U.S. patent application number 16/331494 was filed with the patent office on 2019-06-27 for platform for activation and expansion of virus-specific t-cells.
The applicant listed for this patent is Baylor College of Medicine. Invention is credited to Natalia Lapteva Doyle, Cliona M. Rooney, Sandhya Sharma, Dimitrios Wagner.
Application Number | 20190194619 16/331494 |
Document ID | / |
Family ID | 61619728 |
Filed Date | 2019-06-27 |
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United States Patent
Application |
20190194619 |
Kind Code |
A1 |
Rooney; Cliona M. ; et
al. |
June 27, 2019 |
PLATFORM FOR ACTIVATION AND EXPANSION OF VIRUS-SPECIFIC T-CELLS
Abstract
Embodiments of the disclosure concern methods and compositions
for immunotherapy for diseases and malignancies associated with
viruses other than HPV or with non-virus-associated diseases and
malignancies, such as wherein the VST encodes a CAR specific for a
non-viral cancer and the VST can be stimulated in vitro or in vivo
using viruses, viral vaccines or oncolytic viruses. In specific
embodiments, methods concern production of immune cells that target
one or more antigens of HIV, EBV, CMV, adenovirus, vaccinia virus,
and/or VZV, including methods with stimulation steps that employ
IL-7 and IL-15, but not IL-2, IL-4, or both. Other specific
embodiments utilize stimulations in the presence of certain cells,
such as costimulatory cells and certain antigen presenting
cells.
Inventors: |
Rooney; Cliona M.;
(Bellaire, TX) ; Lapteva Doyle; Natalia; (Houston,
TX) ; Sharma; Sandhya; (Houston, TX) ; Wagner;
Dimitrios; (Houston, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Baylor College of Medicine |
Houston |
TX |
US |
|
|
Family ID: |
61619728 |
Appl. No.: |
16/331494 |
Filed: |
September 13, 2017 |
PCT Filed: |
September 13, 2017 |
PCT NO: |
PCT/US2017/051284 |
371 Date: |
March 7, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62395438 |
Sep 16, 2016 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2740/16034
20130101; A61K 39/12 20130101; C12N 2501/2315 20130101; C12N
2760/16134 20130101; C12N 2501/2307 20130101; A61K 35/17 20130101;
C12N 5/0638 20130101; A61P 31/22 20180101; A61P 31/18 20180101;
Y02A 50/30 20180101; A61P 31/20 20180101; A61P 35/00 20180101; A61K
2039/5158 20130101; Y02A 50/467 20180101; A61P 31/12 20180101; C12N
2710/16234 20130101 |
International
Class: |
C12N 5/0783 20060101
C12N005/0783; A61K 39/12 20060101 A61K039/12 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under
3300028311 and 3300028312 awarded by National Institutes of
Health/National Cancer Institute. The government has certain rights
in the invention.
Claims
1. A method of producing viral antigen-specific T-cells, comprising
the step of stimulating a population of peripheral blood T-cells
with antigen presenting cells in the presence of IL-7 and IL-15,
wherein the antigen presenting cells are or were previously exposed
to a library of peptides, wherein the peptides comprise sequence
that corresponds to at least part of the sequence of one or more
proteins of one or more viruses that are not HPV, wherein the
method comprises one or more of the following: (a) the method
occurs in the absence of IL-4; (b) the concentration of IL-15 is
.gtoreq.100 ng per mL; and (c) the method comprises depletion of
CD45RA-positive cells from the peripheral blood T-cells
2. The method of claim 1, wherein the stimulating occurs in the
absence of IL-6, IL-12, IL-2, IL-21, or a combination thereof.
3. The method of claim 1, wherein the population of peripheral
blood T-cells have reduced levels, compared to normal levels, of
one or more of the following: 1) NK cells; 2) naive cells that can
grow as bystander cells; and/or 3) T-regulatory cells.
4. The method of claim 3, wherein peripheral blood mononuclear
cells (PBMCs) or peripheral blood T-cells obtained therefrom are
subject to a step of reducing the levels of one or more of the
following: 1) NK cells; 2) naive cells that can grow as bystander
cells; 3) T-regulatory cells and/or 4) inhibitory myeloid
cells.
5. The method of claim 1, wherein the virus is from the family
Herpesviridae or is a poxvirus, adenovirus, polyomavirus,
lentivirus, rhabdovirus or other oncolytic virus.
6. The method of claim 1, wherein the virus is selected from the
group consisting of Epstein-Barr virus (EBV), Cytomegalovirus
(CMV), adenovirus, vaccinia virus, and/or Varicella zoster virus
(VZV), HIV, influenza, marabavirus vesicular stomatitis virus and
an oncolytic virus.
7. The method of claim 1, wherein the peripheral blood T-cells are
present in a population of PBMCs or are obtained or isolated
therefrom.
8. The method of claim 4, wherein the PBMCs or apheresis product
are depleted of one or more of the following: 1) NK cells; 2) naive
cells that can grow as bystander cells; and/or 3) T-regulatory
cells.
9. The method of claim 4, wherein the PBMCs in the population are
non-adherent PBMCs
10. The method of claim 1, wherein the antigen presenting cells are
dendritic cells or PBMCs.
11. The method of claim 1, wherein a stimulating step occurs in the
presence of costimulatory cells.
12. The method of claim 11, wherein the costimulatory cells are
CD80+, CD86+, CD83+, 4-1BBL+, or a combination thereof, or wherein
the costimulatory cells are HLA-negative lymphoblastoid cells.
13. The method of claim 1, wherein the stimulating occurs in the
presence of activated T-cells, dendritic cells, PBMCs, or
HLA-negative costimulatory cells.
14. The method of claim 13, wherein when the stimulating occurs in
the presence of activated T-cells, dendritic cells, PBMCs, or
HLA-negative costimulatory cells, the stimulating step is not a
first stimulation step.
15. The method of claim 13, wherein the activated T-cells are
autologous to the individual.
16. The method of claim 1, wherein the stimulating occurs in the
presence of pepmixes, pepmix-pulsed autologous activated T-cells,
in the presence of HLA-negative costimulatory cells, or both.
17. The method of claim 16, wherein when the stimulating occurs in
the presence of pepmix-pulsed autologous activated T-cells, in the
presence of HLA-negative costimulatory cells, or both, the
stimulating step is not a first stimulation step.
18. The method of claim 1, wherein the peptide library comprises
peptides of at least or no more than 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more
amino acids in length.
19. The method of claim 1, wherein the peptide library comprises
peptides of 15 amino acids in length.
20. The method of claim 1, wherein peptides in the library overlap
in sequence with other peptides by 11 amino acids.
21. The method of claim 1, wherein T-cells produced by a first
stimulating step are subject to one or more subsequent stimulating
steps.
22. The method of claim 21, wherein a subsequent stimulating step
occurs in the presence of IL-7 and IL-15.
23. The method of claim 21, wherein a subsequent stimulating step
occurs in the presence of activated T-cells, costimulatory cells,
IL-7, and IL-15.
24. The method of claim 1, wherein the method occurs in the absence
of exposing the T-cells produced by the method to activated B cells
that were previously exposed to a library of peptides.
25. The method of claim 1, wherein the cells are modified to
express a gene product from an expression vector.
26. The method of claim 25, wherein the cells are modified to
express a chimeric antigen receptor, .alpha..beta.T-cell receptor,
or combination thereof.
27. The method of claim 1, wherein a therapeutically effective
amount of T-cells produced by the method are provided to an
individual that has been exposed to EBV, CMV, adenovirus, vaccinia
virus, HIV, and/or VZV, that is seropositive for EBV, CMV,
adenovirus, vaccinia virus, HIV, and/or VZV, or that has disease
associated with EBV, CMV, adenovirus, vaccinia virus, HIV, and/or
VZV.
28. The method of claim 1, wherein the individual is determined as
having a medical condition associated with EBV, CMV, adenovirus,
vaccinia virus, HIV, and/or VZV.
29. The method of claim 1, wherein one or more steps of the method
lack the presence of exogenously added IL-4, IL-2, or both.
30. A method for stimulating T-cells specific for a virus other
than HPV, comprising stimulating T-cells specific for the virus
with antigen presenting cells in the presence of IL-7 and IL-15 but
in the absence of IL-4 and in the presence of co-stimulatory cells,
wherein the antigen presenting cells were previously exposed to one
or more peptides, wherein the peptides comprise sequence that
corresponds to at least part of the sequence of one or more
proteins of a virus other than HPV, and wherein the antigen
presenting cells are depleted of CD45RA-positive cells.
31. A method of producing therapeutic T-cells for a
virus-associated disease or a non-virus-associated disease, the
method comprising the step of stimulating T-cells specific for a
virus other than HPV with antigen presenting cells in the presence
of one or more of IL-7 and IL-15 but in the absence of IL-4 and in
the presence of co-stimulatory cells, wherein the antigen
presenting cells were previously exposed to one or more peptides,
wherein the peptides comprise sequence that corresponds to at least
part of the sequence of one or more proteins of a virus other than
HPV, wherein the stimulating produces T-cells therapeutic for the
virus-associated disease, wherein the antigen presenting cells are
depleted of CD45RA-positive cells.
Description
[0001] This application claims priority to U.S. Provisional Patent
Application Ser. No. 62/395,438, filed Sep. 16, 2016, which is
incorporated by reference herein in its entirety.
TECHNICAL FIELD
[0003] The present disclosure concerns at least the fields of
immunology, cell biology, molecular biology, and medicine,
including cancer medicine.
BACKGROUND
[0004] Antigen-specific T-cell activation and expansion requires 3
signals. Signal 1 requires the T-cell receptor (TCR) to bind its
cognate peptide-MHC complex. Signal 2 requires stimulation of
costimulatory receptors on the T-cell surface, and signal 3 is
derived from cytokines. The signals are required about once every 7
to 14 days to maintain the expansion of antigen-specific T-cells in
vitro. In the absence of any one of these signals, T-cells will
fail to proliferate and may become anergic or die. These
requirements introduce several challenges, especially when
activating tumor antigen-specific T-cells from cancer patients,
whose tumor antigen-specific T-cells are usually anergic
(unresponsive to activation) or otherwise dysfunctional.
[0005] Major challenges for antigen-specific T-cell activation and
expansion that have been addressed include:
[0006] 1. Tumor-induced T-cell anergy. Circulating T-cells have
been anergized by tumors and are difficult to expand from the blood
of cancer patients; and
[0007] 2. Expansion of exclusively antigen-specific CD4+ and CD8+
T-cells requires repeated stimulation of antigen-specific T-cells
with autologous antigen-presenting cells expressing tumor antigens
on both HLA class I and class II molecules, and costimulatory
molecules. If stimulation is too potent, non-specific bystander
cells are expanded diluting the antigen-specific T-cells.
[0008] Autologous dendritic cells (DCs) are potent
antigen-presenting cells, but their numbers are limiting; they do
not divide and their monocyte precursors less than 10% of blood
mononuclear cells (PBMCs). Large amounts of blood would be required
to obtain sufficient DCs for T-cell expansion (300 mLs to 1 liter
of blood).
[0009] Autologous EBV-transformed B-lymphoblastoid cells lines
(LCLs) are also excellent antigen-presenting cells, but it takes at
least 6 weeks to establish LCLs from patients and LCLs express
highly immunogenic EBV antigens that compete with weaker EBV and
non-EBV antigens.
[0010] The present disclosure addresses a variety of issues
associated with antigen-specific T-cell activation and expansion
and provides relief for a long-felt need in the art to treat
virus-associated diseases and malignancies with effective
immunotherapies.
