U.S. patent application number 16/966582 was filed with the patent office on 2021-03-18 for generation of hpv-specific t-cells.
This patent application is currently assigned to Baylor College of Medicine. The applicant listed for this patent is Baylor College of Medicine. Invention is credited to Carlos A. Ramos, Cliona M. Rooney, Alex Salyer, Sandhya Sharma, Benjamin Flyun Joon Shin.
Application Number | 20210077612 16/966582 |
Document ID | / |
Family ID | 1000005239955 |
Filed Date | 2021-03-18 |
View All Diagrams
United States Patent
Application |
20210077612 |
Kind Code |
A1 |
Ramos; Carlos A. ; et
al. |
March 18, 2021 |
GENERATION OF HPV-SPECIFIC T-CELLS
Abstract
Embodiments of the disclosure concern methods and compositions
for immunotherapy for human papillomavirus infection and diseases
associated therewith. In specific embodiments, methods concern
production of immune cells that target one or more antigens of
HPV16 and/or HPV18, including methods with stimulation steps that
employ IL-7 and IL-15, but not IL-6 and/or IL-12. Other specific
embodiments utilize stimulations in the presence of certain cells,
such as costimulatory cells and certain antigen presenting
cells.
Inventors: |
Ramos; Carlos A.; (Houston,
TX) ; Rooney; Cliona M.; (Bellaire, TX) ;
Sharma; Sandhya; (Houston, TX) ; Shin; Benjamin Flyun
Joon; (Houston, TX) ; Salyer; Alex; (Houston,
TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Baylor College of Medicine |
Houston |
TX |
US |
|
|
Assignee: |
Baylor College of Medicine
Houston
TX
|
Family ID: |
1000005239955 |
Appl. No.: |
16/966582 |
Filed: |
January 31, 2019 |
PCT Filed: |
January 31, 2019 |
PCT NO: |
PCT/EP2019/052328 |
371 Date: |
July 31, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15885197 |
Jan 31, 2018 |
10500265 |
|
|
16966582 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2501/2301 20130101;
C12N 2710/20034 20130101; C12N 2501/2312 20130101; A61K 2039/80
20180801; C12N 2501/24 20130101; C12N 5/0638 20130101; C12N
2501/2306 20130101; C12N 2501/02 20130101; C12N 2501/2315 20130101;
A61K 2039/5158 20130101; C12N 5/0636 20130101; C12N 2501/22
20130101; C12N 2501/051 20130101; A61K 39/0011 20130101; C12N
2502/1114 20130101; A61K 39/12 20130101; C12N 5/0639 20130101; C12N
2501/2307 20130101; A61K 2039/55527 20130101; C12N 2501/2304
20130101; C12N 2502/1107 20130101; C12N 2501/999 20130101; C12N
2502/1121 20130101; C12N 2501/05 20130101; C12N 2501/056 20130101;
C12N 2501/25 20130101; A61P 35/00 20180101 |
International
Class: |
A61K 39/12 20060101
A61K039/12; C12N 5/0783 20060101 C12N005/0783; C12N 5/0784 20060101
C12N005/0784 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under P50
CA097007 and P01 CA94237 awarded by National Cancer Institute. The
government has certain rights in the invention.
Claims
1. A method of generating or expanding a population of immune cells
specific for HPV, comprising a step of stimulating a population of
immune cells, optionally PBMCs, by culture in the presence of
antigen-presenting cells (APCs) presenting a peptide of a HPV
antigen, in the presence of HLA-negative LCLs.
2. The method according to claim 1, wherein the HLA-negative LCLs
are EBV replication defective.
3. The method according to claim 1 or claim 2, wherein the APCs are
activated T cells (ATCs), dendritic cells (DCs) or B-Blasts
(BBs).
4. The method according to claim 3, wherein the DCs are derived
from CD14+ cells isolated from a population of peripheral blood
mononuclear cells (PBMCs).
5. The method according to claim 3 or claim 4, wherein the DCs are
derived from CD14+ cells by a method comprising culturing the CD14+
cells in the presence of IL-4 and GM-CSF to produce immature DCs
(iDCs).
6. The method according to claim 5, wherein the method by which the
DCs are derived from CD14+ cells further comprises culturing the
iDCs to produce mature DCs by culture in the presence of: (a)
GM-CSF, IL-4, IL-1.beta., IL-6, TNF.alpha. and CD40L; (b) GM-CSF,
IL-4, MPLA and IFN.gamma.; (c) GM-CSF, IFN.alpha.; or (d) GM-CSF,
IL-4, AmpB and IFN.gamma..
7. The method according to claim 3, wherein the DCs are derived
from immature DCs (iDCs) by a method comprising culture in the
presence of: (a) GM-CSF, IL-4, IL-1.beta., IL-6, TNF.alpha. and
CD40L; (b) GM-CSF, IL-4, MPLA and IFN.gamma.; (c) GM-CSF,
IFN.alpha.; or (d) GM-CSF, IL-4, AmpB and IFN.gamma..
8. The method according to any one of claims 1 to 7, wherein the
population of immune cells stimulated is depleted of CD45RA+
cells.
9. The method according to any one of claims 1 to 8, wherein the
stimulation is performed by culture in the presence of IL-7 and
IL-15, optionally wherein the IL-15 is present in the culture at a
final concentration greater than 15 ng/ml.
10. The method according to any one of claims 1 to 8, wherein the
stimulation is performed by culture in the presence of IL-4, IL-6,
IL-7 and IL-15.
11. A method of generating or expanding a population of immune
cells specific for HPV, comprising stimulating a population of
immune cells by culture in the presence of dendritic cells (DCs)
presenting a peptide of a HPV antigen, wherein the DCs are derived
from CD14+ cells isolated from a population of peripheral blood
mononuclear cells (PBMCs).
12. The method according to claim 11, wherein the DCs are derived
from CD14+ cells by a method comprising culturing the CD14+ cells
in the presence of IL-4 and GM-CSF to produce immature DCs
(iDCs).
13. The method according to claim 12, wherein the method by which
the DCs are derived from CD14+ cells further comprises culturing
the iDCs to produce mature DCs by culture in the presence of: (a)
GM-CSF, IL-4, IL-1.beta., IL-6, TNF.alpha. and CD40L; (b) GM-CSF,
IL-4, MPLA and IFN.gamma.; (c) GM-CSF, IFN.alpha.; or (d) GM-CSF,
IL-4, AmpB and IFN.gamma..
14. A method of generating or expanding a population of immune
cells specific for HPV, comprising stimulating a population of
immune cells by culture in the presence of dendritic cells (DCs)
presenting a peptide of a HPV antigen, wherein the DCs are derived
from immature DCs (iDCs) by a method comprising culture in the
presence of: (a) GM-CSF, IL-4, IL-1.beta., IL-6, TNF.alpha. and
CD40L; (b) GM-CSF, IL-4, MPLA and IFN.gamma.; (c) GM-CSF,
IFN.alpha.; or (d) GM-CSF, IL-4, AmpB and IFN.gamma..
15. The method according to any one of claims 11 to 14, wherein the
stimulation comprises culture in the presence of HLA-negative LCLs,
optionally wherein the HLA-negative LCLs are EBV replication
defective.
16. The method according to any one of claims 11 to 15, wherein the
population of immune cells stimulated is depleted of CD45RA+
cells.
17. The method according to any one of claims 11 to 16, wherein the
stimulation is performed by culture in the presence of IL-7 and
IL-15, optionally wherein the IL-15 is present in the culture at a
final concentration greater than 15 ng/ml.
18. The method according to any one of claims 11 to 16, wherein the
stimulation is performed by culture in the presence of IL-4, IL-6,
IL-7 and IL-15.
19. A method of generating or expanding a population of immune
cells specific for HPV, comprising stimulating a population of
immune cells depleted of CD45RA+ cells by culture in the presence
of antigen-presenting cells (APCs) presenting a peptide of a HPV
antigen.
20. The method according to claim 19, wherein the stimulation
comprises culture in the presence of HLA-negative LCLs, optionally
wherein the HLA-negative LCLs are EBV replication defective.
21. The method according to claim 19 or claim 20, wherein the APCs
are activated T cells (ATCs), dendritic cells (DCs) or B-Blasts
(BBs).
22. The method according to claim 21, wherein the DCs are derived
from CD14+ cells isolated from a population of peripheral blood
mononuclear cells (PBMCs).
23. The method according to claim 21 or claim 22, wherein the DCs
are derived from CD14+ cells by a method comprising culturing the
CD14+ cells in the presence of IL-4 and GM-CSF to produce immature
DCs (iDCs).
24. The method according to claim 23, wherein the method by which
the DCs are derived from CD14+ cells further comprises culturing
the iDCs to produce mature DCs by culture in the presence of: (a)
GM-CSF, IL-4, IL-1.beta., IL-6, TNF.alpha. and CD40L; (b) GM-CSF,
IL-4, MPLA and IFN.gamma.; (c) GM-CSF, IFN.alpha.; or (d) GM-CSF,
IL-4, AmpB and IFN.gamma..
25. The method according to claim 21, wherein the DCs are derived
from immature DCs (iDCs) by a method comprising culture in the
presence of: (a) GM-CSF, IL-4, IL-1.beta., IL-6, TNF.alpha. and
CD40L; (b) GM-CSF, IL-4, MPLA and IFN.gamma.; (c) GM-CSF,
IFN.alpha.; or (d) GM-CSF, IL-4, AmpB and IFN.gamma..
26. The method according to any one of claims 19 to 25, wherein the
stimulation is performed by culture in the presence of IL-7 and
IL-15, optionally wherein the IL-15 is present in the culture at a
final concentration greater than 15 ng/ml.
27. The method according to any one of claims 19 to 25, wherein the
stimulation is performed by culture in the presence of IL-4, IL-6,
IL-7 and IL-15.
28. A method of generating or expanding a population of immune
cells specific for HPV, comprising a step of stimulating a
population of immune cells by culture in the presence of
antigen-presenting cells (APCs) presenting a peptide of a HPV
antigen, in the presence of IL-7 and IL-15.
29. The method according to claim 28, wherein the IL-15 is present
in the culture at a final concentration greater than 15 ng/ml.
30. A method of generating or expanding a population of immune
cells specific for HPV, comprising a step of stimulating a
population of immune cells by culture in the presence of
antigen-presenting cells (APCs) presenting a peptide of a HPV
antigen, in the presence of IL-4, IL-6, IL-7 and IL-15.
31. The method according to any one of claims 28 to 30, wherein the
stimulation comprises culture in the presence of HLA-negative LCLs,
optionally wherein the HLA-negative LCLs are EBV replication
defective.
32. The method according to any one of claims 28 to 31, wherein the
APCs are activated T cells (ATCs), dendritic cells (DCs) or
B-Blasts (BBs).
33. The method according to claim 32, wherein the DCs are derived
from CD14+ cells isolated from a population of peripheral blood
mononuclear cells (PBMCs).
34. The method according to claim 32 or claim 33, wherein the DCs
are derived from CD14+ cells by a method comprising culturing the
CD14+ cells in the presence of IL-4 and GM-CSF to produce immature
DCs (iDCs).
35. The method according to claim 34, wherein the method by which
the DCs are derived from CD14+ cells further comprises culturing
the iDCs to produce mature DCs by culture in the presence of: (a)
GM-CSF, IL-4, IL-1.beta., IL-6, TNF.alpha. and CD40L; (b) GM-CSF,
IL-4, MPLA and IFN.gamma.; (c) GM-CSF, IFN.alpha.; or (d) GM-CSF,
IL-4, AmpB and IFN.gamma..
36. The method according to claim 32, wherein the DCs are derived
from immature DCs (iDCs) by a method comprising culture in the
presence of: (a) GM-CSF, IL-4, IL-1.beta., IL-6, TNF.alpha. and
CD40L; (b) GM-CSF, IL-4, MPLA and IFN.gamma.; (c) GM-CSF,
IFN.alpha.; or (d) GM-CSF, IL-4, AmpB and IFN.gamma..
37. The method according to any one of claims 28 to 36, wherein the
population of immune cells stimulated is depleted of CD45RA+
cells.
38. The method according to any one of claims 1 to 37, wherein the
APCs or DCs have been pulsed with one or more peptides
corresponding to one or more HPV antigens.
39. The method according to claim 38, wherein the one or more HPV
antigens are selected from E1, E2, E3, E4, E5, E6 and E7.
40. The method according to claim 38 or claim 39, wherein the one
or more HPV antigens are antigens of HPV16, HPV18, HPV1, HPV2
and/or HPV3.
41. The method according to any one of claims 1 to 40, wherein the
population of immune cells stimulated is obtained from a prior
stimulation of immune cells by culture in the presence of APCs
presenting a peptide of a HPV antigen, optionally from within a
population of PBMCs.
42. The method according to any one of claims 1 to 41, wherein the
method further comprises one or more further steps comprising
stimulating a population of immune cells by culture in the presence
of APCs presenting a peptide of a HPV antigen.
43. A population of immune cells specific for HPV, wherein the
population of immune cells specific for HPV is obtained according
to the method of any one of claims 1 to 42.
44. The population of immune cells according to claim 43 for use in
a method of treatment or prevention of a HPV-associated
disease.
45. Use of a population of immune cells according to claim 43 in
the manufacture of a medicament for use in the treatment or
prevention of a HPV-associated disease.
46. The population of immune cells for use according to claim 43 of
claim 44, or the use according to claim 45, wherein the treatment
is a method of treatment or prevention of a HPV-associated disease
by adoptive cell transfer (ACT).
47. The population of immune cells for use or the use according to
claim 46, wherein the method of treatment by ACT comprises
administration to a subject of autologous cells or allogeneic
cells.
48. A method of treating or preventing a HPV-associated disease in
a subject, the method comprising administering to a subject a
therapeutically or prophylactically effective quantity of a
population of immune cells according to claim 43.
49. A method of treating or preventing a HPV-associated disease in
a subject, the method comprising: (a) generating or expanding a
population of immune cells specific for HPV according to the method
of any one of claims 1 to 42, and (b) administering a
therapeutically or prophylactically effective quantity of the
population of immune cells specific for HPV obtained at step (a) to
a subject.
50. The population of immune cells for use, the use or the method
according to any one of claims 44 to 49, wherein the HPV-associated
disease is a cancer.
51. The population of immune cells for use, the use or the method
according to claim 50 wherein the cancer is selected from cervical
cancer, anal cancer, vulvar cancer, vaginal cancer, penile cancer,
oropharyngeal cancer, nasopharyngeal carcinoma, laryngeal
papillomatosis, laryngeal cancer, head and neck cancer, or a
dysplasia of any of site thereof.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part application of
International Application Serial No. PCT/EP2017/073274, filed 15
Sep. 2017. International Application Serial No. PCT/EP2017/073274
claims priority to U.S. Provisional Patent Application Ser. No.
62/395,440, filed 16 Sep. 2016 and U.S. Utility application Ser.
No. 15/331,659, filed 21 Oct. 2016. U.S. Utility application Ser.
No. 15/331,659 also claims priority to U.S. Provisional Patent
Application Ser. No. 62/395,440. This application also claims
priority to International Application Serial No. PCT/US17/51284,
filed 13 Sep. 2017. International Application Serial No.
PCT/US17/51284 claims priority to U.S. Provisional Patent
Application Ser. No. 62/395,438, filed 16 Sep. 2016. All of the
referenced applications are incorporated herein by reference.
TECHNICAL FIELD
[0003] The present disclosure concerns at least the fields of
immunology, cell biology, molecular biology, and medicine,
including cancer medicine.
BACKGROUND
[0004] Human papillomavirus (HPV) is a DNA virus that establishes
productive infections in keratinocytes of the skin or mucous
membranes. There are over 170 types of HPV, a subset of which HPV
types are carcinogenic, including high-risk sexually transmitted
types that can develop into genital neoplasias, including cervical
intraepithelial neoplasia (CIN), vulvar intraepithelial neoplasia
(VIN), penile intraepithelial neoplasia (PIN), and/or anal
intraepithelial neoplasia (AIN), for example. HPV-induced cancers
arise when viral sequences are integrated into the cellular DNA of
host cells. Some of the HPV "early" genes, such as E6 and E7, act
as oncogenes that promote tumor growth and malignant
transformation.
[0005] Ramos et al., (J Immunother 2013; 36:66-76) describes a
method for stimulating peripheral blood mononuclear cells to
generate T-cells specific for HPV16 E6 and E7. In brief, the method
comprises stimulation of peripheral blood mononuclear cells with
dendritic cells in which cells are cultured in CTL medium with or
without the combination of cytokines IL-6, IL-7, IL-12 and IL-15, a
second stimulation in which co-cultures are supplemented with IL-2,
and weekly stimulation with pepmix-loaded accessory antigen
presenting cells (e.g., B-blasts) in the presence of IL-15. This
reference teaches the combination of cytokines IL-6, IL-7, IL-12
and IL-15 is required for expansion of the HPV-specific T-cells
from patient samples, for detectable T-cell responses.
[0006] The present disclosure provides relief for a long-felt need
in the art to treat HPV-associated diseases, including at least for
those associated with HPV16 and HPV18, for example.
BRIEF SUMMARY
[0007] The present disclosure is directed to methods and
compositions that concern immune system cells that are modified to
immunogenically recognize particular targets. In some embodiments,
the present disclosure concerns the development of immune cells
(including cytotoxic T-lymphocytes (CTLs, also referred to as
cytotoxic T-cells)) that target a biological moiety that elicits an
immune response in an individual. In specific embodiments, the
present disclosure concerns the development of cytotoxic T-cells
that target a HPV antigen, including a HPV disease-associated
antigen. In some cases, a mixture of cytotoxic T-cells is produced,
and the mixture targets more than one HPV antigen, including more
than one antigen of more than one HPV type, in some cases.
[0008] Embodiments of the disclosure concern methods and
compositions for providing therapy to individuals infected with HPV
or that have HPV-associated diseases, including cancers, for
example. A "HPV-associated disease" may be a disease which is
caused or exacerbated by HPV infection, a disease for which HPV
infection is a risk factor and/or a disease for which HPV infection
is positively associated with disease onset, development,
progression or severity. A HPV-associated disease may be a disease
in which the methods and compositions of the present invention
provide therapeutic effect (e.g. inhibition of the
development/progression of the disease, delayed/prevented onset of
the disease, reduced severity of the symptoms of the disease,
reversal of disease symptoms, and/or increased survival). It will
be clear to the person skilled in the art that the therapeutic
utility of the methods and compositions of the present invention
extends to essentially any disease/condition which would benefit
from a reduction in the number of HPV-infected cells. In specific
embodiments, the disclosure regards methods and compositions for
adoptive cellular immunotherapy that can target HPV-associated,
e.g., HPV16-associated, HPV18-associated, HPV1-associated,
HPV2-associated and/or HPV3-associated medical conditions
(including cancer) and are therapeutic therefor.
