7, IL-15 and IL-21 and an anti-PD-1 antibody to
provide a co-culture; (h) adding an anti-CD3 antibody to the
co-culture at about 3 to 7 days (e.g., about 5 days) after the
co-culturing starts to obtain a population of activated T cells;
and (i) administering an effective amount of the activated T cells
to the individual. In some embodiments, the MPLA is present in the
DC maturation medium at a concentration of at least about 0.5
.mu.g/mL. In some embodiments, the INF.gamma. is present in the DC
maturation medium at a concentration of at least about 100 IU/mL.
In some embodiments, the PGE2 is present in the DC maturation
medium at a concentration of at least about 0.1 .mu.g/mL. In some
embodiments, the IL-2 is present in the initial co-culture medium
at a concentration of at least about 500 IU/mL. In some
embodiments, the anti-PD-1 antibody is present in the initial
co-culture medium at a concentration of at least about 10 .mu.g/mL.
In some embodiments, the population of dendritic cells loaded with
the plurality of tumor antigen peptides and the population of T
cells are co-cultured for at least about 10 days in the presence of
the anti-CD3 antibody. In some embodiments, the activated T cells
are administered to the individual for at least three times. In
some embodiments, the activated T cells are administered
intravenously. In some embodiments, the dendritic cells loaded with
the plurality of tumor antigen peptides are administered for at
least three times. In some embodiments, the dendritic cells loaded
with the plurality of tumor antigen peptides are administered
subcutaneously, intradermally or intravenously. In some
embodiments, the plurality of tumor antigen peptides comprises
tumor antigen peptides derived from hTERT, p53, Survivin, NY-ESO-1,
CEA, CCND1, MUC1, Her2, MAGEA1, MAGEA3, WT-1, RGS5, VEGFR1, VEGFR2,
and CDCA1. In some embodiments, the cancer is a solid cancer
selected from the group consisting of hepatocellular carcinoma,
gastric cancer, bladder cancer, soft tissue sarcoma, colorectal
cancer, endometrial cancer, and lung cancer.
[0128] The methods described herein are suitable for treating
various cancers, including liquid and solid cancers. In some
embodiments, the cancer is selected from the group consisting of
hepatocellular carcinoma, cervical cancer, bladder cancer,
soft-tissue sarcoma, lung cancer, colorectal cancer, endometrial
cancer, lymphoma, renal carcinoma, breast cancer, pancreatic
cancer, gastric cancer, esophageal cancer, ovarian cancer, prostate
cancer, nasopharyngeal carcinoma, melanoma, and brain cancer. The
methods are applicable to cancers of all stages, including early
stage, advanced stage and metastatic cancer.
[0129] In some embodiments, the method reduces the severity of one
or more symptoms associated with the cancer by at least about any
of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%
compared to the corresponding symptom in the same individual prior
to treatment or compared to the corresponding symptom in other
individuals not receiving the treatment method. In some
embodiments, the method delays progression of the cancer.
[0130] In some embodiments, the method is for treating
hepatocellular carcinoma (HCC). In some embodiments, the HCC is
early stage HCC, non-metastatic HCC, primary HCC, advanced HCC,
locally advanced HCC, metastatic HCC, HCC in remission, or
recurrent HCC. In some embodiments, the HCC is localized resectable
(i.e., tumors that are confined to a portion of the liver that
allows for complete surgical removal), localized unresectable
(i.e., the localized tumors may be unresectable because crucial
blood vessel structures are involved or because the liver is
impaired), or unresectable (i.e., the tumors involve all lobes of
the liver and/or has spread to involve other organs (e.g., lung,
lymph nodes, bone). In some embodiments, the HCC is, according to
TNM classifications, a stage I tumor (single tumor without vascular
invasion), a stage II tumor (single tumor with vascular invasion,
or multiple tumors, none greater than 5 cm), a stage III tumor
(multiple tumors, any greater than 5 cm, or tumors involving major
branch of portal or hepatic veins), a stage IV tumor (tumors with
direct invasion of adjacent organs other than the gallbladder, or
perforation of visceral peritoneum), N1 tumor (regional lymph node
metastasis), or M1 tumor (distant metastasis). In some embodiments,
the HCC is, according to AJCC (American Joint Commission on Cancer)
staging criteria, stage T1, T2, T3, or T4 HCC. In some embodiments,
the HCC is any one of liver cell carcinomas, fibrolamellar variants
of HCC, and mixed hepatocellularcholangiocarcinomas. In some
embodiments, the HCC is caused by Hepatitis B Virus (HBV)
infection.
[0131] In some embodiments, the method is for treating lung cancer.
In some embodiments, the lung cancer is a non-small cell lung
cancer (NSCLC). Examples of NCSLC include, but are not limited to,
large-cell carcinoma (e.g., large-cell neuroendocrine carcinoma,
combined large-cell neuroendocrine carcinoma, basaloid carcinoma,
lymphoepithelioma-like carcinoma, clear cell carcinoma, and
large-cell carcinoma with rhabdoid phenotype), adenocarcinoma
(e.g., acinar, papillary (e.g., bronchioloalveolar carcinoma,
nonmucinous, mucinous, mixed mucinous and nonmucinous and
indeterminate cell type), solid adenocarcinoma with mucin,
adenocarcinoma with mixed subtypes, well-differentiated fetal
adenocarcinoma, mucinous (colloid) adenocarcinoma, mucinous
cystadenocarcinoma, signet ring adenocarcinoma, and clear cell
adenocarcinoma), neuroendocrine lung tumors, and squamous cell
carcinoma (e.g., papillary, clear cell, small cell, and basaloid).
In some embodiments, the NSCLC may be, according to TNM
classifications, a stage T tumor (primary tumor), a stage N tumor
(regional lymph nodes), or a stage M tumor (distant
metastasis).
[0132] In some embodiments, the lung cancer is a carcinoid (typical
or atypical), adenosquamous carcinoma, cylindroma, or carcinoma of
the salivary gland (e.g., adenoid cystic carcinoma or
mucoepidermoid carcinoma). In some embodiments, the lung cancer is
a carcinoma with pleomorphic, sarcomatoid, or sarcomatous elements
(e.g., carcinomas with spindle and/or giant cells, spindle cell
carcinoma, giant cell carcinoma, carcinosarcoma, or pulmonary
blastoma). In some embodiments, the lung cancer is small cell lung
cancer (SCLC; also called oat cell carcinoma). The small cell lung
cancer may be limited-stage, extensive stage or recurrent small
cell lung cancer. In some embodiments, the individual may be a
human who has a gene, genetic mutation, or polymorphism suspected
or shown to be associated with lung cancer (e.g., SASH1, LATS1,
IGF2R, PARK2, KRAS, PTEN, Kras2, Krag, Pas1, ERCC1, XPD, IL8RA,
EGFR, .alpha..sub.1-AD, EPHX, MMP1, MMP2, MMP3, MMP12, IL1.beta.,
RAS, and/or AKT) or has one or more extra copies of a gene
associated with lung cancer.
[0133] In some embodiments, the method is for treating cervical
cancer. In some embodiments, the cervical cancer is early stage
cervical cancer, non-metastatic cervical cancer, locally advanced
cervical cancer, metastatic cervical cancer, cervical cancer in
remission, unresectable cervical cancer, cervical cancer in an
adjuvant setting, or cervical cancer in a neoadjuvant setting. In
some embodiments, the cervical cancer is caused by human
papillomavirus (HPV) infection. In some embodiments, the cervical
cancer may be, according to TNM classifications, a stage T tumor
(primary tumor), a stage N tumor (regional lymph nodes), or a stage
M tumor (distant metastasis). In some embodiments, the cervical
cancer is any of stage 0, stage I (Tis, N0, M0), stage IA (T1a, N0,
M0), stage IB (T1b, N0, M0), stage IIA (T2a, N0, M0), stage IIB
(T2b, N0, M0), stage IIIA (T3a, N0, M0), stage IIIB (T3b, N0, M0,
or T1-3, N1, M0) stage IVA (T4, N0, M0), or stage IVB (T1-T3,
N0-N1, M1) cervical cancer. In some embodiments, the cervical
cancer is cervical squamous cell carcinoma, cervical
adenonocarcinoma, or adenosquamous carcinoma.
[0134] In some embodiments, the method is for treating breast
cancer. In some embodiments, the breast cancer is early stage
breast cancer, non-metastatic breast cancer, locally advanced
breast cancer, metastatic breast cancer, hormone receptor positive
metastatic breast cancer, breast cancer in remission, breast cancer
in an adjuvant setting, ductal carcinoma in situ (DCIS), invasive
ductal carcinoma (IDC), or breast cancer in a neoadjuvant setting.
In some embodiments, the breast cancer is hormone receptor positive
metastatic breast cancer. In some embodiments, the breast cancer
(which may be HER2 positive or HER2 negative) is advanced breast
cancer. In some embodiments, the breast cancer is ductal carcinoma
in situ. In some embodiments, the individual may be a human who has
a gene, genetic mutation, or polymorphism associated with breast
cancer (e.g., BRCA1, BRCA2, ATM, CHEK2, RAD51, AR, DIRAS3, ERBB2,
TP53, AKT, PTEN, and/or PI3K) or has one or more extra copies of a
gene (e.g., one or more extra copies of the HER2 gene) associated
with breast cancer.
[0135] In some embodiments, the method is for treating pancreatic
cancer. In some embodiments, the pancreatic cancer includes, but is
not limited to, serous microcystic adenoma, intraductal papillary
mucinous neoplasm, mucinous cystic neoplasm, solid pseudopapillary
neoplasm, pancreatic adenocarcinoma, pancreatic ductal carcinoma,
or pancreatoblastoma. In some embodiments, the pancreatic cancer is
any of early stage pancreatic cancer, non-metastatic pancreatic
cancer, primary pancreatic cancer, resected pancreatic cancer,
advanced pancreatic cancer, locally advanced pancreatic cancer,
metastatic pancreatic cancer, unresectable pancreatic cancer,
pancreatic cancer in remission, recurrent pancreatic cancer,
pancreatic cancer in an adjuvant setting, or pancreatic cancer in a
neoadjuvant setting.
[0136] In some embodiments, the method is for treating ovarian
cancer. In some embodiments, the ovarian cancer is ovarian
epithelial cancer. Exemplary ovarian epithelial cancer histological
classifications include: serous cystomas (e.g., serous benign
cystadenomas, serous cystadenomas with proliferating activity of
the epithelial cells and nuclear abnormalities but with no
infiltrative destructive growth, or serous cystadenocarcinomas),
mucinous cystomas (e.g., mucinous benign cystadenomas, mucinous
cystadenomas with proliferating activity of the epithelial cells
and nuclear abnormalities but with no infiltrative destructive
growth, or mucinous cystadenocarcinomas), endometrioid tumors
(e.g., endometrioid benign cysts, endometrioid tumors with
proliferating activity of the epithelial cells and nuclear
abnormalities but with no infiltrative destructive growth, or
endometrioid adenocarcinomas), clear cell (mesonephroid) tumors
(e.g., benign clear cell tumors, clear cell tumors with
proliferating activity of the epithelial cells and nuclear
abnormalities but with no infiltrative destructive growth, or clear
cell cystadenocarcinomas), unclassified tumors that cannot be
allotted to one of the above groups, or other malignant tumors. In
various embodiments, the ovarian epithelial cancer is stage I
(e.g., stage IA, IB, or IC), stage II (e.g., stage IIA, IIB, or
IIC), stage III (e.g., stage IIIA, IIIB, or IIIC), or stage IV. In
some embodiments, the individual may be a human who has a gene,
genetic mutation, or polymorphism associated with ovarian cancer
(e.g., BRCA1 or BRCA2) or has one or more extra copies of a gene
associated with ovarian cancer (e.g., one or more extra copies of
the HER2 gene). In some embodiments, the ovarian cancer is an
ovarian germ cell tumor. Exemplary histologic subtypes include
dysgerminomas or other germ cell tumors (e.g., endodermal sinus
tumors such as hepatoid or intestinal tumors, embryonal carcinomas,
olyembryomas, choriocarcinomas, teratomas, or mixed form tumors).
Exemplary teratomas are immature teratomas, mature teratomas, solid
teratomas, and cystic teratomas (e.g., dermoid cysts such as mature
cystic teratomas, and dermoid cysts with malignant transformation).
Some teratomas are monodermal and highly specialized, such as
struma ovarii, carcinoid, struma ovarii and carcinoid, or others
(e.g., malignant neuroectodermal and ependymomas). In some
embodiments, the ovarian germ cell tumor is stage I (e.g., stage
IA, IB, or IC), stage II (e.g., stage IIA, IIB, or IIC), stage III
(e.g., stage IIIA, IIIB, or IIIC), or stage IV.
[0137] The improved MASCT methods described herein in some
embodiments are not applicable to patients with cancers of T-cell
origin, such as T-cell lymphoma.