BRIEF SUMMARY
[0011] The present disclosure is directed to methods and
compositions that concern immune system cells that immunogenically
recognize particular targets. In some embodiments, the present
invention concerns the development of virus-specific T cells (VSTs)
(that may also be referred to as virus antigen-specific T cells or
antigen-specific T cell) that target a biological moiety that
elicits an immune response in an individual. In specific
embodiments, the present disclosure concerns the development of
VSTs that target a viral antigen, including a viral
disease-associated antigen. Embodiments of the disclosure include
production of cells that are CD8+ T cells, CD4+ T cells, and cells
that do no kill but produce one or more cytokines. In some cases, a
mixture of cytotoxic T-cells is produced, and the mixture targets
more than one viral antigen, including more than one antigen of
more than one virus, in some cases. In particular embodiments, the
virus is not human papilloma virus (HPV). Embodiments of the
disclosure concern the generation and/or expansion of
non-HPV-specific T-cells.
[0012] Embodiments of the disclosure concern methods and
compositions for providing therapy to individuals infected with a
non-HPV virus or that have virus-associated diseases and
malignancies that are not associated with HPV, including cancers
associated with certain viruses. In specific embodiments, the
disclosure regards methods and compositions for adoptive cellular
immunotherapy that can target virus-associated medical conditions
and are therapeutic therefor.
[0013] In certain aspects, the present disclosure concerns the
development of a plurality of T-cells that target antigens from
EBV, CMV, adenovirus, vaccinia virus, HIV, and/or VZV, for example.
The present disclosure provides significant and non-obvious
improvements on methods for generating T cell lines with
specificity against antigens from EBV, CMV, adenovirus, vaccinia
virus, and/or VZV, for example, wherein the virus is not HPV.
[0014] In some embodiments of the disclosure, an individual is in
need of the methods and/or compositions of the invention. In
certain embodiments, an individual has been exposed to EBV, CMV,
adenovirus, vaccinia, and/or VZV, for example (the presence of
which may or may not be known for the individual), or the
individual is suspected of having been exposed to or at risk for
being exposed to EBV, CMV, adenovirus, vaccinia virus, and/or VZV,
for example. In certain embodiments, the individual has or is
suspected of having or is at risk for having disease associated
with EBV, CMV, adenovirus, vaccinia, and/or VZV, or has been
vaccinated with EBV, CMV, adenovirus, VZV or vaccinia virus, for
example.
[0015] In specific embodiments of at least part of the method,
certain antigen(s) associated with EBV, CMV, adenovirus, vaccinia,
and/or VZV are presented to APCs in the form of one or more
peptides that span some or all of certain antigen(s). The antigenic
peptides may be provided to the APCs in a library of peptide
mixtures, which may be referred to as pepmixes. In certain aspects
of the disclosure, there is pooling of a variety of pepmixes for
exposure to the APCs. APCs that present the antigens on MHC
molecules may be exposed to peripheral blood T-cells under certain
conditions to result in stimulation of T-cells specific for the
certain viral antigen(s).
[0016] In some embodiments, there are methods for stimulating
peripheral blood cells, such as peripheral blood T-cells, wherein
the method comprises stimulating peripheral blood T-cells with
antigen presenting cells in the presence of interleukin (IL)-7 and
IL-15 and, in at least some cases, in the absence of one or more
other cytokines, such as IL-6 and/or IL-12, wherein the antigen
presenting cells were previously exposed to one or more peptides,
wherein the peptides comprise sequence that corresponds to at least
part of the sequence of one or more proteins of a virus that is not
HPV.
[0017] Methods for stimulating T-cells specific for a non-HPV virus
or for an antigen from a non-HPV virus include stimulating T-cells
specific for the virus or for the antigen with antigen presenting
cells in the presence of IL-7 and IL-15, and optionally in the
presence of co-stimulatory cells, wherein the antigen presenting
cells were previously exposed to one or more peptides, wherein the
peptides comprise sequence that corresponds to at least part of the
sequence of one or more proteins of the virus.
[0018] In some cases there are methods of producing therapeutic
T-cells for non-HPV virus-associated disease(s), comprising the
step of stimulating peripheral blood T-cells with antigen
presenting cells in the presence of one or more of interleukin IL-7
and IL-15 and, in at least some cases, optionally in the absence of
one or more other cytokines, such as IL-6 and/or IL-12, wherein the
antigen presenting cells were previously exposed to one or more
peptides, wherein the peptides comprise sequence that corresponds
to at least part of the sequence of one or more proteins of a virus
that is not HPV, wherein the stimulating produces T-cells
therapeutic for virus-associated diseases and malignancies that are
not HPV-associated diseases.
[0019] In specific embodiments, there are methods of producing
therapeutic T-cells for virus-associated diseases and malignancies
other than HPV-associated diseases, comprising the step of
stimulating T-cells specific for the virus or for a viral antigen
with antigen presenting cells in the presence of one or more of
interleukin IL-7 and IL-15, and optionally in the presence of
co-stimulatory cells, wherein the antigen presenting cells were
previously exposed to one or more peptides, wherein the peptides
comprise sequences that corresponds to at least part of the
sequence of one or more proteins of a virus that is not HPV,
wherein the stimulating produces T-cells therapeutic for one or
more virus-associated diseases and malignancies.
[0020] In some cases, the peripheral blood T-cells being stimulated
are obtained from a prior stimulation of peripheral blood cells,
such as stimulating peripheral blood cells with antigen presenting
cells in the presence of IL-7 and IL-15, and in at least some cases
in the presence of one or more other cytokines, such as IL-6 and/or
IL-12, wherein the antigen presenting cells were previously exposed
to one or more peptides, wherein the peptides comprise sequence
that corresponds to at least part of the sequence of one or more
proteins of a virus that is not HPV. As such, prior to stimulating
the peripheral blood T-cells, the methods may further comprise
stimulating peripheral blood cells with antigen presenting cells in
the presence of IL-7 and IL-15, and in at least some cases in the
presence of IL-6 and/or IL-12, wherein the antigen presenting cells
were previously exposed to one or more peptides, wherein the
peptides comprise sequence that corresponds to at least part of the
sequence of one or more proteins of a virus that is not HPV, to
produce peripheral blood T-cells.
[0021] In some embodiments the one or more peptides comprise
sequence that corresponds to at least part of the sequence of one
or more proteins of a virus that is not HPV. In some embodiments
the one or more peptides may be a library of peptides, which may
also be referred to as a collection of peptides. In specific
embodiments the method may produce T-cells specific for EBV, CMV,
adenovirus, vaccinia, and/or VZV or for an antigen from EBV, CMV,
adenovirus, Vaccinia, and/or VZV. In some embodiments the method
may expand a population of T-cells present in the peripheral blood
T-cells that are specific for EBV, CMV, adenovirus, vaccinia,
and/or VZV or for an antigen from EBV, CMV, adenovirus, vaccinia,
and/or VZV.
[0022] APCs used in one or more methods of the disclosure include
monocytes, dendritic cells (DC), B-Blasts (BB), and/or PBMCs, for
example. In particular embodiments the antigen presenting cells are
activated T-cells.
[0023] In some embodiments the stimulation of T-cells specific for
a virus other than HPV or for an antigen other than one from HPV is
not a first stimulation step. The T-cells being stimulated cells
may be the product of a prior stimulation. In certain embodiments
stimulation of T-cells specific for a virus other than HPV or for
an antigen other than HPV comprises stimulating T-cells specific
for the virus or for the viral antigen with antigen presenting
cells in the presence of interleukin (IL)-7, IL-15, and in the
presence of one or more types of co-stimulatory cells.
[0024] In some embodiments the method may produce T-cells specific
for a non-HPV virus or for an antigen that is not an HPV antigen.
In some embodiments the method may expand a population of T-cells
specific for a virus or for an antigen other than HPV.
[0025] In specific embodiments, stimulation of peripheral blood
T-cells in the presence of IL-7 and IL-15 optionally occurs in the
absence of IL-2. In some embodiments stimulation of peripheral
blood T-cells in the presence of IL-7 and IL-15 optionally occurs
in the absence of at least IL-4; although in some cases IL-4 is
added, for example to increase CD4+ T cells. In some embodiments
stimulation of peripheral blood T-cells in the presence of IL-7 and
IL-15 occurs optionally in the absence of IL-6. In some embodiments
stimulation of peripheral blood T-cells in the presence of IL-7 and
IL-15 but alternatively may occur in the absence of IL-7 and/or
IL-15. In some embodiments stimulation of peripheral blood T-cells
in the presence of IL-7 and IL-15 optionally occurs in the absence
of IL-12. In some embodiments stimulation of peripheral blood
T-cells in the presence of IL-7 and IL-15 optionally occurs in the
absence of IL-21.
[0026] In some embodiments, the peripheral blood T-cells utilized
in methods of the disclosure may be present in a population of
peripheral blood mononuclear cells (PBMCs) or are obtained or
isolated therefrom. The PBMCs in the population may be non-adherent
PBMCs, or may be CD45RA-depleted PBMCs (for example, to eliminate a
combination of Tregs, NK cells and naive T-cells). The antigen
presenting cells may be dendritic cells, B-blasts, or PBMCs, for
example.
[0027] Methods of the present disclosure include methods of
producing therapeutic T-cells for virus-associated diseases and
malignanices that are not HPV. The stimulation of cells may produce
T-cells that are therapeutic for virus-associated diseases and
malignancies that are not HPV-associated diseases. in some
embodiments a method of producing therapeutic T-cells for
HPV-associated diseases is provided, the method comprising:
[0028] (i) stimulating peripheral blood cells, wherein the method
comprises stimulating peripheral blood T-cells with antigen
presenting cells in the presence of interleukin (IL)-7 and IL-15
and optionally in the absence of IL-6 and/or IL-12, wherein the
antigen presenting cells were previously exposed to one or more
peptides, wherein the peptides comprise sequence that corresponds
to at least part of the sequence of one or more proteins of a
non-HPV virus; or
[0029] (ii) stimulating T-cells obtained from (i) with antigen
presenting cells in the presence of interleukin (IL)-7 and IL-15
and optionally in the absence of IL-6 and/or IL-12, wherein the
antigen presenting cells were previously exposed to one or more
peptides, wherein the peptides comprise sequence that corresponds
to at least part of the sequence of one or more proteins of the
virus, wherein (ii) is optionally repeated one or more times;
[0030] (iii) stimulating T-cells obtained from (ii) with antigen
presenting cells in the presence of IL-7 and IL-15, and optionally
in the presence of co-stimulatory cells, wherein the antigen
presenting cells were previously exposed to one or more peptides,
wherein the peptides comprise sequence that corresponds to at least
part of the sequence of one or more proteins of the virus, wherein
(iii) is optionally repeated one or more times.
[0031] In some embodiments the antigen presenting cells used in (i)
and (ii) are monocytes, dendritic cells (DC) B-Blasts (BB) or
PBMCs. In some embodiments the antigen presenting cells used in
(iii) are activated T-cells, dendritic cells (DC), B-Blasts (BB) or
PBMCs. In some embodiments the antigen presenting cells used in
(iii) are different than the antigen presenting cells used in (i)
and/or (ii). In preferred embodiments the antigen presenting cells
used in (iii) are activated T-cells.
[0032] In particular embodiments, stimulation occurs in the
presence of co-stimulatory cells. In some embodiments the
co-stimulatory cells are one or more cell types selected from the
group consisting of CD80+ cells, CD86+ cells, CD83+ cells, 4-1BBL+
cells, and a combination thereof. The co-stimulatory cells may be
CD80+/CD86+/CD83+/4-1BBL+ cells. The co-stimulatory cells may be
HLA-negative lymphoblastoid cells.
[0033] In some particular embodiments, methods of the present
disclosure are for producing T-cells specific for EBV, CMV,
adenovirus, Vaccinia, and/or VZV. In some particular embodiments
methods of the present disclosure are for producing T-cells
specific for diseases and malignanices associated with EBV, CMV,
adenovirus, Vaccinia, and/or VZV.