[0009] In certain aspects, the present disclosure concerns the
development of a plurality of T-cells that target antigens from
HPV, e.g., HPV16, HPV18, HPV1, HPV2 and/or HPV3. The present
disclosure provides significant and non-obvious improvements on
methods for generating T cell lines with specificity against HPV,
e.g., HPV16, HPV18, HPV1, HPV2 and/or HPV3 antigens.
[0010] In some embodiments of the disclosure, an individual is in
need of the methods and/or compositions of the disclosure. In
certain embodiments, an individual has been exposed to HPV, e.g.,
HPV16, HPV18, HPV1, HPV2 and/or HPV3 (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
HPV, e.g., HPV16, HPV18, HPV1, HPV2 and/or HPV3. In certain
embodiments, the individual has or is suspected of having or is at
risk for having HPV-associated disease, e.g., HPV16-associated,
HPV18-associated, HPV1-associated, HPV2-associated and/or
HPV3-associated disease, including cancer.
[0011] In specific embodiments of part of the method, certain HPV,
e.g., HPV16, HPV18, HPV1, HPV2 and/or HPV3, antigen(s) are
presented to antigen-presenting cells (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 antigen-presenting cells
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 express
the antigens may be exposed to peripheral blood T-cells under
certain conditions to result in stimulation of T-cells specific for
the certain HPV antigen(s).
[0012] Some aspects and embodiments of the present disclosure
concern the generation and/or expansion of HPV-specific
T-cells.
[0013] In a first aspect, the present disclosure provides a method
for stimulating peripheral blood cells, preferably 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 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 HPV. In accordance with various
aspects disclosed herein, where a stimulation/culture is performed
in the "presence of" a given cytokine, the relevant cytokine (e.g.
recombinant and/or exogenous cytokine) may have been added to the
stimulation/culture. Where a stimulation/culture is performed in
the "absence of" a given cytokine, the relevant cytokine (e.g.
recombinant and/or exogenous cytokine) will not have been added to
the stimulation/culture.
[0014] In some embodiments a method of producing therapeutic
T-cells for human papillomavirus (HPV)-associated disease(s) is
provided, the method 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,
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 HPV, wherein the stimulating
produces T-cells therapeutic for HPV-associated diseases.
[0015] In some embodiments, the peripheral blood T-cells being
stimulated are obtained from a prior stimulation of peripheral
blood cells. The prior stimulation may 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 HPV.
[0016] 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 HPV, to produce peripheral blood T-cells.
[0017] 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 HPV16; one or more proteins of HPV18; or both
of one or more proteins of HPV16 and one or more proteins of HPV18.
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 HPV1; one or more proteins of HPV2; one or more
proteins of HPV3; or one or more proteins of HPV1, HPV2 and/or
HPV3. In some embodiments the one or more peptides comprise
sequence that corresponds to one or more of proteins E5, E6, E7, L1
and L2. In some embodiments the one or more peptides may be a
library of peptides, including E1, E2, E3, E4, E5, E6, E7, L1,
and/or L2 peptides.
[0018] In some embodiments the method may produce immune cells,
such as T-cells, specific for HPV or for an HPV antigen. In some
embodiments the method may expand a population of T-cells present
in the peripheral blood T-cells that are specific for HPV or for at
least one HPV antigen. Immune cells other than T cells that may be
produced by methods of the disclosure including NK cells and NKT
cells.
[0019] In some embodiments the antigen presenting cells are
activated T-cells, dendritic cells (DC), B-Blasts (BB), or PBMCs,
for example.
[0020] In some embodiments stimulation of peripheral blood T-cells
in the presence of IL-7 and IL-15 occurs in the absence of at least
IL-2. In some embodiments stimulation of peripheral blood T-cells
in the presence of IL-7 and IL-15 occurs in the absence of at least
IL-4. In some embodiments stimulation of peripheral blood T-cells
in the presence of IL-7 and IL-15 occurs in the absence of at least
IL-6. In some embodiments stimulation of peripheral blood T-cells
in the presence of IL-7 and IL-15 occurs in the absence of at least
IL-12. In some embodiments stimulation of peripheral blood T-cells
in the presence of IL-7 and IL-15 occurs in the absence of at least
IL-21. In some embodiments stimulation of peripheral blood T-cells
in the presence of IL-7 and IL-15 occurs in the absence of IL-6 and
IL-12.
[0021] In some particular embodiments stimulation of cells in the
method of the first aspect of the present invention occurs in the
absence of IL-6 and IL-12.
[0022] In some embodiments, peripheral blood T-cells 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. The antigen presenting cells may be
activated T-cells, dendritic cells, B-blasts, or PBMCs, for
example.
[0023] In a second aspect, the present disclosure provides a method
for stimulating T-cells specific for HPV or for an HPV antigen,
wherein the method comprises stimulating T-cells specific for HPV
or for an HPV 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 HPV.
[0024] In some embodiments a method of producing therapeutic
T-cells for human papillomavirus (HPV)-associated diseases is
provided, the method comprising the step of stimulating T-cells
specific for HPV or for an HPV 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 sequence that corresponds
to at least part of the sequence of one or more proteins of HPV,
wherein the stimulating produces T-cells therapeutic for one or
more HPV-associated diseases.
[0025] In some embodiments the antigen presenting cells are
activated T-cells, dendritic cells (DC), B-Blasts (BB) or PBMCs. In
particular embodiments the antigen presenting cells are activated
T-cells.
[0026] 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, CD40+ cells, OX40+ cells, and a
combination thereof. The co-stimulatory cells may be
CD80+/CD86+/CD83+/4-1BBL+ cells.
[0027] In some embodiments the stimulation of T-cells specific for
HPV or for an HPV antigen is not a first stimulation step. The
T-cells being stimulated cells may be the product of a prior
stimulation, e.g. using the method of the first aspect of the
present disclosure.
[0028] 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 HPV16; one or more proteins of HPV18; or one or
more proteins of HPV16 and one or more proteins of HPV18. 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 HPV1; one or more proteins of HPV2; one or more
proteins of HPV3; or one or more proteins of HPV1, HPV2 and/or
HPV3. In some embodiments the one or more peptides comprise
sequence that corresponds to one or more of proteins E5, E6, E7, L1
and L2. In some embodiments the one or more peptides may be a
library of peptides, including E1, E2, E3, E4, E5, E6, E7, L1,
and/or L2 peptides.
[0029] In some embodiments the method may produce T-cells specific
for HPV or for an HPV antigen. In some embodiments the method may
expand a population of T-cells specific for HPV or for an HPV
antigen.
[0030] In certain embodiments stimulation of T-cells specific for
HPV or for an HPV antigen comprises stimulating T-cells specific
for HPV or for an HPV antigen with antigen presenting cells in the
presence of IL-7, IL-15, and in the presence of one or more types
of co-stimulatory cells.
[0031] In some embodiments stimulation of T-cells in the presence
of IL-7 and IL-15 is in the absence of IL-2. In some embodiments
stimulation of T-cells in the presence of IL-7 and IL-15 is in the
absence of IL-4. In some embodiments stimulation of T-cells in the
presence of IL-7 and IL-15 is in the absence of IL-6. In some
embodiments stimulation of T-cells in the presence of IL-7 and
IL-15 is in the absence of IL-7. In some embodiments stimulation of
T-cells in the presence of IL-7 and IL-15 is in the absence of
IL-12. In some embodiments stimulation of T-cells in the presence
of IL-7 and IL-15 is in the absence of IL-21. In some embodiments
stimulation of T-cells in the method of the first aspect of the
present invention is in the absence of IL-6 and IL-12.
[0032] Methods according to the first and second aspect of the
present disclosure may be methods of producing therapeutic T-cells
for HPV-associated diseases. The stimulation of cells may produce
T-cells that are therapeutic for HPV-associated diseases.
[0033] In a third aspect, the methods of the first and second
aspects may be combined to provide a method of producing
therapeutic T-cells for HPV-associated diseases, the method
comprising:
[0034] stimulating peripheral blood cells, preferably 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 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 HPV;
[0035] stimulating T-cells obtained from (i) with antigen
presenting cells in the presence of interleukin (IL)-7 and IL-15,
and optionally in the presence of one or more types 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 HPV.
[0036] In some embodiments prior to step (ii), T-cells obtained
from (i) may be re-stimulated in the presence of IL-7 and IL-15 but
not in the presence of co-stimulatory cells, and optionally in the
absence of IL-6 and/or IL-12. Such re-stimulation may occur for
one, two, three, four, five or more rounds, as required.
[0037] In some embodiments the antigen presenting cells used in (i)
are dendritic cells (DC), B-Blasts (BB) or PBMCs. In some
embodiments the antigen presenting cells used in (ii) are activated
T-cells, dendritic cells (DC), B-Blasts (BB) or PBMCs. In some
embodiments the antigen presenting cells used in (i) are different
to the antigen presenting cells used in (ii), although they may be
the same in certain cases. In particular embodiments the antigen
presenting cells used in (ii) are activated T-cells.
[0038] 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, CD40+ cells, OX40+ cells, and a
combination thereof. The co-stimulatory cells may be
CD80+/CD86+/CD83+/4-1BBL+ cells.
[0039] In some embodiments stimulation of cells in the presence of
IL-7 and IL-15 is in the absence of IL-2. In some embodiments
stimulation of cells in the presence of IL-7 and IL-15 is in the
absence of IL-4. In some embodiments stimulation of cells in the
presence of IL-7 and IL-15 is in the absence of IL-6. In some
embodiments stimulation of cells in the presence of IL-7 and IL-15
is in the absence of IL-12. In some embodiments stimulation of
cells in the presence of IL-7 and IL-15 is in the absence of IL-21.
In some embodiments stimulation of cells in the presence of IL-7
and IL-15 is in the absence of IL-6 and IL-12.
[0040] In some preferred embodiments stimulation of cells in step
(i) is in the absence of IL-6 and IL-12.
[0041] In some embodiments stimulation of cells in step (ii) is in
the absence of IL-6 and IL-12.
[0042] Accordingly, in some embodiments a method of producing
therapeutic T-cells for HPV-associated diseases is provided, the
method comprising:
[0043] (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
HPV;
[0044] (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 HPV,
wherein (ii) is optionally repeated one or more times; and
[0045] (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 HPV, wherein (iii)
is optionally repeated one or more times.
[0046] In some embodiments the antigen presenting cells used in (i)
and (ii) are 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 to the antigen presenting cells used in (i) and/or (ii).
In preferred embodiments the antigen presenting cells used in (iii)
are activated T-cells.
[0047] In preferred embodiments the stimulation in (iii) is 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, CD40+ cells, OX40+ cells, and a combination thereof. The
co-stimulatory cells may be CD80+/CD86+/CD83+/4-1BBL+ cells.
[0048] In some embodiments stimulation of cells in the presence of
IL-7 and IL-15 is in the absence of IL-2. In some embodiments
stimulation of cells in the presence of IL-7 and IL-15 is in the
absence of IL-4. In some embodiments stimulation of cells in the
presence of IL-7 and IL-15 is in the absence of IL-6. In some
embodiments stimulation of cells in the presence of IL-7 and IL-15
is in the absence of IL-12. In some embodiments stimulation of
cells in the presence of IL-7 and IL-15 is in the absence of IL-21.
In some embodiments stimulation of cells in the presence of IL-7
and IL-15 is in the absence of IL-6 and IL-12.
[0049] In some embodiments stimulation of cells in step (i) is in
the absence of IL-6 and IL-12. In some other embodiments
stimulation of cells in step (i) is in the presence of IL-6 and
IL-12.
[0050] In some particular embodiments stimulation of cells in step
(ii) is in the absence of IL-6 and IL-12. In some particular
embodiments stimulation of cells in step (iii) is in the absence of
IL-6 and IL-12.
[0051] In some particular embodiments methods of the present
disclosure are for producing T-cells specific for HPV16 and/or
HPV18. In some particular embodiments methods of the present
invention are for producing T-cells specific for HPV16-associated
and/or HPV18-associated diseases.
[0052] In some embodiments, peripheral blood T-cells may be
obtained from an individual that is known to be infected or
suspected of being infected with HPV; HPV16 or HPV18; both HPV16
and HPV18; HPV1, HPV2 and/or HPV3.
[0053] In some embodiments, antigen presenting cells may be
obtained from an individual that is known to be infected or
suspected of being infected with HPV; HPV16 or HPV18; both HPV16
and HPV18; HPV1, HPV2 and/or HPV3.
[0054] 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.
[0055] In some embodiments, antigen presenting cells may be
autologous or allogeneic to an individual intended to be treated
with the therapeutic T-cells obtained.
[0056] 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 HPV16; one or more proteins of HPV18; or one or
more proteins of HPV16 and one or more proteins of HPV18. 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 HPV1; one or more proteins of HPV2; one or more
proteins of HPV3; or one or more proteins of HPV1, HPV2 and/or
HPV3. In some embodiments the one or more peptides comprise
sequence that corresponds to one or more of proteins E1, E2, E3,
E4, E5, E6, E7, L1, and/or L2 that come from HPV16, HPV18, or HPV16
and HPV18.
[0057] In embodiments of the present disclosure the peptides may
comprise sequence that corresponds to one or more of HPV proteins
E1, E2, E3, E4, E5, E6, E7, L1, and/or L2. In some embodiments, the
peptides may comprise sequence that corresponds to one or more HPV
proteins which are expressed following proviral integration (e.g.
E1, E2, E3, E4, E5, E6 and/or E7), e.g. in a cell infected HPV. In
some embodiments, the peptides may comprise sequence that
corresponds to one or more transforming HPV proteins (e.g. E6,
and/or E7). The peptides may correspond to a contiguous amino acid
sequence present within said HPV 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: a) the HPV18 E6 protein and/or the
HPV18 E7 protein, and/or b) the HPV16 E6 protein and/or the HPV16
E7 protein.
[0058] In embodiments of the present disclosure the HPV may be
HPV16 or HPV18, or both. In embodiments concerned with treatment of
an HPV-associated disease, the disease may be cancer and the
peptides may comprise a sequence that corresponds to one or both of
E6 and E7. When the HPV-associated disease is a pre-cancerous
lesion, the peptides may comprise sequence that corresponds to one,
some, or all of E1, E2, E3, E4, E5, E6, E7, L1, and L2.
[0059] T-cells produced by the methods of the present disclosure
may be isolated and/or purified, e.g., isolated/purified from other
cells.
[0060] 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 HPV, or that has
HPV-associated disease. In a related aspect T-cells produced by the
method of the present disclosure are provided for use in the
treatment of HPV-associated disease. 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 HPV-associated disease.
[0061] In one aspect of the present invention T-cells for use in a
method of adoptive cellular immunotherapy are provided, wherein the
T-cells are obtained by, obtainable by, or are the product of, a
method for stimulating peripheral blood or T-cells or a method of
producing therapeutic T-cells described herein, the method of
adoptive cellular immunotherapy comprising administering the
T-cells to the subject.
[0062] In one aspect of the present invention the use of T-cells in
the manufacture of a medicament for use in a method of adoptive
cellular immunotherapy comprising administering the T-cells to the
subject is provided, wherein the T-cells are obtained by,
obtainable by, or are the product of, a method for stimulating
peripheral blood or T-cells or a method of producing therapeutic
T-cells described herein.
[0063] In one aspect of the present invention a method of preparing
a pharmaceutical composition, medicament or vaccine is provided,
the method comprising stimulating peripheral blood or T-cells
according to a method described herein, or producing therapeutic
T-cells according to a method described herein, and mixing the
cells obtained, with a pharmaceutically acceptable carrier,
adjuvant, diluent or excipient.
[0064] The disease to be treated may be a neoplasm. The neoplasm
may be a cancer. The cancer may be an HPV-positive cancer, e.g. a
HPV16-positive cancer and/or HPV18-positive cancer.
[0065] The individual to be treated may be a human. The individual
may be a patient. The individual may have been exposed to HPV, such
as HPV16, HPV18, or both HPV16 and HPV18, or has an HPV-, HPV16-
and/or HPV18-associated disease. The HPV-, HPV16- and/or
HPV18-associated disease may be a neoplasm. The neoplasm may be a
cancer.
[0066] A cancer may be of any kind. In some embodiments the cancer
is a cervical cancer, anal cancer, vulvar cancer, vaginal cancer,
penile cancer, or oropharyngeal cancer. In some embodiments the
cancer may be a HPV-associated cancer. A "HPV-associated cancer"
may be a cancer which is caused or exacerbated by HPV infection, a
cancer for which HPV infection is a risk factor and/or a cancer for
which HPV-infection is positively associated with onset,
development, progression, severity or metastasis. A HPV-associated
cancer may be a cancer in which the methods and compositions of the
present invention provide therapeutic effect (e.g. inhibition of
the development/progression of the cancer, delayed/prevented onset
of the cancer, reduced/delayed/prevented metastasis, reduced
severity of the symptoms of the cancer, reduction in number of
cancer cells, reduction in tumour size, and/or increased survival).
In some embodiments the cancer is a HPV-related carcinoma,
HPV-positive oropharyngeal carcinoma, HPV-positive cervical
carcinoma, HPV-positive anal carcinoma, HPV-positive vulvar
carcinoma, nasopharyngeal carcinoma, HPV-positive penile carcinoma,
HPV-positive dysplasias of any site, or laryngeal
papillomatosis.
[0067] The individual or subject may have received, be receiving,
or will receive an additional cancer therapy. The additional cancer
therapy may be surgery, radiation, hormone therapy, chemotherapy,
immunotherapy, or a combination thereof.
[0068] The individual or subject may be determined as having
HPV-associated cancer or HPV-positive cancer. The individual may be
determined as having HPV16-associated cancer or HPV16-positive
cancer. The individual may be determined as having HPV18-associated
cancer or HPV18-positive cancer. The individual or subject may be
any animal or human. The individual or subject is preferably
mammalian, more preferably human. The individual or subject may be
a non-human mammal, but is more preferably human. The individual or
subject may be male or female. The individual or subject may be a
patient.
[0069] 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.
[0070] 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 and 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 human papillomavirus (HPV). The peripheral
blood T-cells may be obtained from a prior stimulation of
peripheral blood cells, such as wherein the prior stimulation of
peripheral blood cells comprises stimulating peripheral blood cells
with antigen presenting cells in the presence of IL-7 and IL-15,
and 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 HPV. In specific
cases, prior to stimulating the peripheral blood T-cells, the
method further comprises stimulating peripheral blood cells with
antigen presenting cells in the presence of IL-7 and IL-15, and 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 HPV, to produce peripheral
blood T-cells.