[0138] Several viruses are related to cancer in humans. For
example, Hepatitis B virus (HBV) can cause chronic infection of the
liver, increasing an individual's chance of liver cancer, or
hepatocellular carcinoma (HCC). Human papilloma viruses (HPVs) are
a group of more than 150 related viruses, which cause papilloma, or
warts, when they infect and grow in skin or mucous membranes, such
as the mouth, throat, or vagina. Several types of HPV (including
types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 6) are
known to cause cervical cancer. HPVs also play a role in inducing
or causing other cancers of the genitalia, and are linked to some
cancers of the mouth and throat. Epstein-Barr virus (EBV) is a type
of herpes virus, which chronically infects and remains latent in B
lymphocytes. EBV infection increases an individual's risk of
developing nasopharyngeal carcinoma and certain types of
fast-growing lymphomas such as Burkitt lymphoma. EBV is also linked
to Hodgkin lymphoma and some cases of gastric cancer. In addition
to causing cancer or increasing risk of developing cancer, viral
infections, such as infections with HBV, HPV, and EBV, may result
in damage to tissues or organs, which can increase the disease
burden of an individual suffering from a cancer, and contribute to
cancer progression. It is known in the art that the human body can
be induced to mount effective and specific immune response,
including cytotoxic T cell response, against several cancer-related
viruses, such as HBV, HPV and EBV, including their various
subtypes. Therefore, in some embodiments, there is provided a
method of treating a virus-related cancer in an individual,
comprising administering to the individual an effective amount of
activated T cells, wherein the activated T cells are prepared by
co-culturing a population of T cells with a population of dendritic
cells loaded with a plurality of tumor antigen peptides, wherein
the plurality of tumor antigen peptides comprise one or more tumor
antigen peptides derived from the virus. In some embodiments, the
cancer is HBV-related hepatocellular carcinoma, HPV-related
cervical cancer, or EBV-related nasopharyngeal carcinoma.
[0139] The methods described herein can be used for any one or more
of the following purposes: alleviating one or more symptoms of
cancer, delaying progression of cancer, shrinking cancer tumor
size, disrupting (such as destroying) cancer stroma, inhibiting
cancer tumor growth, prolonging overall survival, prolonging
disease-free survival, prolonging time to cancer disease
progression, preventing or delaying cancer tumor metastasis,
reducing (such as eradiating) preexisting cancer tumor metastasis,
reducing incidence or burden of preexisting cancer tumor
metastasis, preventing recurrence of cancer, and/or improving
clinical benefit of cancer.
[0140] In some embodiments, there is provided a method of
inhibiting cancer cell proliferation (such as tumor growth) in an
individual, comprising administering to the individual an effective
amount of activated T cells. In some embodiments, the method
further comprises administering to the individual an effective
amount of dendritic cells loaded with a plurality of tumor antigen
peptides. In some embodiments, at least about 10% (including for
example at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or
100%) cell proliferation is inhibited.
[0141] In some embodiments, there is provided a method of
inhibiting tumor metastasis in an individual, comprising
administering to the individual an effective amount of activated T
cells. In some embodiments, the method further comprises
administering to the individual an effective amount of dendritic
cells loaded with a plurality of tumor antigen peptides. In some
embodiments, at least about 10% (including for example at least
about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) metastasis
is inhibited. In some embodiments, method of inhibiting metastasis
to lymph node is provided.
[0142] In some embodiments, there is provided a method of reducing
tumor size in an individual, comprising administering to the
individual an effective amount of activated T cells. In some
embodiments, the method further comprises administering to the
individual an effective amount of dendritic cells loaded with a
plurality of tumor antigen peptides. In some embodiments, the tumor
size is reduced at least about 10% (including for example at least
about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%).
[0143] In some embodiments, there is provided a method of
prolonging progression-free survival of cancer in an individual,
comprising administering to the individual an effective amount of
activated T cells. In some embodiments, the method further
comprises administering to the individual an effective amount of
dendritic cells loaded with a plurality of tumor antigen peptides.
In some embodiments, the method prolongs the time to disease
progression by at least any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
or 12 weeks.
[0144] In some embodiments, there is provided a method of
prolonging survival of an individual having cancer, comprising
administering to the individual an effective amount of activated T
cells. In some embodiments, the method further comprises
administering to the individual an effective amount of dendritic
cells loaded with a plurality of tumor antigen peptides. In some
embodiments, the method prolongs the time to disease progression by
at least about any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12
weeks. In some embodiments, the method prolongs the survival of the
individual by at least about any one of 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 18, or 24 months.
[0145] In some embodiments, there is provided a method of reducing
AEs and SAEs in an individual having cancer, comprising
administering to the individual an effective amount of activated T
cells. In some embodiments, the method further comprises
administering to the individual an effective amount of dendritic
cells loaded with a plurality of tumor antigen peptides.
[0146] In some embodiments, the method is predictive of and/or
results in an objective response (such as a partial response or
complete response). In some embodiments, the method is predictive
of and/or results in improved quality of life.
[0147] Some cancer immunotherapies are associated with
immune-related adverse events (irAEs) in additional to common
adverse events generally associated with other cancer therapies.
IrAEs are usually mechanistically related to either on-target
T-cell toxicity against target antigens that are expressed in
normal, non-tumor tissue, so called on-target off-tumor effect, or
off-target effects such as breaking of self-tolerance or epitope
cross-reaction. IrAEs can lead to severe symptoms and conditions on
the dermatologic, gastrointestinal, endocrine, hepatic, ocular,
neurologic, and other tissues or organs. Typical irAEs reported for
cancer immunotherapy methods known in the art include fatal
immune-mediated dermatitis, pneumonia, colitis, lymphocytic
hypophysitis, pancreatitis, lymphadenopathy, endocrine disorders,
CNS toxicity, and the like. In some embodiments, the improved MASCT
method is associated with low incidence of adverse events, such as
irAEs. In some embodiments, less than about any one of 50%, 40%,
30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% of individuals experience
irAEs, such as irAEs of Grade 2-5.
[0148] Generally, dosages, schedules, and routes of administration
of the activated T cells and the population of dendritic cells
loaded with the plurality of tumor antigen peptides may be
determined according to the size and condition of the individual,
and according to standard pharmaceutical practice. Exemplary routes
of administration include intravenous, intra-arterial,
intraperitoneal, intrapulmonary, intravesicular, intramuscular,
intra-tracheal, subcutaneous, intraocular, intrathecal, or
transdermal. In some embodiments, the dendritic cells loaded with
the plurality of tumor antigen peptides are administered
subcutaneously. In some embodiments, the activated T cells are
administered intravenously.
[0149] The dose of the cells administered to an individual may vary
according to, for example, the particular type of cells being
administered, the route of administration, and the particular type
and stage of cancer being treated. The amount should be sufficient
to produce a desirable response, such as a therapeutic response
against cancer, but without severe toxicity or adverse events. In
some embodiments, the amount of the activated T cells or the
dendritic cells to be administered is a therapeutically effective
amount. In some embodiments, the amount of the cells (such as
multiple-antigen loaded dendritic cells, or the activated T cells)
is an amount sufficient to decrease the size of a tumor, decrease
the number of cancer cells, or decrease the growth rate of a tumor
by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, 95% or 100% compared to the corresponding tumor size, number
of cancer cells, or tumor growth rate in the same individual prior
to treatment or compared to the corresponding activity in other
individuals not receiving the treatment. Standard methods can be
used to measure the magnitude of this effect, such as in vitro
assays with purified enzyme, cell-based assays, animal models, or
human testing.
[0150] In some embodiments, the population of dendritic cells
loaded with the plurality of tumor antigen peptides is administered
at a dose at least about any one of 1.times.10.sup.5,
5.times.10.sup.5, 1.times.10.sup.6, 1.5.times.10.sup.6,
2.times.10.sup.6, 3.times.10.sup.6, 4.times.10.sup.6,
5.times.10.sup.6, 6.times.10.sup.6, 7.times.10.sup.6,
8.times.10.sup.6, 9.times.10.sup.6, 1.times.10.sup.7 or
5.times.10.sup.7 cells/individual. In some embodiments, the
population of dendritic cells loaded with the plurality of tumor
antigen peptides are administered at a dose about any one of
1.times.10.sup.5-5.times.10.sup.5,
5.times.10.sup.5-1.times.10.sup.6,
1.times.10.sup.6-2.times.10.sup.6,
2.times.10.sup.6-3.times.10.sup.6,
3.times.10.sup.6-4.times.10.sup.6,
4.times.10.sup.6-5.times.10.sup.6,
5.times.10.sup.6-6.times.10.sup.6,
6.times.10.sup.6-7.times.10.sup.6,
7.times.10.sup.6-8.times.10.sup.6,
8.times.10.sup.6-1.times.10.sup.8,
1.times.10.sup.6-3.times.10.sup.6,
3.times.10.sup.6-5.times.10.sup.6,
5.times.10.sup.6-7.times.10.sup.6,
2.times.10.sup.6-2.times.10.sup.7,
5.times.10.sup.6-2.times.10.sup.7 or
1.times.10.sup.6-2.times.10.sup.7 cells/individual. In some
embodiments, the dendritic cells loaded with the plurality of tumor
antigen peptides are administered at a dose of at least about
1.times.10.sup.6 cells/individual. In some embodiments, the
dendritic cells loaded with the plurality of tumor antigen peptides
are administered at a dose of about 1.5.times.10.sup.6 to about
1.5.times.10.sup.7 cells/individual.
[0151] In some embodiments, the population of dendritic cells
loaded with the plurality of tumor antigen peptides are
administered at a dose at least about any one of 1.times.10.sup.4,
2.5.times.10.sup.4, 5.times.10.sup.4, 1.times.10.sup.5,
2.times.10.sup.5, 2.5.times.10.sup.5, 4.times.10.sup.5,
6.times.10.sup.5, 8.times.10.sup.5, 1.times.10.sup.6,
2.times.10.sup.6 or 1.times.10.sup.7 cells/kg. In some embodiments,
the population of dendritic cells loaded with the plurality of
tumor antigen peptides are administered at a dose about any one of
1.times.10.sup.4-5.times.10.sup.4,
5.times.10.sup.4-1.times.10.sup.5,
1.times.10.sup.5-2.times.10.sup.5,
2.times.10.sup.5-4.times.10.sup.5,
4.times.10.sup.5-6.times.10.sup.5,
6.times.10.sup.5-8.times.10.sup.5,
8.times.10.sup.5-1.times.10.sup.6,
1.times.10.sup.6-2.times.10.sup.6,
2.times.10.sup.6-1.times.10.sup.7,
1.times.10.sup.4-1.times.10.sup.5,
1.times.10.sup.5-1.times.10.sup.6,
1.times.10.sup.6-1.times.10.sup.7,
1.times.10.sup.4-1.times.10.sup.6, or
1.times.10.sup.5-1.times.10.sup.7 cells/kg. In some embodiments,
the dendritic cells loaded with the plurality of tumor antigen
peptides are administered at a dose of at least about
2.times.10.sup.5 cells/kg. In some embodiments, the dendritic cells
loaded with the plurality of tumor antigen peptides are
administered at a dose of about 2.5.times.10.sup.4 to about
2.5.times.10.sup.5 cells/kg.
[0152] In some embodiments, the activated T cells are administered
at a dose of at least about any one of 1.times.10.sup.8,
5.times.10.sup.8, 1.times.10.sup.9, 2.times.10.sup.9,
3.times.10.sup.9, 4.times.10.sup.9, 5.times.10.sup.9,
6.times.10.sup.9, 7.times.10.sup.9, 8.times.10.sup.9,
9.times.10.sup.9, 1.times.10.sup.15, 1.5.times.10.sup.15,
2.times.10.sup.15, or 5.times.10.sup.15 cells/individual. In some
embodiments, the activated T cells are administered at a dose of
about any one of 1.times.10.sup.8-5.times.10.sup.8,
5.times.10.sup.8-1.times.10.sup.9,
1.times.10.sup.9-5.times.10.sup.9,
5.times.10.sup.9-1.times.10.sup.15,
3.times.10.sup.9-7.times.10.sup.9,
1.times.10.sup.15-2.times.10.sup.15, or
1.times.10.sup.9-1.times.10.sup.15 cells/individual. In some
embodiments, the activated T cells are administered at a dose of at
least about 3.times.10.sup.9 cells/individual. In some embodiments,
the activated T cells are administered at a dose of about
1.times.10.sup.9 to about 1.times.10.sup.15 cells/individual.
[0153] In some embodiments, the activated T cells are administered
at a dose of at least about any one of 1.times.10.sup.7,
2.times.10.sup.7, 4.times.10.sup.7, 6.times.10.sup.7,
8.times.10.sup.7, 1.times.10.sup.8, 2.times.10.sup.8,
4.times.10.sup.8, 6.times.10.sup.8, 8.times.10.sup.8,
1.times.10.sup.9 cells/kg. In some embodiments, the activated T
cells are administered at a dose of about any one of
1.times.10.sup.7-1.times.10.sup.8,
1.times.10.sup.7-5.times.10.sup.7,
2.times.10.sup.7-4.times.10.sup.7, 5'10.sup.7-1.times.10.sup.8,
1.times.10.sup.8-2.times.10.sup.8,
5.times.10.sup.7-1.times.10.sup.8,
1.times.10.sup.8-2.times.10.sup.8,
2.times.10.sup.8-5.times.10.sup.8,
1.times.10.sup.8-1.times.10.sup.9, or
1.times.10.sup.7-1.times.10.sup.9 cells/kg. In some embodiments,
the activated T cells are administered at a dose of at least about
6.times.10.sup.7 cells/kg. In some embodiments, the activated T
cells are administered at a dose of about 1.5.times.10.sup.7 to
about 2.times.10.sup.8 cells/kg.