[0034] In some embodiments, peripheral blood T-cells may be
obtained from an individual that is known to be infected or
suspected of being infected or vaccinated with EBV, CMV,
adenovirus, vaccinia, and/or VZV. Antigen presenting cells may be
obtained from an individual that is known to be infected or
suspected of being infected or vaccinated with EBV, CMV,
adenovirus, vaccinia virus, and/or VZV.
[0035] In some embodiments, the method may occur in the absence of
exposing the T-cells produced by the method to activated B cells
that were previously exposed to a library of peptides.
[0036] In some embodiments, antigen presenting cells may be
autologous or allogeneic to an individual intended to be treated
with the therapeutic T-cells obtained.
[0037] In some embodiments, the one or more peptides comprise
sequence that corresponds at least part to the sequence of one or
more proteins of EBV, CMV, adenovirus, vaccinia virus, and/or VZV.
The peptides may correspond to a contiguous amino acid sequence
present within the viral protein. A peptide may have a length of at
least or no more than 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 amino acids in length, or of 15 amino acids in length. The
collection of peptides may form a library and peptides in the
library may overlap in sequence with other peptides by any suitable
amount, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino
acids, for example. The peptides may comprise sequence that
corresponds to the viral protein.
[0038] T-cells produced by the methods of the present disclosure
may be isolated and/or purified, e.g., isolated/purified from other
cells.
[0039] In some embodiments, a therapeutically effective amount of
T-cells produced by the methods of the present disclosure are
provided to an individual that has been exposed to a virus that is
not HPV, or that has a virus-associated disease that is not from
HPV. In a related aspect T-cells produced by the methods of the
present disclosure are provided for use in the treatment of
virus-associated disease that is not from HPV. In another related
aspect the use of T-cells produced by the method of the present
disclosure are provided for use in the manufacture of a medicament
for use in the treatment of virus-associated disease that is not
HPV.
[0040] In particular embodiments, the methods of the disclosure
encompass the use of viruses to promote the expansion of VSTs
modified with one or more engineered receptors, for example
chimeric antigen receptors (CARs). For example, if the virus was
VZV and the VSTs were VZV-specific (VZVSTs), then a VZV vaccine
(e.g., ZOSTAVAX or VARIVAX) could be used to stimulate
proliferation of CAR-modified VZVSTs after infusion. If the VSTs
were specific for an oncolytic virus such as adenovirus, maraba
virus or vaccinia or VSV, then the oncolytic virus (OV) could not
only kill tumor cells, but also stimulate CAR-modified T-cells
specific for that oncolytic virus. The CAR-modified OV-specific T
cells (CAR-OVSTs) could then kill uninfected or metastatic tumors
cells via the CAR.
[0041] The individual to be treated may be a human. The individual
may be a patient. The individual may have been exposed to EBV, CMV,
adenovirus, vaccinia virus, and/or VZV, or has a disease associated
with EBV, CMV, adenovirus, vaccinia virus, and/or VZV. The disease
may be a neoplasm, such as a cancer of any kind.
[0042] The individual may have received, be receiving, or will
receive an additional therapy for the disease including, where
appropriate, an additional cancer therapy, such as surgery,
radiation, hormone therapy, chemotherapy, immunotherapy, or a
combination thereof.
[0043] The individual may be determined as having virus-associated
cancer that is not from HPV.
[0044] Methods according to the present disclosure that involve
steps of cell stimulation may be performed in vitro or ex vivo. The
term "in vitro" is intended to encompass studies with materials,
biological substances, cells and/or tissues in laboratory
conditions or in culture. "Ex vivo" refers to something present or
taking place outside an organism, e.g. outside the human or animal
body, which may be on tissue (e.g. whole organs) or cells taken
from the organism.
[0045] In one embodiment, there is a method for stimulating
peripheral blood cells, the method comprising stimulating
peripheral blood T-cells with antigen presenting cells in the
presence of interleukin (IL)-7 and IL-15, wherein the antigen
presenting cells were previously exposed to one or more peptides,
wherein the peptides comprise sequence that corresponds to at least
part of the sequence of one or more proteins of one or more viruses
that are not HPV.
[0046] In a particular embodiment, there is a method of producing
viral antigen-specific T-cells, comprising the step of stimulating
a population of peripheral blood T-cells with antigen presenting
cells in the presence of IL-7 and IL-15 (for example, .gtoreq.100
ng/mL), wherein the antigen presenting cells are or were previously
exposed to a library of peptides, wherein the peptides comprise
sequence that corresponds to at least part of the sequence of one
or more proteins of one or more viruses that are not HPV.
[0047] In particular embodiments of the method, a population of
peripheral blood T-cells have reduced levels, compared to normal
levels, of one or more of the following: 1) NK cells; 2) naive
cells that can grow as bystander cells; and/or 3) T-regulatory
cells, for example when CD45RA+ cells have been depleted from a
starting PBMC or apheresis product. In certain cases, PBMCs or
peripheral blood T-cells obtained therefrom are subject to a step
of reducing the levels of one or more of the following: 1) NK
cells; 2) naive cells that can grow as bystander cells; and/or 3)
T-regulatory cells. Peripheral blood T-cells may be present in a
population of peripheral blood mononuclear cells (PBMCs) or are
obtained or isolated therefrom. PBMCs or an apheresis product may
be depleted of one or more of the following: 1) NK cells; 2) naive
cells that can grow as bystander cells; and/or 3) T-regulatory
cells. The PBMCs may be CD45RA-depleted PBMCs. PBMCs in the
population may be non-adherent PBMCs. In specific embodiments, the
PBMCs may be depleted of myeloid cells that are suppressive myeloid
cells, for example by adherence or by other depletion methods.
[0048] Viruses encompassed by the disclosure include those from the
family Herpesviridae or is a poxvirus, adenovirus, polyomavirus,
lentivirus, rhabdovirus or other oncolytic virus. In specific
embodiments, the virus is selected from the group consisting of
Epstein-Barr virus (EBV), Cytomegalovirus (CMV), adenovirus,
vaccinia virus, and/or Varicella zoster virus (VZV), HIV,
influenza, marabavirus vesicular stomatitis virus or any other
oncolytic virus.
[0049] Antigen presenting cells utilized in methods of the
disclosure may be dendritic cells or PBMCs, in specific
embodiments.
[0050] In methods encompassed by the disclosure a stimulating step
occurs in the absence of IL-6, IL-12, IL-2, IL-4, IL-21, or a
combination thereof. In certain embodiments, a stimulating step
occurs in the presence of costimulatory cells, such as
costimulatory cells that are CD80+, CD86+, CD83+, 4-1BBL+, or a
combination thereof, or wherein the costimulatory cells are
HLV-negative lymphoblastoid cells. The stimulating may occur in the
presence of activated T-cells, dendritic cells, PBMCs, or
HLA-negative costimulatory cells. The stimulating may occur in the
presence of activated T-cells, dendritic cells, PBMCs, or
HLA-negative costimulatory cells, the stimulating step is not a
first stimulation step. Activated T-cells may be autologous to the
individual. Stimulating may occur in the presence of pepmixes,
pepmix-pulsed autologous activated T-cells, in the presence of
HLA-negative costimulatory cells, or both. In specific cases, when
the stimulating occurs in the presence of pepmix-pulsed autologous
activated T-cells, in the presence of HLA-negative costimulatory
cells, or both, the stimulating step is not a first stimulation
step. In such cases, two stimulations are performed and the second
stimulation occurs in the presence of costimulatory cells and
pepmix-pulsed (autologous activated T cells (AATCs).
[0051] Peptide libraries utilized in methods of the disclosure may
comprise peptides of at least or no more than 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
or more amino acids in length. In specific cases, the peptide
library comprises peptides of 15 amino acids in length and/or
peptides in the library overlap in sequence with other peptides by
11 amino acids.
[0052] In certain embodiments, T-cells produced by a first
stimulating step are subject to one or more subsequent stimulating
steps, such as a subsequent stimulating step that occurs in the
presence of IL-7 and IL-15. A subsequent stimulating step may occur
in the presence of activated T-cells, costimulatory cells, IL-7,
and/or IL-15. In specific cases, the method occurs in the absence
of exposing the T-cells produced by the method to activated B cells
that were previously exposed to a library of peptides.
[0053] In some cases, cells produced by the method are modified to
express a gene product from an expression vector, such as modified
to express a chimeric antigen receptor, .alpha..beta. T-cell
receptor, or combination thereof.
[0054] In certain cases, a therapeutically effective amount of
T-cells produced by the method are provided to an individual that
has been exposed to EBV, CMV, adenovirus, vaccinia virus, HIV,
and/or VZV, that is seropositive for EBV, CMV, adenovirus, vaccinia
virus, HIV, and/or VZV, or that has disease associated with EBV,
CMV, adenovirus, vaccinia virus, HIV, and/or VZV. In specific
aspects, the individual is determined as having a medical condition
associated with EBV, CMV, adenovirus, vaccinia virus, HIV, and/or
VZV. In certain embodiments, the cancer is a non-viral cancer.
[0055] In specific cases, one or more steps of the method lack the
presence of exogenously added IL-4, IL-2, or both.
[0056] In an embodiment, there is a method for stimulating T-cells
specific for a virus other than HPV, comprising stimulating T-cells
specific for the virus with antigen presenting cells in the
presence of IL-7 and IL-15 and in the presence of co-stimulatory
cells, wherein the antigen presenting cells were previously exposed
to one or more peptides, wherein the peptides comprise sequence
that corresponds to at least part of the sequence of one or more
proteins of a virus other than HPV.
[0057] In another embodiment, there is a method of producing
therapeutic T-cells for a virus-associated disease or a
non-virus-associated disease, the method comprising the step of
stimulating T-cells specific for a virus other than HPV with
antigen presenting cells in the presence of one or more of IL-7 and
IL-15 and in the presence of co-stimulatory cells, wherein the
antigen presenting cells were previously exposed to one or more
peptides, wherein the peptides comprise sequence that corresponds
to at least part of the sequence of one or more proteins of a virus
other than HPV, wherein the stimulating produces T-cells
therapeutic for the virus-associated disease or malignancy.
[0058] The disclosure encompasses at least the following:
[0059] 1. A method for stimulating peripheral blood cells, the
method comprising stimulating peripheral blood T-cells with antigen
presenting cells in the presence of interleukin (IL)-7 and IL-15,
wherein the antigen presenting cells were previously exposed to one
or more peptides, wherein the peptides comprise sequence that
corresponds to at least part of the sequence of one or more
proteins of one or more viruses that are not human papillomavirus
(HPV).
[0060] 2. A method of producing viral antigen-specific T-cells,
comprising the step of stimulating a population of peripheral blood
T-cells with antigen presenting cells in the presence of IL-7 and
IL-15, wherein the antigen presenting cells are or were previously
exposed to a library of peptides, wherein the peptides comprise
sequence that corresponds to at least part of the sequence of one
or more proteins of one or more viruses that are not HPV.
[0061] 3. The method of paragraph 1 or 2, wherein the concentration
of IL-15 is .gtoreq.100 ng per mL.
[0062] 4. The method of paragraph 1, 2, or 3, wherein the
stimulating occurs in the absence of IL-6, IL-12, IL-2, IL-4, IL-7,
IL-21, or a combination thereof.
[0063] 5. The method of any one of paragraphs 1-4, wherein the
population of peripheral blood T-cells have reduced levels,
compared to normal levels, of one or more of the following: 1) NK
cells; 2) naive cells that can grow as bystander cells; and/or 3)
T-regulatory cells.