[0071] In an embodiment, there is a method of producing therapeutic
T-cells for HPV-associated diseases, the method comprising the step
of: stimulating peripheral blood T-cells with antigen presenting
cells in the presence of one or more of IL-7 and IL-15 and 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 HPV, wherein the stimulating
produces T-cells therapeutic for HPV-associated diseases. The
peripheral blood T-cells may be obtained from a prior stimulation
of peripheral blood cells, such as wherein the prior stimulation of
peripheral blood cells comprises stimulating peripheral blood cells
with antigen presenting cells in the presence of IL-7 and IL-15,
and 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 HPV. In specific
cases, prior to stimulating the peripheral blood T-cells, the
method further comprises stimulating peripheral blood cells with
antigen presenting cells in the presence of IL-7 and IL-15, and 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 HPV, to produce peripheral
blood T-cells.
[0072] In embodiments of methods encompassed by the disclosure,
antigen presenting cells are activated T-cells, dendritic cells,
B-blasts, or PBMCs. Peripheral blood T-cells may be present in a
population of peripheral blood mononuclear cells (PBMCs) or are
obtained or isolated therefrom, in at least some cases, and the
PBMCs in the population may be non-adherent PBMCs. When employed,
co-stimulatory cells may be CD80+, CD86+, CD83+, 4-1BBL+, CD40+
cells, OX40+ cells, or a combination thereof.
[0073] In a particular embodiment, there is a method for
stimulating T-cells specific for HPV or for an HPV antigen, the
method comprising stimulating T-cells specific for HPV or for an
HPV antigen 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
HPV.
[0074] In certain embodiments, there is a method of producing
therapeutic T-cells for HPV-associated diseases, the method
comprising the step of stimulating T-cells specific for HPV or for
an HPV antigen 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 HPV, wherein the stimulating produces T-cells
therapeutic for HPV-associated diseases.
[0075] In one embodiment, there is a method of producing
therapeutic T-cells for HPV-associated diseases, the method
comprising: (i) stimulating peripheral blood cells, wherein the
method comprises stimulating peripheral blood T-cells with antigen
presenting cells in the presence of 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 HPV; and (ii) stimulating
T-cells obtained from (i) 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 HPV. In specific embodiments, prior to step (ii)
T-cells obtained from (i) may be re-stimulated in the presence of
IL-7 and IL-15 but not in the presence of co-stimulatory cells.
[0076] In an embodiment, a method of producing therapeutic T-cells
for HPV-associated diseases is provided, the method comprising: (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 HPV; (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 HPV, wherein (ii) is optionally repeated one or
more times; and (iii) stimulating T-cells obtained from (ii) with
antigen presenting cells in the presence of interleukin (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 HPV,
wherein (iii) is optionally repeated one or more times.
[0077] In any method of the disclosure, the HPV may be HPV16,
HPV18, HPV1, HPV2 and HPV3. Peptides comprising sequence that
corresponds to one or more of E1, E2, E3, E4, E5, E6, E7, L1, and
L2 may be utilized in any method of the disclosure. The peptides
may comprise sequence that corresponds to: a) the HPV18 E6 protein
and/or the HPV18 E7 protein, and/or b) the HPV16 E6 protein and/or
the HPV16 E7 protein. In some cases, an individual being provided
with an effective amount of cells as described herein has an
HPV-associated disease, such as cancer, and the peptides comprise
sequence that corresponds to one or both of E6 and E7. In specific
aspects, the one or more peptides comprises peptides 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, and in particular the one or more peptides
comprises peptides of 15 amino acids in length. In specific
embodiments, one or more peptides form a library and peptides in
the library overlap in sequence with other peptides by 11 amino
acids.
[0078] In particular embodiments, a therapeutically effective
amount of T-cells produced by the method are provided to an
individual that has been exposed to HPV or that has HPV-associated
disease. In specific embodiments, an HPV-associated disease
comprises a neoplasm.
[0079] A therapeutically effective amount of T-cells produced by a
method of the disclosure may be provided to an individual that has
been exposed to HPV16, HPV18, or both, or that has HPV16-associated
and/or HPV18-associated disease, including a neoplasm such as
cancer.
[0080] In particular embodiments, the cancer is a cervical cancer,
anal cancer, vulvar cancer, vaginal cancer, penile cancer,
oropharyngeal cancer, nasopharyngeal carcinoma, laryngeal
papillomatosis, laryngeal cancer, head and neck cancer, or a
dysplasia of any site thereof.
[0081] In some cases, an individual that has received and/or will
receive cells of the disclosure has also received, is receiving, or
will receive an additional cancer therapy, such as surgery,
radiation, hormone therapy, chemotherapy, immunotherapy, or a
combination thereof.
[0082] In certain aspects, an individual that has received and/or
will receive cells of the disclosure is determined as having
HPV-associated cancer, such as HPV16-associated cancer or
HPV18-associated cancer.
[0083] The following numbered paragraphs contain statements of
broad combinations of the inventive technical features herein
disclosed:
[0084] 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
and 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 human
papillomavirus (HPV).
[0085] 2. A method of producing therapeutic T-cells for
HPV-associated diseases, the method comprising the step of: [0086]
stimulating peripheral blood T-cells with antigen presenting cells
in the presence of one or more of IL-7 and IL-15 and 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 HPV, [0087] wherein the stimulating
produces T-cells therapeutic for HPV-associated diseases.
[0088] 3. The method of paragraph 1 or 2, wherein the peripheral
blood T-cells are obtained from a prior stimulation of peripheral
blood cells.
[0089] 4. The method of paragraph 3, wherein the prior stimulation
of peripheral blood cells comprises stimulating peripheral blood
cells with antigen presenting cells in the presence of IL-7 and
IL-15, and 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
HPV.
[0090] 5. The method of paragraph 1 or 2, wherein prior to
stimulating said peripheral blood T-cells, the method further
comprises stimulating peripheral blood cells with antigen
presenting cells in the presence of IL-7 and IL-15, and 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 HPV, to produce peripheral
blood T-cells.
[0091] 6. The method of any one of paragraphs 1-7, wherein the
antigen presenting cells are dendritic cells, B-blasts, or
PBMCs.
[0092] 7. The method of any one of paragraphs 1-6, wherein the
peripheral blood T-cells are present in a population of peripheral
blood mononuclear cells (PBMCs) or are obtained or isolated
therefrom.
[0093] 8. The method of paragraph 7, wherein the PBMCs in the
population are non-adherent PBMCs.
[0094] 9. A method for stimulating T-cells specific for HPV or for
an HPV antigen, the method comprising stimulating T-cells specific
for HPV or for an HPV antigen 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 HPV.
[0095] 10. A method of producing therapeutic T-cells for
HPV-associated diseases, the method comprising the step of
stimulating T-cells specific for HPV or for an HPV antigen 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 HPV,
wherein the stimulating produces T-cells therapeutic for
HPV-associated diseases.
[0096] 11. The method of paragraph 9 or 10, wherein the antigen
presenting cells are activated T cells, dendritic cells, B-blasts,
or PBMCs.
[0097] 12. The method of any one of paragraphs 9 to 11, wherein the
co-stimulatory cells are CD80+, CD86+, CD83+, 4-1BBL+, CD40+ cells,
OX40+ cells, or a combination thereof 13. A method of producing
therapeutic T-cells for HPV-associated diseases, the method
comprising: [0098] (i) stimulating peripheral blood cells, wherein
the method comprises stimulating peripheral blood T-cells with
antigen presenting cells in the presence of 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 HPV; and [0099]
(ii) stimulating T-cells obtained from (i) 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 HPV.
[0100] 14. The method of paragraph 13, wherein prior to step (ii)
T-cells obtained from (i) may be re-stimulated in the presence of
IL-7 and IL-15 but not in the presence of co-stimulatory cells.
[0101] 15. The method of paragraph 13 or 14, wherein the antigen
presenting cells used in (i) are dendritic cells (DC), B-Blasts
(BB) or PBMCs.
[0102] 16. The method of any one of paragraphs 13 to 15, wherein
the antigen presenting cells used in (ii) are activated T cells,
dendritic cells (DC) or B-Blasts (BB).
[0103] 17. The method of any one of paragraphs 13 to 16, wherein
the co-stimulatory cells are CD80+, CD86+, CD83+, 4-1BBL+, CD40+
cells, OX40+ cells, or a combination thereof.
[0104] 18. A method of producing therapeutic T-cells for
HPV-associated diseases is provided, the method comprising: [0105]
(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 HPV;
[0106] (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 HPV,
wherein (ii) is optionally repeated one or more times; and [0107]
(iii) stimulating T-cells obtained from (ii) with antigen
presenting cells in the presence of interleukin (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 HPV, wherein (iii)
is optionally repeated one or more times.
[0108] 19. The method of paragraph 18, wherein the antigen
presenting cells used in (i) and (ii) are DC, BB, or PBMCs.
[0109] 20. The method of paragraph 18 or 19, wherein the antigen
presenting cells used in (iii) are activated T cells, DC, BB, or
PBMCs.
[0110] 21. The method of any one of paragraphs 18 to 20, wherein
the co-stimulatory cells are CD80+, CD86+, CD83+, 4-1BBL+, CD40+
cells, OX40+ cells or a combination thereof.
[0111] 22. The method of any one of paragraphs 1-21, wherein the
HPV is HPV16 or HPV18.
[0112] 23. The method of any one of paragraphs 1-22, wherein the
peptides comprise sequence that corresponds to one or more of E1,
E2, E3, E4, E5, E6, E7, L1, and L2.
[0113] 24. The method of any one of paragraphs 1-23, wherein the
HPV-associated disease is cancer and the peptides comprise sequence
that corresponds to one or both of E6 and E7.
[0114] 25. The method of any one of paragraphs 1-25, wherein the
peptides comprise sequence that corresponds to: [0115] a) the HPV18
E6 protein and/or the HPV18 E7 protein, and/or [0116] b) the HPV16
E6 protein and/or the HPV16 E7 protein.
[0117] 26. The method of any one of paragraphs 1-25, wherein the
one or more peptides comprises peptides 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.
[0118] 27. The method of any one of paragraphs 1-26, wherein the
one or more peptides comprises peptides of 15 amino acids in
length.
[0119] 28. The method of any one of paragraphs 1-27, wherein one or
more peptides form a library and peptides in the library overlap in
sequence with other peptides by 11 amino acids.
[0120] 29. The method of any one of paragraphs 1-28, wherein a
therapeutically effective amount of T-cells produced by the method
are provided to an individual that has been exposed to HPV or that
has HPV-associated disease.
[0121] 30. The method of paragraph 29, wherein the HPV-associated
disease comprises a neoplasm.
[0122] 31. The method of any one of paragraphs 1 to 30, wherein a
therapeutically effective amount of T-cells produced by the method
are provided to an individual that has been exposed to HPV16, HPV18
or both, or that has HPV16-associated and/or HPV18-associated
disease.
[0123] 32. The method of paragraph 31, wherein the HPV16-associated
and/or HPV18-associated disease comprises a neoplasm.
[0124] 33. The method of paragraph 31 or 32, wherein the neoplasm
is cancer.
[0125] 34. The method of paragraph 33, wherein the cancer is
cervical cancer, anal cancer, vulvar cancer, vaginal cancer, penile
cancer, oropharyngeal cancer, nasopharyngeal carcinoma, laryngeal
papillomatosis, laryngeal cancer, head and neck cancer, or a
dysplasia of any of site thereof.
[0126] 35. The method of paragraph 33 or 34, wherein the individual
has received, is receiving, or will receive an additional cancer
therapy.
[0127] 36. The method of paragraph 35, wherein the additional
cancer therapy is surgery, radiation, hormone therapy,
chemotherapy, immunotherapy, or a combination thereof.
[0128] 37. The method of any one of paragraphs 33 to 36, wherein
the individual is determined as having HPV-associated cancer.
[0129] 38. The method of any one of paragraphs 33 to 37, wherein
the individual is determined as having HPV16-associated cancer.
[0130] 39. The method of any one of paragraphs 33 to 38, wherein
the individual is determined as having HPV18-associated cancer.
[0131] 40. T-cells for use in a method of adoptive cellular
immunotherapy, wherein the T-cells are obtained by, obtainable by,
or are the product of, a method for stimulating peripheral blood or
T-cells or a method of producing therapeutic T-cells according to
any one of paragraphs 1 to 39, wherein the method of adoptive
cellular immunotherapy comprises administering the T-cells to the
subject.
[0132] 41. Use of T-cells in the manufacture of a medicament for
use in a method of adoptive cellular immunotherapy comprising
administering the T-cells to the subject, wherein the T-cells are
obtained by, obtainable by, or are the product of, a method for
stimulating peripheral blood or T-cells or a method of producing
therapeutic T-cells according to any one of claims 1 to 39.
[0133] 42. A method of preparing a pharmaceutical composition,
medicament or vaccine, the method comprising stimulating peripheral
blood or T-cells or producing therapeutic T-cells according to any
one of claims 1 to 39, and mixing the cells obtained with a
pharmaceutically acceptable carrier, adjuvant, diluent or
excipient.
[0134] 43. A method of treating a cancer in a subject, the method
comprising: [0135] (1) isolating T cells from a subject; [0136] (2)
generating or expanding a population of T cells specific for a
human papillomavirus (HPV) by a method comprising: stimulating the
T-cells with antigen presenting cells in the presence of
interleukin (IL)-7 and IL-15 and 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 HPV; and [0137] (3) administering the generated or
expanded population of T cells to a subject.
[0138] 44. The method of paragraph 43, wherein the T-cells
stimulated in (2) are obtained from a prior stimulation of
peripheral blood cells or T-cells.
[0139] 45. The method of paragraph 43, wherein prior to stimulating
said T-cells, the method comprises stimulating peripheral blood
cells or T-cells with antigen presenting cells in the presence of
IL-7 and IL-15, and 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
HPV.
[0140] 46. The method of paragraph 43, wherein after (2) and before
(3) the method comprises stimulating the T-cells obtained from (2)
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 HPV.
[0141] 47. The method of paragraph 46 wherein the co-stimulatory
cells are CD80+, CD86+, CD83+, 4-1BBL+, CD40+ cells, OX40+ cells,
or a combination thereof.
[0142] 48. The method of paragraph 43, wherein after (2) and before
(3) the method comprises (i) re-stimulating the T-cells obtained
from (2) in the presence of IL-7 and IL-15 but not in the presence
of co-stimulatory cells, and (ii) stimulating the T-cells obtained
after (i) 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 HPV
[0143] 49. The method of paragraph 43, wherein the antigen
presenting cells are dendritic cells (DC), B-blasts (BB), or
peripheral blood mononuclear cells (PBMCs).
[0144] 50. The method of paragraph 43, wherein in (1) the T-cells
are isolated from a population of peripheral blood mononuclear
cells (PBMCs).
[0145] 51. The method of paragraph 43, wherein the cancer is
cervical cancer, anal cancer, vulvar cancer, vaginal cancer, penile
cancer, oropharyngeal cancer, nasopharyngeal carcinoma, laryngeal
papillomatosis, laryngeal cancer, head and neck cancer, or a
dysplasia of any of site thereof.
[0146] 52. The method of paragraph 43, wherein the cancer is
HPV-positive.
[0147] 53. The method of paragraph 43, wherein the subject is
determined as having HPV-associated cancer.
[0148] 54. A method of treating a cancer in a subject, the method
comprising: [0149] (1) isolating T cells from a subject; [0150] (2)
generating or expanding a population of T cells specific for a
human papillomavirus (HPV) by a method comprising: [0151] (i)
stimulating the 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 HPV; [0152] (ii)
stimulating T-cells obtained from (i) with antigen presenting cells
in the presence of interleukin (IL)-7 and IL-15 and 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 HPV, wherein (ii) is optionally repeated
one or more times; and [0153] (iii) stimulating T-cells obtained
from (ii) with antigen presenting cells in the presence of
interleukin (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 HPV, wherein (iii) is optionally repeated one or more
times. [0154] (3) administering the generated or expanded
population of T cells to a subject.
[0155] 55. The method of paragraph 54, wherein stimulation of
T-cells in (i) is in the presence of IL-6 and/or IL-12.
[0156] 56. The method of paragraph 54, wherein the antigen
presenting cells used in (i) and (ii) are dendritic cells (DC),
B-blasts (BB), or peripheral blood mononuclear cells (PBMCs).
[0157] 57. The method of paragraph 54, wherein the antigen
presenting cells used in (iii) are activated T cells, dendritic
cells (DC), B-blasts (BB), or peripheral blood mononuclear cells
(PBMCs).
[0158] 58. The method of paragraph 54, wherein the co-stimulatory
cells are CD80+, CD86+, CD83+, 4-1BBL+, CD40+ cells, OX40+ cells or
a combination thereof.
[0159] 59. A method of treating a cancer in a subject, the method
comprising: [0160] (1) isolating T cells from a subject; [0161] (2)
generating or expanding a population of T cells specific for a
human papillomavirus (HPV) by a method comprising: stimulating
T-cells specific for HPV or for an HPV antigen 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 HPV; and [0162] (3)
administering the generated or expanded population of T cells to a
subject.
[0163] 60. The method of paragraph 59, wherein the antigen
presenting cells are activated T cells, dendritic cells (DC),
B-blasts (BB), or peripheral blood mononuclear cells (PBMCs).
[0164] 61. The method of paragraph 59, wherein the co-stimulatory
cells are CD80+, CD86+, CD83+, 4-1BBL+, CD40+ cells, OX40+ cells,
or a combination thereof.
[0165] 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.
[0166] The invention includes the combination of the aspects and
preferred features described except where such a combination is
clearly impermissible or expressly avoided.
[0167] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described.
[0168] Aspects and embodiments of the present invention will now be
illustrated, by way of example, with reference to the accompanying
figures. Further aspects and embodiments will be apparent to those
skilled in the art. All documents mentioned in this text are
incorporated herein by reference.
[0169] Throughout this specification, including the claims which
follow, unless the context requires otherwise, the word "comprise,"
and variations such as "comprises" and "comprising," will be
understood to imply the inclusion of a stated integer or step or
group of integers or steps but not the exclusion of any other
integer or step or group of integers or steps.
BRIEF DESCRIPTION OF THE DRAWINGS
[0170] FIG. 1A demonstrates a method in the art that utilizes
certain conditions for the production of HPV16-specific T-cells.
FIG. 1A is a bar chart showing production of spot forming colonies
(SFC) on stimulation of PBMCs from three HPV-associated cancer
patients (identified as OCV, HND and HNC) with autologous DCs
loaded with no pepmix (Neg), HPV16 E6 pepmix (HPV16 E6) or HPV16 E7
pepmix (HPV16 E7). For patient OCV the three bars present from left
to right are Neg, HPV16 E6 and HPV16 E7. For patients HND and HNC
the two bars present from left to right are HPV16 E6 and HPV16
E7.