[0154] In some embodiments, a stabilizing agent or an excipient,
such as human albumin, is used together with the activated T cells,
and/or the dendritic cells loaded with the plurality of tumor
antigen peptides.
[0155] The dosage and dosing schedule of the cells in the improved
MASCT method may be adjusted over the course of the treatment,
based on the judgment of the administering physician. In some
embodiments, the activated T cells are administered at least about
any one of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8
days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days,
16 days, 17 days, 18 days, 19 days, 20 days, 21 days, or 28 days,
after the dendritic cells loaded with the plurality of tumor
antigen peptides are administered. In some embodiments, the
activated T cells are administered concurrently with the dendritic
cells. In some embodiments, the activated T cells are administered
about 17-26 days after the dendritic cells are administered. In
some embodiments, the activated T cells are administered about 17
days after the dendritic cells are administered.
[0156] The improved MASCT method may comprise a single treatment,
or repeated treatments. In some embodiments, the activated T cells
are administered for at least about any one of 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, or more than 10 times. In some embodiments, the activated
T cells are administered at least 3 times. In some embodiments, the
dendritic cells are administered for at least about any one of 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times. In some
embodiments, the dendritic cells are administered at least 3 times.
In some embodiments, one or more cell (such as antigen-loaded
dendritic cell or activated T cells) preparation steps are repeated
prior to the repeated administration of the dendritic cells, the
activated T cells, or both. In some embodiments, the improved MASCT
method is repeated once per week, once 2 weeks, once 3 weeks, once
4 weeks, once per month, once per 2 months, once per 3 months, once
per 4 months, once per 5 months, once per 6 months, once per 7
months, once per 8 months, once per 9 months, or once per year. In
some embodiments, the interval between each administration of the
dendritic cells, or the activated T cells is about any one of 1
week to 2 weeks, 2 weeks to 1 month, 2 weeks to 2 months, 1 month
to 2 months, 1 month to 3 months, 3 months to 6 months, or 6 months
to a year. In some embodiments, the interval between each
administration of the dendritic cells or the activated T cells is
about 1 day to about 5 months, such as about 2 weeks to about 2
months, or about 2 months to about 5 months. In some embodiments,
all steps of the improved MASCT method are repeated once per month
during the first 6 months of treatment, every two months for the
second 6 months of treatment, and every half a year after first 12
months of treatment if the individual has stable disease. Any
embodiment of the improved MASCT method described herein can be
combined with any other embodiment of the improved MASCT method
during the full course of a repeated treatment.
[0157] The improved MASCT method provided herein may be used as a
first therapy, second therapy, third therapy, or combination
therapy with other types of cancer therapies known in the art, such
as chemotherapy, surgery, radiation, gene therapy, immunotherapy,
bone marrow transplantation, stem cell transplantation, targeted
therapy, cryotherapy, ultrasound therapy, photodynamic therapy,
radio-frequency ablation or the like, in an adjuvant setting or a
neoadjuvant setting. In some embodiments, the improved MASCT method
is used as a first therapy. In some embodiments, there exists no
other approved anti-cancer therapy for the individual. In some
embodiments, the improved MASCT method is used as a second therapy,
wherein the individual has previously received resection,
radio-frequency ablation, chemotherapy, radiation therapy, or other
types of cancer therapy. In some embodiments, the individual has
progressed or has not been able to tolerate standard anti-cancer
therapy. In some embodiments, the individual receives other types
of cancer therapy prior to, concurrently with, or after the
improved MASCT treatment(s). For example, the improved MASCT method
may precede or follow the other cancer therapy (such as
chemotherapy, radiation, surgery or combination thereof) by
intervals ranging from minutes, days, weeks to months. In some
embodiments, the interval between the first and the second therapy
is such that the activated T cells of the improved MASCT method and
the other cancer therapy (such as chemotherapy, radiation, surgery,
or combination thereof) would be able to exert an advantageously
combined effect on the individual. In some embodiments, the
improved MASCT method is used in conjunction with other cancer
therapy (such as chemotherapy, radiation, surgery, or combination
thereof) treat cancer in an individual. The combination therapy
methods described herein may be performed alone or in conjunction
with another therapy, such as surgery, radiation, gene therapy,
immunotherapy, bone marrow transplantation, stem cell
transplantation, hormone therapy, targeted therapy, cryotherapy,
ultrasound therapy, photodynamic therapy, chemotherapy or the like.
Additionally, a person having a greater risk of developing a
proliferative disease may receive treatments to inhibit and/or
delay the development of the disease.
[0158] The methods described herein for treating cancer can be used
in monotherapy as well as in combination therapy with another
agent. For example, any of the treatment methods described herein
may be combined with administration of one or more (such as any of
1, 2, 3, 4, or more) immune checkpoint inhibitors. In some
embodiments, the immune checkpoint inhibitor is selected from the
group consisting of inhibitors of PD-1, PD-L1, CTLA-4, IDO, TIM-3,
BTLA, VISTA, and LAG-3.
[0159] In some embodiments, the immune checkpoint inhibitor is an
inhibitor of PD-1. In some embodiments, the immune checkpoint
inhibitor is an anti-PD-1 antibody. Exemplary anti-PD-1 antibodies
include, but are not limited to, Nivolumab, pembrolizumab,
pidilizumab, BMS-936559, and atezolizumab, Pembrolizumab, MK-3475,
AMP-224, AMP-514, STI-A1110, and TSR-042. In some embodiments, the
immune checkpoint inhibitor is nivolumab (for example,
OPDIVO.RTM.). In some embodiments, the immune checkpoint inhibitor
is Pembrolizumab (for example, KEYTRUDA.RTM.). In some embodiments,
the immune checkpoint inhibitor is SHR-1210.
[0160] In some embodiments, the immune checkpoint inhibitor is an
inhibitor of PD-L1. In some embodiments, the immune checkpoint
inhibitor is an anti-PD-L1 antibody. Exemplary anti-PD-L1
antibodies include, but are not limited to, KY-1003, MCLA-145,
RG7446, BMS935559, MPDL3280A, MEDI4736, Avelumab, or STI-A1010.
[0161] In some embodiments, the immune checkpoint inhibitor is an
inhibitor of CTLA-4. In some embodiments, the immune checkpoint
inhibitor is an anti-CTLA-4 antibody. Exemplary anti-CTLA-4
antibodies include, but are not limited to, Ipilimumab,
Tremelimumab, and KAHR-102. In some embodiments, the immune
checkpoint inhibitor is Ipilimumab (for example, YERVOY.RTM.).
[0162] In some embodiments, the activated T cells and the immune
checkpoint inhibitor are administered simultaneously. In some
embodiments, the activated T cells and the immune checkpoint
inhibitor are administered in a single composition. In some
embodiments, the immune checkpoint inhibitor is present in the
co-culture. In some embodiments, the activated T cells and the
immune checkpoint inhibitor are admixed prior to (such as
immediately prior to) the administration. In some embodiments, the
activated T cells and the immune checkpoint inhibitor are
administered simultaneously via separate compositions.
[0163] In some embodiments, the activated T cells and the immune
checkpoint inhibitor are administered sequentially. In some
embodiments, the immune checkpoint inhibitor is administered prior
to the administration of the activated T cells. In some
embodiments, the immune checkpoint inhibitor is administered after
the administration of the activated T cells.
[0164] Exemplary routes of administration of the immune checkpoint
inhibitor include, but are not limited to, intratumoral,
intravesical, intramuscular, intraperitoneal, intravenous,
intra-arterial, intracranial, intrapleural, subcutaneous, and
epidermal routes, or be delivered into lymph glands, body spaces,
organs or tissues known to contain such live cancer cells. In some
embodiments, the immune checkpoint inhibitor is administered
intravenously. In some embodiments, the immune checkpoint inhibitor
is administered by infusion. In some embodiments, the immune
checkpoint inhibitor is infused over at least about any of 10
minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, or more. In
some embodiments, the immune checkpoint inhibitor is administered
via the same administration route as the activated T cells. In some
embodiments, the immune checkpoint inhibitor is administered via a
different administration route as the activated T cells.
[0165] Suitable dose of the immune checkpoint inhibitor include,
but are not limited to, about any one of 1 mg/m.sup.2, 5
mg/m.sup.2, 10 mg/m.sup.2, 20 mg/m.sup.2, 50 mg/m.sup.2, 100
mg/m.sup.2, 200 mg/m.sup.2, 300 mg/m.sup.2, 400 mg/m.sup.2, 500
mg/m.sup.2, 750 mg/m.sup.2, 1000 mg/m.sup.2, or more. In some
embodiments, the dose of immune checkpoint inhibitor is any one of
about 1 to about 5 mg/m.sup.2, about 5 to about 10 mg/m.sup.2,
about 10 to about 20 mg/m.sup.2, about 20 to about 50 mg/m.sup.2,
about 50 to about 100 mg/m.sup.2, about 100 mg/m.sup.2 to about 200
mg/m.sup.2, about 200 to about 300 mg/m.sup.2, about 300 to about
400 mg/m.sup.2, about 400 to about 500 mg/m.sup.2, about 500 to
about 750 mg/m.sup.2, or about 750 to about 1000 mg/m.sup.2. In
some embodiments, the dose of immune checkpoint inhibitor is about
any one of 1 .mu.g/kg, 2 .mu.g/kg, 5 .mu.g/kg, 10 .mu.g/kg, 20
.mu.g/kg, 50 .mu.g/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg,
0.5 mg/kg, 1 mg/kg, 2 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 50 mg/kg,
100 mg/kg, or more. In some embodiments, the dose of the immune
checkpoint inhibitor is any one of about 1 .mu.g/kg to about 5
.mu.g/kg, about 5 .mu.g/kg to about 10 .mu.g/kg, about 10 .mu.g/kg
to about 50 .mu.g/kg, about 50 .mu.g/kg to about 0.1 mg/kg, about
0.1 mg/kg to about 0.2 mg/kg, about 0.2 mg/kg to about 0.3 mg/kg,
about 0.3 mg/kg to about 0.4 mg/kg, about 0.4 mg/kg to about 0.5
mg/kg, about 0.5 mg/kg to about 1 mg/kg, about 1 mg/kg to about 5
mg/kg, about 5 mg/kg to about 10 mg/kg, about 10 mg/kg to about 20
mg/kg, about 20 mg/kg to about 50 mg/kg, about 50 mg/kg to about
100 mg/kg, or about 1 mg/kg to about 100 mg/kg.
[0166] In some embodiments, the immune checkpoint inhibitor is
administered daily. In some embodiments, the immune checkpoint
inhibitor is administered is administered at least about any one of
1.times., 2.times., 3.times., 4.times., 5.times., 6.times., or
7.times. (i.e., daily) a week. In some embodiments, the immune
checkpoint inhibitor is administered weekly. In some embodiments,
the immune checkpoint inhibitor is administered weekly without
break; weekly, two out of three weeks; weekly three out of four
weeks; once every two weeks; once every 3 weeks; once every 4
weeks; once every 6 weeks; once every 8 weeks, monthly, or every
two to 12 months. In some embodiments, the intervals between each
administration are less than about any one of 6 months, 3 months, 1
month, 20 days, 15, days, 12 days, 10 days, 9 days, 8 days, 7 days,
6 days, 5 days, 4 days, 3 days, 2 days, or 1 day. In some
embodiments, the intervals between each administration are more
than about any one of 1 month, 2 months, 3 months, 4 months, 5
months, 6 months, 8 months, or 12 months. In some embodiments, the
immune checkpoint inhibitor is administered once every 3 months. In
some embodiments, there is no break in the dosing schedule. In some
embodiments, the interval between each administration is no more
than about a week. In some embodiments, the immune checkpoint
inhibitor is administered with the same dosing schedule as the
activated T cells. In some embodiments, the immune checkpoint
inhibitor is administered with a different dosing schedule as the
activated T cells.
[0167] In some embodiments, the immune checkpoint inhibitor is
administered in every improved MASCT treatment cycle. For example,
the immune checkpoint inhibitor may be administered about any of 1,
2, 3, 4, 5, 6, or more times every improved MASCT treatment cycle.
In some embodiments, the immune checkpoint inhibitor is not
administered in every improved MASCT treatment cycle. For example,
the immune checkpoint inhibitor may be administered about once
every 1, 2, 3, 4, 5, or more improved MASCT treatment cycles.
[0168] The administration of the immune checkpoint inhibitor can be
over an extended period of time, such as from about a month up to
about seven years. In some embodiments, the immune checkpoint
inhibitor is administered over a period of at least about any one
of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 48, 60,
72, or 84 months. In some embodiments, the immune checkpoint
inhibitor is administered for a single time. In some embodiments,
the immune checkpoint inhibitor is administered repeatedly. In some
embodiments, the immune checkpoint inhibitor is administered
repeatedly until disease progression.
IV. PRECISION IMPROVED MASCT METHODS
[0169] Further provided herein are precision improved MASCT methods
that are customized to the individual being treated based on the
genetics and therapeutic response of the individual. Any of the
methods of treatment described above in Section III may be
customized to provide a precision improved MASCT method.