[0064] 6. The method of paragraph 5, wherein the PBMCs or
peripheral blood T-cells obtained therefrom are subject to a step
of reducing the levels of one or more of the following: 1) NK
cells; 2) naive cells that can grow as bystander cells; and/or 3)
T-regulatory cells.
[0065] 7. The method of any one of paragraphs 1-6, wherein the
virus is from the family Herpesviridae or is a poxvirus,
adenovirus, polyomavirus, lentivirus, rhabdovirus or other
oncolytic virus.
[0066] 8. The method of any one of paragraphs 1-7, wherein the
virus is selected from the group consisting of Epstein-Barr virus
(EBV), Cytomegalovirus (CMV), adenovirus, Vaccinia, and/or
Varicella zoster virus (VZV), HIV, influenza, marabavirus vesicular
stomatitis virus and an oncolytic virus.
[0067] 9. The method of any one of paragraphs 1-8, wherein the
peripheral blood T-cells are present in a population of peripheral
blood mononuclear cells (PBMCs) or are obtained or isolated
therefrom.
[0068] 10. The method of paragraph 6 or 9, wherein the PBMCs or
apheresis product are depleted of one or more of the following: 1)
NK cells; 2) naive cells that can grow as bystander cells; and/or
3) T-regulatory cells.
[0069] 11. The method of paragraph 10, wherein the PBMCs are
CD45RA-depleted PBMCs and/or are CD45RO-depleted PBMCs.
[0070] 12. The method of any one of paragraphs 6 or 9-11, wherein
the PBMCs in the population are non-adherent PBMCs
[0071] 13. The method of any one of paragraphs 1-12, wherein the
antigen presenting cells are dendritic cells or PBMCs.
[0072] 14. The method of any one of paragraphs 1-12, wherein a
stimulating step occurs in the presence of costimulatory cells.
[0073] 15. The method of paragraph 14, wherein the costimulatory
cells are CD80+, CD86+, CD83+, 4-1BBL+, or a combination thereof,
or wherein the costimulatory cells are HLV-negative lymphoblastoid
cells.
[0074] 16. The method of any one of paragraphs 1-15, wherein the
stimulating occurs in the presence of activated T-cells, dendritic
cells, PBMCs, or HLA-negative costimulatory cells.
[0075] 17. The method of paragraph 16, wherein when the stimulating
occurs in the presence of activated T-cells, dendritic cells,
PBMCs, or HLA-negative costimulatory cells, the stimulating step is
not a first stimulation step.
[0076] 18. The method of paragraph 16 or 17, wherein the activated
T-cells are autologous to the individual.
[0077] 19. The method of any one of paragraphs 1-18, wherein the
stimulating occurs in the presence of pepmixes, pepmix-pulsed
autologous activated T-cells, in the presence of HLA-negative
costimulatory cells, or both.
[0078] 20. The method of paragraph 19, wherein when the stimulating
occurs in the presence of pepmix-pulsed autologous activated
T-cells, in the presence of HLA-negative costimulatory cells, or
both, the stimulating step is not a first stimulation step.
[0079] 21. The method of any one of paragraphs 1-20, wherein the
peptide library comprises peptides of at least or no more than 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, or more amino acids in length.
[0080] 22. The method of any one of paragraphs 1-21, wherein the
peptide library comprises peptides of 15 amino acids in length.
[0081] 23. The method of any one of paragraphs 1-22, wherein
peptides in the library overlap in sequence with other peptides by
11 amino acids.
[0082] 24. The method of any one of paragraphs 1-23, wherein
T-cells produced by a first stimulating step are subject to one or
more subsequent stimulating steps.
[0083] 25. The method of paragraph 24, wherein a subsequent
stimulating step occurs in the presence of IL-7 and IL-15.
[0084] 26. The method of paragraph 24 or 25, wherein a subsequent
stimulating step occurs in the presence of activated T-cells,
costimulatory cells, IL-7, and IL-15.
[0085] 27. The method of any one of paragraphs 1-26, wherein the
method occurs in the absence of exposing the T-cells produced by
the method to activated B cells that were previously exposed to a
library of peptides.
[0086] 28. The method of any one of paragraphs 1-27, wherein the
cells are modified to express a gene product from an expression
vector.
[0087] 29. The method of paragraph 29, wherein the cells are
modified to express a chimeric antigen receptor,
.gamma..delta.T-cell receptor, or combination thereof.
[0088] 30. The method of any one of paragraphs 1-29, wherein a
therapeutically effective amount of T-cells produced by the method
are provided to an individual that has been exposed to EBV, CMV,
adenovirus, Vaccinia, HIV, and/or VZV, that is seropositive for
EBV, CMV, adenovirus, Vaccinia, HIV, and/or VZV, or that has
disease associated with EBV, CMV, adenovirus, Vaccinia, HIV, and/or
VZV.
[0089] 31. The method of any one of paragraphs 1-30, wherein the
individual is determined as having a medical condition associated
with EBV, CMV, adenovirus, Vaccinia, HIV, and/or VZV.
[0090] 32. The method of any one of paragraphs 1-31, wherein one or
more steps of the method lack the presence of exogenously added
IL-4, IL-2, or both.
[0091] 33. A method for stimulating T-cells specific for a virus
other than HPV, comprising stimulating T-cells specific for the
virus with antigen presenting cells in the presence of IL-7 and
IL-15 and in the presence of co-stimulatory cells, wherein the
antigen presenting cells were previously exposed to one or more
peptides, wherein the peptides comprise sequence that corresponds
to at least part of the sequence of one or more proteins of a virus
other than HPV.
[0092] 34. A method of producing therapeutic T-cells for a
virus-associated disease or a non-virus-associated disease, the
method comprising the step of stimulating T-cells specific for a
virus other than HPV with antigen presenting cells in the presence
of one or more of IL-7 and IL-15 and in the presence of
co-stimulatory cells, wherein the antigen presenting cells were
previously exposed to one or more peptides, wherein the peptides
comprise sequence that corresponds to at least part of the sequence
of one or more proteins of a virus other than HPV, wherein the
stimulating produces T-cells therapeutic for the viral-associated
disease.
[0093] The foregoing has outlined rather broadly the features and
technical advantages of the present invention in order that the
detailed description of the invention that follows may be better
understood. Additional features and advantages of the invention
will be described hereinafter which form the subject of the claims
of the invention. It should be appreciated by those skilled in the
art that the conception and specific embodiment disclosed may be
readily utilized as a basis for modifying or designing other
structures for carrying out the same purposes of the present
invention. It should also be realized by those skilled in the art
that such equivalent constructions do not depart from the spirit
and scope of the invention as set forth in the appended claims. The
novel features which are believed to be characteristic of the
invention, both as to its organization and method of operation,
together with further objects and advantages will be better
understood from the following description when considered in
connection with the accompanying figures. It is to be expressly
understood, however, that each of the figures is provided for the
purpose of illustration and description only and is not intended as
a definition of the limits of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0094] For a more complete understanding of the present invention,
reference is now made to the following descriptions taken in
conjunction with the accompanying drawings.
[0095] FIG. 1 illustrates general embodiments of virus-specific
T-cell (VST) generation methods of the disclosure.
[0096] FIG. 2 demonstrates improved specificity of methods of the
disclosure that employ IL-7 and IL-15 as compared to known methods
that employ IL-4 and IL-7.
[0097] FIG. 3 shows improved specificity of lymphoma patient EBVSTs
grown in the presence of IL-7 and IL-15.
[0098] FIG. 4 demonstrates that high doses of IL-15 increase
specificity of VSTs.
[0099] FIG. 5 shows that high dose of IL-15 increases central
memory EBVSTs.
[0100] FIG. 6 shows excessive NK-cell outgrowth in EBVSTs from some
patients.
[0101] FIG. 7 illustrates generation of pepmix-activated EBVSTs
from CD45RA-depleted PBMCs.
[0102] FIG. 8 demonstrates that CD45RA depletion decreases the
frequency of CD3-CD56+ NK cells in EBVSTs expanded from healthy
donors.
[0103] FIG. 9 shows that removal of CD45RA+ cells increases
proliferation of EBVSTs.
[0104] FIG. 10 demonstrates that CD45RA depletion enhanced the fold
expansion of EBVSTs.
[0105] FIG. 11 shows CD45RA depletion enhances antigen specificity
of EBVSTs at the end of a second stimulation (at day 16).
[0106] FIG. 12 demonstrates CD45RA depletion enhances antigen
specificity of EBVSTs.
[0107] FIG. 13 demonstrates increased antigen specificity of CD45RA
depleted EBVSTs sustained after a third stimulation.
[0108] FIG. 14 demonstrates CD45RA depletion decreases NK cell
population outgrowth in lymphoma patient EBVSTs.
[0109] FIG. 15 shows CD45RA depletion increased the frequency of
antigen specific T-cells in lymphoma patient EBVSTs.
[0110] FIG. 16 demonstrates CD45RA depletion increased antigen
specificity in EBVSTs from lymphoma patients.
[0111] FIG. 17 shows the effect of CD45RA depletion on
proliferation of lymphoma patients' EBVSTs.
[0112] FIG. 18 demonstrates that CD45RA-depletion enhanced
cytolytic activity against pepmix-pulsed autologous activated
T-cells (aATCs).
[0113] FIG. 19 illustrates an embodiment of generation of T-cells
specific for EBV, CMV, Adenovirus, BK virus, and HHV6
(multivirus-specific T-cells).
[0114] FIG. 20 demonstrates expansion of multivirus-specific
T-cells.
[0115] FIG. 21 shows antigen specificity of multivirus-specific
T-cells.
[0116] FIG. 22 demonstrates proliferation of VZV-specific VSTs
after a first stimulation.
[0117] FIG. 23 shows expansion of VZVSTs after a second
stimulation.
[0118] FIG. 24 demonstrates specificity of the VZVSTs after a first
stimulation (at day 8).
[0119] FIG. 25 demonstrates specificity of VZVSTs after a second
stimulation (at day 16).
[0120] FIG. 26 illustrates manufacturing of HIV-specific T-cells
from HIV seropositive donors.
[0121] FIG. 27 shows optimal expansion with K562 cells in a second
stimulation.
[0122] FIG. 28 demonstrates that in the presence of K562, HIV
antigen-specific T cells (HIVSTs) expanded to clinically relevant
numbers after only two stimulations.
[0123] FIG. 29 shows that HIVSTs are specific for multiple HIV
antigens.
[0124] FIG. 30 demonstrates that HIVSTs comprise mixed CD4+ and
CD8+ T cells.
[0125] FIG. 31 demonstrate that HIVSTs can lyse antigen-pulsed and
HIV-infected targets.
DETAILED DESCRIPTION
[0126] The scope of the present application is not intended to be
limited to the particular embodiments of the process, machine,
manufacture, composition of matter, means, methods and steps
described in the specification.
[0127] In keeping with long-standing patent law convention, the
words "a" and "an" when used in the present specification in
concert with the word comprising, including the claims, denote "one
or more." Some embodiments of the invention may consist of or
consist essentially of one or more elements, method steps, and/or
methods of the invention. It is contemplated that any method or
composition described herein can be implemented with respect to any
other method or composition described herein.
[0128] The present disclosure concerns the production and use of
therapeutic T-cells for individuals that are in need of EBV-, CMV-,
adenovirus-, vaccinia virus-, and/or VZV-specific T-cells,
including for treating medical conditions associated with one or
more of these viruses in an individual. In some embodiments, the
disclosure concerns the production and use of therapeutic T-cells
for a non-viral cancer; in such cases, one or more CARs are
expressed in the VSTs and vaccination or oncolytic viruses are used
to stimulate the CAR-VSTs via their T-cell receptors. In particular
embodiments, therapeutic T-cells are generated upon stimulation of
APCs in the presence of IL-7 and IL-15, wherein the APCs were
previously exposed to a peptide library directed to one or more
viral antigens.