[0171] FIG. 1B demonstrates a method of the disclosure that
utilizes, under certain novel conditions, the production of a
mixture of T-cells specific for HPV16 or HPV18 by stimulation of
T-cells in the presence of IL-7 and IL-15 and in the absence of
IL-6 and IL-12. FIG. 1B is a bar chart showing production of spot
forming colonies (SFC) on stimulation of PBMCs from three
HPV-associated cancer patients (identified as OCV, PCV and HND).
For patient OCV the five bars present from left to right are no
pepmix (Neg), HPV16 E6 pepmix (HPV16 E6), HPV16 E7 pepmix (HPV16
E7), HPV18 E6 pepmix (HPV18 E6), and HPV18 E7 pepmix (HPV18 E7).
For patient PCD the two bars present from left to right are HPV16
E6 and HPV16 E7. For patient HND the four bars present from left to
right are HPV16 E6, HPV16 E7, HPV18 E6 and HPV18 E7.
[0172] FIG. 2 is a chart showing in vivo expansion and persistence
of infused HPV stimulated T-cells transduced with a dominant
negative receptor for TGF-beta (DNRII) in patient #1 at time points
post infusion.
[0173] FIG. 3 is a chart showing in vivo expansion and persistence
of infused HPV stimulated T cells transduced with a dominant
negative receptor for TGF-beta (DNRII) in patient #2 at time points
post infusion.
[0174] FIG. 4 shows PET scans (left and center) and photographs of
physical examination (right) for patient #2. Top row:
pre-treatment. Bottom row: 6 weeks after treatment with HPV
stimulated T cells produced according to the present invention.
[0175] FIGS. 5A and 5B are scatterplots showing surface expression
of (FIG. 5A) CD83 and CD80, and (FIG. 5B) CCR7 and PD-L1 expression
by monocyte-derived DCs derived from Donor 1 CD14+ PBMCs following
culture according to experimental conditions 1, 2, 3, 4, 5, 6, 7,
8, 9, 10 or 11 (see Table 4).
[0176] FIGS. 6A and 6B are scatterplots showing surface expression
of CCR7 and CD45RO by (FIG. 6A) CD4+ T cells, and (FIG. 6B) CD8+ T
cells obtained following stimulation of autologous CD14- PBMCs
derived from Donor 1 for 9 days with EBV peptide-pulsed mature DCs
cultured according to experimental conditions 1, 2, 3, 4, 5, 6, 7,
8, 9, 10 or 11 (see Table 4).
[0177] FIG. 7 is a bar chart showing the total number of
virus-specific T cells obtained following stimulation of autologous
CD14- PBMCs derived from Donor 1 for 9 days with EBV peptide-pulsed
mature DCs cultured according to experimental conditions 1, 2, 3,
4, 5, 6, 7, 8, 9, 10 or 11 (see Table 4).
[0178] FIG. 8 is a bar chart shows the proportions of
IFN.gamma.+CD8+ CTLs and IFN.gamma.+CD4+ Th cells (background
subtracted) obtained following stimulation of autologous CD14-
PBMCs derived from Donor 1 for 9 days with EBV peptide-pulsed
mature DCs cultured according to experimental conditions 1, 2, 3,
4, 5, 6, 7, 8, 9, 10 or 11 (see Table 4).
[0179] FIG. 9 is a bar chart showing the proportions of EBV
antigen-reactive IFN.gamma.+CD8+ CTLs and IFN.gamma.+CD4+ Th cells
(background subtracted) obtained following stimulation of
autologous CD14- PBMCs derived from Donor 1 for 9 days with EBV
peptide-pulsed mature DCs cultured according to experimental
conditions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 (see Table 4).
[0180] FIG. 10 is a graph showing the total number of
virus-specific T cells plotted against the proportion of
IFN.gamma.+CD8+ CTLs obtained following stimulation of autologous
CD14- PBMCs derived from Donor 1 for 9 days with EBV peptide-pulsed
mature DCs cultured under different experimental conditions (see
Table 4).
[0181] FIGS. 11A and 11B are scatterplots showing surface
expression of (FIG. 11A) CD83 and CD80, and (FIG. 11B) CCR7 and
PD-L1 expression by monocyte-derived DCs derived from Donor 2 CD14+
PBMCs following culture according to experimental conditions 1, 2,
3, 12, 13, 14, 15, 16, 17, 18, 19 or 20 (see Table 4).
[0182] FIGS. 12A and 12B are scatterplots showing surface
expression of CCR7 and CD45RO by (FIG. 12A) CD4+ T cells, and (FIG.
12B) CD8+ T cells obtained following stimulation of autologous
CD14- PBMCs derived from Donor 2 for 9 days with EBV peptide-pulsed
mature DCs cultured according to experimental conditions 1, 2, 3,
12, 13, 14, 15, 16, 17, 18, 19 or 20 (see Table 4).
[0183] FIG. 13 is a bar chart showing the total number of
virus-specific T cells obtained following stimulation of autologous
CD14- PBMCs derived from Donor 2 for 9 days with EBV peptide-pulsed
mature DCs cultured according to experimental conditions 1, 2, 3,
12, 13, 14, 15, 16, 17, 18, 19 or 20 (see Table 4).
[0184] FIG. 14 is a bar chart shows the proportions of
IFN.gamma.+CD8+ CTLs and IFN.gamma.+CD4+ Th cells (background
subtracted) obtained following stimulation of autologous CD14-
PBMCs derived from Donor 2 for 9 days with EBV peptide-pulsed
mature DCs cultured according to experimental conditions 1, 2, 3,
12, 13, 14, 15, 16, 17, 18, 19 or 20 (see Table 4).
[0185] FIG. 15 is a bar chart showing the proportions of EBV
antigen-reactive IFN.gamma.+CD8+ CTLs and IFN.gamma.+CD4+ Th cells
(background subtracted) obtained following stimulation of
autologous CD14- PBMCs derived from Donor 2 for 9 days with EBV
peptide-pulsed mature DCs cultured according to experimental
conditions 1, 2, 3, 12, 13, 14, 15, 16, 17, 18, 19 or 20 (see Table
4).
[0186] FIG. 16 is a graph showing the total number of
virus-specific T cells plotted against the proportion of
IFN.gamma.+CD8+ CTLs obtained following stimulation of autologous
CD14- PBMCs derived from Donor 2 for 9 days with EBV peptide-pulsed
mature DCs cultured under different experimental conditions (see
Table 4).
[0187] FIGS. 17A, 17B, 17C, 17D and 17E are scatterplots and bar
charts showing surface expression of (FIG. 17A) CD80 and CD83, and
(FIG. 17B) CCR7 and PD-L1 by monocyte-derived DCs derived from
three different donors following culture according to the
experimental conditions 1, 2, 5, 18, 19 and 20 (see Table 4). FIG.
17C shows the proportions of CD80+CD83- cells, FIG. 17D shows the
proportions of CD80+CD83+ cells, and FIG. 17E shows the proportions
of PD-L1+ cells.
[0188] FIG. 18 is a bar chart showing the total number of
virus-specific T cells obtained following stimulation of autologous
CD14- PBMCs derived from the three different donors for 9 days with
EBV peptide-pulsed mature DCs cultured according to the
experimental conditions 2, 5, 18, 19 and 20 (see Table 4).
[0189] FIG. 19 is a bar chart showing the proportions of
IFN.gamma.+CD8+ CTLs and IFN.gamma.+CD4+ Th cells (background
subtracted) obtained following stimulation of autologous CD14-
PBMCs derived from the different donors for 9 days with EBV
peptide-pulsed mature DCs cultured according to the experimental
conditions 2, 5, 18, 19 and 20 (see Table 4).
[0190] FIG. 20 shows the proportions of EBV antigen-reactive
IFN.gamma.+CD8+ CTLs and IFN.gamma.+CD4+ Th cells (background
subtracted) obtained following stimulation of autologous CD14-
PBMCs derived from the different donors for 9 days with EBV
peptide-pulsed mature DCs cultured according to the experimental
conditions 2, 5, 18, 19 and 20 (see Table 4).
[0191] FIG. 21 is a graph showing the scaled total number of
virus-specific T cells and the scaled frequency of IFN.gamma.+ T
cells obtained following stimulation of autologous CD14- PBMCs
derived from the three donors for 9 days with EBV peptide-pulsed
mature DCs cultured under different experimental conditions (see
Table 4).
[0192] FIGS. 22A and 22B are graphs showing the results of two
separate experiments investigating overall fold expansion of cells
following stimulations using K562-cs cells as costimulatory cells
as compared to ULCL clones #5 and #13.
[0193] FIGS. 23A and 23B are graphs showing the results of two
separate experiments investigating overall fold expansion of
virus-specific T cells following stimulations using K562-cs cells
as costimulatory cells as compared to ULCL clones #5 and #13, as
determined by ELISPOT analysis of the number of IFN.gamma.
producing cells.
[0194] FIGS. 24A, 24B and 24C provide scatterplots from a
representative donor (n=7) showing the proportions of (FIG. 24A)
CD3-CD56+ NK cells, (FIG. 24B) CD4+ T cells and CD8+ T cells, and
(FIG. 24C) gamma delta T cells and alpha beta T cells in
populations expanded using K562-cs cells or ULCL clones #5 and #13
as costimulatory cells.
[0195] FIG. 25 provides histograms showing representative results
of characterization of gamma delta TCR expression by ULCL clones #5
and #13, as compared to expression by K562cs cells.
[0196] FIGS. 26A and 26B are bar charts for two different donors
showing the frequency of virus-specific T cells in populations
expanded using K562-cs cells or ULCL clones #5 and #13 as
costimulatory cells, after the indicated number of stimulations, as
determined by ELISPOT analysis of the number of IFN.gamma.
producing cells.
[0197] FIGS. 27A, 27B, 27C and 27D are bar charts for four
different donors showing the frequency of T cells specific for the
indicated EBV antigens, after the indicated number of stimulations
using K562cs cells (K), ULCL clone #5 (5) or LCLs (L) as
costimulatory cells, as determined by ELISPOT analysis of the
number of IFN.gamma. producing cells.
[0198] FIGS. 28A, 28B, 28C and 28D are bar charts and graphs
showing the frequency and fold expansion of virus-specific T cells
in populations expanded using K562-cs cells or ULCL clones #4, #5
and #13 as costimulatory cells, after the indicated number of
stimulations, as determined by ELISPOT analysis of the number of
IFN.gamma. producing cells.
[0199] FIGS. 29A and 29B are bar charts showing the frequency of
virus-specific T cells in populations expanded using ULCL clone #4
or parental ULCL cells as costimulatory cells, after the indicated
number of stimulations, as determined by ELISPOT analysis of the
number of IFN.gamma. producing cells.
[0200] FIGS. 30A and 30B are bar charts for two different donors
showing the frequency of virus-specific T cells in populations
expanded using ULCL clone #5 or K562cs cells as costimulatory
cells, after the indicated number of stimulations, as determined by
ELISPOT analysis of the number of IFN.gamma. producing cells.
[0201] FIGS. 31A and 31B are bar charts for two different donors
showing the fold expansion of virus-specific T cells in populations
expanded using ULCL clone #5 or K562cs cells as costimulatory
cells, after the indicated number of stimulations, as determined by
ELISPOT analysis of the number of IFN.gamma. producing cells.
[0202] FIG. 32 provides scatterplots showing surface expression of
CD3 and CD56 by cells expanded using ULCL clone #5 or K562cs cells
as costimulatory cells, after the indicated number of stimulations,
as determined by flow cytometry, for donor PB.
[0203] FIG. 33 provides scatterplots showing surface expression of
CD3 and CD56 by cells expanded using ULCL clone #5 or K562cs cells
as costimulatory cells, after the indicated number of stimulations,
as determined by flow cytometry, for donor KP.
[0204] FIG. 34 provides scatterplots showing surface expression of
CD45RO and CCR7 by cells expanded using ULCL clone #5 or K562cs
cells as costimulatory cells, after the indicated number of
stimulations, as determined by flow cytometry, for donor PB.
[0205] FIG. 35 provides scatterplots showing surface expression of
CD45RO and CCR7 by cells expanded using ULCL clone #5 or K562cs
cells as costimulatory cells, after the indicated number of
stimulations, as determined by flow cytometry, for donor KP.
[0206] FIGS. 36A and 36B are bar charts for two different donors
showing the frequency of virus-specific T cells in populations
expanded using ULCL clone #5 or K562cs cells as costimulatory
cells, after the indicated number of stimulations, as determined by
ELISPOT analysis of the number of IFN.gamma. producing cells.
[0207] FIGS. 37A and 37B are bar charts for two different donors
showing the fold expansion of virus-specific T cells in populations
expanded using ULCL clone #5 or K562cs cells as costimulatory
cells, after the indicated number of stimulations, as determined by
ELISPOT analysis of the number of IFN.gamma. producing cells.
[0208] FIG. 38 provides scatterplots showing surface expression of
CD3 and CD56 by cells expanded using ULCL clone #5 or K562cs cells
as costimulatory cells, after the indicated number of stimulations,
as determined by flow cytometry, for one donor.
[0209] FIG. 39 provides scatterplots showing surface expression of
CD3 and CD56 by cells expanded using ULCL clone #5 or K562cs cells
as costimulatory cells, after the indicated number of stimulations,
as determined by flow cytometry, for one donor.
[0210] FIG. 40 provides scatterplots showing surface expression of
CD45RO and CCR7 by cells expanded using ULCL clone #5 or K562cs
cells as costimulatory cells, after the indicated number of
stimulations, as determined by flow cytometry, for one donor.
[0211] FIG. 41 provides scatterplots showing surface expression of
CD45RO and CCR7 by cells expanded using ULCL clone #5 or K562cs
cells as costimulatory cells, after the indicated number of
stimulations, as determined by flow cytometry, for one donor.
[0212] FIG. 42 is a bar chart showing the frequency of HPV-specific
T cells in populations expanded using ULCL clone #5 or K562cs cells
as costimulatory cells in stimulations comprising the indicated
cytokines, and after the indicated number of stimulations, as
determined by ELISPOT analysis of the number of IFN.gamma.
producing cells.
[0213] FIG. 43 is a graph showing the fold expansion of cells in
populations expanded using ULCL clone #5 or K562cs cells as
costimulatory cells in stimulations comprising the indicated
cytokines, and after the indicated number of stimulations.
[0214] FIG. 44 provides scatterplots showing surface expression of
CD3 and CD56 by cells expanded using ULCL clone #5 or K562cs cells
as costimulatory cells in stimulations comprising the indicated
cytokines, and after the indicated number of stimulations, as
determined by flow cytometry.
[0215] FIG. 45 provides scatterplots showing surface expression of
CD4 and CD8 by cells expanded using ULCL clone #5 or K562cs cells
as costimulatory cells in stimulations comprising the indicated
cytokines, and after the indicated number of stimulations, as
determined by flow cytometry.
[0216] FIG. 46 provides scatterplots showing surface expression of
CD45RO and CCR7 by cells expanded using ULCL clone #5 or K562cs
cells as costimulatory cells in stimulations comprising the
indicated cytokines, and after the indicated number of
stimulations, as determined by flow cytometry.
[0217] FIG. 47 is a schematic illustration of general embodiments
of virus-specific T-cell (VST) generation methods of the
disclosure.
[0218] FIG. 48 provides bar charts demonstrating 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.
[0219] FIG. 49 is a bar chart showing improved EBV antigen
specificity of lymphoma patient EBVSTs obtained by stimulations
performed in the presence of IL-7 and IL-15, as determined by
ELISPOT.
[0220] FIG. 50 is a bar chart demonstrating that stimulation in the
presence of high doses of IL-15 increases frequency of VSTs.
[0221] FIG. 51 is a bar chart showing shows that stimulation in the
presence of high doses of IL-15 increases the proportion of central
memory EBVSTs.
[0222] FIG. 52 provides bar charts showing excessive NK-cell
outgrowth in EBVSTs from some patients.
[0223] FIG. 53 is a schematic illustration of the generation of
pepmix-activated EBVSTs from CD45RA+ cell-depleted PBMCs.
[0224] FIG. 54 is a bar chart showing that CD45RA depletion
decreases the frequency of CD3-CD56+ NK cells in EBVSTs expanded
from healthy donors.
[0225] FIG. 55 is a bar chart showing that removal of CD45RA+ cells
increases proliferation of EBVSTs.
[0226] FIG. 56 is a graph that demonstrates that CD45RA depletion
enhances the fold expansion of EBVSTs.
[0227] FIG. 57 is a bar chart showing that CD45RA depletion
enhances antigen specificity of EBVSTs at the end of a second
stimulation (at day 16).
[0228] FIG. 58 is a bar chart which demonstrates that CD45RA
depletion enhances antigen specificity of EBVSTs.
[0229] FIG. 59 is a bar chart which demonstrates increased antigen
specificity of CD45RA depleted EBVSTs is sustained after a third
stimulation.
[0230] FIG. 60 is a bar chart which demonstrates that CD45RA
depletion decreases NK cell population outgrowth in lymphoma
patient EBVSTs.
[0231] FIG. 61 is a bar chart showing that CD45RA depletion
increases the frequency of antigen specific T-cells in lymphoma
patient EBVSTs.
[0232] FIG. 62 is a bar chart demonstrating that CD45RA depletion
increases antigen specificity in EBVSTs from lymphoma patients.
[0233] FIG. 63 is a bar chart showing the effect of CD45RA
depletion on proliferation of lymphoma patients' EBVSTs.
[0234] FIG. 64 is a chart which demonstrates that CD45RA- depletion
enhances cytolytic activity against pepmix-pulsed autologous
activated T-cells (aATCs).
DETAILED DESCRIPTION
[0235] 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.
[0236] 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.
[0237] The present disclosure concerns the production and use of
therapeutic T-cells for individuals that are in need of
HPV-specific T-cells, e.g., HPV16- and/or HPV18-specific T-cells,
including for treating HPV infection and HPV-associated medical
conditions. In particular embodiments, the methods and compositions
are useful for treating neoplasms that are indirectly or directly
related to HPV infection, and such neoplasms may be benign or
malignant. Between 13 and 18 HPV strains have been characterized as
conferring a high oncogenic risk, with 12 of these strains
belonging to the HPV species 7 (HPV-18, -39, -45, -59, -68) and
species 9 (HPV-16, -31, -33, -35, -52, -58, -67). HPV Types 6 and
11 cause laryngeal papillomatisis.
[0238] I. HPV Antigen(s) and Generation of Pepmixes
[0239] 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 of the peripheral blood T-cells, 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 CTLs. 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, although in some cases the library
comprises pepmixes that span part or all of one or more antigens,
and the one or more antigens may or may not be from the same HPV.