[0170] The improved MASCT methods described herein in some
embodiments are particularly suitable for a certain population of
individuals, such as individuals with a low mutation load (such as
in the MHC genes) in the cancer (such as all or a subset of cancer
cells), and/or individuals with one or more neoantigens.
Mutation Load
[0171] In some embodiments, the improved MASCT method is
particularly suitable for an individual with a low total mutation
load in the cancer of the individual. In some embodiments, the
improved MASCT method is particularly suitable for an individual
with a low mutation load in the cancer-associated genes in the
cancer of the individual. In some embodiments, the improved MASCT
method is particularly suitable for an individual with a low
mutation load in immune genes related to T cell response in the
cancer of the individual. In some embodiments, the improved MASCT
method is particularly suitable for an individual with a low
mutation load in the MHC genes in the cancer of the individual. The
mutation load may be mutation load in all cancer cells, or a subset
of cancer cells, such as a primary or metastatic tumor site, for
example, cells in a tumor biopsy sample.
[0172] Thus, in some embodiments, there is provided a method of
treating a cancer in an individual, comprising: (a) optionally
administering an effective amount of dendritic cells loaded with a
plurality of tumor antigen peptides; and (b) administering to the
individual an effective amount of activated T cells, wherein the
activated T cells are prepared by any one of the methods of
preparing activated T cells described above in Section II, and
wherein the individual has a low mutation load in the cancer.
[0173] In some embodiments, there is provided a method of treating
a cancer in an individual, comprising: (a) selecting the individual
for the method based on the mutation load in the cancer; (b)
optionally administering an effective amount of dendritic cells
loaded with a plurality of tumor antigen peptides; and (c)
administering to the individual an effective amount of activated T
cells, wherein the activated T cells are prepared by any one of the
methods of preparing activated T cells described above in Section
II.
[0174] In some embodiments, there is provided a method of treating
a cancer in an individual, comprising: (a) optionally administering
an effective amount of dendritic cells loaded with a plurality of
tumor antigen peptides; and (b) administering to the individual an
effective amount of activated T cells, wherein the activated T
cells are prepared by any one of the methods of preparing activated
T cells described above in Section II, and wherein the individual
is selected for treatment based on having a low mutation load in
the cancer.
[0175] In some embodiments, a low mutation load of one or more
genes is a low number of mutations accumulated on the one or more
genes. In some embodiments, a total number of no more than about
any of 500, 400, 300, 200, 100, 50, 40, 30, 20, 10, 5 or fewer
mutations indicate a low mutation load. In some embodiments, no
more than about any of 50, 40, 30, 25, 20, 15, 14, 13, 12, 11, 10,
9, 8, 7, 6, 5, 4, 3, 2 or 1 mutations in the one or more MHC genes
indicate a low mutation load of the one or more MHC genes. In some
embodiments, a low mutation load of one or more genes is a low
ratio between the number of mutations accumulated on the one or
more genes (such as MHC genes) and the total number of mutations in
a selected set of genes (such as cancer-associated genes) or the
full genome.
[0176] In some embodiments, the one or more MHC genes comprise MHC
class I genes (or loci). In some embodiments, the one or more MHC
genes comprise MHC class II genes (or loci). In some embodiments,
wherein the individual is a human individual, the one or more MHC
genes are selected from the group consisting of HLA-A, HLA-B, HLA-C
and B2M.
[0177] Exemplary mutations include, but are not limited to,
deletion, frameshift, insertion, indel, missense mutation, nonsense
mutation, point mutation, copy number variation, single nucleotide
variation (SNV), silent mutation, splice site mutation, splice
variant, gene fusion, and translocation. In some embodiments, the
copy number variation of the MHC gene is caused by structural
rearrangement of the genome, including deletions, duplications,
inversion, and translocation of a chromosome or a fragment thereof.
In some embodiments, the mutations in the one or more MHC genes are
selected from point mutations, frameshift mutations, gene fusions,
and copy number variations. In some embodiments, the mutations are
in the protein-coding region of the MHC genes. In some embodiments,
the mutation is a nonsynonymous mutation. In some embodiments, the
mutation is not a polymorphism. In some embodiments, the mutation
is present in normal cells of the individual. In some embodiments,
the mutation is not present in normal cells of the individual. In
some embodiments, the mutation affects the physiochemical or
functional properties, such as stability or binding affinity, of
the MHC molecule encoded by the affected gene. In some embodiments,
the mutation results in an irreversible deficiency in the MHC
molecule. In some embodiments, the mutation reduces the binding
affinity of the MHC molecule to T cell epitopes and/or T cell
receptors. In some embodiments, the mutation is a loss-of-function
mutation. In some embodiments, the mutation results in reversible
deficiency in the MHC molecule. In some embodiments, the mutation
does not affect the binding affinity of the MHC molecule to T cell
epitopes and/or T cell receptors. In some embodiments, the mutation
is a somatic mutation. In some embodiments, the mutation is a
germline mutation.
[0178] The mutations counted towards the mutation load may be
present in all cancer cells or in a subset of cancer cells. In some
embodiments, the mutations are present in all cancer cells in the
individual. In some embodiments, the mutations are present in all
cancer cells of a tumor site. In some embodiments, the mutations
are clonal. In some embodiments, the mutations are subclonal. In
some embodiments, the mutations are present in at least about any
of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more cancer
cells of the individual.
[0179] The mutations in certain MHC genes and/or in certain domains
or positions of the one or more MHC genes may have more profound
influence on the clinical response of the individual to the
improved MASCT methods described herein. For example,
loss-of-function mutations may occur in the leader peptide
sequence, a3 domain (which binds the CD8 co-receptor of T cells),
a1 peptide binding domain, or a2 peptide binding domain of the HLA
molecule; see, for example, Shukla S. et al. Nature Biotechnology
33, 1152-1158 (2015), incorporated herein by reference. Mutations
in B2M (.beta.2-macroglobulin) gene may also promote tumor escape
phenotypes. See, for example, Monica B et al. Cancer Immunol.
Immu., (2012) 61: 1359-1371. In some embodiments, presence of any
number (such as 1, 2, 3, 4, 5, or more) of mutations in the
functional regions of the one or more MHC genes, such as the leader
peptide sequence, a1 domain, a2 domain, or a3 domain, indicates a
high mutation load. In some embodiments, presence of any number
(such as 1, 2, 3, 4, 5, or more) loss-of-function mutations in the
one or more MHC genes (such as HLA-A, HLA-B or HLA-C genes in human
individuals) indicates a high mutation load. In some embodiments, a
low mutation load in the one or more MHC genes comprises no
mutation in the functional regions, including leader peptide
sequence, a1 domain (for example, residues in direct contact with
the CD8 co-receptor), a2 domain, and a3 domain (for example,
residues in direct contact with the epitope), of the one or more
MHC genes (such as HLA-A, HLA-B or HLA-C genes). In some
embodiments, presence of any number of mutations (such as
loss-of-function mutations) in the B2M gene indicates a high
mutation load. In some embodiments, a low mutation load in the one
or more MHC genes comprises no mutation in the B2M gene.
[0180] The mutation load of one or more genes (such as MHC genes)
may be determined by any known methods in the art, including, but
not limited to, genomic DNA sequencing, exome sequencing, or other
DNA sequencing-based methods using Sanger sequencing or next
generation sequencing platforms; polymerase chain reaction assays;
in situ hybridization assays; and DNA microarrays.
[0181] In some embodiments, the mutation load of the one or more
MHC genes is determined by sequencing a tumor sample from the
individual. In some embodiments, the sequencing is next generation
sequencing. In some embodiments, the sequencing is full genome
sequencing. In some embodiments, the sequencing is exome
sequencing. In some embodiments, the sequencing is targeted
sequencing of candidate genes, such as cancer-associated genes plus
HLA genes. For example, ONCOGXONE.TM. Plus (Admera Health), are
available to sequence cancer-associated genes and HLA loci with
high sequencing depth. In some embodiments, the same sequencing
data can be used to determine the mutation load of the one or more
MHC genes and to identify neoantigens in the individual.
[0182] In some embodiments, the tumor sample is a tissue sample. In
some embodiments, the tumor sample is a tumor biopsy sample, such
as fine needle aspiration of tumor cells or laparoscopy obtained
tumor cells (such as including tumor stroma). In some embodiments,
the tumor sample is freshly obtained. In some embodiments, the
tumor sample is frozen. In some embodiments, the tumor sample is a
Formaldehyde Fixed-Paraffin Embedded (FFPE) sample. In some
embodiments, the tumor sample is a cell sample. In some
embodiments, the tumor sample comprises a circulating metastatic
cancer cell. In some embodiments, the tumor sample is obtained by
sorting circulating tumor cells (CTCs) from blood. In some
embodiments, nucleic acids (such as DNA and/or RNA) are extracted
from the tumor sample for the sequencing analysis. In some
embodiments, the sequencing data of the tumor sample is compared to
the sequencing data of a reference sample, such as a sample of a
healthy tissue from the same individual, or a sample of a healthy
individual, to identify mutations and determine mutation load in
the tumor cells. In some embodiments, the sequencing data of the
tumor sample is compared to the reference sequences from a genome
database to identify mutations and determine mutation load in the
tumor cells.
Neoantigen Peptides
[0183] In some embodiments, the improved MASCT method is
particularly suitable for treating an individual with one or more
neoantigens. Any of the improved MASCT methods described herein
using one or more neoantigen peptides in the plurality of tumor
antigen peptides may further comprise the steps of selecting the
individual for the method of treating based on having one or more
(such as at least 5) neoantigens in the individual, and/or the
steps of: (i) identifying a neoantigen of the individual; and (ii)
incorporating a neoantigen peptide derived from the neoantigen in
the plurality of tumor antigen peptides for use in the improved
MASCT method.
[0184] Thus, in some embodiments, there is provided a method of
treating a cancer in an individual, comprising: (a) identifying a
neoantigen of the individual; (b) incorporating a neoantigen
peptide in a plurality of tumor antigen peptides, wherein the
neoantigen peptide comprises a neoepitope in the neoantigen; (c)
optionally administering an effective amount of dendritic cells
loaded with the plurality of tumor antigen peptides; (d) preparing
a population of activated T cells using any one of the methods of
preparing activated T cells described above in Section II; and (e)
administering to the individual an effective amount of activated T
cells, wherein the individual has one or more neoantigens.
[0185] The individual may have any number (such as at least about
any one of 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 50,
100 or more) of neoantigens in order to benefit from the improved
MASCT method using a plurality of tumor antigen peptides comprising
a neoantigen peptide. In some embodiments, the improved MASCT
method is particularly suitable for an individual having at least
about any one of 4, 5, 6, 7, 8, 10, 15, 20, 50, 100 or more
neoantigens. In some embodiments, the neoantigen comprises one or
more neoepitopes. In some embodiments, the improved MASCT method is
particularly suitable for an individual having at least about any
one of 4, 5, 6, 7, 8, 10, 15, 20, 50, 100 or more neoepitopes. In
some embodiments, the T cell epitopes are MHC-I restricted
epitopes. In some embodiments, the neoepitope has a higher affinity
to the MHC molecules of the individual than the corresponding
wildtype T cell epitope. In some embodiments, the neoepitope has
higher affinity to a model T cell receptor than the corresponding
wildtype T cell epitope. In some embodiments, the neoantigen (or
neoepitope) is a clonal neoantigen. In some embodiments, the
neoantigen (or neoepitope) is a subclonal neoantigen. In some
embodiments, the neoantigen (or neoepitope) is present in at least
about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%
or more tumor cells in the individual.
[0186] The number of neoantigens may be combined with other
biomarkers or selection criteria to select an individual for any
one of the improved MASCT methods described herein. In some
embodiments, the improved MASCT method is particularly suitable for
an individual having a low mutation load (such as in one or more
MHC genes) in the cancer cells, and at least about any of 4, 5, 6,
7, 8, 10 or more neoantigens (such as neoantigens with high
affinity MHC-I restricted neoepitopes).
[0187] Any number (such as any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or
more) of neoantigen peptides may be designed based on the
neoantigens of the individual and to be incorporated in the
plurality of tumor antigen peptides for use in any of the improved
MASCT methods described herein. In some embodiments, the plurality
of tumor antigen peptides comprises a single neoantigen peptide. In
some embodiments, the plurality of tumor antigen peptides comprises
a plurality of neoantigen peptides. Each neoantigen peptide may
comprise one or more neoepitopes from a neoantigen of the
individual. In some embodiments, the neoepitope is a T cell
epitope. Methods of designing a neoantigen peptide based on a
neoantigen are described in the section "Plurality of tumor antigen
peptides."
[0188] The neoantigens in the individual may be identified using
any known methods in the art. In some embodiments, the neoantigen
is identified based on the genetic profile of a tumor sample from
the individual. Each neoantigen comprises one or more neoepitopes.
In some embodiments, the one or more neoepitopes in the neoantigen
are identified based on the genetic profile of the tumor sample.
Any known genetic profiling methods, such as next generation
sequencing (NGS) methods, microarrays, or proteomic methods may be
used to provide the genetic profile of the tumor sample.