[0129] In particular aspects, the present disclosure addresses
challenges related to adoptive T cell transfer. For example, to
address tumor induced T-cell anergy, the inventors have evaluated
various combinations of cytokines and determined that at least in
some cases a high dose of IL-15 combined with IL-7 facilitates the
expansion of anergic antigen-specific T-cells (FIG. 2). Thus for
the first stimulation antigen-specific T-cells are activated by
pulsing PBMCs with overlapping peptide libraries (pepmixes that
comprise 15mer amino acids overlapping by 11 amino acids and
spanning the proteins of interest, for example) in the presence of
IL7 (10 ng per mL) and IL15 (100 ng per mL). Herein the inventors
have evaluated antigens from several different viruses to make
virus-specific T-cells (VSTs).
[0130] To address one problem of the antigen-presenting cell, the
inventors have evaluated an antigen-presenting cell complex in
which autologous activated T-cells pulsed with peptides provide
signal 1 (binding of T-cell receptor (TCR) to its cognate
peptide-MHC complex), and an HLA-negative LCL, provides
co-stimulation (signal 2). An alternative artificial costimulatory
cell line is the HLA-negative K562 cell line genetically modified
to express CD80, CD86, CD83 and 4-1BB ligand, for example. In this
case co-stimulation is provided in trans, on a different cell type.
In such cases, HLA antigens must be absent because these molecules
are potent antigens and can activate allospecific T-cells, and
EV-LCLs naturally express a range of costimulatory molecules.
[0131] This strategy is able to induce log-fold expansion of VSTs
with specificity for the stimulating antigens. Because in some
donors this strategy potently expands NKcells, the inventors have
also introduced a depletion step. For example, PBMCs may be
depleted of CD45RA+ T-cells. This depletes not only NK cells but
also naive T-cells and natural T regulatory cells. VSTs grown from
CD45RA-depleted PBMCs have higher antigen-specificity, show greater
fold expansion and have minimal NK cells. Further from some
individuals the inventors were able to grow VSTs only if they first
depleted RA+ T-cells from PBMCs.
[0132] I. Viral Antigen(s) and Generation of Pepmixes
[0133] Methods of the disclosure utilize antigen-presenting cells
that present mixtures of peptides to T-cells. Such "loaded" APCs
are generated prior to exposure to peripheral blood T-cells for
stimulation, and the generation of the loaded APCs may or may not
be performed by the individual or entity that performs the
stimulation step for the peripheral blood T-cells. Thus, in some
embodiments, an effective amount of a library of peptides is
provided to APCs as part of methods that ultimately generate
therapeutic virus-specific T cells (VSTs) or antigen-specific
T-cells. In methods of the disclosure, prior to a stimulation step,
APCs are exposed to a sufficient amount of the library of peptides.
The library, in particular cases, comprises a mixture of peptides
("pepmixes") that span part or all of the same antigen. In
particular embodiments, peptides for the APCs are non-natural.
[0134] In utilizing a library of mixtures of peptides from one or
more antigens, the various peptides may come from any part of a
given protein, but in specific cases the peptides span the length
of the majority or all of the protein, wherein the sequence of the
peptides overlap at least in part to facilitate coverage of the
entire desired region of the specific antigen(s). In some cases the
peptides span the length or one or more known epitopes or domains
of the respective antigen to which the peptides correspond. Certain
regions may be covered by peptides that span the length of the
region, including a region such as a N-terminal domain, C-terminal
domain, extracellular domain, or intracellular domain, for
example.
[0135] The antigens from which the peptides are derived are
antigens for EBV, CMV, adenovirus, vaccinia virus, and/or VZV that
may be of any kind, but in specific embodiments the antigens are
such that they allow for targeting for cytotoxic T-cells to medical
conditions associated with EBV, CMV, adenovirus, vaccinia virus,
and/or VZV infection, respectively. In particular embodiments, the
peptides are derived from, or have sequence that corresponds to, at
least part of one or more antigens of at least one type of EBV,
CMV, adenovirus, vaccinia virus, or VZV. In some cases, a pepmix
library includes peptides corresponding to one or more antigens
from a single virus, and those peptides may or may not provide
sequence coverage across the entire antigen(s) in question. In
other cases, a pepmix library includes peptides corresponding to
one or more antigens from more than one virus, and those peptides
may or may not provide sequence coverage across the entire
antigen(s) in question. The pepmix may or may not be enriched for
peptides corresponding to one or more certain regions of one or
more certain antigens or corresponding to the entirety of one or
more certain antigens.
[0136] Pepmixes utilized in the disclosure may be from commercially
available peptide libraries and/or may be synthetically generated.
Examples of available libraries include those from JPT Technologies
(Springfield, Va.) or Miltenyi Biotec (Auburn, Calif.). The skilled
artisan based on known sequences of viral antigens, for example,
would have sufficient information to be able to generate peptides
that correspond to their exemplary, respective sequences. The
skilled artisan, based on known sequences of antigens from these
well-known viruses, for example, would have sufficient information
to be able to generate peptides that correspond to their exemplary,
respective sequences.
[0137] In particular embodiments, a library is comprised of
peptides of a certain length that correspond to their respective
antigens, although in some cases a library is comprised of a
mixture of peptides with two or more different lengths. The
peptides may be of a certain length(s) and they may overlap in
sequence of a certain amount, although there may be variability of
length of overlap in some libraries. In particular embodiments, the
peptides are at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or
35 or more amino acids in length, for example. In particular
embodiments, there is overlap among the peptides of at least 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 amino acids in
length, for example. In specific embodiments, the peptides are 15
amino acids long and overlap one another by 11 amino acids. A
mixture of different peptides may include any ratio of the
different peptides, although in some embodiments each particular
peptide is present at substantially the same quantities in the
mixture as another particular peptide. Although coverage of an
antigen in sequence for the peptides may be random and
substantially even over a given region of an antigen, in some
embodiments a library may be enriched for one or more particular
peptides, such as one or more peptides that are known to encode an
epitope or a part thereof, for example.
[0138] In particular embodiments, the pepmix for a particular
antigen protein comprise all possible HLA class I epitopes that are
8 to 10 amino acids long, for example. In specific embodiments,
longer peptides are utilized to cover all class II epitopes for a
particular peptide. In some cases, a range of length of epitopes is
12-25 amino acids.
[0139] II. Methods of Producing and Using Therapeutic VSTs
[0140] A. Producing Therapeutic VSTs
[0141] In certain aspects, the present disclosure concerns the
development of VSTs that target one or more antigens from at least
one of EBV, CMV, adenovirus, vaccinia virus, and/or VZV.
[0142] In methods of producing the T-cells, peripheral blood
T-cells are initially stimulated with APCs that have been exposed
to one or more peptides that span some or all of at least one viral
antigen. The antigenic peptides may be provided to the APCs as a
library of peptide mixtures, and multiple libraries of pepmixes may
be provided to the same collection of APCs. In some embodiments,
the collection includes both immunodominant and subdominant
antigens.
[0143] In embodiments of the disclosure, therapeutic T-cells are
generated and may be provided to an individual that has a viral
infection or is at risk of having a virus-associated medical
condition that results indirectly or directly from a viral
infection or are provided to an individual that has a non-virally
infected tumor. In methods of producing the therapeutic T-cells,
under certain conditions peripheral blood T-cells are mixed with
APCs that are loaded with a library of peptides that span part or
all of one or more viral antigens from one or more viruses. In
specific embodiments, for the stimulating step the T-cells reside
within a population of PBMCs.
[0144] Thus, although the source of the peripheral blood T-cells
may be of any kind, in specific embodiments the source is PBMCs,
and in some cases a plurality of PBMCs are utilized in the methods,
wherein the plurality comprises the peripheral blood T-cells.
Peripheral blood T-cells may be isolated or purified at least in
part from PBMCs. In some cases, the PBMCs are non-adherent, and in
some cases the PBMCs are CD45RA-depleted (wherein the depletion
occurs prior to exposure of the PBMCs to the APCs). In specific
embodiments, the peripheral blood T-cells have reduced numbers of
CD45RA-positive cells in comparison to a normal standard. The
peripheral blood T-cells or PBMCs may be depleted for certain cells
by using standard means in the art including, for example, magnetic
labeling and separation (for example, using Miltenyi.RTM. Biotec
columns or StemSep.TM. magnetic beads). The term "depleted" as used
herein refers to the peripheral blood T-cells or PBMCs having
substantially no CD45RA-positive cells therein. In some cases,
"depleted" refers to there being a reduction of a certain
percentage of CD45RA-positive cells compared to the numbers of
those cells in an original collection of peripheral blood T-cells
or PBMCs. The number of CD45RA-positive cells are reduced from an
original collection of peripheral blood T-cells or PBMCs because
they are manipulated specifically to remove CD45RA-positive cells.
In some cases, there is at least a 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% reduction of
CD45RA-positive and/or CD45RO-positive cells from an original
collection of peripheral blood T-cells or PBMCs following
manipulation of the original collection to remove the
CD45RA-positive cells. In some embodiments, myeloid cells may be
removed using magnetic beads or plastic adherence.
[0145] In some embodiments, there is a method of generating T-cells
that target at least one antigen from EBV, CMV, adenovirus,
Vaccinia, and/or VZV, and this occurs generally by contacting a
plurality of PBMCs with a plurality of APCs loaded for peptides
from a library of peptides that correspond to one or more
particular viral antigens from EBV, CMV, adenovirus, Vaccinia,
and/or VZV. In specific embodiments, the exposure of the two
populations of cells allows for expansion of the T-cells. In
particular embodiments, the stimulation step(s) occurs in the
presence of one or more particular cytokines. In certain
embodiments, the one or more cytokines are IL-7 and/or IL-15,
although in alternative embodiments the cytokine(s) are selected
from the group consisting of IL-2, IL-15, IL-7, IL-21, IL-12, IL-6,
IL-4, and a combination thereof. In specific embodiments, one or
more steps of the methods do not occur in the presence of IL-2,
IL-4, IL-6, IL-7, IL-12, and/or IL-21, although alternatively IL-2,
IL-4, IL-6, IL-7, IL-12, and/or IL-21 may be utilized. Reference to
the presence of a cytokine is to presence of exogenously added
cytokine, i.e. excluding any cytokine present within or secreted by
the culture of cells. In some embodiments, the peptides are further
defined as peptides that overlap in sequence to span part or all of
a viral antigen that is not a HPV antigen. For example, in certain
aspects the peptides overlap by at least 10 amino acids, and
particularly 11, and in some embodiments the peptides are at least
12 or more amino acids in length, and particularly 15 amino acids
in length.
[0146] The selection of an appropriate amount or concentration of a
given cytokine for inclusion in a cell culture is within the
ability of the person or ordinary skill in the art. By way of
example, the following is a list of certain interleukins and
examples of appropriate concentrations that may be used:
[0147] Interleukin 6 (IL-6): 50 to 150 ng/ml, one of about 50
ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml, 90 ng/ml, 100 ng/ml, 110
ng/ml, 120 ng/ml, 130 ng/ml, 140 ng/ml or 150 ng/ml;
[0148] Interleukin 7 (IL-7): 5 to 15 ng/ml, one of about 5 ng/ml, 6
ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13
ng/ml, 14 ng/ml or 15 ng/ml;
[0149] Interleukin 12 (IL-12): 5 to 15 ng/ml, one of about 5 ng/ml,
6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml,
13 ng/ml, 14 ng/ml or 15 ng/ml;
[0150] Interleukin 15 (IL-15): 5 to 15 ng/ml, one of about 5 ng/ml,
6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml,
13 ng/ml, 14 ng/ml or 15 ng/ml.
[0151] Table 1 below provides examples of certain embodiments of
methods of the disclosure.