In particular embodiments, peptides for the APCs are non-natural,
and they may or may not be chemically synthesized or produced by
recombinant means.
[0240] In utilizing a library of mixtures of peptides from one or
more HPV antigens, the various peptides may come from any part of a
given protein, but in specific cases the peptides collectively 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 of 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.
[0241] The antigens from which the peptides are derived may be
antigens for HPVs that may be of any kind, but in specific
embodiments the antigens are such that they allow for direction of
cytotoxic T-cells to neoplasms, including cancers, associated with
HPV infection. 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 HPV type, including HPV16 and/or
HPV18. For example, in late stage cervical cancer, the HPV virus
integrates into a tumor cell genome and loses all of its other
genes except E6 and E7, so in some cases these antigens are
targeted. In embodiments wherein one would treat an earlier stage
of cancer, such as before the virus integrated, one could utilize
peptides from antigens other than E6 and E7, including E5 and L1
and L2, for example. However, given that the two primary
oncoproteins of high risk HPV types are E6 and E7, in specific
embodiments the sequence of the peptides are obtained from E6
and/or E7 from any HPV, but HPV16 and/or HPV18, in particular.
Peptides from any of antigens E1, E2, E3, E4, E5, E6, E7, L1,
and/or L2 may be utilized in methods of the disclosure.
[0242] In some cases, a pepmix library includes peptides
corresponding to one or more antigens from a single type of HPV
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 HPV 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.
[0243] Pepmixes utilized in the disclosure may be from commercially
available peptide libraries or may be synthetically generated, for
example. Examples of available libraries include those from JPT
Technologies (Springfield, Va.) or Miltenyi Biotec (Auburn,
Calif.). The skilled artisan, based on known sequences of HPV16 E6,
HPV16 E7, HPV18 E6, and HPV18 E7, for example, would have
sufficient information to be able to generate peptides that
correspond to their exemplary, respective sequences. An example of
sequence of the HPV16 E6 protein is available at the National
Center for Biotechnology Information's GenBank.RTM. database at
GenBank.RTM. Accession No. AIQ82776.1 GI:688010703. An example of
sequence of the HPV16 E7 protein is at GenBank.RTM. Accession No.
AIQ82814.1 GI:688010789. An example of sequence of the HPV18 E6
protein is at GenBank.RTM. Accession No. AGU90423.1 GI:537801975.
An example of sequence of the HPV18 E7 protein is at GenBank.RTM.
Accession No. AGU90424.1 GI:537801976.
[0244] 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 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 numbers 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.
[0245] 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 certain aspects, the peptides are at a
maximum of 30 amino acids in length with overlapping of 25 amino
acids.
[0246] II. Methods of Producing and Using Therapeutic T-Cells
[0247] A. Producing Therapeutic T-Cells
[0248] In certain aspects, the present disclosure concerns the
development of immune cells, such as cytotoxic T-cells, that target
one or more antigens from at least one HPV virus.
[0249] Methods disclosed herein may involve the stimulation and/or
expansion of immune cells. The methods may involve the stimulation
and/or expansion of peripheral blood cells, such as peripheral
blood mononuclear cells. The methods may involve expansion of an
immune cell population (e.g. a population of T-cells) from within a
population of immune cells (e.g. PBMCs). For example, a population
of T-cells may be expanded from within a population of PBMCs, by
stimulation of the T-cells within the population of PBMCs.
Accordingly, in embodiments of the methods disclosed herein,
stimulation and/or expansion of T cells may involve stimulation of
a population of PBMCs. In some embodiments, a population of T-cells
may be expanded from within a population of tumor-infiltrating
lymphocytes, by stimulation of the T-cells within the population of
tumor-infiltrating lymphocytes. Accordingly, in embodiments of the
methods disclosed herein, stimulation and/or expansion of T cells
may involve stimulation of a population of tumor-infiltrating
lymphocytes. In some embodiments, a population of T-cells may be
expanded from within a population of T-cells (e.g. a population of
T cells of heterogeneous specificity), which may have been obtained
from a blood sample, a population of PBMCs, or from a population of
tumor-infiltrating lymphocytes. The stimulations/expansions may
result in an increase the number HPV-specific immune cells (e.g.
HPV-specific T cells, such as HPV-specific CTLs), and/or result in
an increased proportion of such cells in the cell population at the
end of the stimulation/expansion. The methods may involve the
stimulation and/or expansion of T cells. The cells may have been
obtained from the patient to be treated (i.e., autologous cells),
or from another individual (i.e., allogeneic cells). The methods
involve stimulation and/or expansion of isolated immune cells, in
certain embodiments. That is, specific methods may be performed on
a population of cells that contains substantially no non-immune
cells, such as erythrocytes. In some cases, the immune cells are
isolated PBMCs, or isolated T cells. The cells may have been
obtained from a sample of blood, such as a sample of blood obtained
from the patient or individual. The cells may have been obtained
from tissue sample or biopsy. The cells may have been obtained from
a tumor (e.g. tumor-infiltrating lymphocytes). Certain methods
disclosed herein involve a step of obtaining PBMCs and/or T cells
from a sample obtained from the patient. Certain embodiments of
methods do not involve the step of obtaining a sample of blood or
cells from the patient or individual, but instead are performed on
a sample or cells that have been previously obtained. The method
may involve processing the sample, such as enriching the sample for
immune cells, such as PBMCs and/or T cells. Such methods may
involve removing or substantially reducing the amount of,
erythrocytes, platelets, serum and/or plasma in a sample. This may
result in a population of immune cells containing substantially no
other cells, such as erythrocytes. Methods disclosed herein may be
performed on isolated immune cells, or a sample containing immune
cells in addition to other cells.
[0250] In methods of producing the T-cells, peripheral blood
T-cells may be initially stimulated with APCs that have been
exposed to one or more peptides that span some or all of at least
one HPV antigen. The antigenic peptides may be provided to the APCs
in 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.
[0251] In embodiments of the disclosure, therapeutic T-cells are
generated and may be provided to an individual that has an HPV
infection or is at risk of having an HPV-associated medical
condition that results indirectly or directly from an HPV
infection. 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 antigens, including part or all of a HPV16 and/or HPV18
antigen, including E6 and/or E7, for example. In specific
embodiments, for the stimulating step the T-cells reside within a
population of PBMCs.
[0252] In some embodiments, the APCs used in certain steps may be
dendritic cells (DCs). Methods for generation of DCs are well known
in the art, e.g. see Ramos et al., supra. Monocytes may be isolated
from PBMCs by CD14 selection and cultured in DC medium and 2 mM
alanyl-glutamine with 800 U/ml granulocyte/macrophage colony
stimulating factor (GM-CSF) and 1000 U/ml interleukin 4 (IL-4) for
5 days. GM-CSF and IL-4 may be replenished on day 3. On day 5, DCs
are matured in DC media with 10 ng/ml interleukin-1.beta.
(IL-1.beta.), 100 ng/ml interleukin 6 (IL-6), 10 ng/ml
prostaglandin E2, 800 U/ml GM-CSF and 1000 U/ml IL-4. DC maturation
may be assessed by flow cytometry to detect upregulation of CD80.
CD83, CD86 and HLA-DR.
[0253] In some embodiments, the APCs used in certain steps are
activated T-cells. Activated T-cells may be polyclonal T-cells
(T-APCs) generated using a portion of the autologous PBMC isolated
from the venesected blood. The cells may be activated by culturing
in cell culture plates that are coated with anti-CD3 and anti-CD28
antibodies. The cells are then cultured to expand in the presence
of IL-2 for 2 weeks. The expanded T-APC can be cryopreserved for
later use. 2-3 days prior to using T-APC for stimulation (e.g., for
the 3rd cycle of stimulation and optionally for subsequent
stimulations), cryopreserved cells are thawed and re-stimulated in
anti-CD3 and anti-CD28 antibody-coated cell culture plates. On the
day of stimulation, the T-APC cells are harvested and pulsed with
the HPV E6/E7 peptides, followed by adding to the on-going culture
of HPV stimulated T-cells at 1:1 ratio.
[0254] In some embodiments, the APCs used in certain steps and/or
methods may be B-blasts (BBs). B-blasts may be generated from a
patient's autologous PBMC, for example. The B lymphocytes within
the PBMCs are activated by co-culturing with an irradiated
allogeneic CD40L-expressing MRCS epithelial cell line and expanded
in media containing 100 U/ml IL-4 and 1 microgram/ml cyclosporin
A.
[0255] In some embodiments, there is a method of generating T-cells
that target at least one antigen from one or both of HPV16 and
HPV18, 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 HPV16 and/or
HPV18 viral antigens. 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, which may be
mammalian (e.g. murine, human) or human cytokines. In certain
embodiments, the one or more cytokines are IL-7 and IL-15, although
in alternative embodiments the cytokine(s) are selected from the
group consisting of 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 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.
[0256] 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:
[0257] 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;
[0258] 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;
[0259] 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;
[0260] 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.
[0261] 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 First Stimulation Source of T-cells
Peripheral blood mononuclear cells (PBMC) Non-adherent PBMC
Antigen-presenting Dendritic cells (DC)s, PBMCs or B-blasts cells
(APC) Cytokines Combinations of IL-15 and IL-7, optionally with
IL-6 and/or IL-12 and/or IL-21 and/or IL-4 Antigen Viral pepmixes
for HPV Second stimulation Source of T-cells Product of first
stimulation APCs DC PBMCs Autologous activated T-cells (AATC)
Cytokines IL-15 and IL-7, preferably no IL-6 or IL-12 and
optionally IL-15 and IL-7 are the only interleukins Antigen Viral
pepmixes for HPV Third stimulation Source of T-cells Product of
second stimulation (and subsequent stimulations as desired) APCs
Pepmix-loaded AATCs + costimulatory cells Cytokines IL-15 and IL-7,
preferably no IL-6 or IL-12 and optionally IL-15 and IL-7 are the
only interleukins Costimulatory cells Cells expressing CD86, 4-1BB,
and CD83, e.g., K562 cells
[0262] Thus, in particular embodiments, a population of T-cells
(wherein the population may comprise 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 HPV16 E6 and/or E7 and/or
effectiveness at targeting HPV18 E6 and/or E7; b) polyclonality; c)
TH1 bias; or d) a combination thereof. The generated T cell lines
may be produced to be effective at targeting HPV species 7 (HPV-18,
-39, -45, -59, -68) and species 9 (HPV-16, -31, -33, -35, -52, -58,
-67), and types 6 and 11, and this may be the results of pepmixes
directed to any one or more of the following antigens: E1, E2, E3,
E4, E5, E6, E7, L1, and/or L2.
[0263] 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. This stimulation step may optionally be
repeated one or more times.
[0264] 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 not later, and then a
subsequent re-stimulation may occur on day 15, day 16, or day
17.
[0265] In 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 be in a heterogeneous population of
cells) are exposed to pepmix-loaded DCs or pepmix-loaded PBMCs
and/or autologous activated T-cells. In a stimulation that is
subsequent to first and second stimulation steps, T-cells obtained
after the second or later stimulation (and which may reside in a
heterogeneous population of cells) are exposed to pepmix-autologous
activated T-cells. Costimulatory cells that may be utilized in any
stimulation step include at least cells that express CD86, 4-1BB,
CD83, CD40, OX40, and/or CD80. In specific cases, the costimulatory
cells may be K562 cells.
[0266] 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, and/or modified to resist tumor
antigen-specific cellular immunity, for example.
[0267] In some 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. The cells may be modified to express a
chimeric antigen receptor (CAR), an .alpha..beta. T-cell receptor,
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)
during the method at specific time points, such as the vector being
introduced between day 2 and 5 of culture, for example. In some
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).
[0268] 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 .alpha., 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.
[0269] In some embodiments the cells are modified to resist tumor
antigen-specific cellular immunity, e.g. mediated by transforming
growth factor beta (TGF-.beta.). For example, the cells may be
modified to express a dominant negative receptor for TGF-beta
(DNRII), e.g. as described in Foster et al., (Antitumor activity of
EBV-specific T lymphocytes transduced with a dominant negative
TGF-beta receptor. J Immunother. 2008; 31:500-505, incorporated
herein by reference). This may comprise transfecting the cells with
a retroviral expression vector encoding a dominant negative
TGF-.beta. type II receptor (DNRII) modified by removal of the
immunogenic hemagglutinin tag. Such modified T-cells have been
shown to have a functional advantage over unmodified T-cells in the
presence of TGF-.beta.--secreting tumor, including enhanced
antitumor activity (Foster et al., supra).
[0270] Methods according to the present invention may improve the
rate of expansion for populations of virus-specific T-cells as
compared to prior art methods. The rate of expansion for a T-cell
population can be analysed by methods well known to the skilled
person. Methods include measuring the number of T-cells at one or
more time points. For example, the number of T-cells can be
determined after performing a method according to the invention and
compared to the number of T-cells at the beginning of the method;
fold expansion in the number of T-cells can then be calculated.
[0271] Rates of expansion can also be determined by analysing cell
division by T-cells over a period of time. Cell division for a
given population of T-cells can be analysed, for example, by in
vitro analysis of incorporation of .sup.3H-thymidine or by CFSE
dilution assay, e.g. as described in Fulcher and Wong, Immunol Cell
Biol (1999) 77(6): 559-564, hereby incorporated by reference in
entirety.
[0272] The improvement in the rate of expansion achieved by the
methods according to the present invention can be determined by
performing a method according to the invention, and comparing the
expansion for T-cells in that method to a comparable, control
method, e.g. as per the method of Ramos et al., (J Immunother 2013;
36:66-76).
[0273] In some embodiments, the rate of expansion for a population
of T-cells in a method according to the present invention is one of
at least 1.001 times, 1.002 times, 1.003 times, 1.004 times, 1.005
times, 1.006 times, 1.007 times, 1.008 times, 1.009 times, 1.01
times, 1.02 times, 1.03 times, 1.04 times, 1.05 times, 1.06 times,
1.07 times, 1.08 times, 1.09 times, 1.1 times, 1.2 times, 1.3
times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9
times, or 2 times the rate of expansion in a comparable control
method.
[0274] The rate of expansion may be of the virus-specific T-cell
population, or the total T-cell population.
[0275] The virus-specific T-cells generated/expanded according to
the method of the present invention may at least retain the same
functional properties as virus-specific T-cells generated/expanded
according to prior art methods. That is, the accelerated rate of
expansion does not negatively influence the functional properties
of the expanded T-cells.
[0276] For example, in embodiments wherein the methods
generate/expand a population of virus-specific T-cells, the T-cells
display similar cytotoxicity to cells infected with or
comprising/expressing a peptide of the virus as virus-specific
T-cells expanded according to prior art methods.
[0277] Cytotoxicity of expanded T-cells can be analysed e.g. by
culturing the expanded T-cell population with APCs presenting a
peptide of the virus for which the T-cell is specific at different
effector (i.e. T-cell) to target (i.e. APC) ratios, and measuring
specific lysis of the APCs. For example, cytotoxicity of an
HPV-specific CTL population can be analysed by measuring specific
lysis of HPV-transformed LCL cells at different effector to target
ratios.
[0278] B. Using Therapeutic T-Cells
[0279] In certain embodiments, cells produced by methods of the
disclosure are provided to an individual in need thereof for
treatment of a medical condition, including one caused by a viral
infection, or to target a viral infection 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, e.g.,
cancer. 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.
[0280] In the methods encompassed by the disclosure, the
therapeutic T-cells are utilized to treat viral-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 viral-associated disease(s) caused directly or
indirectly by more than one virus or are otherwise provided to an
individual that is seropositive for 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.
[0281] In at least some methods of the disclosure, a
therapeutically effective amount of the CTLs generated thereby are
administered to an individual, for example, an individual known to
have or suspected of having or susceptible to having HPV16 and/or
HPV18-associated disease. 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 CTLs are further defined as
polyclonal CD4+ and CD8+ CTLs. The PBMCs may be allogeneic to the
individual or may be autologous to the individual.
[0282] 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.
[0283] In certain cases, premalignant lesions that can lead to
cancers, such as premalignant lesions of the cervix, vulva, vagina,
penis, larynx, oropharynx anus, and other upper aerodigestive
areas, for example, are treated with cells produced by methods of
the disclosure. 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. HPV-associated
medical conditions that may be treated with cells produced by
methods of the disclosure include at least dysplasias of the
genital area(s), cervical intraepithelial neoplasia, vulvar
intraepithelial neoplasia, penile intraepithelial neoplasia, anal
intraepithelial neoplasia, cervical cancer, anal cancer, vulvar
cancer, vaginal cancer, penile cancer, genital cancers,
oropharyngeal cancer, nasopharyngeal carcinoma, oral papillomas and
other upper aerodigestive lesions.
[0284] In some cases, one can determine the serotype that is
associated with a cancer before administration of the cells,
although in some cases the serotype is not determined. In specific
embodiments, HPV16-specific or HPV18-specific cells have activity
for tumors that are HPV16 or HPV18-positive, respectively, although
in some cases there is cross-reactivity with different HPV
serotypes. The ability to cross-react may or may not be known, and
in certain cases, for example, an individual with HPV16 infection
or HPV16-associated medical condition is administered
HPV18-specific T-cells, and vice versa. In such cases, an
individual may be treated with cells specific for a serotype in
which it is unknown if the individual has that serotype, yet the
cells still are therapeutically effective because of
cross-reactivity.
[0285] In cases wherein the APCs of the stimulation steps of the
method are loaded with HPV16 and HPV18 pepmixes together, the
outcome of administration of T-cells expanded through such APCs is
determined by whether the individual has been exposed to the virus
in question. For example, if an individual is infected with HPV18
and not HPV16, only HPV18-specific T-cells will respond, and this
is because the infection will initially have stimulated a T-cell
response to HPV 18. 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 HPV16 that have never been activated, for
example.
[0286] The individual being treated may be known to have cancer,
suspected of having cancer, or at risk for having cancer (such as
personal or family history; being sexual active, including sexually
promiscuous; and/or having a genetic predisposition, including one
or more specific markers). An individual being treated may have the
presence of the HPV virus but there are not yet any deleterious
symptoms of a HPV-related medical condition. The individual may
have a benign or malignant neoplasm. 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 HPV-associated disease is malignant
cancer of the mouth or genital region. In specific embodiments, the
cancer may be primary, metastatic, recurrent, refractory, and so
forth. The individual may be infected with HPV16 and/or HPV18 as a
result of sexual acts of any kind or intimate physical contact of
any kind.