[0189] In some embodiments, the neoantigen is identified by
sequencing a tumor sample from the individual. In some embodiments,
the sequencing is next generation sequencing. In some embodiments,
the sequencing is full-genome sequencing. In some embodiments, the
sequencing is exome sequencing, such as whole exome sequencing
("WES"). In some embodiments, the sequencing is RNA sequencing. In
some embodiments, the sequencing is targeted sequencing of
candidate genes, such as cancer-associated genes. Many commercial
NGS cancer panels, for example, ONCOGXONE.TM. Plus (Admera Health),
are available to sequence cancer-associated genes with high
sequencing depth.
[0190] In some embodiments, the tumor sample is a tissue sample. In
some embodiments, the tumor sample is a tumor biopsy sample, such
as fine needle aspiration of tumor cells or laparoscopy obtained
tumor cells (such as including tumor stroma). In some embodiments,
the tumor sample is freshly obtained. In some embodiments, the
tumor sample is frozen. In some embodiments, the tumor sample is a
Formaldehyde Fixed-Paraffin Embedded (FFPE) sample. In some
embodiments, the tumor sample is a cell sample. In some
embodiments, the tumor sample comprises a circulating metastatic
cancer cell. In some embodiments, the tumor sample is obtained by
sorting circulating tumor cells (CTCs) from blood. In some
embodiments, nucleic acids (such as DNA and/or RNA) are extracted
from the tumor sample for the sequencing analysis. In some
embodiments, proteins are extracted from the tumor sample for the
sequencing analysis.
[0191] In some embodiments, the genetic profile of the tumor sample
is compared to the genetic profile of a reference sample, such as a
sample of a healthy tissue from the same individual, or a sample of
a healthy individual, to identify candidate mutant genes in the
tumor cells. In some embodiments, the genetic profile of the tumor
sample is compared to the reference sequences from a genome
database to identify candidate mutant genes in the tumor cells. In
some embodiments, the candidate mutant genes are cancer-associated
genes. In some embodiments, each candidate mutant gene comprises
one or more mutations, such as non-synonymous substitutions, single
nucleotide variation (SNV), indel (insertion or deletion, e.g.,
non-frame shift indel), new open reading frame (ORF), or gene
fusion, which may give rise to a neoantigen. Common Single
Nucleotide Polymorphisms (SNPs) are excluded from the candidate
mutations.
[0192] In some embodiments, neoepitopes in neoantigens are
identified from the candidate mutant proteins. In some embodiments,
the neoepitopes are predicted in silico. Exemplary bioinformatics
tools for T cell epitope prediction are known in the art, for
example, see Yang X. and Yu X. (2009) "An introduction to epitope
prediction methods and software" Rev. Med. Virol. 19(2): 77-96.
Factors considered in the T cell epitope prediction algorithms
include, but are not limited to, MHC subtype of the individual,
sequence-derived physiochemical properties of the T cell epitope,
MHC binding motifs, proteasomal cleavage pattern, transporter
associated with antigen processing (TAP) transport efficiency, MHC
binding affinity, peptide-MHC stability, and T-cell receptor
binding affinity. In some embodiments, the neoepitope is an MHC-I
restricted epitope. In some embodiments, the neoepitope is an
MHC-II restricted epitope.
[0193] In some embodiments, the neoepitope has high affinity to the
MHC molecules of the individual. In some embodiments, the method
further comprises determining the MHC subtype of the individual,
for example, from the sequencing data, to identify one or more MHC
molecules of the individual. In some embodiments, the method
further comprises determining the affinity of the neoepitope to an
MHC molecule, such as an MHC class I molecule. In some embodiments,
the method comprises determining the affinity of the neoepitope to
one or more MHC (such as MHC class I) molecules of the individual.
In some embodiments, the affinity of the neoepitope to one or more
MHC molecules of the individual is compared to the affinity of the
corresponding wildtype epitope to the one or more MHC molecules of
the individual. In some embodiments, the neoepitope is selected for
having a higher (such as at least about any of 1.5, 2, 5, 10, 15,
20, 25, 50, 100, or more times) affinity to the one or more MHC
molecules (such as MHC-I molecules) of the individual than the
corresponding wildtype epitope. In some embodiments, the MHC
binding affinity is predicted in silico using any known tools or
methods in the art. In some embodiments, the MHC binding affinity
is determined experimentally, such as using an in vitro binding
assay.
[0194] In some embodiments, the method further comprises
determining the affinity of the complex comprising the neoepitope
and an MHC molecule (such as an MHC class I molecule of the
individual) to a T cell receptor. In some embodiments, the affinity
of the complex comprising the neoepitope and the MHC molecule to
the T cell receptor is compared to that of the complex comprising
the corresponding wildtype epitope and the MHC molecule. In some
embodiments, the MHC molecule is from the individual. In some
embodiments, the T cell receptor is on the surface of one or more T
cells of the individual. In some embodiments, the neoepitope is
selected for having a higher (such as at least about any one of
1.5, 2, 5, 10, 15, 20, 25, 50, 100, or more times) affinity in a
complex comprising the neoepitope and an MHC molecule to a T cell
receptor model than the corresponding wildtype epitope. In some
embodiments, the TCR binding affinity is predicted in silico using
any known tools or methods in the art. In some embodiments, the TCR
binding affinity is determined experimentally, for example, by
determining the T cell response against the neoepitope.
[0195] In some embodiments, the neoantigen (or the neoepitope) is
identified further based on the expression level of the neoantigen
(or the neoepitope) in the tumor sample. Expression level of the
neoantigen (or the neoepitope) may be determined using any methods
for quantification of mRNA or protein levels known in the art, such
as RT-PCR, antibody-based assays, mass spectrometry. In some
embodiments, the expression level of the neoantigen (or the
neoepitope) is determined from the sequencing data of the tumor
sample. In some embodiments, the neoantigen (or the neoepitope) is
expressed in the tumor cells at a level of at least about any one
of 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10.sup.4, or more
copies per cell. In some embodiments, the neoantigen (or the
neoepitope) is expressed at a level of more than about any one of
1.5, 2, 5, 10, 20, 50, 100, or more times than the corresponding
wildtype protein (or the corresponding wildtype epitope) in the
tumor cells.
[0196] In some embodiments, the neoantigen peptide is selected or
identified by the steps comprising: (a) sequencing a tumor sample
from the individual to identify a neoantigen; (b) identifying a
neoepitope in the neoantigen; optionally (c) determining the MHC
subtype of the individual (e.g., using the sequencing data) to
identify an MHC molecule of the individual; optionally (d)
determining the affinity of the neoepitope to the MHC molecule of
the individual; optionally (e) determining the affinity of the
complex comprising the neoepitope and the MHC molecule to a T cell
receptor; and (f) obtaining a peptide comprising the neoepitope to
provide the neoantigen peptide. In some embodiments, the neoepitope
has higher affinity to the MHC molecule (such as MHC-I molecule) of
the individual and/or higher affinity in the complex comprising the
neoepitope and the MHC molecule to the TCR as compared to the
complex comprising the corresponding wildtype T cell epitope and
the MHC molecule. In some embodiments, the neoepitope is extended
at the N terminus or the C terminus or both termini according to
the natural sequence of the neoantigen harboring the epitope to
obtain an extended sequence, wherein the extended sequence is
amenable for presentation by both class I and class II MHC
molecules. Any of the improved MASCT methods described herein using
one or more neoantigen peptides may further comprise any one or
more of the neoantigen selection/identification steps.
Monitoring After Improved MASCT
[0197] Any of the improved MASCT methods described herein may
further comprise a monitoring step after the individual receives
the improved MASCT treatment. Post-treatment monitoring may be
beneficial for adjusting the treatment regimen of the individual to
optimize treatment outcome.
[0198] For example, the plurality of tumor antigen peptides
described herein may be adjusted or customized based on the
specific immune response of the individual against each of the
plurality of tumor antigen peptides and/or the clinical response of
the individual to the activated T cells in order to provide a
plurality of customized tumor antigen peptides, which may be used
for repeated improved MASCT treatment(s). In some embodiments,
tumor antigen peptides that do not elicit a strong specific immune
response can be removed from the antigen peptide pool for future
preparations of the pulsed DCs or activated T cells. In some
embodiments, if the individual does not respond (such as having
signs of disease progression, metastasis, etc.) to the improved
MASCT treatment using one antigen peptide pool, the antigen peptide
pool may be adjusted, or neoantigens may be incorporated in the
antigen peptide pool for use in a second cycle of the improved
MASCT treatment.
[0199] Specific immune response against one or more tumor antigen
peptides may be determined using any known methods in the art, for
example, by measuring levels of cytotoxic factor (such as perforin
or granzyme B), or cytokine release (such as IFN.gamma. or
TNF.alpha.) from T cells (or PBMCs) after stimulation by the
individual tumor antigen peptide. An antibody-based assay, such as
ELISPOT, may be used to quantify the cytotoxic factor, or cytokine
(such as IFN.gamma.) levels. In some embodiments, the cytokine
(such as IFN.gamma.) release level from T cells (or PBMCs) in
response to a tumor antigen peptide is normalized to a reference,
such as a baseline cytokine release level, or a nonspecific
cytokine release level of from T cells (or PBMCs) in response to an
irrelevant peptide, to provide a cytokine (such as IFN.gamma.) fold
change value. In some embodiments, a cytokine (such as IFN.gamma.)
fold change value of more than about any one of 1.2, 1.5, 2, 2.5,
3, 4, 5, 6, 8, 10, or more in an ELISPOT assay indicate strong
specific immune response against the tumor antigen peptide. In some
embodiments, a tumor antigen peptide with a cytokine (such as
IFN.gamma.) fold change value of less than about any one of 10, 8,
6, 5, 4, 3, 2.5, 2, 1.5, 1.2 or less in an ELISPOT assay is removed
from the plurality of tumor antigen peptides to provide a plurality
of customized tumor antigen peptides for future improved MASCT
treatments.
[0200] Clinical response of the individual to the improved MASCT
methods may be assessed by known methods in the art by a physician,
such as by imaging methods, blood tests, biomarker assessment, and
biopsy. In some embodiments, the clinical response is monitored by
determining the number of circulating tumor cells (CTC) in the
individual before and after receiving the improved MASCT treatment.
In some embodiments, the CTCs have detached from a primary tumor
and circulate in a bodily fluid. In some embodiments, the CTCs have
detached from a primary tumor and circulate in the bloodstream. In
some embodiments, the CTCs are an indication of metastasis. CTC
numbers can be determined by a variety of methods known in the art,
including, but not limited to, CellSearch method, Epic Science
method, isoflux, and maintrac. In some embodiments, the number of
single CTCs, including specific subtypes of CTCs, in a blood sample
of the individual is determined. In some embodiments, a number of
more than about any of 10, 20, 50, 100, 150, 200, 300 or more of
single CTCs per mL of the blood sample in the individual after
receiving the improved MASCT treatment indicates an increased risk
of metastasis, and/or poor clinical response to the improved MASCT
treatment. In some embodiments, an increased number (such as at
least about any one of 1.5, 2, 3, 4, 5, 10, or more fold increase)
of single CTCs of the individual after receiving the improved MASCT
treatment compared to before receiving the improved MASCT treatment
indicates poor clinical response to the improved MASCT treatment.
In some embodiments, the number of CTC clusters in a blood sample
of the individual is determined. In some embodiments, detection of
at least about any of 1, 5, 10, 50, 100, or more CTC clusters in a
blood sample of the individual after receiving the improved MASCT
treatment indicates an increased risk of metastasis, and/or poor
clinical response to the improved MASCT treatment. In some
embodiments, an increased number (such as at least about any one of
1.5, 2, 3, 4, 5, 10, or more fold increase) of CTC clusters of the
individual after receiving the improved MASCT treatment compared to
before receiving the improved MASCT treatment indicates poor
clinical response to the improved MASCT treatment.
V. COMPOSITIONS, KITS AND ARTICLES OF MANUFACTURE
[0201] The present application further provides kits, compositions
(such as pharmaceutical compositions), and articles of manufacture
for use in any embodiment of the improved MASCT methods (including
the precision improved MASCT methods) or the cell (such as
antigen-loaded DCs or activated T cells) preparation methods
described herein.
[0202] In some embodiments, there is provided a kit useful for
cancer immunotherapy, comprising at least 10 tumor antigen
peptides. A person skilled in the art may use any combinations of
tumor antigen peptides from the first core group and optionally any
combinations of cancer-type specific antigen peptides from the
second group, and/or neoantigen peptides to load a population of
dendritic cells, which can further be used to prepare activated T
cells useful for treating cancer in an individual.
[0203] The kit may contain additional components, such as
containers, reagents, culturing media, cytokines, immune checkpoint
inhibitors, TLR agonists, buffers, antibodies, and the like to
facilitate execution of any embodiment of the treatment methods or
cell preparation methods described herein. For example, in some
embodiments, the kit further comprises a peripheral blood
collection and storage apparatus, which can be used to collect an
individual's peripheral blood. In some embodiments, the kit further
comprises containers and reagents for density gradient
centrifugation of peripheral blood, which can be used to isolate
PBMCs from a sample of human peripheral blood. In some embodiments,
the kit further comprises culturing media, cytokines, or buffers
for obtaining dendritic cells from peripheral blood. In some
embodiments, the kit further comprises culturing media, TLR
agonists (e.g., MPLA), IFN.gamma., PGE2, reagents and buffers for
loading the plurality of tumor antigen peptides into dendritic
cells. In some embodiments, the kit further comprises cytokines
(e.g., IL-2, IL-7, IL-15 and IL-21), immune checkpoint inhibitors
(e.g., anti-PD1 antibody), anti-CD3 antibody, buffers, or culturing
media for co-culturing T cells obtained from the peripheral blood
with the dendritic cells loaded with the plurality of tumor antigen
peptides. In some embodiments, the kit further comprises reagents
for determining the mutation load (such as in one or more MHC
genes) in cancer cells. In some embodiments, the kit further
comprises an immune checkpoint inhibitor for combination therapy
with the improved MASCT. In some embodiments, the kit further
comprises reagents for identifying a neoantigen (such as by
sequencing) in a tumor sample. In some embodiments, the kit further
comprises an ELISPOT assay for assessing specific immune response
against the plurality of tumor antigen peptides.