TABLE-US-00001 TABLE 1 Examples of Elements of a Method Embodiments
Examples for Embodiments Source of T-cells Leukapheresis product
Apheresis product Peripheral blood mononuclear cells (PBMC)
Non-adherent PBMC CD45RA-depleted PBMC Depleted of myeloid cells
Antigen-presenting Dendritic cells (DC)s or PBMCs or monocytes
cells (APC) for first stimulation Cytokines for first Combinations
of IL15, IL-7, IL21, IL12, IL-6, IL-4 stimulation (stim) (although
in specific embodiments IL-4 is not utilized) Antigen Viral
pepmixes for EBV, CMV, adenovirus, vaccinia, and/or VZV APCs for
2.sup.nd stim DC PBMCs Autologous activated T-cells (AATC) +
HLA-negative costimulatory cells APCs for 3.sup.rd and Px-AATCs +
HLA-negative costimulatory cells subsequent stims Costimulatory
cells K562 expressing CD86, 4-1BB-L, CD83 and CD80 Or HLA-negative
Lymphoblastoid cell lines (LCL)
[0152] Thus, in particular embodiments, a population of T-cells
(wherein the population may comprise some, a majority, or
substantially all T-cells or wherein the population of T-cells is
within another population of cells, such as within PBMCs) is
exposed to a population of APCs to generate T cell lines having
particular characteristics, including at least: a) effectiveness at
targeting viral antigen(s); b) polyclonality; c) TH1 bias; d)
minimally differentiated memory type; or e) a combination thereof.
In specific embodiments, the cells may be minimally differentiated,
but in some cases they may not all be and a majority may be
somewhat differentiated.
[0153] In some cases, T-cells are stimulated more than once, and
different stimulation steps may or may not expose the population of
cells to the same conditions. In specific embodiments, a first
stimulation has conditions different from a subsequent stimulation,
including a second stimulation and/or a third stimulation. In
specific embodiments, a first stimulation step of the method
utilizes APCs that are pepmix-loaded DCs or pepmix-loaded PBMCs and
utilizes IL-7 and IL-15, although in alternative embodiments the
step utilizes one or more cytokines selected from IL15, IL-7, IL21,
IL12, IL-6, and/or IL-4. This stimulation step may optionally be
repeated one or more times.
[0154] In certain embodiments of the methods, between days 8 and 10
following an initial exposure of the peripheral blood T-cells (or
PBMCs) to the pepmix or APCs, there may be a re-stimulation of the
PBMCs on day 8, day 9, or day 10, but rarely later, and then a
subsequent re-stimulation may occur on day 15, day 16, or day 17
(see FIG. 7 as an example of one specific embodiment).
[0155] In some cases, a stimulation step that is subsequent to the
first stimulation step (including optional repeats of the first
stimulation step), the resultant T-cells obtained after the first
stimulation (and which may reside in a heterogeneous population of
cells) may be exposed to pepmix-loaded DCs or pepmix-loaded PBMCs
and/or pepmix-pulsed autologous activated T-cells and/or
HLA-negative costimulatory cells. Generally sufficient cells are
produced after a second stimulation on day 8 to 10 with
pepmix-pulsed AATCs combined with HLA-ve costimulatory cells.
Occasionally a third stimulation may be required using the same
antigen-presenting complex. Costimulatory cells that may be
utilized in any stimulation step include at least cells that
express CD86, 4-1BB, and/or CD83 and/or cells that are HLA-negative
lymphoblastoid cells. In specific cases, the costimulatory cells
may be gene-modified K562 cells.
[0156] In some embodiments, during the steps of the method the
cells in culture are modified. In specific embodiments, the cells
are modified to harbor a polynucleotide that expresses a gene
product that renders the cells effective or more effective for a
specific purpose or function, such as effective or more effective
for targeting a particular target and/or enhanced in function for
T-cell-mediated cytotoxicity, for example. In specific embodiments,
the cells are modified to express a certain non-natural receptor
that allows the T-cells to effectively or more effectively target a
desired target cell, such as one that expresses a certain antigen.
In specific embodiments, the cells are modified to express a
chimeric antigen receptor (CAR), and so forth. The cells may be
modified to express an expression vector (that may be viral
(including retroviral, lentiviral, adenoviral, adeno-associated
viral, and so forth) or non-viral, e.g., a transposon such as
piggyBac) during the method at specific time points, such as the
vector being introduced between day 2 and 5 of culture, for example
so that the vector is expressed in T-cells with long-term
repopulation potential. In specific embodiments the cells are
exposed to the expression vector within about 3 days after each
stimulation, but in such cases the modification occurs in more
differentiated T-cells that have less long term potential (which in
specific circumstances is desirable, such as if long term
expression of gene-modified cells is not desirable, as in the case
where the transgene has potential toxicity, for example).
[0157] In specific embodiments, the cells are modified to express a
CAR that targets a cancer antigen, such as EphA2, HER2, GD2,
Glypican-3, 5T4, 8H9, .alpha..sub.v.beta..sub.6 integrin, B cell
maturation antigen (BCMA) B7-H3, B7-H6, CAIX, CA9, CD19, CD20,
CD22, kappa light chain, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8,
CD70, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFRvIII, EGP2, EGP40,
EPCAM, ERBB3, ERBB4, ErbB3/4, FAP, FAR, FBP, fetal AchR, Folate
Receptor a, GD2, GD3, HLA-AI MAGE A1, HLA-A2, IL11Ra, IL13Ra2, KDR,
Lambda, Lewis-Y, MCSP, Mesothelin, Muc1, Muc16, NCAM, NKG2D
ligands, NY-ESO-1, PRAME, PSCA, PSC1, PSMA, ROR1, Sp17, SURVIVIN,
TAG72, TEM1, TEM8, VEGRR2, carcinoembryonic antigen, HMW-MAA, VEGF
receptors, and/or other exemplary antigens that are present with in
the extracellular matrix of tumors, such as oncofetal variants of
fibronectin, tenascin, or necrotic regions of tumors and other
tumor-associated antigens or actionable mutations that are
identified through genomic analysis and or differential expression
studies of tumors, for example.
[0158] B. Using Therapeutic VSTs
[0159] In certain embodiments, cells produced by methods of the
disclosure are provided to an individual in need thereof for
treatment of a medical condition or to target a viral infection or
virus-associated cancer or non-virus-associated cancer in which no
symptoms of a medical condition are detectable or have manifested.
As used herein "treatment" or "treating," includes any beneficial
or desirable effect on the symptoms or pathology of a disease or
pathological condition, and may include even minimal reductions in
one or more measurable markers of the disease or condition being
treated. Treatment can involve optionally either the reduction or
amelioration of symptoms of the disease or condition, or the
delaying of the progression of the disease or condition.
"Treatment" does not necessarily indicate complete eradication or
cure of the disease or condition, or associated symptoms
thereof.
[0160] In the methods encompassed by the disclosure, the
therapeutic T-cells are utilized to treat virus-associated disease
caused directly or indirectly by a single non-HPV virus or are
otherwise provided to an individual that is seropositive for a
single non-HPV virus. In other cases, the therapeutic T-cells are
utilized to treat disease(s) caused directly or indirectly by more
than one virus In the collection of therapeutic T-cells, each
T-cell and its progeny has specificity for only one peptide in one
antigen from one virus, and upon production of the collection of
therapeutic T-cells, one expands a population of T-cell clones that
together have multi-specificity, such as for multiple epitopes in
each viral antigen, for example.
[0161] In at least some methods of the disclosure, a
therapeutically effective amount of the VSTs generated thereby are
administered to an individual, for example, an individual known to
have or suspected of having or susceptible to having disease
associated with EBV, CMV, adenovirus, vaccinia virus, and/or VZV.
In specific embodiments, the cells are administered by injection,
such as intravenous, intramuscular, intradermal, subcutaneous,
intraperitoneal injection, and so forth, for example. In some
embodiments, the VSTs may be polyclonal CD4+ and CD8+ VSTs. The
PBMCs may be allogeneic to the individual or are autologous to the
individual.
[0162] In certain cases, neoplasms are treated with cells of the
disclosure, and the neoplasm may be benign, malignant, or a
premalignant lesion that can lead to cancer. Thus, an individual
may be treated with cells produced by methods of the disclosure at
the premalignant lesion stage and/or after the lesion becomes
malignant. The individual may have early or late stage cancer, and
the skilled artisan is aware that the methods of producing the
cells may be tailored for such different stages of cancer, such as
by utilizing peptides for the APCs that are from antigens
associated with early vs. late stage cancer. In specific
embodiments, the cancer may be primary, metastatic, recurrent,
refractory, and so forth.
[0163] In some cases, one can determine the virus or viruses that
are associated with the medical condition before administration of
the cells, although in some cases the virus type(s) is not
determined. In specific embodiments, cells specific for EBV, CMV,
adenovirus, vaccinia virus, and/or VZV have activity for
individuals that are positive for EBV, CMV, adenovirus, vaccinia
virus, and/or VZV, respectively. In some cases cells specific for
one of the viruses encompassed herein are gene-modified with a
receptor for a non-viral tumor and then infused to treat that
non-viral tumor, so that the virus or viral antigen can be used to
stimulate expansion of the T-cells in vivo, for example by
vaccination or using oncolytic viruses or endogenous viruses.
[0164] In cases wherein the APCs of the stimulation steps of the
method are loaded with pepmixes of different viral antigens
together, the outcome of administration of T-cells expanded through
such APCs is determined by whether the individual has been exposed
to the virus. For example, in particular embodiments if an
individual is infected with a certain virus, only T-cells specific
for that virus will respond, and this is because the infection will
initially have stimulated a T-cell response to that virus. Those
T-cells will expand in the individual and then become memory
T-cells and would be at higher numbers than T-cells specific for
another virus that have never been activated.
[0165] The individual being treated may be known to have a
virus-associated disease, suspected of having a virus-associated
disease, or at risk for a virus-associated disease. An individual
being treated may have the presence of the virus but there are not
yet any deleterious symptoms of a virus-related medical condition.
The individual may be at risk for a non-HPV virus-associated
disease given an environment or event that exposes them to the
non-HPV virus.
[0166] In some embodiments, one or more administrations of the
cells produced by methods of the disclosure are provided to an
individual in need thereof. The length of time between multiple
administrations may be of any suitable duration so long as
subsequent administrations are effective against the cancer,
including on the order of days, weeks, months, or years. In cases
wherein more than one administration of cells are provided to the
individual, the antigen to which the cells are targeted may or may
not be the same antigen as the cells utilized in earlier
administration(s). For example, in a first administration of cells,
they may target one viral antigen, whereas in another
administration of cells, the cells target a different antigen,
including from a different virus, in at least some cases.
[0167] In some cases, an individual is optionally determined to
have viral infection by any suitable means in the art. One may
employ infection diagnosis methods such as DNA tests utilizing PCR,
Southern blot hybridization, in situ hybridization, and these
methods may or may not be used in conjunction with other methods,
for example.
[0168] In specific embodiments, the individual is immunocompromised
(which for example, may be defined as an individual whose ability
to fight infectious disease or cancer with the immune system is
compromised or entirely absent). In specific embodiments, the
immunocompromised individual has had a stem cell transplant
(including hematopoietic stem cell transplantation), has had an
organ transplant and/or has received one or more cancer treatments,
including chemotherapy or radiation, or has been infected with HIV,
for example. In some cases, the individual has acquired or
inherited immune deficiency disorder. In some embodiments, those
that are immunocompromised by their disease and/or its treatment
are provided methods and/or compositions of the disclosure.