[0287] Any stage of HPV infection may be treated with cells
encompassed by the disclosure. The individual with established
HPV-associated cancer being treated with methods of the disclosure
include Carcinoma in Situ (Stage 0), Stage I, Stage II, Stage III,
or Stage IV (which may be determined by MRI, CT scan, PET scan,
etc.). Additionally, individuals with pre-cancer lesions
(dysplasia) may also be treated.
[0288] 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 different
administrations may be of any suitable duration, including on the
order of 1-7 days, 1-4 weeks, 1-12 months, or 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, or more years. Multiple infusions within about a year may
be employed, in some cases. 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
that was targeted with the cells utilized in earlier
administration(s). For example, in a first administration of cells,
the cells may target HPV18 E6, whereas in another administration of
cells, the cells target HPV18 E7, or vice versa. Additional
administration(s) may be required in cancers that become
refractory, for example. Additional stimulations may be employed in
conjunction with one or more other types of cancer treatments.
[0289] In some cases, an individual is optionally determined to
have HPV infection by any suitable means in the art. Because HPV
cannot be cultured in cell cultures, one may utilize HPV infection
diagnosis methods such as DNA tests utilizing PCR, Southern blot
hybridization, and/or in situ hybridization, and these methods may
or may not be used in conjunction with colposcopy; acetic acid
test; biopsy; physical examination; and/or Pap smear, for
example.
[0290] In specific embodiments, a male individual is provided an
effective amount of cells produced by methods of the disclosure to
target HPV with which he is infected, and in such a case the
individual thereafter has a reduced chance of infecting another,
such as a female individual through sexual activity. The male
individual may or may not be determined to be infected with HPV
prior to exposure to the cells of the methods of the disclosure. In
some cases, if an individual is shown to be infected with an
oncogenic HPV, it would be worth treating him with cells to
eliminate his risk. If the cells were effective, they would also
reduce the chances of him transmitting the virus to his
partner.
[0291] 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, 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.
[0292] Methods of medical treatment may involve treatment of cancer
by a method of ameliorating, treating, or preventing a malignancy
in a human subject wherein the steps of the method assist or boost
the immune system in eradicating cancerous cells. Such methods may
include the administration of cells, according to the present
invention that invoke an active (or achieve a passive) immune
response to destroy cancerous cells. Methods of treatment may
optionally include the co-administration of biological adjuvants
(e.g., interleukins, cytokines, Bacillus Comette-Guerin,
monophosphoryl lipid A, etc.) in combination with conventional
therapies for treating cancer such as chemotherapy, radiation, or
surgery. Methods of treatment may involve administering a
composition according to the present invention as a vaccine that
works by activating the immune system to prevent or destroy cancer
cell growth. Methods of medical treatment may also involve in vivo,
ex vivo, and adoptive immunotherapies, including those using
autologous and/or heterologous cells or immortalized cell
lines.
[0293] III. Pharmaceutical Compositions
[0294] In accordance with this disclosure, the term "pharmaceutical
composition" relates to a composition for administration to an
individual. In a particular 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 neoplasm, such as 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.
[0295] 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, water, emulsions, such
as oil/water emulsions, various types of wetting agents, sterile
solutions, etc. Compositions comprising such carriers can be
formulated by well-known conventional methods. These pharmaceutical
compositions can be administered to the subject at a suitable
dose.
[0296] The dosage regimen will be determined by the attending
physician and clinical factors. As is well known in the medical
arts, dosages for any one patient depends upon many factors,
including the patient's size, body surface area, age, the
particular compound to be administered, sex, time and route of
administration, general health, and other drugs being administered
concurrently. A particular dosage for administration might be in
the range of 2.times.10.sup.7 cells per m.sup.2 to
1.times.10.sup.10 cells per m.sup.2 of body surface area. Progress
can be monitored by periodic assessment.
[0297] The compositions of the disclosure may be administered
locally or systemically. In a preferred embodiment, the
pharmaceutical composition is administered subcutaneously and in an
even more preferred embodiment intravenously. Preparations for
parenteral administration include sterile aqueous or non-aqueous
solutions, suspensions, and emulsions. Examples of non-aqueous
solvents are propylene glycol, polyethylene glycol, vegetable oils
such as olive oil, and injectable organic esters such as ethyl
oleate. Aqueous carriers include water, alcoholic/aqueous
solutions, emulsions or suspensions, including saline and buffered
media. Parenteral vehicles include sodium chloride solution,
Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's,
or fixed oils. Intravenous vehicles include fluid and nutrient
replenishes, electrolyte replenishers (such as those based on
Ringer's dextrose), and the like. Preservatives and other additives
may also be present such as, for example, antimicrobials,
anti-oxidants, chelating agents, and inert gases and the like. In
addition, the pharmaceutical composition of the present disclosure
might comprise proteinaceous carriers, like, e.g., serum albumin or
immunoglobulin, preferably of human origin. It is envisaged that
the pharmaceutical composition of the disclosure might comprise, in
addition to the cells as described in this disclosure, further
biologically active agents, depending on the intended use of the
pharmaceutical composition.
[0298] IV. Combination Therapy
[0299] In certain embodiments of the disclosure that concern CTLs
generated against HPV antigen(s), methods of the present disclosure
for clinical aspects are combined with other agents effective in
the treatment of hyperproliferative disease, such as anti-cancer
agents. An "anti-cancer" agent is capable of negatively affecting
cancer in a subject, for example, by killing cancer cells, inducing
apoptosis in cancer cells, reducing the growth rate of cancer
cells, reducing the incidence or number of metastases, reducing
tumor size, inhibiting tumor growth, reducing the blood supply to a
tumor or cancer cells, promoting an immune response against cancer
cells or a tumor, preventing or inhibiting the progression of
cancer, or increasing the lifespan of a subject with cancer. More
generally, these other compositions may be provided in a combined
amount effective to kill or inhibit proliferation of the cell. This
may be achieved by contacting the cancer cell with a single
composition or pharmacological formulation that includes both
agents, or by contacting the cancer cell with two distinct
compositions or formulations, at the same time, wherein one
composition includes the expression construct and the other
includes the second agent(s). In other cases, administration of the
cells and a second composition may be separate and may have
separate administration routes and/or carriers.
[0300] Tumor cell resistance to chemotherapy and radiotherapy
agents represents a major problem in clinical oncology. One goal of
current cancer research is to find ways to improve the efficacy of
chemo- and/or radiotherapy by combining it with additional therapy.
In the context of the present disclosure, it is contemplated that
cell therapy could be used similarly in conjunction with
chemotherapeutic, radiotherapeutic, and/or immunotherapeutic
intervention, for example.
[0301] Alternatively, the present inventive therapy may precede or
follow the other agent treatment by intervals ranging from minutes
to weeks, months, or years. In embodiments where the other agent
and present invention are applied separately to the individual, one
would generally ensure that a significant period of time did not
expire between the time of each delivery, such that the agent and
inventive therapy would still be able to exert an advantageously
combined effect on the cell. In such instances, it is contemplated
that one may contact the cell with both modalities within about
12-24 h of each other and, more preferably, within about 6-12 h of
each other. In some situations, it may be desirable to extend the
time period for treatment significantly, however, where several
days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or
8) lapse between the respective administrations.
[0302] It is expected that the treatment cycles would be repeated
as necessary. It also is contemplated that various standard
therapies, as well as surgical intervention, may be applied in
combination with the inventive cell therapy.
[0303] Examples of HPV-associated cancer treatments (as an example)
that may be used in conjunction with cells produced from methods of
the disclosure include at least the following: 1) surgery (tumor
resection, neck dissection, conization, hysterectomy, and so
forth.); 2) drug therapy that may include Avastin.RTM.
(Bevacizumab); Blenoxane (Bleomycin); Hycamtin.RTM. (Topotecan
Hydrochloride); or a combination thereof; 3) radiotherapy; 4)
immunotherapy other than that of the disclosure; 5) hormone
therapy; or 6) a combination thereof.
[0304] V. Kits of the Disclosure
[0305] 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. Cytokine(s) 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.
[0306] In one embodiment a kit may comprise a container comprising
a quantity of T-cells obtained by a method of the present invention
formulated for administration to a subject (e.g. by admixture with
a suitable carrier, excipient, diluent, or adjuvant) preferably by
infusion, more preferably for administration by infusion in a
method of autologous adoptive cellular immunotherapy. The kit may
be maintained at a predetermined temperature, e.g. less than about
4.degree. C., less than about -2.degree. C. or less than about
-50.degree. C. The kit may further comprise instructions for the
storage and/or transport of the kit and/or for the administration
of the T-cells.
[0307] The kits may comprise a suitably aliquoted compositions of
the present invention. 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.
[0308] 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.
[0309] In some cases, reagents and/or devices to detect HPV
infection may be included in the kit. Examples include swabs,
spatulas, cytobrushes, slides, cover slips, cytology sample
collection receptacle(s), and so forth. Additional drugs for HPV
infection or cancer may be included in the kit, such as
Bevacizumab; Bleomycin; Topotecan Hydrochloride; or a combination
thereof.
[0310] VI. High IL-15
[0311] In some embodiments of aspects of the present disclosure
stimulations are performed in the presence of IL-15. In some
embodiments stimulations are performed in the presence of IL-7 and
IL-15. In some embodiments stimulations are performed in the
presence of IL-7 and IL-15 only. In some embodiments stimulations
are performed in the presence of IL-7 and IL-15 and in the absence
of IL-6 and/or IL-12).
[0312] In embodiments of the present disclosure where stimulations
are performed in the presence of IL-15, the IL-15 may be present in
the culture at a final concentration greater than 15 ng/ml. Final
IL-15 concentrations greater than 15 ng/ml may be referred to
herein as high IL-15. In some embodiments, the IL-15 may be present
in the culture at a final concentration of one of 20-1000 ng/ml,
20-900 ng/ml, 20-800 ng/ml, 20-700 ng/ml, 20-600 ng/ml, 20-500
ng/ml, 30-500 ng/ml, 40-500 ng/ml, 50-500 ng/ml, 50-400 ng/ml,
50-300 ng/ml, 50-200 ng/ml, 50-175 ng/ml, 50-150 ng/ml, 75-150
ng/ml, 75-125 ng/ml, 80-120 ng/ml, 90-110 ng/ml or 100 ng/ml.
Stimulations using high IL-15 are contemplated in particular in
connection with methods in which the populations of HPV-specific
immune cells are generated/expanded from within a population of
cells (e.g. blood cells, immune cells or PBMCs) depleted of CD45RA+
cells.
[0313] Stimulations using high IL-15 may be provided with
advantageous properties. For example, methods comprising
stimulations using high IL-15 may be useful to generate/expand
populations of immune cells specific for HPV with improved
efficiency (e.g. greater fold expansion within the same period of
time). Methods comprising stimulations using high IL-15 may be
useful for generating/expanding populations of immune cells
specific for HPV comprising a reduced number/frequency of
undesirable immune cell subsets (e.g. NK cells, regulatory T cells
(Tregs) and/or naive T cells), an increased proportion/frequency of
desirable immune cell subsets (e.g. CD8+ T cells, CD8+ cytotoxic T
lymphocytes, CD4+ T cells, CD4+ T helper cells,
IFN.gamma.-producing cells, memory T cells, central memory T cells,
antigen-experienced T cells, CD45RO+ T cells), and/or an increased
proportion/frequency of virus- and/or antigen-specific cells in the
generated/expanded population, as compared to populations
generated/expanded in accordance with prior art methods.
[0314] VII. HLA-Negative LCLs
[0315] In aspects of the present disclosure the costimulatory cells
used in a stimulation step described herein may be cells of a
lymphoblastoid cell line (LCL) lacking gene and/or protein
expression of MHC class I and/or MHC class II. In some embodiment
LCLs may lack surface expression of MHC class I and MHC class II;
such LCLs may be referred to herein as "HLA-negative LCLs",
"universal LCLs" or "ULCLs".
[0316] LCLs can be prepared by viral transformation of B cells.
LCLs are typically produced by transformation of B cells with
Epstein-Barr virus (EBV). Generation and characteristics of LCLs is
described in detail, for example, in Hui-Yuen et al., J Vis Exp
(2011) 57: 3321, and Hussain and Mulherkar, Int J Mol Cell Med
(2012) 1(2): 75-87, both hereby incorporated by reference in their
entirety. Briefly, LCLs can be produced by incubation of PBMCs with
concentrated cell culture supernatant of cells producing EBV, for
example B95-8 cells, in the presence of cyclosporin A.
[0317] HLA-negative LCLs may lack surface expression of an MHC
class I polypeptide and an MHC class II polypeptide. An "MHC class
I polypeptide" refers to a constituent polypeptide of an MHC class
I molecule (i.e. a polypeptide complex of an MHC class I .alpha.
chain polypeptide and a B2M polypeptide). An "MHC class II
polypeptide" refers to a constituent polypeptide of an MHC class II
molecule (i.e. a polypeptide complex of an MHC class II .alpha.
chain polypeptide and a MHC class II.beta. chain polypeptide).
Surface expression refers to expression of the relevant
polypeptide/polypeptide complex which is detectable at the cell
surface (i.e. in or at the cell membrane). Surface expression can
be analyzed e.g. on intact cells using an antigen-binding molecule
specific for a region of the polypeptide/polypeptide complex which
is extracellular to the cell when the polypeptide/polypeptide
complex is expressed at the cell surface.
[0318] In some embodiments the HLA-negative LCLs display
substantially no gene/protein expression of MHC class I and MHC
class II, e.g. as determined by an appropriate method for detecting
gene and/or protein expression. In some embodiments the
HLA-negative LCLs display substantially no surface expression of
MHC class I and MHC class II, e.g. as determined by analysis by
flow cytometry using an antibody capable of binding to MHC class I
and an antibody capable of binding to MHC class II. In such assays,
the level of staining of the HLA-negative LCLs by the relevant
antibodies may not be significantly greater than the level of
staining of the cells by appropriate negative control antibodies of
the same isotype.
[0319] HLA-negative LCLs may have been obtained by modification
(e.g. to a nucleic acid, e.g. by insertion, substitution or
deletion of one or more nucleotides) to reduce/prevent gene and/or
protein expression of one or more polypeptides of an MHC class I
molecule and an MHC class I molecule (e.g. B2M polypeptide, MHC
class I .alpha. chain polypeptide (e.g. HLA-A, HLA-B or HLA-C), MHC
class II .alpha. chain polypeptide (e.g. HLA-DPA1, HLA-DQA1,
HLA-DQA2 or HLA-DRA) and/or MHC class II .beta. chain polypeptide
(e.g. HLA-DPB1, HLA-DQB1, HLA-DQB2, HLA-DRB1, HLA-DRB3, HLA-DRB4 or
HLA-DRB5)). In some embodiments the HLA-negative LCLs comprise
modification to reduce/prevent gene and/or protein expression of an
MHC class I polypeptide (e.g. B2M) and modification to
reduce/prevent gene and/or protein expression of one or more MHC
class II polypeptides (e.g. HLA-DR, HLA-DQ, and HLA-DP) as compared
to gene and/or protein expression by an unmodified LCL. In some
embodiments the HLA-negative LCLs comprise modification to
reduce/prevent gene and/or protein expression of B2M, HLA-DRA,
HLA-DQA1, HLA-DQA2, and HLA-DP. In some embodiments the
HLA-negative LCLs may be obtained by targeted knockout of genes
encoding B2M, HLA-DRA, HLA-DQA1, HLA-DQA2, and HLA-DP, e.g. using
sequence specific nucleases (SSNs); gene editing using SSNs is
reviewed e.g. in Eid and Mahfouz, Exp Mol Med. 2016 October;
48(10): e265, which is hereby incorporated by reference in its
entirety. In some embodiments modification to reduce/prevent gene
and/or protein expression of an MHC class I polypeptide (e.g. B2M)
and/or modification to reduce/prevent gene and/or protein
expression of one or more MHC class II polypeptides (e.g. HLA-DR,
HLA-DQ, and HLA-DP) is achieved using CRISPR/Cas-9 systems
comprising crRNA targeting nucleic acid encoding the relevant
polypeptide(s). In some embodiments the HLA-negative LCLs are
obtained by sequential knockout of genes encoding B2M, HLA-DRA,
HLA-DQA1, HLA-DQA2, and HLA-DP.
[0320] In some embodiments the HLA-negative LCLs additionally
comprise modification to nucleic acid encoding one or more
polypeptides necessary for EBV replication/infection. LCLs
comprising modification to reduce/prevent EBV replication/infection
may be described herein as being EBV replication defective.
Accordingly, in some embodiments the HLA-negative LCLs are EBV
replication defective. In some embodiments the HLA-negative LCLs
comprise modification to nucleic acid (e.g. by insertion,
substitution or deletion of one or more nucleotides) encoding one
or more of BFLF1, BFLF2, BFRF1, BFRF2 and BFRF3 e.g. using SSNs. In
some embodiments the HLA-negative LCLs comprise modification to
nucleic acid encoding BFLF1 and/or nucleic acid encoding BFRF1. In
some embodiments modification is achieved using CRISPR/Cas-9
systems comprising crRNA targeting nucleic acid encoding the
relevant polypeptide(s). In some embodiments the HLA-negative LCLs
are obtained by a method comprising culture in the presence of an
agent suppressing viral replication (e.g. acyclovir). In some
embodiments the EBV replication defective HLA-negative LCLs
stimulate less proliferation of B cells from within a population of
PBMCs following co-culture with the PBMCs as compared to the level
of proliferation of B cells from within a population of PBMCs
following co-culture of the PBMCs with LCLs described in the prior
art. In some embodiments the EBV replication defective HLA-negative
LCLs lack the ability to promote outgrowth of B cells in a
co-culture with PBMCs. HLA-negative LCLs modified to reduce/prevent
gene and/or protein expression of one or more polypeptides
necessary for EBV replication may have an improved safety profile
as compared to LCLs lacking modification to reduce/prevent gene
and/or protein expression of one or more polypeptides necessary for
EBV replication.
[0321] The HLA-negative LCLs of the present disclosure are employed
as costimulatory cells in the manner in which costimulatory cells
are typically employed in methods for generating/expanding
virus-specific T cells. For example, the HLA-negative LCLs may be
provided as an irradiated cell population, and at appropriate
ratios to responder cells and antigen presenting cells (APCs). In
some embodiments the HLA-negative LCLs are irradiated or treated
with a substance (e.g. mitomycin C) to prevent their proliferation,
prior to use in stimulations. Irradiation of LCLs in accordance
with the present methods is typically at 6000 to 12000 rads. In
some embodiments the HLA-negative LCLs are present in a stimulation
comprising responder cells, APCs and HLA-negative LCLs at a ratio
of responder cells:APCs:HLA-negative LCLs of one of 1:1:1 to
1:1:10, e.g. about 1:1:5.