[0204] The kits of the present application are in suitable
packaging. Suitable packaging include, but is not limited to,
vials, bottles, jars, flexible packaging (e.g., Mylar or plastic
bags), and the like. Kits may optionally provide additional
components such as buffers and interpretative information. The
present application thus also provides articles of manufacture,
which include vials (such as sealed vials), bottles, jars, flexible
packaging, and the like.
[0205] The instructions may also comprise instructions relating to
the use of the tumor antigen peptides (and optionally additional
components described above). In some embodiments, the kit further
comprises an instructional manual, such as a manual describing a
protocol of an embodiment of the improved MASCT methods (including
the precision improved MASCT methods), or an embodiment of the cell
preparation methods as described herein. The instructions may also
include information on dosage, dosing schedule, and routes of
administration of the dendritic cells and/or the activated T cells
prepared using the kit for the intended treatment. In some
embodiments, the kit further comprises instructions for selecting
an individual for the improved MASCT method. In some embodiments,
the kit further comprises instructions for determining the mutation
load of cancer cells, and/or determining the number of neoantigens
in an individual. In some embodiments, the kit further comprises
instructions for administering an immune checkpoint inhibitor in
combination with the improved MASCT, including, for example,
information on dosage, dosing schedule, and route of administration
of the immune checkpoint inhibitor. In some embodiments, the kit
further comprises instructions for identifying a neoantigen (such
as by sequencing) in a tumor sample. In some embodiments, the kit
further comprises instructions for monitoring an individual after
receiving the improved MASCT treatment.
[0206] The containers may be unit doses, bulk packages (e.g.,
multi-dose packages) or sub-unit doses. For example, kits may be
provided that contain sufficient tumor antigen peptides as
disclosed herein to prepare sufficient activated T cells and/or
antigen-loaded dendritic cells (such as dendritic cells) to provide
effective treatment of an individual for an extended period, such
as any of 3 weeks, 6 weeks, 9 weeks, 3 months, 4 months, 5 months,
6 months, 8 months, 9 months, 1 year or more.
[0207] Kits may also include multiple unit doses of tumor antigen
peptides and instructions for use and packaged in quantities
sufficient for storage and use in pharmacies, for example, hospital
pharmacies and compounding pharmacies.
[0208] Further provided are kits, compositions (such as
pharmaceutical compositions), and articles of manufacture of any
one of the isolated population of cells (such as dendritic cells,
or activated T cells) described herein.
[0209] The isolated population of cells described herein may be
used in pharmaceutical compositions or formulations, by combining
the isolated population of cells described with a pharmaceutically
acceptable carrier, excipients, stabilizing agents and/or other
agents, which are known in the art, for use in the methods of
treatment, methods of administration, and dosage regimens described
herein. In some embodiments, human albumin is used as a
pharmaceutically acceptable carrier.
[0210] Suitable pharmaceutical carriers include sterile water;
saline, dextrose; dextrose in water or saline; condensation
products of castor oil and ethylene oxide combining about 30 to
about 35 moles of ethylene oxide per mole of castor oil; liquid
acid; lower alkanols; oils such as corn oil; peanut oil, sesame oil
and the like, with emulsifiers such as mono- or di-glyceride of a
fatty acid, or a phosphatide, e.g., lecithin, and the like;
glycols; polyalkylene glycols; aqueous media in the presence of a
suspending agent, for example, sodium carboxymethylcellulose;
sodium alginate; poly(vinylpyrolidone); and the like, alone, or
with suitable dispensing agents such as lecithin; polyoxyethylene
stearate; and the like. The carrier may also contain adjuvants such
as preserving stabilizing, wetting, emulsifying agents and the like
together with the penetration enhancer. The final form may be
sterile and may also be able to pass readily through an injection
device such as a hollow needle. The proper viscosity may be
achieved and maintained by the proper choice of solvents or
excipients.
[0211] The pharmaceutical compositions described herein may include
other agents, excipients, or stabilizers to improve properties of
the composition. Examples of suitable excipients and diluents
include, but are not limited to, lactose, dextrose, sucrose,
sorbitol, mannitol, starches, gum acacia, calcium phosphate,
alginates, tragacanth, gelatin, calcium silicate, microcrystalline
cellulose, polyvinylpyrrolidone, cellulose, water, saline solution,
syrup, methylcellulose, methyl- and propylhydroxybenzoates, talc,
magnesium stearate and mineral oil. In some embodiments, the
pharmaceutical composition is formulated to have a pH in the range
of about 4.5 to about 9.0, including for example pH ranges of about
any one of 5.0 to about 8.0, about 6.5 to about 7.5, or about 6.5
to about 7.0. In some embodiments, the pharmaceutical composition
can also be made to be isotonic with blood by the addition of a
suitable tonicity modifier, such as glycerol.
[0212] In some embodiments, the isolated cell compositions (such as
pharmaceutical compositions) is suitable for administration to a
human. In some embodiments, the compositions (such as
pharmaceutical compositions) is suitable for administration to a
human by parenteral administration. Formulations suitable for
parenteral administration include aqueous and non-aqueous, isotonic
sterile injection solutions, which can contain anti-oxidants,
buffers, bacteriostats, and solutes that render the formulation
compatible with the blood of the intended recipient, and aqueous
and non-aqueous sterile suspensions that can include suspending
agents, solubilizers, thickening agents, stabilizing agents, and
preservatives. The formulations can be presented in unit-dose or
multi-dose sealed containers, such as ampules and vials, and can be
stored in a condition requiring only the addition of the sterile
liquid excipient methods of treatment, methods of administration,
and dosage regimens described herein (i.e., water) for injection,
immediately prior to use. In some embodiments, the compositions
(such as pharmaceutical compositions) is contained in a single-use
vial, such as a single-use sealed vial. In some embodiments, each
single-use vial contains about 10.sup.9 activated T cells. In some
embodiments, each single-use vial contains enough activated T cells
to be expanded to about 10.sup.9 activated T cells. In some
embodiments, the composition (such as pharmaceutical composition)
is contained in a multi-use vial. In some embodiments, the
composition (such as pharmaceutical composition) is contained in
bulk in a container.
[0213] Also provided are unit dosage forms comprising the isolated
cell compositions (such as pharmaceutical compositions) and
formulations described herein. These unit dosage forms can be
stored in a suitable packaging in single or multiple unit dosages
and may also be further sterilized and sealed. In some embodiments,
the composition (such as pharmaceutical composition) also includes
one or more other compounds (or pharmaceutically acceptable salts
thereof) that are useful for treating cancer.
[0214] The present application further provides kits comprising any
of the isolated population of cells, compositions (such as
pharmaceutical compositions), formulations, unit dosages, and
articles of manufacture described herein for use in the methods of
treatment, methods of administration, and dosage regimens described
herein. Kits described herein include one or more containers
comprising the activated T cells.
VI. EXEMPLARY EMBODIMENTS
[0215] Among the embodiments provided herein are:
1. A method of preparing a population of activated T cells, the
method comprising: [0216] a) contacting a population of dendritic
cells with a plurality of tumor antigen peptides to obtain a
population of dendritic cells loaded with the plurality of tumor
antigen peptides; [0217] b) co-culturing the population of
dendritic cells loaded with the plurality of tumor antigen peptides
and a population of T cells in an initial co-culture medium
comprising a plurality of cytokines and an immune checkpoint
inhibitor to provide a co-culture; and [0218] c) adding an anti-CD3
antibody to the co-culture at about 3 to 7 days after the
co-culturing starts, thereby obtaining the population of activated
T cells. 2. The method of embodiment 1, wherein step a) further
comprises culturing the population of dendritic cells loaded with
the plurality of tumor antigen peptides in a DC maturation medium
comprising a toll-like receptor (TLR) agonist. 3. The method of
embodiment 2, wherein the TLR agonist is selected from the group
consisting of MPLA, Poly I: C, resquimod, gardiquimod, and CL075.
4. A method of preparing a population of activated T cells, the
method comprising: [0219] a) contacting a population of dendritic
cells with a plurality of tumor antigen peptides to obtain a
population of dendritic cells loaded with the plurality of tumor
antigen peptides; [0220] b) culturing the population of dendritic
cells loaded with the plurality of tumor antigen peptides in a DC
maturation medium comprising MPLA; and [0221] c) co-culturing the
population of dendritic cells loaded with the plurality of tumor
antigen peptides and a population of T cells, thereby obtaining the
population of activated T cells. 5. The method of embodiment 3 or
4, wherein the DC maturation medium comprises INF.gamma. and MPLA.
6. The method of embodiment 5, wherein the DC maturation medium
further comprises PGE2. 7. The method of embodiment 5 or 6, wherein
the INF.gamma. is present in the DC maturation medium at a
concentration of at least about 100 IU/mL. 8. The method of any one
of embodiments 3-7, wherein the MPLA is present in the DC
maturation medium at a concentration of at least about 0.5
.mu.g/mL. 9. The method of any one of embodiments 6-8, wherein the
PGE2 is present in the DC maturation medium at a concentration of
at least about 0.1 .mu.g/mL. 10. The method of any one of
embodiments 4-9, wherein step c) comprises: co-culturing the
population of dendritic cells loaded with the plurality of tumor
antigen peptides and a population of T cells in an initial
co-culture medium comprising a plurality of cytokines and an immune
checkpoint inhibitor to provide a co-culture; and adding an
anti-CD3 antibody to the co-culture, thereby obtaining the
population of activated T cells. 11. The method of any one of
embodiments 1-3 and 5-10, wherein the plurality of cytokines
comprises IL-2, IL-7, IL-15 and IL-21. 12. The method of embodiment
11, wherein the IL-2 is present in the initial co-culture medium at
a concentration of at least about 500 IU/mL. 13. The method of any
one of embodiments 1-3 and 5-12, wherein the immune checkpoint
inhibitor is an anti-PD-1 antibody. 14. The method of embodiment
13, wherein the anti-PD-1 antibody is present in the initial
co-culture medium at a concentration of at least about 10 .mu.g/mL.
15. The method of any one of embodiments 10-14, wherein the
anti-CD3 antibody is added to the co-culture at about 3 to 7 days
after the co-culturing starts. 16. The method of any one of
embodiments 1-3 and 5-15, wherein the anti-CD3 antibody is added to
the co-culture at about 5 days after the co-culturing starts. 17.
The method of any one of embodiments 1-16, wherein the population
of dendritic cells loaded with the plurality of tumor antigen
peptides and the population of T cells are co-cultured for at least
about 10 days in the presence of the anti-CD3 antibody. 18. The
method of any one of embodiments 1-17, wherein the population of T
cells is present in a population of PBMCs. 19. The method of any
one of embodiments 1-18, wherein the population of dendritic cells
is obtained by inducing differentiation of a population of
monocytes from PBMCs. 20. The method of any one of embodiments
1-19, wherein the population of dendritic cells and the population
of T cells are obtained from the same individual. 21. The method of
any one of claims 1-20, wherein the plurality of tumor antigen
peptides comprises a neoantigen peptide, optionally wherein the
plurality of tumor antigen peptides consists of neoantigen
peptides. 22. The method of any one of embodiments 1-21, wherein
the plurality of tumor antigen peptides is a plurality of synthetic
tumor antigen peptides. 23. The method of any one of embodiments
1-22, wherein the plurality of tumor antigen peptides is not
obtained from a cell sample. 24. An isolated population of
activated T cells prepared using the method of any one of
embodiments 1-23. 25. A method of treating a cancer in an
individual, comprising administering to the individual an effective
amount of the activated T cells of embodiment 24. 26. The method of
embodiment 25, further comprising administering to the individual
an effective amount of dendritic cells loaded with the plurality of
tumor antigen peptides. 27. The method of embodiment 26, wherein
the population of dendritic cells and the population of T cells are
obtained from the individual being treated. 28. The method of any
one of embodiments 25-27, wherein the activated T cells are
administered to the individual for at least three times. 29. The
method of any one of embodiments 25-28, wherein the activated T
cells are administered intravenously. 30. The method of any one of
embodiments 25-29, wherein the dendritic cells loaded with the
plurality of tumor antigen peptides are administered for at least
three times. 31. The method of any one of embodiments 25-30,
wherein the dendritic cells loaded with the plurality of tumor
antigen peptides are administered subcutaneously, intradermally or
intravenously. 32. The method of any one of embodiments 25-31,
wherein the cancer is a solid cancer. 33. The method of embodiment
32, wherein the solid cancer is selected from the group consisting
of hepatocellular carcinoma, gastric cancer, bladder cancer, soft
tissue sarcoma, colorectal cancer, endometrial cancer, and lung
cancer. 34. The method of any one of embodiments 25-33, wherein the
individual is a human individual. 35. A composition comprising the
activated T cells of embodiment 24 for treating a cancer in an
individual. 36. Use of the activated T cells of embodiment 24 in
the preparation of a medicament for treating a cancer in an
individual.