[0169] A. Epstein-Barr Virus
[0170] The Epstein-Barr virus (EBV), also called human herpesvirus
4 (HHV-4), is one of eight known viruses in the human Herpesviridae
family. EBV causes infectious mononucleosis, some forms of cancer
(including Hodgkin's lymphoma, Burkitt's lymphoma, gastric cancer,
nasopharyngeal cancer, NK/T lymphoma, diffuse large B-cell
lymphoma, and leiomyosarcoma, at least), and certain conditions
associated with human immunodeficiency virus (HIV), including hairy
leukoplakia and central nervous system lymphomas, as examples. In
methods of the disclosure, EBV infection or one or more
EBV-associated medical conditions are treated using the cells.
[0171] Although EBV encodes about 90 proteins, a limited fraction
of these are expressed in EBV-associated malignancies. About 80
genes are involved in the viral lytic cycle and about 9 genes are
associated with virus latency. In some tumors only 4 EBV genes are
expressed and in others only 2 are expressed. Recently some tumors
have been show to undergo abortive virus replication and express
early viral proteins and they have also been show to express
unexpected transcripts from the lytic cycle. All of these provide
transcripts may encode potential target antigens for VSTs.
Generally one can target LMP1, LMP2, BARF1 and EBNA1 that are
considered EBV type 2 latency antigens. However, in some cases one
may also target early viral proteins, such as those encoded by
BZLF1, BRLF1, BMLF1 or unexpected transcripts such as BXLF1/2, for
example.
[0172] B. Cytomegalovirus
[0173] Cytomegalovirus (CMV), also known as human herpesvirus-5, is
a virus in the family Herpesviridae. CMV infection normally does
not result in disease unless the infected individual is an infant
or is immune-compromised (such as an organ or tissue or cell
transplant recipient, including after allogeneic marrow
transplantation, for example). Although any antigen of CMV may be
targeted with cells produced by methods of the disclosure, in
specific embodiments the antigen is the immediate early antigen,
IE1, and the tegument protein, pp65.
[0174] C. Adenovirus
[0175] Adenoviruses can cause common cold, sore throat
(pharyngitis), bronchitis, pneumonia, diarrhea, pink eye
(conjunctivitis), fever, cystitis, gastroenteritis, and neurologic
disease. Healthy individuals are rarely subject to
adenovirus-associated serious illness or death. However, infants
and people with weakened immune systems, or existing respiratory or
cardiac disease, are at higher risk of developing severe illness
from an adenovirus infection.
[0176] Cells of the disclosure may be targeted for any adenovirus
antigen, but in specific embodiments the antigen is hexon and/or
penton.
[0177] D. Vaccinia Virus
[0178] vaccinia virus (also called VACV or VV) is a poxvirus that
causes cow pox in cows but has provided an effective vaccine for
smallpox, which may be treated with cells produced by methods of
the disclosure. Although any antigen of vaccinia virus may be
targeted with cells produced by methods of the disclosure, in
particular cases the antigens are E3L, A10L/121L, H3L/093L,
G5R/074R, C7L/018R, B22R/189R, D8L, E5R, E4L, f17R, A17L, and/or
L4R.
[0179] E. Varicella Zoster Virus
[0180] Varicella zoster virus (VZV) is one of eight herpesviruses
known to infect humans and vertebrates. VZV causes chickenpox in
children, teens and young adults, and it causes herpes zoster
(shingles) in adults and some children. VZV is known by many names,
including chickenpox virus, varicella virus, zoster virus, and
human herpesvirus type 3 (HHV-3).
[0181] VZV antigens that may be utilized as a source of peptides
(or sequence thereof) include any of the capsid, envelope, or
soluble antigens. Specific examples include V antigen, S antigen,
1E61, 1E62, 1E63, gE and ORF10.
[0182] Vaccines for VZV (VARIVAX.RTM. and ZOSTAVAX.RTM.) comprise
live attenuated viruses that cause limited infection in humans.
[0183] III. Pharmaceutical Compositions
[0184] In accordance with this disclosure, the term "pharmaceutical
composition" relates to a composition for administration to an
individual. In a preferred embodiment, the pharmaceutical
composition comprises a composition comprising therapeutic immune
cells for parenteral, transdermal, intraluminal, intra-arterial,
intrathecal or intravenous administration or for direct injection
into a cancer. It is in particular envisaged that the
pharmaceutical composition is administered to the individual via
infusion or injection. Administration of the suitable compositions
may be effected by different ways, e.g., by intravenous,
subcutaneous, intraperitoneal, intramuscular, topical or
intradermal administration.
[0185] The pharmaceutical composition of the present disclosure may
further comprise a pharmaceutically acceptable carrier. Examples of
suitable pharmaceutical carriers are well known in the art and
include phosphate buffered saline solutions, and the cells may be
in a sterile buffer suitable for infusion with a protein such as
human serum albumin for infusion.
[0186] The dosage regimen will be determined by the clinical
protocol that has been approved by the appropriate local and
federal regulatory agents. As is well known in the medical arts,
dosages for any one patient depends upon many factors, including
the patient's size and body surface area, for example. A particular
dosage for administration might be in the range of from
5.times.10.sup.6 per m.sup.2 to 5.times.10.sup.9 per m.sup.2.
Progress can be monitored by periodic assessment.
[0187] In certain embodiments of the disclosure that concern VSTs
generated against viral antigen(s), methods of the present
disclosure for clinical aspects are combined with other agents
effective in the treatment of one or more medical conditions with
which the virus is associated.
[0188] IV. Kits of the Disclosure
[0189] Any of the compositions described herein may be comprised in
a kit. In a non-limiting example, a library of pepmixes may be
comprised in a kit, any type of cells may be provided in the kit,
and/or reagents for manipulation of pepmixes and/or cells may be
provided in the kit. Cytokines or means of producing them (such as
vectors that encode them) may be included in the kit. Cell culture
reagents and/or apparatus(es) may be included. The component(s) are
provided in suitable container means.
[0190] The kits may comprise a suitably aliquoted compositions of
the present disclosure. The components of the kits may be packaged
either in aqueous media or in lyophilized form. The container means
of the kits will generally include at least one vial, test tube,
flask, bottle, syringe or other container means, into which a
component may be placed, and preferably, suitably aliquoted. Where
there are more than one component in the kit, the kit also will
generally contain a second, third or other additional container
into which the additional components may be separately placed.
However, various combinations of components may be comprised in a
vial. The kits of the present invention also will typically include
a means for containing the components in close confinement for
commercial sale. Such containers may include injection or blow
molded plastic containers into which the desired vials are
retained.
[0191] However, the components of the kit may be provided as dried
powder(s). When reagents and/or components are provided as a dry
powder, the powder can be reconstituted by the addition of a
suitable solvent. It is envisioned that the solvent may also be
provided in another container means.
[0192] In some cases, reagents and/or devices to detect viral
infection may be included in the kit. Examples include swabs,
spatulas, cytobrushes, slides, cover slips, cytology sample
collection receptacle, and so forth. Additional drugs for viral
infection may be included in the kit.
EXAMPLES
[0193] The following examples are presented in order to more fully
illustrate the preferred embodiments of the invention. They should
in no way, however, be construed as limiting the broad scope of the
invention.
Example 1
Production of Therapeutic T Cells
[0194] In some embodiments of the disclosure, there is a mechanism
by which one can rapidly generate a single preparation of T-cells,
including polyclonal (for example, CD4+ and CD8+) VSTs, that are
consistently specific for a variety of antigens derived from one or
more human viruses that can prove fatal. The disclosure is readily
adaptable to clinical implementation and can be used as an "off the
shelf" antiviral agent, including for EBV, CMV, adenovirus,
vaccinia virus, and/or VZV. The methods and compositions are
readily adaptable to clinical implementation and are useful as a
safe and effective antiviral agent for individuals.
[0195] In specific embodiments, peripheral blood T-cells are
stimulated with monocyte-derived dendritic cells loaded with
pepmixes (peptide libraries of 15-mers overlapping by 11 amino
acids (aa)) spanning the antigen protein, in the presence or
absence of specific accessory cytokines. The resulting T-cell lines
may be further expanded with pepmix-loaded activated cells.
[0196] The presence of the cytokines IL-7 and IL-15 is useful, in
specific embodiments of the methods. These T-cell lines possess the
desirable characteristics of polyclonality, multiple T-cell subset
representation (including the memory compartment) and a TH1 bias,
and eliminate viral targets. The disclosure provides that it is
possible to robustly generate viral-directed T-cell lines from
patients with virus-associated cancers. Because the technique is
scalable and good-manufacturing procedures-compliant, these lines
are useful for adoptive cellular immunotherapy of patients with
virus-related medical condition(s).
[0197] Turning to specifics of the methods, in certain cases DCs
are loaded with viral antigen(s) pepmix libraries. In such cases,
the cell lines are able to recognize one or more viral antigens. In
at least certain cases, expansion of the T-cells occurs in the
presence of IL-7 and IL-15 but not IL-2. The presence of IL-7 and
IL-15 in conditions for the method may or may not be at each step
of stimulation and expansion. In some embodiments, expansion of the
virus-specific T-cells after initial generation/expansion with DCs
utilizes autologous, polyclonal activated T-cells loaded with
pepmixes, in the presence of costimulatory cells
(CD80/CD86/CD83/4-1BBL or others), and IL-7 and IL-15. Employing
these conditions, T-cell expansion occurs at a more rapid rate than
in the absence of such conditions and occurs without loss of
specificity.
Example 2
Generation of Non-HPV Antigen-Specific T-Cells
[0198] Turning to particular embodiments of the disclosure, methods
for generating antigen-specific immune cells, such as T-cells, that
are specific for viruses other than HPV are described herein. In
particular embodiments, the method(s) are effective for at least
EBV, CMV, adenovirus, VZV, vaccinia virus, HIV, BK and HHV6,
although the methods may be effective for other viruses.
Modifications of these methods compared to those known in the art
address deficiencies, such as low frequency of viral-specific
antigen-specific T-cells and/or high frequency of NK cells in some
viral-specific antigen-specific T cell lines, for example.
[0199] In the first stimulation, in specific embodiments there are
no DCs, as with other methods in the art. PBMCs utilized as a
source of T-cells in steps of the method may be depleted for
certain cells, such as depletion of CD45RA+ cells, for example.
[0200] Culture of the cells at any step of the method may occur in
the presence of certain cytokines or combinations thereof, and
certain levels of these cytokine(s) may need to be achieved. In
specific embodiments, one or more steps of the method occurs in
high (100 ng to 1000 ng per mL) doses of IL15 and IL-7. In specific
cases, in one or more steps of the method costimulatory cells are
utilized. Although a variety of costimulatory cells may be useful,
in specific embodiments the cells are HLA-negative LCLs.
[0201] FIG. 1 describes steps of the methods of the disclosure for
generating virus-specific T-cells. As illustrated therein, on day 0
PBMCs (which may or may not be depleted for certain cells, such as
CD45RA+ T-cells), are pulsed with viral peptides; as an example for
EBV, peptides that encompass part or all of LMP1, LMP2, EBNA1,
and/or BARF1 are utilized. In this first step where PBMCs of any
kind are pulsed with peptides, there may be present cytokines,
including certain one or more cytokines, including certain
combinations of cytokines. As an example, in the original pulse
step, IL7 and/or IL15, including human IL15 (IL15H), are employed.
In this first step, there may or may not also be costimulatory
cells, including irradiated HLA-ve LCLs. In a second stimulation,
the cells are exposed to peptide-pulsed irradiated autologous
antigen T-cells and in at least some cases also costimulatory
cells, such as HLA-ve LCLs. This step may or may not occur in the
presence of one or more cytokines, including certain combinations,
such as IL7 and/or IL15, including IL15H. Following continued
culturing for an appropriate time span, such as number of days, the
cells may be used or may be cryopreserved.
[0202] As noted above, embodiments of the disclosure provide
improvements to certain methods utilized in the art. FIG. 2 shows
results of improved specificity of EB viral-specific T-cells
(EBVSTs) developed in the presence of IL-7 and IL-15 instead of
IL-4 and IL-7.