[0322] HLA-negative LCLs described herein may be provided with
advantageous properties relevant to their use as costimulatory
cells in methods for generating/expanding populations of
HPV-specific immune cells. For example, the HLA-negative LCLs may
be useful to generate/expand populations of immune cells specific
for HPV with improved efficiency (e.g. greater fold expansion
within the same period of time). The HLA-negative LCLs may be
useful in methods for generating/expanding populations of immune
cells specific for HPV comprising a reduced number/frequency of
undesirable immune cell subsets (e.g. NK cells, regulatory T cells
(Tregs) and/or naive T cells), an increased proportion/frequency of
desirable immune cell subsets (e.g. CD8+ T cells, CD8+ cytotoxic T
lymphocytes, CD4+ T cells, CD4+ T helper cells,
IFN.gamma.-producing cells, memory T cells, central memory T cells,
antigen-experienced T cells, CD45RO+ T cells), and/or an increased
proportion/frequency of virus- and/or antigen-specific cells in the
generated/expanded population, as compared to populations
generated/expanded in accordance with prior art methods.
[0323] HLA-negative LCLs are useful for generating/expanding
populations of HPV-specific immune cells having reduced
alloreactivity as compared to prior art methods for
generating/expanding populations of HPV-specific immune cells.
Accordingly, HLA-negative LCLs are useful for generating/expanding
populations of HPV-specific immune cells for use in both autologous
and allogeneic applications. In some embodiments the HLA-negative
LCLs stimulate less proliferation of PBMCs in a coculture
comprising the HLA-negative LCLs and PBMCs obtained from an
allogeneic donor as compared to HLA-positive LCLs (i.e. LCLs
described in the prior art). In some embodiments the HLA-negative
LCLs stimulate less proliferation of PBMCs in a coculture
comprising the HLA-negative LCLs and PBMCs obtained from a
HLA-matched donor as compared to HLA-positive LCLs.
[0324] VIII. Dendritic Cells
[0325] In aspects of the present disclosure the dendritic cells
used in a stimulation step described herein may be derived from
cells within a population of blood cells obtained from a subject.
In some embodiments the dendritic cells are derived from cells
within a population of peripheral blood mononuclear cells (PBMCs).
In some embodiments the dendritic cells are derived from CD14+
positive cells within a population of PBMCs. In some embodiments
the dendritic cells are monocyte-derived dendritic cells (also
referred to herein as moDCs).
[0326] In some embodiments the dendritic cells are obtained by a
process comprising separating CD14+ cells from other cells (i.e.
CD14- cells), e.g. from within a population of PBMCs. Separation
may be performed by appropriate positive or negative selection. In
some embodiments, CD14+ positive cells may be positively selected
from within a population of cells using beads coated with antibody
capable of binding specifically to CD14. For example, CD14+ cells
may be isolated by MACS cell separation using CD14 MicroBeads
(Miltenyi Biotec).
[0327] In some embodiments the dendritic cells are obtained by a
process comprising culturing cells in the presence of factors
promoting differentiation of immature dendritic cells (also
referred to herein as iDCs) from CD14+ cells. The cells cultured in
the presence of factors promoting differentiation of iDCs may be
e.g. PBMCs, or CD14+ cells isolated from a population of PBMCs.
Cells may be cultured at any suitable density, e.g. one of
0.1.times.10.sup.6 to 1.times.10.sup.6 cells/ml, 0.2.times.10.sup.6
to 0.9.times.10.sup.6 cells/ml, 0.3.times.10.sup.6 to
0.8.times.10.sup.6 cells/ml, 0.4.times.10.sup.6 to
0.7.times.10.sup.6 cells/ml or 0.5.times.10.sup.6 cells/ml. Factors
promoting differentiation of immature dendritic cells may include
IL-4 and/or GM-CSF. In some embodiments the culture comprises IL-4
and GM-CSF. The IL-4 may be present in the culture at a final
concentration of one of 50-1000 IU/ml, 100-800 IU/ml, 150-700
IU/ml, 200-600 IU/ml, 250-500 IU/ml, 300-500 IU/ml or 400 IU/ml.
The GM-CSF may be present in the culture at a final concentration
of one of 100-1500 IU/ml, 200-1400 IU/ml, 300-1300 IU/ml, 400-1200
IU/ml, 500-1100 IU/ml, 600-1000 IU/ml, 700-900 IU/ml or 800 IU/ml.
The culture may be for any suitable period of time for
differentiation of immature DCs, e.g. one of 1 day, 2 days, 3 days,
4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days.
[0328] In some embodiments the dendritic cells used in a
stimulation step described herein are obtained by a process
comprising culturing cells (i.e. precursor cells of mature
dendritic cells, e.g. immature dendritic cells) to mature dendritic
cells. In some embodiments the process comprises culturing the
cells in the presence of factors promoting differentiation of
mature DCs (e.g. moDCs). The cells cultured in the presence of
factors promoting differentiation of mature DCs may be e.g. PBMCs,
CD14+ cells isolated (i.e. separated and/or purified) from a
population of PBMCs, or immature dendritic cells (iDCs). Cells may
be cultured at any suitable density, e.g. one of 0.1.times.10.sup.6
to 1.times.10.sup.6 cells/ml, 0.2.times.10.sup.6 to
0.9.times.10.sup.6 cells/ml, 0.3.times.10.sup.6 to
0.8.times.10.sup.6 cells/ml, 0.4.times.10.sup.6 to
0.7.times.10.sup.6 cells/ml or 0.5.times.10.sup.6 cells/ml. Factors
promoting differentiation of immature dendritic cells to mature DCs
may be one or more of GM-CSF, IL-4, IL-1.beta., IL-6, TNF.alpha.,
PGE-1, CD40L, Poly (I:C), MPLA, Resiquimod, IFN.alpha., IFN.gamma.
or AmpB. In some embodiments the culture comprises IL-4 and GM-CSF.
In some embodiments the culture comprises GM-CSF, IL-4, IL-1.beta.,
IL-6, TNF.alpha. and PGE-1. In some embodiments the culture
comprises GM-CSF, IL-4, IL-1.beta., IL-6 and TNF.alpha.. In some
embodiments the culture comprises GM-CSF, IL-4, IL-1.beta., IL-6,
TNF.alpha., PGE-1 and CD40L. In some embodiments the culture
comprises GM-CSF, IL-4, IL-1.beta., IL-6, TNF.alpha. and CD40L. In
some embodiments the culture comprises GM-CSF, IL-4, IL-1.beta.,
IL-6, TNF.alpha., PGE-1 and Poly (I:C). In some embodiments the
culture comprises GM-CSF, IL-4, IL-1.beta., IL-6, TNF.alpha. and
Poly (I:C). In some embodiments the culture comprises GM-CSF, IL-4,
IL-1.beta., IL-6, TNF.alpha., PGE-1 and MPLA. In some embodiments
the culture comprises GM-CSF, IL-4, IL-1.beta., IL-6, TNF.alpha.
and MPLA. In some embodiments the culture comprises GM-CSF, IL-4,
IL-1.beta., IL-6, TNF.alpha., PGE-1 and Resiquimod. In some
embodiments the culture comprises GM-CSF, IL-4, IL-1.beta., IL-6,
TNF.alpha. and Resiquimod. In some embodiments the culture
comprises GM-CSF, IL-4, IL-1.beta., IL-6, TNF.alpha., PGE-1 and
IFN.alpha.. In some embodiments the culture comprises GM-CSF, IL-4,
IL-1.beta., IL-6, TNF.alpha. and IFN.alpha.. In some embodiments
the culture comprises GM-CSF, IL-4, IL-1.beta., IL-6, TNF.alpha.,
PGE-1 and IFN.gamma.. In some embodiments the culture comprises
GM-CSF, IL-4, IL-1.beta., IL-6, TNF.alpha. and IFN.gamma.. In some
embodiments the culture comprises GM-CSF, IL-4, IL-1.beta., IL-6,
TNF.alpha., PGE-1, IFN.alpha. and IFN.gamma.. In some embodiments
the culture comprises GM-CSF, IL-4, IL-1.beta., IL-6, TNF.alpha.,
IFN.alpha. and IFN.gamma.. In some embodiments the culture
comprises GM-CSF, IL-4, IL-1.beta., IL-6, TNF.alpha., PGE-1, Poly
(I:C) and MPLA. In some embodiments the culture comprises GM-CSF,
IL-4, IL-1.beta., IL-6, TNF.alpha., Poly (I:C) and MPLA. In some
embodiments the culture comprises GM-CSF, IL-4, MPLA and
IFN.gamma.. In some embodiments the culture comprises GM-CSF and
IFN.alpha.. In some embodiments the culture comprises GM-CSF, IL-4,
IFN.gamma. and AmpB. The IL-4 may be present in the culture at a
final concentration of one of 50-1000 IU/ml, 100-800 IU/ml, 150-700
IU/ml, 200-600 IU/ml, 250-500 IU/ml, 300-500 IU/ml or 400 IU/ml.
The GM-CSF may be present in the culture at a final concentration
of one of 100-1500 IU/ml, 200-1400 IU/ml, 300-1300 IU/ml, 400-1200
IU/ml, 500-1100 IU/ml, 600-1000 IU/ml, 700-900 IU/ml or 800 IU/ml.
The IL-1.beta. may be present in the culture at a final
concentration of one of 1-100 pg/ml, 1-75 pg/ml, 5-50 pg/ml, 5-40
pg/ml, 5-30 pg/ml, 5-25 pg/ml, 5-20 pg/ml, 5-15 pg/ml or 10 pg/ml.
The IL-6 may be present in the culture at a final concentration of
one of 10-1000 pg/ml, 10-750 pg/ml, 50-500 pg/ml, 50-400 pg/ml,
50-300 pg/ml, 50-250 pg/ml, 50-200 pg/ml, 50-150 pg/ml or 100
pg/ml. The TNF.alpha. may be present in the culture at a final
concentration of one of 1-100 pg/ml, 1-75 pg/ml, 5-50 pg/ml, 5-40
pg/ml, 5-30 pg/ml, 5-25 pg/ml, 5-20 pg/ml, 5-15 pg/ml or 10 pg/ml.
The PGE-1 may be present in the culture at a final concentration of
one of 0.1-10 ng/ml, 0.1-7.5 ng/ml, 0.5-5 ng/ml, 0.5-4 ng/ml, 0.5-3
ng/ml, 0.5-2.5 ng/ml, 0.5-2 ng/ml, 0.5-1.5 ng/ml or 1 ng/ml. The
CD40L may be present in the culture at a final concentration of one
of 10-1000 pg/ml, 10-750 pg/ml, 50-500 pg/ml, 50-400 pg/ml, 50-300
pg/ml, 50-250 pg/ml, 50-200 pg/ml, 50-150 pg/ml or 100 pg/ml. The
Poly (I:C) may be present in the culture at a final concentration
of one of 0.1-10 .mu.g/ml, 0.1-7.5 .mu.g/ml, 0.5-5 .mu.g/ml, 0.5-4
.mu.g/ml, 0.5-3 .mu.g/ml, 0.5-2.5 .mu.g/ml, 0.5-2 .mu.g/ml, 0.5-1.5
.mu.g/ml or 1 .mu.g/ml. The MPLA may be present in the culture at a
final concentration of one of 0.1-10 .mu.g/ml, 0.1-7.5 .mu.g/ml,
0.5-5 .mu.g/ml, 0.5-4 .mu.g/ml, 0.5-3 .mu.g/ml, 0.5-2.5 .mu.g/ml,
0.5-2 .mu.g/ml, 0.5-1.5 .mu.g/ml or 1 .mu.g/ml. The Resiquimod may
be present in the culture at a final concentration of one of 0.1-10
.mu.g/ml, 0.1-7.5 .mu.g/ml, 0.5-5 .mu.g/ml, 0.5-4 .mu.g/ml, 0.5-3
.mu.g/ml, 0.5-2.5 .mu.g/ml, 0.5-2 .mu.g/ml, 0.5-1.5 .mu.g/ml or 1
.mu.g/ml. The IFN.alpha. may be present in the culture at a final
concentration of one of 10-1000 ng/ml, 10-750 ng/ml, 50-500 ng/ml,
50-400 ng/ml, 50-300 ng/ml, 50-250 ng/ml, 50-200 ng/ml, 50-150
ng/ml or 100 ng/ml. The IFN.gamma. may be present in the culture at
a final concentration of one of 10-1000 ng/ml, 10-750 ng/ml, 50-500
ng/ml, 50-400 ng/ml, 50-300 ng/ml, 50-250 ng/ml, 50-200 ng/ml,
50-150 ng/ml or 100 ng/ml. The AmpB may be present in the culture
at a final concentration of one of 0.1-10 .mu.g/ml, 0.1-7.5
.mu.g/ml, 0.5-5 .mu.g/ml, 0.5-4 .mu.g/ml, 0.5-3 .mu.g/ml, 0.5-2.5
.mu.g/ml, 0.5-2 .mu.g/ml, 0.5-1.5 .mu.g/ml or 1 .mu.g/ml. The
culture may be for any suitable period of time for differentiation
of mature DCs, e.g. one of 6 hours to 5 days, 12 hours to 4 days,
12 hours to 72 hours, 12 hours to 48 hours, 12 hours to 36 hours,
or 24 hours. In some embodiments the dendritic cells used in
stimulations are obtained by a process comprising culturing
immature DCs in the presence of GM-CSF, IL-4, IL-1.beta., IL-6,
TNF.alpha. and CD40L. In some embodiments the dendritic cells used
in stimulations are obtained by a process comprising culturing
immature DCs in the presence of GM-CSF, IL-4, MPLA and
IFN.gamma..
[0329] Dendritic cells obtained by culture according to the methods
described herein may display higher surface expression of CD80
and/or CD83 as compared to dendritic cells obtained by culture
according to methods disclosed in the prior art. Populations of
dendritic cells obtained by culture according to the methods
described herein may comprise a higher proportion of cells
displaying surface expression of CD80 and/or CD83 as compared to
populations of dendritic cells obtained by culture according to
methods disclosed in the prior art.
[0330] Dendritic cells obtained by culture according to the methods
described herein may be provided with advantageous properties
relevant to their use as antigen-presenting cells (APCs) in methods
for expanding HPV-specific immune cells. For example, the DCs may
be useful to generate/expand populations of immune cells specific
for HPV with improved efficiency (e.g. greater fold expansion
within the same period of time). The DCs obtained by culture
according to the methods described herein may be useful in methods
for generating/expanding populations of immune cells specific for
HPV comprising a reduced number/frequency of undesirable immune
cell subsets (e.g. NK cells, regulatory T cells (Tregs) and/or
naive T cells), an increased proportion/frequency of desirable
immune cell subsets (e.g. CD8+ T cells, CD8+ cytotoxic T
lymphocytes, CD4+ T cells, CD4+ T helper cells,
IFN.gamma.-producing cells, memory T cells, central memory T cells,
antigen-experienced T cells, CD45RO+ T cells), and/or an increased
proportion/frequency of virus- and/or antigen-specific cells in the
generated/expanded population, as compared to populations
generated/expanded in accordance with prior art methods.
[0331] VIII. CD45RA+ Cell Depletion
[0332] In aspects of the present disclosure, populations of
HPV-specific immune cells are generated/expanded from a population
of cells (e.g. blood cells, immune cells or PBMCs) depleted of
CD45RA+ cells (i.e. a CD45RA- cell population). In some embodiments
the populations of HPV-specific immune cells are generated/expanded
from a population of PBMCs depleted of CD45RA+ cells. In some
embodiments the depletion of CD45RA+ cells may be achieved by
separation of CD45RA- cells from other cells (i.e. CD45RA+ cells).
Separation may be performed by appropriate positive or negative
selection. In some embodiments, CD45RA- cells may be separated from
CD45RA+ cells using beads coated with antibody capable of binding
specifically to CD45RA. For example, CD45RA- cells may be separated
from CD45RA+ cells by MACS cell separation using CD45RA MicroBeads
(Miltenyi Biotec). In some embodiments the population of cells may
additionally be depleted of CD14+ cells.
[0333] Depletion of CD45RA+ cells may remove NK cells, Tregs and/or
naive T cells, preventing substantial outgrowth of these cell types
during stimulations according to the present disclosure. Depletion
of CD45RA+ cells may also enrich the initial population of cells
for CD45RO+ cells and/or antigen-experienced T cells, promoting
preferential expansion of these desirable cell types in
stimulations according to the present disclosure.
[0334] In some embodiments depletion of CD45RA+ cells is
contemplated especially in methods employing IL-7 and IL-15 in one
or more stimulations, and in particular where high concentrations
of IL-15 are used (i.e. concentrations greater than 15 ng/ml, e.g.
.about.100 ng/ml), because such methods may be particularly
susceptible to the problem of NK cell outgrowth. Depletion of
CD45RA+ cells from the starting population of cells used in
expansions is also contemplated in particular in methods employing
K562cs costimulatory cells in methods for generating/expanding
populations of HPV-specific immune cells, and/or methods in which
HPV-specific immune cells are expanded from populations of immune
cells (e.g. PBMCs) obtained from a patient, e.g. a patient having
an HPV-associated disease.
[0335] Generation/expansion of populations of HPV-specific immune
cells from within a population of cells (e.g. blood cells, immune
cells or PBMCs) depleted of CD45RA+ cells may be provided with
advantages over prior art methods for generating/expanding
populations of HPV-specific immune cells. For example, the immune
cell populations depleted of CD45RA+ cells may be useful to
generate/expand populations of immune cells specific for HPV with
improved efficiency (e.g. greater fold expansion within the same
period of time). Immune cell populations depleted of CD45RA+ cells
(e.g. PBMCs depleted of CD45RA+ cells) may be useful in methods for
generating/expanding populations of immune cells specific for HPV
comprising a reduced number/frequency of undesirable immune cell
subsets (e.g. NK cells, regulatory T cells (Tregs) and/or naive T
cells), an increased proportion/frequency of desirable immune cell
subsets (e.g. CD8+ T cells, CD8+ cytotoxic T lymphocytes, CD4+ T
cells, CD4+ T helper cells, IFN.gamma.-producing cells, memory T
cells, central memory T cells, antigen-experienced T cells, CD45RO+
T cells), and/or an increased proportion/frequency of virus- and/or
antigen-specific cells in the generated/expanded population, as
compared to populations generated/expanded in accordance with prior
art methods.
[0336] IX. Allogeneic and Autologous Applications
[0337] The methods disclosed herein are contemplated in the context
of generating/expanding HPV-specific immune cells for use in both
autologous and allogeneic applications, e.g. cellular
immunotherapies. Populations of immune cells specific for HPV
prepared in accordance with the methods disclosed herein may be
used after their generation/expansion, or may be frozen for use at
a later date.