EXAMPLES
[0222] The examples below are intended to be purely exemplary of
the present application and should therefore not be considered to
limit the invention in any way. The following examples and detailed
description are offered by way of illustration and not by way of
limitation.
Example 1: Optimized DC Maturation Media
Experimental Methods
[0223] Peripheral blood mononuclear cells (PBMCs) from healthy
volunteers were obtained by density gradient centrifugation on
Lymphoprep (Nycomed Pharma, Oslo, Norway). The adherent monocytes
were continued to be cultured in AIM-V medium with 1000 U/mL GM-CSF
and 500 U/mL IL-4 to differentiate into immature dendritic cells
(DCs). The resulting immature DCs were pulsed by multiple tumor
antigens peptide pool (1 .mu.g/mL/peptide), followed by incubation
in a DC maturation medium for 2 days to differentiate into mature
DCs.
[0224] The cell culture was subject to flow cytometry to determine
the number of mature DCs (CD11C+ cells) and subpopulations thereof
that expressed various co-stimulatory molecules. The number of
mature DCs was normalized to the total number of cells. The number
of cells in each DC subpopulation was normalized to the total
number of mature DCs. Antibodies for dendritic cell surface
staining were obtained from BD Biosciences (anti-human CD86-FITC,
CD40-FITC, CD11C-PE, CCR7-FITC, PD-L1-FITC, HLA-DR-FITC). Flow
cytometry was performed using FACS CantoII (BD Biosciences) flow
cytometers and data was analyzed with the Flowjo program.
[0225] Cytokine secretion by mature DCs was assessed by ELISA. The
supernatants of mature DCs were centrifuged to remove particulate
debris and stored at -80.degree. C. until use. IL-12p70 and IL-10
secretion levels were measured by specific ELISA kits (eBioscience)
according to the manufacturer's protocols. TNF-.alpha. secretion
level was determined using Procarta Plex Multiplex Immunoassays
(Affymetrix).
Effects of Different TLRs
[0226] The antigen-loaded immature DCs were incubated in DC
maturation media comprising different Toll-like receptors (TLRs):
DC3/4 indicates a DC maturation medium from a previously reported
exemplary MASCT method, including IL6, TNF.alpha., IL1.beta., and
Poly I:C; M+I medium, comprising MPLA ("M") and INF.gamma. ("I");
I+P+R medium, comprising INF.gamma., Poly I:C ("P"") and resquimod
("R"); M+I+G medium, comprising MPLA, INF.gamma. and gardiquimod
("G"); M+I+P medium, comprising MPLA, INF.gamma., and Poly I:C; and
M+I+C medium, comprising MPLA, INF.gamma., and CL075. In the
situation where two DC maturation media contain the same
ingredient, the concentration of that ingredient was identical in
the two DC maturation media.
[0227] As shown in FIG. 1A, different TLRs and combinations thereof
did not have significant impact on the expression of co-stimulatory
molecules on mature DCs. However, as shown in FIG. 1B, M+I and
M+I+G media led to significantly increased secretion levels of IL12
and TNF.alpha. by the mature DCs.
Effects of TLRs at Different Concentrations
[0228] The antigen-loaded immature DCs were incubated in DC
maturation media comprising TLRs at different concentrations: DC3/4
indicates a DC maturation medium from a previously reported
exemplary MASCT method, including IL6, TNF.alpha., IL1.beta., and
Poly I:C; I+M medium, comprising INF.gamma. and a low concentration
of MPLA; I+M2 medium, comprising INF.gamma. and a high
concentration of MPLA; I+M+G1 medium, comprising MPLA, INF.gamma.
and a low concentration of gardiquimod; I+M+G2 medium, comprising
MPLA, INF.gamma. and a high concentration of gardiquimod; I+M+R1
medium, comprising MPLA, INF.gamma. and a low concentration of
resquimod; I+M+R2 medium, comprising MPLA, INF.gamma. and a high
concentration of resquimod; I+M+CL1 medium, comprising MPLA,
INF.gamma., and a low concentration of CL075; and I+M+CL2 medium,
comprising MPLA, INF.gamma., and a high concentration of CL075. The
concentration of M was between 1 .mu.g/mL and 10 .mu.g/mL. The
concentration of gardiquimod was between 1 .mu.g/mL and 10
.mu.g/mL. The concentration of resquimod was between 1 .mu.g/mL and
10 .mu.g/mL. The concentration of CL075 was between 1 .mu.g/mL and
5 .mu.g/mL.
[0229] As shown in FIG. 2A, the DC3/4 medium resulted in a higher
number of mature DCs. However, as shown in FIG. 2B, the various DC
maturation media did not result in significant difference in the
expression of co-stimulatory molecules on the mature DCs. As shown
in FIG. 2C, the I+M, I+M1, and I+M+G1 media significantly increased
the IL12/IL10 ratio by the mature DCs. The DC maturation media
ranked from the highest efficacy to lowest efficacy for induction
of DC maturation are as follows: I+M>I+M+G1>I+M+R1.
Effects of PGE2
[0230] The antigen-loaded immature DCs were incubated in DC
maturation media comprising MPLA, INF.gamma., and different
concentrations of PGE2: DC3/4 indicates a DC maturation medium from
a previously reported exemplary MASCT method, including IL6,
TNF.alpha., IL1.beta., and Poly I:C; M+I medium, comprising
INF.gamma. and MPLA, but no PGE2; M+I+P1 medium, comprising
INF.gamma., MPLA, and a low concentration of PGE2; M+I+P2 medium,
comprising INF.gamma., MPLA, and a medium concentration of PGE2;
M+I+P3 medium, comprising INF.gamma., MPLA, and a high
concentration of PGE2. The concentration of PGE2 was between 0.1
.mu.g/mL and 5 .mu.g/mL.
[0231] The effects of the DC maturation media on induction of DC
maturation are shown in FIGS. 3A-3C, and summarized below in Table
1. The DC maturation medium having the highest efficacy on
induction of DC maturation is M+I+P1.
TABLE-US-00001 TABLE 1 Effects of PGE2 on DC maturation M + I + M +
I + M + I + Parameter DC3/4 M + I PI P2 P3 Number of DCs 2 1 2 2 2
Expression of co- 2 1 3 2 3 stimulatory molecules on DCs Secretion
of IL-12 1 3 3 2 1 Total 5 5 8 6 6
Example 2: Optimized Co-Culturing Conditions
Effects of Cytokine Cocktail
[0232] Thawed T cells from a frozen stock were mixed with
antigen-loaded mature DCs prepared in Example 1 in an initial
co-culture medium (AIM-V medium) to provide a co-culture. The
initial co-culture medium contained either IL-2 or an interleukin
cocktail (including IL-2, IL-7, IL-15 and IL-21), and an anti-PD-1
antibody SHR-1210 (Jiangsu Hengrui). The co-culture was cultured
for 19 days.
[0233] To determine the proliferation of tumor antigen-specific T
cells, FACS analysis was performed as described in the Click-iT EdU
Alexa Fluor 488 Flow Cytometry Assay Kit (Invitrogen). IFN.gamma.
production of tumor antigen-specific T cells was detected by
intracellular cytokine staining and FACS analysis. Anti-human
CD3-PE antibody for cell surface staining and anti-human
IFN-.gamma.-APC antibody for intracellular cytokine staining were
obtained from BD Biosciences. Intracellular cytokine staining was
performed by fixing and permeabilizing cells with cytofix/cytoperm
(BD Biosciences). Flow cytometry was performed using FACS Canton
(BD Biosciences) flow cytometers and data was analyzed with the
Flowjo program.
[0234] FIG. 4 shows results from co-culture using DCs and T cells
from PBMC samples of three healthy donors. Compared to an initial
co-culture medium having IL-2 alone, an initial co-culture medium
having an interleukin cocktail yielded higher level of
proliferation and percentage of IFN.gamma.-secreting tumor
antigen-specific T cells in the co-culture.
Effects of anti-PD-1, IL-2 and Anti-CD3 Antibody
[0235] Thawed T cells from a frozen stock were mixed with
antigen-loaded mature DCs prepared in Example 1 in an initial
co-culture medium (AIM-V medium) to provide a co-culture. The
co-culture was subject to various conditions as shown in Table 2
below. For example, initial co-culture media with or without
anti-PD-1 antibody SHR-1210 (Jiangsu Hengrui), with a low
concentration or high concentration of IL-2 (rIL-2; R&D
Systems, Minneapolis, Minn.), and addition of an anti-CD3 antibody
(eBioscience, San Diego, Calif.) to the co-culture after 3 days or
5 days from the start of the co-culture were tested. The DCs and T
cells were co-cultured for a total of 19 days. The numbers of
tumor-antigen specific T cells and percentages of
IFN.gamma.-secreting T cells in the co-culture were determined as
described above.
TABLE-US-00002 TABLE 2 Co-culturing conditions. Interleukin
Anti-CD3 cocktail IL-2 Add at Add at Conditions Anti-PD1 (-IL-2)
low high 3 days 5 days CIK - - + + Previous - - + + MASCT 1 + + + +
2 + + + +
[0236] The "CIK" condition resembles a standard condition for
preparing cytokine-induced killer cells, except the anti-CD3
antibody was added 3 days after the co-culture started. The
"previous MASCT" condition resembles an exemplary condition for
preparing activated T cells as disclosed previously in
WO2016145578A1, except the anti-CD3 antibody was added 3 days after
the co-culture started. As shown in FIG. 5A, condition 2 yielded
the highest number of tumor antigen-specific T cells, and highest
percentage of IFN.gamma.-secreting T cells in the co-culture.
Effects of Different Concentrations of IL-2
[0237] Thawed T cells from a frozen stock were mixed with
antigen-loaded mature DCs prepared in Example 1 in an initial
co-culture medium (AIM-V medium) to provide a co-culture. The
initial co-culture media contained an anti-PD-1 antibody SHR-1210
(Jiangsu Hengrui), interleukin cocktail, a low concentration or
high concentration of IL-2 (rIL-2; R&D Systems, Minneapolis,
Minn.), and addition of an anti-CD3 antibody (eBioscience, San
Diego, Calif.) to the co-culture after 3 days or 5 days from the
start of the co-culture. The DCs and T cells were co-cultured for a
total of 19 days. The concentration of IL-2 was between 100 IU/mL
and 1000 IU/mL. The number of tumor-antigen specific T cells, and
percentage of IFN.gamma.-secreting T cells in the co-culture were
determined as described above.
[0238] As shown in FIG. 5B, compared to a low concentration of
IL-2, a high concentration of IL-2 yielded a higher number of tumor
antigen-specific T cells, and a higher percentage of
IFN.gamma.-secreting T cells in the co-culture.
Example 3: Comparison of Exemplary Improved MASCT and Previous
MASCT Cell Preparation Methods
[0239] This example provides a head-to-head comparison of immune
cells prepared using exemplary cell preparation methods described
in the present application ("improved MASCT protocol") versus
exemplary cell preparation methods disclosed in WO2016145578A1
("previous MASCT protocol").
Effects on IL-12 Secretion by Mature DCs
[0240] Peripheral blood mononuclear cells (PBMCs) from healthy
volunteers and cancer patients were obtained by density gradient
centrifugation on Lymphoprep (Nycomed Pharma, Oslo, Norway). The
adherent monocytes were continued to be cultured in AIM-V medium
with 1000 U/mL GM-CSF and 500 U/mL IL-4 to differentiate into
immature dendritic cells (DCs). The resulting immature DCs were
pulsed by multiple tumor antigens peptide pool (1
.mu.g/mL/peptide), followed by incubation in either "improved
MASCT" DC maturation medium or "previous MASCT" DC maturation
medium for two days to differentiate into mature DCs. The improved
MASCT DC maturation medium comprises IFN-.gamma., MPLA, and PGE2.
The previous MASCT DC maturation medium comprises IL-6,
TNF-.alpha., IL-1.beta., POLY(I:C) and PGE2. IL-12p secretion
levels from the mature DCs were determined.
[0241] Cytokine secretion by mature DCs was assessed by ELISA. As
shown in FIGS. 6A-6B, DCs induced by the improved MASCT DC
maturation medium secreted significantly higher levels of IL-12p70
than DCs induced by the previous MASCT DC maturation medium.
Notably, using PBMCs from cancer patients, the improved MASCT DC
maturation medium led to more than 70 fold increase in the IL-12p70
secretion level than the previous MASCT DC maturation medium. This
increase in IL-12 secretion was more pronounced with DCs derived
from cancer patients than DCs derived from healthy volunteers. The
enhanced cytokine secretion level by mature DCs prepared using the
improved MASCT DC maturation medium increased the cytotoxicity of
DCs against solid tumors.
Effects on Tumor-Specific T Cells
[0242] T cells and antigen-loaded mature DCs derived from PBMCs of
healthy volunteers (n=5) were co-cultured according to an exemplary
improved MASCT protocol or an exemplary previous MASCT protocol.