[0203] As merely an example, methods of the disclosure are
developed for lymphoma patients, whose T-cells are anergic to viral
antigens expressed in their tumor cells. Specific embodiments of
methods of the disclosure improve the antigen-specificity of
lymphoma patient EBVSTs and allow anergy to be overcome by
utilizing at least IL-15 in one or more steps (FIG. 3). Thus, using
IL-15 instead of IL-4 increases the frequency of antigen-specific
T-cells in patient EBVSTs. In at least some cases, however, EBVSTs
in patients wherein IL-15 had been utilized in culture lacked
specificity, so the dosage or IL-15 was optimized. It was
questioned whether the IL-15 dose was too high, inducing expansion
of non-specific T-cells, for example. In fact, an optimization
study (FIG. 4) revealed that a higher dose of IL-15 was better for
increasing specificity (100 ng per mL compared to standard dose of
5 ng per mL). FIG. 5 demonstrates that high doses of IL-15 increase
central memory EBVSTs.
[0204] Excessive NK cell outgrowth in EBVSTs from some patients and
healthy donors was addressed. In cases wherein there is
preferential outgrowth of NK cells, in specific cases it could be
attributable to the presence of IL-15 (NK cell populations seem to
be exacerbated in lines grown in IL-15 and IL-7) and/or could be
attributable to the use of K562cs (irradiated HLA-negative K562
cells genetically modified to express CD80, CD83, CD86, and 4-1BBL)
(FIG. 6) or the combination of IL15 and K562 cells. To address
this, conditions were developed to avoid excessive NK cell
outgrowth. In specific embodiments, there was utilized depletion of
CD45RA+ cells from the PBMCs prior to T-cell activation. CD45RA is
a naive T-cell marker that is also expressed on natural
T-regulatory cells and NK cells, so such depletion should remove
the NK cells. In other embodiments, CD45RO-positive cells are
enriched through depletion of cells expressing CD45RA. In addition,
this step should remove T regulatory cells that can inhibit the
outgrowth of antigen-specific T-cells, especially in cancer
patients, and also removes naive cells that can grow as bystander
cells and dilute the antigen-specific T-cells. Depletion may occur
by any suitable method, but in specific embodiments depletion
occurs using magnetic labeling and separation (for example, using
Miltenyi.RTM. Biotec columns). Use of antibody to deplete cells
using magnetic beads or nanobubbles may also occur.
[0205] Generation of pepmix-activated EBVSTs from CD45RA-depleted
PBMCs is illustrated in FIG. 7. As shown therein, whole PBMCs are
depleted of CD45RA, and beginning day 0 or day 1 the first
stimulation (S1) EBVpepmix is added to the depleted cells in the
presence of IL-7 and IL-15 to produce EBVSTs. At the end of S1 and
beginning of the second stimulation S2 (for example, between day 8
and day 10), the EBVSTs are exposed to sufficient amounts of
EBV-Pepmix pulsed ATCs and sufficient amounts of costimulatory
cells (such as K562cs cells) in the presence of IL-7 and IL-15, but
in the absence of IL-2, to produce the desired pepmix-activated
EBVSTs. FIG. 8 demonstrates the results that CD45RA depletion
(CD45RA+ PBMC from healthy donors were depleted using Miltenyi.RTM.
columns and GMP grade CD45RA-conjugated beads) decreases the
frequency of CD3-CD56+ NK cells in EBVSTs. Following this, there is
increased proliferation of EBVSTs (FIG. 9). FIG. 10 illustrates the
enhanced fold expansion of EBVSTs following CD45RA depletion from
healthy donors at the end of a second stimulation step. In
addition, the CD45RA depletion enhances antigen specificity of
EBVSTs at the end of a second stimulation (for example, day 16)
(FIGS. 11 and 12, both showing data for healthy donors). The
increased antigen specificity of CD45RA depleted EBVSTs is
sustained after a third stimulation (FIG. 13).
[0206] The effects of CD45RA depletion was characterized in
lymphoma patients, chosen either because their EBVSTs grown without
depletion showed high frequencies of NK cells or because they had
failed to grow or show antigen-specificity. FIG. 14 shows the total
NK cell population at the end of a second stimulation step in five
lymphoma patients, demonstrating that CD45RA depletion decreases NK
cell population outgrowth in lymphoma patient EBVSTs, and this
depletion increased the frequency of the antigen-specific T-cells
(illustrated by IFN-.gamma. release ELIspot assay at the end of a
second stimulation) (FIG. 15). This experiment was performed in the
absence of dendritic cells for the first stimulation. Analogous to
results in healthy donors, CD45RA depletion increased antigen
specificity in EBVSTs from lymphoma patients (FIG. 16).
Proliferation of the lymphoma patients' EBVSTs is demonstrated in
FIG. 17. Furthermore, CD45RA depletion enhanced cytolytic activity
against pepmix-pulsed autologous activated T-cells (aATCs);
percentage lysis was observed at effector to target ratio of 20:1
(FIG. 18).
[0207] FIG. 19 illustrates the generation of multivirus-specific
T-cells specific for EBV, CMV, Adenovirus, BKG virus, and HHV6. As
merely an example, the pepmixes include peptides from EBNA1, LMP2,
and BZLF1 for EBV; IE and pp65 for CMV; hexon and penton for
Adenovirus, LT and VP1 for BK, and U11, U14, and U90 for HHV6. As
illustrated therein, a pepmix comprising peptides that span part or
all of each of these viral antigens is exposed to PBMCs at day 0 in
the presence of IL7 and high dose IL15. Upon the second stimulation
beginning at day 9, pepmix-activated ATCs and costimulatory cells
(such as K562cs cells) are exposed to the cells in the presence of
IL7 and high dose IL15. At a third stimulation step, such as
beginning at day 16, another round of pepmix-activated ATCs and
costimulatory cells (such as K562cs cells) are exposed to the cells
in the presence of IL7 and IL15H, ultimately producing multivirus
(m)VSTs (D23) cells.
[0208] FIG. 20 demonstrates the total fold expansion at and after
subsequent stimulation steps of embodiments of the method. The
antigen specificity of the multivirus-specific T-cells was
examined, and T-cells with specificity for all 5 viruses expanded
(FIG. 21).
[0209] In specific embodiments, CAR-modified T-cells may be
generated using methods of the disclosure. FIG. 22 demonstrates
proliferation of VZV-specific VSTs after a first stimulation, again
comparing methods of the disclosure to those that employ dendritic
cells; there was no significant difference in proliferation between
DC-initiated VSTs and PBMC-initiated VSTs or in expansion of the
VZVSTs after the second stimulation (FIG. 23). FIGS. 24 and 25
demonstrate specificity of the VZV-specific VSTs after a first
stimulation at day 8 (FIG. 24) and after a second stimulation at
day 16 (FIG. 25). In specific cases, and without being bound by
theory, such an outcome is because if the antigens are not
expressed in tumor cells they are not anergic.
[0210] In summary, in specific demonstrations in the present
Example, provided herein is a comparison of EBVSTs expanded with
dendritic cells in IL4/7 to methods encompassed herein. As
demonstrated, a high dose IL-15 and IL-7 increased the fold
expansion of EBVST in healthy donors and patients as well as
increased the frequency of EBV-antigen-specific T-cells in both
healthy donors and patients, in addition to eliminating the
requirement for dendritic cells in first stimulations. The strategy
was effective for multiple viruses (EBV, CMV, adenovirus, BK virus,
HHV6 and VZV) and was effective for retrovirally-transduced
virus-specific T-cells. Finally, demonstrated herein are data to
show that CD45RA-depletion leads to broader and higher antigen
specificity and decreased NK cell population in EBVSTs, for
example.
Example 3
Generation of HIV Antigen-Specific T-Cells
[0211] In one embodiment, T cells specific for HIV antigen are
produced with methods of the disclosure. FIG. 26 illustrates one
embodiment of manufacturing of HIV-specific T cells from HIV
seropositive donors. FIG. 27 shows optimal expansion of the cells
with K562 cells in a second stimulation. Therein, results are shown
after only 2 stimulations (15-16 days)+1 week for DCs. Expansion in
presence of K562 is higher than without K562 during the second
stimulation. FIG. 28 demonstrates that in the presence of K562, HIV
antigen-specific T cells (HIVSTs) expanded to clinically relevant
numbers after only 2 stimulations.
[0212] FIG. 29 shows that HIVSTs are specific for multiple HIV
antigens. The specificity of HIVSTs was assessed by interferon
(IFN)-.gamma. secretion in response to individual pepmixes for each
HIV antigen in an ELIspot assay. The specificity of HIVSTs was
assessed by interferon (IFN)-.gamma. secretion in response to
individual PepMixes for each HIV antigen in ELIspot assay. All of
the lines were multispecific for all 3 antigens. In the first
validation, equivalent antigen specificity was observed post first
stimulation. Specificity was not lost by adding K562 during a
second stimulation. There was an advantage for High dose IL-15 by
the end of the second stimulation. For the second validation, there
was high background after the first stimulation. Greater
specificity was observed for GAG, POL and NEF in ARM2 (High IL-15)
than in ARM1 (low IL-15). A negative control included <10 spots
per 1e5 HIVSTs.
[0213] FIG. 30 shows that HIVSTs comprise mixed CD4+ and CD8+ T
cells. The lines were predominantly CD3+CD8+ T cells, although
there were a proportion of CD4+ T cells. The T cell lines contained
a subpopulation of CD3-CD56+ NK cells. In the first validation,
there was a similar phenotype with more CD3-CD56+CD16+ NK cells and
fewer CD3+ T-cells in ARM1. In the second validation, there was a
higher ratio of CD4+ to CD8+ cells in ARM2 (High IL-15) (more
balanced and few NK Cells). Because CD4 T cells are known to
provide CD8 T cell help, aiding in memory, persistence, and
effector functions, it is critical that any T cell immunotherapy
approach for HIV utilizes CD4 T cells. While the infusion of a
small percentage of CD4 T cells may seem counterintuitive,
non-CD4-depleted T cells have been infused in the past, and no
significant increase in viral load was observed.
[0214] FIG. 31 demonstrates that HIVSTscan lyse antigen-pulsed and
HIV-infected targets. To evaluate the cytolytic specificity of the
HIVSTs, the inventors incubated the HIVSTs with a panel of
.sup.51Chromium (.sup.51Cr)-labeled autologous pepmix-ATCs target
cells. Importantly, HIVSTs were specific because lysis of activated
autologous target cells alone was not observed. The ability of
HIVSTs to lyse antigen-expressing targets was measured using an
4-hour .sup.51Chromium-release assay. Target cells consisted of
autologous PHA-blasts that were pulsed with either media or Gag,
Pol, or Nef PepMixes and were loaded with chromium. HIVSTs were
cultured with target cells pulsed with only the antigens they were
specific for, which was predetermined by IFN-.gamma. ELIspot
assay.
[0215] Although the present invention and its advantages have been
described in detail, it should be understood that various changes,
substitutions and alterations can be made herein without departing
from the spirit and scope of the invention as defined by the
appended claims. Moreover, the scope of the present application is
not intended to be limited to the particular embodiments of the
process, machine, manufacture, composition of matter, means,
methods and steps described in the specification. As one of
ordinary skill in the art will readily appreciate from the
disclosure of the present invention, processes, machines,
manufacture, compositions of matter, means, methods, or steps,
presently existing or later to be developed that perform
substantially the same function or achieve substantially the same
result as the corresponding embodiments described herein may be
utilized according to the present invention. Accordingly, the
appended claims are intended to include within their scope such
processes, machines, manufacture, compositions of matter, means,
methods, or steps.
* * * * *