The improved MASCT protocol involved co-culturing T cells with
antigen-loaded mature DCs in an initial co-culture medium
containing an interleukin cocktail (including IL-2, IL-7, IL-15 and
IL-21), and an anti-PD-1 antibody SHR-1210 (Jiangsu Hengrui). The
co-culture was incubated for 5 days when an anti-CD3 antibody was
added to the co-culture. The cells were co-cultured for a total of
19 days to provide activated T cells. The previous MACT protocol
involved co-culturing T cells with antigen-loaded mature DCs in a
co-culture medium containing IL-2 and an anti-CD3 antibody. The
cells were co-cultured for a total of 19 days to provide activated
T cells.
[0243] IFN production by the activated T cells in response to tumor
antigens was detected by intracellular cytokine staining and FACS
analysis as described in Example 2. As shown in FIG. 7, the
percentage of IFN.gamma.-producing activated T cells in the
co-culture increased significantly (about 2-4 times) using the
improved MASCT protocol compared to that using the previous MASCT
protocol.
[0244] Anti-tumor effects of the activated T cells prepared using
the improved MASCT protocol and the previous MASCT protocol was
determined using various solid tumor cell lines, including MBA231
(breast cancer cells), CNE1 (nasopharyngeal carcinoma cells), HepG2
(liver carcinoma cells) and Saos-2 (osteosarcoma cells). Briefly,
tumor cells were cultured in DMEM supplemented with 10% inactivated
fetal bovine serum, 100 U/mL penicillin, 100 .mu.g/mL streptomycin,
Glutamax, MEM NEAA, (Gibico, Carlsbad, Calif.). Tumor cells were
washed with D-PBS (Invitrogen) and co-cultured with the activated T
cells at a T cells: target (T: Target) ratio of 1:10 or 1:30 in
96-well round-bottom plates in triplicates in AIM-V for 4 hours.
Cytotoxicity was shown as the percentage of maximal LDH released
after lysis and measured by the Cytotox 96 Assay kit (Promega
G1780, Canada).
[0245] As shown in FIG. 8, activated T cells prepared using the
improved MASCT protocol had significantly enhanced cytotoxicity
against all four types of tumor cells lines, compared to activated
T cells prepared using the previous MASCT protocol.
Example 4: Improved MASCT Clinical Study
[0246] The improved MASCT study is an open-label, multi-center
study that aims to investigate the safety and efficacy of an
embodiment of the improved MASCT method in treating patients having
solid tumors, including hepatocellular carcinoma, gastric cancer,
bladder cancer, soft tissue sarcoma, endometrial cancer, colorectal
cancer, and lung cancer. Enrolled patients may have previously
received curative resection, such as resection or RFA, or
first-line chemotherapy.
[0247] Patients receive one or more cycles of improved MASCT
treatment. For example, patients may receive one cycle of improved
MASCT treatment every 1-3 months for 1-2 years. In each cycle of
improved MASCT treatment, PBMCs are obtained from each patient.
Immature dendritic cells are obtained from the PBMCs. On day 1,
immature dendritic cells are pulsed with a pool of antigen
peptides, including up to 44 antigen peptides derived from hTERT,
p53, Survivin, NY-ESO-1, CEA, CCND1, MUC1, Her2, MAGEA1, MAGEA3,
WT-1, RGS5, VEGFR1, VEGFR2, CDCA1, HBcAg, HBV polymerase, GPC3,
SSX, and AFP. The pool of antigen peptides may also contain up to
10 antigen peptides derived from neoantigens. The immature
dendritic cells loaded with the pool of antigen peptides are
cultured in a DC maturation medium containing IFN-.gamma., MPLA,
and PGE2 to provide mature dendritic cells loaded with the pool of
antigen peptides. On day 8, the patient receives subcutaneous
injection of the mature dendritic cells loaded with the pool of
antigen peptides. From day 8 to day 13, T cells in PBMCs and mature
dendritic cells loaded with the pool of antigen peptides are
co-cultured in the presence of a cytokine cocktail (IL-2, IL-7,
IL-15, IL-21) and anti-PD-1 antibody (e.g., SHR-1210). On day 13,
anti-CD3 antibody (e.g., OKT-3) is added to the co-culture. On day
28, the co-culture containing activated T cells are infused to the
patient. In the combination therapy group, patients received
anti-PD-1 antibody treatment or chemotherapy treatment.
[0248] Each patient receives improved MASCT treatment for up to 18
cycles unless the patient experiences disease progression or
unacceptable toxicity. Patients are followed for about 2.5 years or
until death or disease progression of all patients, whichever
occurs earlier.
[0249] Patients must fulfill all of the following criteria to be
eligible for admission to the study. [0250] 1. The patient is
diagnosed with solid tumors, including hepatocellular carcinoma,
gastric cancer, bladder cancer, soft tissue sarcoma, endometrial
cancer, colorectal cancer, and NSCLC; [0251] 2. At least one
measurable lesion as defined by RECIST criteria 1.1 for solid
tumors; [0252] 3. No cancer embolus in the main portal vein, first
branch of hepatic duct, first branch of hepatic vein, or inferior
vena cava; [0253] 4. ECOG Performance status (ECOG-PS).ltoreq.2;
[0254] 5. The expected survival time is more than 6 months; [0255]
6. Tests of blood, liver and kidney meeting the following criteria:
[0256] a. WBC>3.times.10.sup.9/L [0257] b. Neutrophil
counts>1.5.times.10.sup.9/L [0258] c. Hemoglobin.gtoreq.85 g/L
[0259] d. Platelet counts.gtoreq.50.times.10.sup.9/L [0260] e. PT
is normal or The extend time<3 s [0261] f. BUN.ltoreq.1.5 times
the upper-limit, [0262] g. Serum creatinine<1.5 times of the
upper-limit [0263] 7. Patient consent obtained and signed according
to local Institutional and/or University Human Experimentation
Committee requirements and/or a central Institutional Review Board
(IRB) or other as appropriate.
[0264] Patients who meet any of the following criteria are not
eligible for admission to the study: [0265] 1. Women who are
pregnant or during breast feeding or plan to be pregnant within 2
years; [0266] 2. Known active brain metastases as determined by CT
or MRI evaluation; [0267] 3. Know the period of systemic and
continuous use of immunomodulatory agents (such as interferon,
thymosin, traditional Chinese medicine) within 6 months; [0268] 4.
Positive for HIV antibody or HCV antibody; [0269] 5. Have a history
of immunodeficiency disease or autoimmune diseases (such as
rheumatoid arthritis, Buerger's disease, multiple sclerosis or
diabetes type 1); [0270] 6. Patients with organ failure; [0271] 7.
Patients with serious mental disease; [0272] 8. Drug addiction
within 1 year before enrollment, including alcoholism; [0273] 9.
Participated in other clinical trials within 3 months before
screening; [0274] 10. Other reasons the researchers deem unsuitable
for the study.
[0275] The primary outcome measures include safety and efficacy of
the improved MASCT treatment. Secondary outcome measures include
overall survival (OS), progression-free survival (PFS), objective
response rate (ORR), complete response (CR), partial response (PR),
stable disease rate (SDR), and disease-related biomarker
measurements. Tumor response and progression are assessed using
RECIST criteria (v1.1). Safety is assessed on the basis of vital
signs, clinical laboratory findings, and adverse events graded
according to the NCI CTCAE version 4.02. ELISPOT assays are
performed to determine antigen peptide-specific response of the
activated T cells of each patient in each cycle of the improved
MASCT treatment.
Example 5: Immune Response Against Neoantigens by Neo-MASCT-Treated
Patients
[0276] To investigate whether neoantigens could induce tumor
specific immune responses in patients with solid tumor (e.g., HCC,
endometrial cancer, and colon cancer), neoantigen stimulating
cellular therapy using the improved MASCT preparation protocol
("neo-MASCT") was applied to patients after radical resection.
[0277] Patients received neo-MASCT treatments according to the
clinical protocol described in Example 4. As shown in FIG. 9, in
each cycle of neo-MASCT treatment, each patient received injection
of mature dendritic cells (DCs) loaded with a peptide pool of
multiple shared tumor associated antigens (TAAs) and several
personalized neoantigens, followed by infusion of autologous T
cells stimulated by these DCs. The shared tumor associated antigens
(TAAs) included both general tumor antigens and cancer-type
specific tumor antigens. The patients received 1 to 10 cycles of
neo-MASCT treatments.
[0278] FIG. 10 shows a schematic flowchart for designing neoantigen
peptides. To obtain neoantigen sequences, fresh tumor samples were
acquired immediately after biopsy or surgery for subsequent Whole
Exosome Sequencing (WES) and RNAseq. All mutations including SNV,
non-frame shift insert/deletion (Indel), and neoORF were detected
by comparing the next-generation sequencing data from tumors to
normal tissues. All neo-epitope candidates were predicted by the
MASNEO.sup.Tm algorithm according to the patient's HLA class I
genotypes. Long neoantigen peptides containing 25-31 amino acids
with higher affinities were selected for further synthesis and
treatment. Peptides containing multiple neo-epitopes were
preferred. A smaller number of neoantigen peptides could be
predicted using the NetMHC algorithm (Andreatta M, Nielsen M.
Bioinformatics (2016) February 15; 32(4):511-7) from the same
next-generation sequencing results.
[0279] ELISPOT assays were carried out to determine whether the
patients developed MHC-restricted T cell response against each of
the tumor antigen peptides, including neoantigen peptides. Briefly,
PBMCs from patients were plated (1.times.10.sup.6 cells/well) in
AIM-V medium without any cytokines on cell culture plate, and
further stimulated with individual antigen peptides for 48 h. PBMCs
were then transferred onto a 96-well ELISPOT assay plate (U-CyTech
Biosciences) for IFN.gamma. detection. PBMCs were further
stimulated with peptides for another 16 h. The ELISPOT assay was
performed and analyzed according to the manufacturer's
instructions. The number of spot-forming units was determined with
computer-assisted image analysis software (ChampSpot; Saizhi). The
responses were shown as spot-forming units per 10.sup.5 PBMC/well.
Results were demonstrated as an IFN.gamma.-producing fold index
compute by specific peptide group: irrelevant peptide group.
Exemplary raw ELISPOT results are shown in FIG. 14.
[0280] FIG. 11 summarizes the ELISPOT results of eight neoantigen
peptide-responding patients, including six HCC patients, one
endometrial cancer patient, and one colon cancer patient. Each
patient responded to 25% to 100% of the neoantigen peptides as
predicted by MASNEO.TM.. An overall response rate of 66% was
achieved among the patients using neoantigen peptides predicted by
MASNEO.TM..
[0281] Within the HCC patient group, eight HCC patients have
received neo-MASCT, and seven of them were tested for specific
immune responses against both TAAs and neoantigens using the
ELISPOT assay. The results showed that Neo-MASCT induced
TAA-specific immune responses in five patients (5/7, 71%), and
induced neoantigen-specific immune responses in six patients (6/7,
86%), respectively. One patient (Patient #2) demonstrated specific
T cell responses against all five neoantigen peptides (5/5, 100%)
after neo-MASCT treatment. The average responsive rate of
neoantigens was 65% (22/34 peptides) among the six responding HCC
patients.
[0282] FIG. 12 shows ELISPOT results using PMBCs from Patient #2
before the neo-MASCT treatment and after four cycles of neo-MASCT
treatment. "Base-pep" indicates results using a pool of general
tumor antigen peptides, including hTERT, p53, Survivin, NY-ESO-1,
CEA, CDCA1, VEGFR1, VEGFR2, RGS5, CCND1, MUC1, Her2, MAGEA1, MAGEA3
and WT-1. "HCC-pep" indicates results using a pool of HCC-specific
tumor antigen peptides, including HBcAg, HBV polymerase, GPC3, SSX
and AFP. Enhanced T-cell response against the Base-pep pool, the
HCC-pep pool, as well as individual tumor antigen peptides (e.g.,
p53, Survivin, CEA, CDCA1, VEGFR1, VEGFR2, MUC1, Her2, GPC3, and
SSC) was observed after the neo-MASCT treatment.
[0283] FIG. 13 shows ELISPOT results using PMBCs from Patient #2
before the neo-MASCT treatment, after 2 cycles of neo-MASCT
treatment, and after 5 cycles of neo-MASCT treatment. "Neo-pep"
indicates results using a pool of neoantigen peptides, including
ZGQ-1, ZGQ-2, ZGQ-4, ZGQ-5, ZGQ-6 and ZGQ-7. Enhanced T-cell
response against the Neo-pep pool and individual neoantigen
peptides was observed after multiple rounds of neo-MASCT
treatment.
[0284] In conclusion, the results demonstrate that neo-MASCT with
improved MASCT preparation protocol is well-tolerated in cancer
patients and elicits immune responses against multiple tumor
antigens, especially personalized neoantigens. The neo-MASCT
treatment in this example uses both neoantigen peptides and
tumor-associated antigen peptides. In view of the high rate of
specific T-cell response to neoantigen peptides, we plan to conduct
clinical studies using a pool of neoantigen peptides without any
general tumor antigen peptides to prepare activated T cells for
neo-MASCT treatments of patients with solid tumors.
* * * * *