[0338] In some embodiments the generated/expanded populations of
immune cells specific for HPV are prepared for use in the subject
from which the initial population of immune cells from which the
population is generated/expanded was obtained/derived. In some
embodiments the generated/expanded populations of immune cells
specific for HPV are prepared for use with any subject, e.g. a
different subject to the subject from which the initial population
of immune cells from which the population is generated/expanded was
obtained/derived.
[0339] Accordingly, in some embodiments the generated/expanded
populations of immune cells specific for HPV are adoptively
transferred to the subject from which the initial population of
immune cells from which the population is generated/expanded was
obtained/derived. In some embodiments the generated/expanded
populations of immune cells specific for HPV are adoptively
transferred to a different subject from the subject from which the
initial population of immune cells from which the population is
generated/expanded was obtained/derived.
EXAMPLES
[0340] 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
[0341] 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+) CTLs, that are
consistently specific for a variety of antigens derived from one or
more human papillomaviruses that can prove fatal. The disclosure is
readily adaptable to clinical implementation and can be used as an
"off the shelf" HPV antiviral agent. The methods and compositions
are readily adaptable to clinical implementation and are useful as
a safe and effective HPV antiviral agent for individuals.
[0342] In specific embodiments, peripheral blood T-cells were
stimulated with monocyte-derived dendritic cells loaded with
pepmixes [peptide libraries of 15-mers overlapping by 11 amino
acids (aa)] spanning E6/E7, in the presence or absence of specific
accessory cytokines. The resulting T-cell lines were further
expanded with pepmix-loaded activated B-cell blasts. There was
successfully reactivation and expansion (>1200-fold) of
E6-specific/E7-specific T-cells from 8/16 cervical and 33/52
oropharyngeal cancer patients.
[0343] The presence of the cytokines interleukin (IL)-6, IL-7,
IL-12, and IL-15 is useful in the method, in specific embodiments
of the methods. The produced T-cell lines possess the desirable
characteristics of polyclonality, multiple T-cell subset
representation (including the memory compartment) and a TH1 bias,
and eliminate E6/E7 targets. The disclosure has shown that it is
possible to robustly generate HPV16 E6/E7-directed T-cell lines
from patients with HPV16-associated cancers. Because the technique
is scalable and good-manufacturing procedures-compliant, these
lines are useful for adoptive cellular immunotherapy of patients
with HPV16 cancers and may be applied to HPV18 cancers also.
[0344] Known methods for producing T-cells for HPV16 are
demonstrated in FIG. 1A, showing results for 3 HPV-associated
cancer patients (.gamma.IFN ELISpot assay obtained in cell lines
obtained after stimulation of PBMCs by DCs loaded with only
HPV16-pepmix). In FIG. 1B, results are shown for 2 of the patients
whose results are also demonstrated in FIG. 1A, in addition to a
third individual. FIG. 1B shows results of .gamma.IFN ELISpot assay
for cell lines obtained after stimulation of PBMCs by DCs loaded
with HPV16-pepmix and HPV18-pepmix. Reactivity against both HPV16
and HPV18 antigens can be detected (not all patients will have
reactivity against both serotypes).
[0345] Turning to specifics of the methods, in certain cases DCs
are loaded with HPV16-E6/E7 and HPV18-E6/E7 pepmix libraries. In
such cases, the cell lines are able to recognize both HPV16 and
HPV18 E6 and E7 antigens (instead of only HPV16 antigens, for
example). 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 HPV-specific T-cells after initial
generation/expansion with DCs occurs not with autologous B-blasts
loaded with pepmixes in the presence of IL-15 but instead utilizes
autologous, polyclonal activated T-cells loaded with pepmix, in the
presence of costimulatory cells (CD80/CD86/CD83/4-1BBL), and IL-7
and IL-15. Employing these conditions, T-cell expansion occurs at a
more rapid rate, at least 10-fold as that obtained by known
methods, with successful demonstration having occurred after 3
rounds of stimulation and without loss of specificity.
[0346] Summary of fold cellular expansion using the known method
with 3 HPV-associated cancer patients is provided in Table 2.
TABLE-US-00002 TABLE 2 Fold expansion at the end of each
stimulation with known method After 1.sup.st stimulation After
2.sup.nd stimulation After 3.sup.rd stimulation Patient ID (with DC
and IL-2/15) (with DC and IL-2/15) (with B-blasts and IL-2/15) OPA
3.38 2.63 4.96 OPE 1.59 4.60 1.60 OPY 2.20 2.50 0.38
[0347] A summary of fold cellular expansion using a novel method of
the disclosure with 3 HPV-associated cancer patients is shown in
Table 3. Fold expansion after 3 rounds of stimulation is on average
approximately 50 times higher than using a known method.
Specificity is maintained (illustrated in #1).
TABLE-US-00003 TABLE 3 Fold expansion at the end of each
stimulation with a method of the disclosure After 3.sup.rd
stimulation After 1.sup.st stimulation After 2.sup.nd stimulation
(with activated T-cells, Patient ID (with DC and IL-7/15) (with DC
and IL-7/15) costim cells and IL-7/15) PDC 2.60 5.35 151.20 PGD
5.00 6.36 120.00 PJK 3.58 5.53 60.00
Example 2
Protocol for the Expansion of HPV T-Cells
[0348] HPV stimulated T-cells (HPVST) are first activated by HPV
E6/E7 peptide-pulsed autologous dendritic cells (DCs) at 10-20:1
PBMC:DC ratio, and cultured for 8 days in culture medium containing
IL-6 (100 ng/ml), IL-7 (10 ng/ml), IL-12 (10 ng/ml), IL-15 (10
ng/ml) (e.g. per the first stimulation step described by Ramos et
al., (J Immunother 2013; 36:66-76)).
[0349] A second stimulation step on day 9 is carried out using
peptide-pulsed DCs at 5-10:1 PBMC:DC ratio in media containing IL-7
(10 ng/ml) and IL-15 (100 ng/ml).
[0350] Subsequent weekly stimulation/expansion steps are then
carried to achieve a desired number of HPVSTs out using HPV E6/E7
peptide-pulsed autologous T-APC at 1:1 ratio, in the presence of
equal number of irradiated allogeneic K562-cs co-stimulatory cells,
and in media containing IL-7 (10 ng/ml) and IL-15 (100 ng/ml). The
polyclonal T cells (T-APCs) are generated using a portion of the
autologous PBMC isolated from the venesected blood. The cells are
activated by culturing in cell culture plates that are coated with
anti-CD3 and anti-CD28 antibodies. The cells are then cultured to
expand in the presence of IL-2 for 2 weeks. The expanded T-APC can
be cryopreserved for later use. 2-3 days prior to using T-APC for
HPVST re-stimulation (3rd cycle of HPVST re-stimulation and
onward), cryopreserved cells are thawed and re-stimulated in
anti-CD3 and anti-CD28 antibody-coated cell culture plates. On the
day of HPVST re-stimulation, the T-APC cells are harvested and
pulsed with the HPV E6/E7 peptides, followed by adding to the
on-going culture of HPVST at 1:1 ratio.
Example 3
In Vivo Expansion and Persistence of Infused HPVSTs in Human
Patients
[0351] HPVSTs obtained from Example 2 were transduced with a
dominant negative receptor for TGF-beta (DNRII) [see Foster et al.,
Antitumor activity of EBV-specific T lymphocytes transduced with a
dominant negative TGF-beta receptor. J Immunother. 2008;
31:500-505].
[0352] The HPVSTs were administered to two human patients and
tested for in vivo expansion and persistence. Patient #1 had widely
metastatic oropharyngeal cancer and had discontinued prior therapy.
Patient #2 had oropharyngeal cancer metastatic to the neck and was
receiving concomitant nivolumab treatment, without prior response
to nivolumab alone.
[0353] In vivo expansion and persistence of infused HPVSTs was
assessed by qPCR for the DNRII gene performed in PBMCs isolated
from peripheral blood from the patient. Data points in FIGS. 2 and
3 represent critical post-infusion intervals after the infusion of
HPVSTs.
[0354] In patient #1 progressive expansion was observed (FIG. 2).
In patient #2 although expansion was limited (FIG. 3), with a peak
at 6 weeks coinciding with re-infusion of HPVSTs, the patient had a
partial clinical response. Six weeks after HPVST infusion patient
#2 exhibited a decrease in disease burden measured by PET scan and
physical examination compared to a pre-treatment baseline (FIG.
4).
Example 4
Optimization of DC Maturation
[0355] Blood samples were collected from EBV-reactive donors in
heparin vacutainers and PBMCs were isolated from the blood samples
by Ficoll density gradient centrifugation. CD14 MicroBeads
(Miltenyi Biotec) were then used to isolate CD14+ cells from within
the PBMCs by positive selection. The number of cells was counted,
and the CD14- cell population was frozen and stored. [0275] The
CD14+ cells were then differentiated to immature dendritic cells
(iDCs). Briefly, CD14+ cells were cultured in wells of a 24-well
plate at a concentration of 0.5.times.10.sup.6 cells/ml in DC cell
culture medium containing 400 IU/ml IL-4 and 800 IU/ml GM-CSF, for
5 days.
[0356] The iDCs were then matured to mature DCs by culture for 24
hours in DC cell culture medium according to the conditions shown
in one of 1 to 20 in Table 4 below:
TABLE-US-00004 TABLE 4 DC Maturation Culture Conditions Condition
No. Conditions 1 800 U/ml GM-CSF, 400 U/ml IL-4 2 800 U/ml GM-CSF,
400 U/ml IL-4, 10 pg/ml IL-1.beta., 100 pg/ml IL-6, 10 pg/ml
TNF.alpha., 1 ng/ml PGE-1 3 800 U/ml GM-CSF, 400 U/ml IL-4, 10
pg/ml IL-1.beta., 100 pg/ml IL-6, 10 pg/ml TNF.alpha. 4 800 U/ml
GM-CSF, 400 U/ml IL-4, 10 pg/ml IL-1.beta., 100 pg/ml IL-6, 10
pg/ml TNF.alpha., 1 ng/ml PGE-1, 100 pg/ml CD40L 5 800 U/ml GM-CSF,
400 U/ml IL-4, 10 pg/ml IL-1.beta., 100 pg/ml IL-6, 10 pg/ml
TNF.alpha., 100 pg/ml CD40L 6 800 U/ml GM-CSF, 400 U/ml IL-4, 10
pg/ml IL-1.beta., 100 pg/ml IL-6, 10 pg/ml TNF.alpha., 1 ng/ml
PGE-1, 1 .mu.g/ml Poly (I:C) 7 800 U/ml GM-CSF, 400 U/ml IL-4, 10
pg/ml IL-1.beta., 100 pg/ml IL-6, 10 pg/ml TNF.alpha., 1 .mu.g/ml
Poly (I:C) 8 800 U/ml GM-CSF, 400 U/ml IL-4, 10 pg/ml IL-1.beta.,
100 pg/ml IL-6, 10 pg/ml TNF.alpha., 1 ng/ml PGE-1, 1 .mu.g/ml MPLA
9 800 U/ml GM-CSF, 400 U/ml IL-4, 10 pg/ml IL-1.beta., 100 pg/ml
IL-6, 10 pg/ml TNF.alpha., 1 .mu.g/ml MPLA 10 800 U/ml GM-CSF, 400
U/ml IL-4, 10 pg/ml IL-1.beta., 100 pg/ml IL-6, 10 pg/ml
TNF.alpha., 1 ng/ml PGE-1, 1 .mu.g/ml Resiquimod 11 800 U/ml
GM-CSF, 400 U/ml IL-4, 10 pg/ml IL-1.beta., 100 pg/ml IL-6, 10
pg/ml TNF.alpha., 1 .mu.g/ml Resiquimod 12 800 U/ml GM-CSF, 400
U/ml IL-4, 10 pg/ml IL-1.beta., 100 pg/ml IL-6, 10 pg/ml
TNF.alpha., 1 ng/ml PGE-1, 100 ng/ml IFN.alpha. 13 800 U/ml GM-CSF,
400 U/ml IL-4, 10 pg/ml IL-1.beta., 100 pg/ml IL-6, 10 pg/ml
TNF.alpha., 100 ng/ml IFN.alpha. 14 800 U/ml GM-CSF, 400 U/ml IL-4,
10 pg/ml IL-1.beta., 100 pg/ml IL-6, 10 pg/ml TNF.alpha., 1 ng/ml
PGE-1, 100 ng/ml IFN.gamma. 15 800 U/ml GM-CSF, 400 U/ml IL-4, 10
pg/ml IL-1.beta., 100 pg/ml IL-6, 10 pg/ml TNF.alpha., 100 ng/ml
IFN.gamma. 16 800 U/ml GM-CSF, 400 U/ml IL-4, 10 pg/ml IL-1.beta.,
100 pg/ml IL-6, 10 pg/ml TNF.alpha., 1 ng/ml PGE-1, 100 ng/ml
IFN.alpha., 100 ng/ml IFN.gamma. 17 800 U/ml GM-CSF, 400 U/ml IL-4,
10 pg/ml IL-1.beta., 100 pg/ml IL-6, 10 pg/ml TNF.alpha., 100 ng/ml
IFN.alpha., 100 ng/ml IFN.gamma. 18 800 U/ml GM-CSF, 400 U/ml IL-4,
10 pg/ml IL-1.beta., 100 pg/ml IL-6, 10 pg/ml TNF.alpha., 1 ng/ml
PGE-1, 1 .mu.g/ml Poly (I:C), 1 .mu.g/ml MPLA 19 800 U/ml GM-CSF,
400 U/ml IL-4, 10 pg/ml IL-1.beta., 100 pg/ml IL-6, 10 pg/ml
TNF.alpha., 1 .mu.g/ml Poly (I:C), 1 .mu.g/ml MPLA 20 800 U/ml
GM-CSF, 400 U/ml IL-4, 1 .mu.g/ml MPLA, 100 ng/ml IFN.gamma.
[0357] The mature DCs were then analyzed by flow cytometry for
expression of CD80, CD83, CCR7 and PD-L1. The previously frozen
CD14- fraction was thawed and rested in cell culture medium for 24
hours.
[0358] Immature and mature DCs were then pulsed with individual EBV
antigen pepmixes (LMP1, LMP2, BRAF1 or EBNA1), and the
peptide-pulsed DCs were used to stimulate the autologous CD14-
cells by coculture for 9 days in the presence of IL-7 (10 ng/ml)
and IL-15 (100 ng/ml). The expanded T cell populations were then
analyzed by flow cytometry for expression of CCR7, CD45RO, EBV
peptide reactivity and intracellular staining for IFN.gamma..
[0359] FIGS. 5A and 5B show CD83, CD80, CCR7 and PD-L1 expression
by monocyte-derived DCs derived from Donor 1 CD14+ PBMCs following
culture according to experimental conditions 1, 2, 3, 4, 5, 6, 7,
8, 9, 10 or 11 (see Table 4).
[0360] FIGS. 6A and 6B show CCR7 and CD45RO expression by CD4+ T
cell and CD8+ T cells obtained following stimulation of autologous
CD14- PBMCs derived from Donor 1 for 9 days with EBV peptide-pulsed
mature DCs cultured according to experimental conditions 1, 2, 3,
4, 5, 6, 7, 8, 9, 10 or 11. No major differences in T cell memory
phenotype were observed for the expanded T cell populations.
[0361] FIG. 7 shows the total number of virus-specific T cells
obtained following stimulation of autologous CD14- PBMCs derived
from Donor 1 for 9 days with EBV peptide-pulsed mature DCs cultured
according to experimental conditions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10
or 11.
[0362] FIG. 8 shows the proportions of IFN.gamma.+CD8+ CTLs and
IFN.gamma.+CD4+ Th cells obtained following stimulation of
autologous CD14- PBMCs derived from Donor 1 for 9 days with EBV
peptide-pulsed mature DCs cultured according to experimental
conditions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, as determined by
ELISPOT analysis. Frequencies shown are subtracted for background
signal obtained in control conditions performed in the absence of
EBV peptide stimulation.
[0363] FIG. 9 shows the proportions of EBV antigen-reactive
IFN.gamma.+CD8+ CTLs and IFN.gamma.+CD4+ Th cells obtained
following stimulation of autologous CD14- PBMCs derived from Donor
1 for 9 days with EBV peptide-pulsed mature DCs cultured according
to experimental conditions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, as
determined by ELISPOT analysis. Frequencies shown are subtracted
for background signal obtained in control conditions performed in
the absence of EBV peptide stimulation. Donor 1 was strongly
responsive to LMP2.
[0364] FIG. 10 shows the total number of virus-specific T cells and
the proportion of IFN.gamma.+CD8+ CTLs obtained following
stimulation of autologous CD14- PBMCs derived from Donor 1 for 9
days with EBV peptide-pulsed mature DCs cultured under different
experimental conditions. Condition number 5 (`CD40L wo`) was
identified as a promising candidate for further evaluation because
it was shown to perform better than condition 2 (`Standard
PGE`).
[0365] FIGS. 11A and 11B show CD83, CD80, CCR7 and PD-L1 expression
by monocyte-derived DCs derived from Donor 2 CD14+ PBMCs following
culture according to experimental conditions 1, 2, 3, 12, 13, 14,
15, 16, 17, 18, 19 or 20.
[0366] FIGS. 12A and 12B show CCR7 and CD45RO expression by CD4+ T
cell and CD8+ T cells obtained following stimulation of autologous
CD14- PBMCs derived from Donor 2 for 9 days with EBV peptide-pulsed
mature DCs cultured according to experimental conditions 1, 2, 3,
12, 13, 14, 15, 16, 17, 18, 19 or 20. No major differences in T
cell memory phenotype were observed for the expanded T cell
populations.
[0367] FIG. 13 shows the total number of virus-specific T cells
obtained following stimulation of autologous CD14- PBMCs derived
from Donor 2 for 9 days with EBV peptide-pulsed mature DCs cultured
according to experimental conditions 1, 2, 3, 12, 13, 14, 15, 16,
17, 18, 19 or 20.
[0368] FIG. 14 shows the proportions of IFN.gamma.+CD8+ CTLs and
IFN.gamma.+CD4+ Th cells obtained following stimulation of
autologous CD14- PBMCs derived from Donor 2 for 9 days with EBV
peptide-pulsed mature DCs cultured according to experimental
conditions 1, 2, 3, 12, 13, 14, 15, 16, 17, 18, 19 or 20, as
determined by ELISPOT analysis. Frequencies shown are subtracted
for background signal obtained in control conditions performed in
the absence of EBV peptide stimulation.
[0369] FIG. 15 shows the proportions of EBV antigen-reactive
IFN.gamma.+CD8+ CTLs and IFN.gamma.+CD4+ Th cells obtained
following stimulation of autologous CD14- PBMCs derived from Donor
2 for 9 days with EBV peptide-pulsed mature DCs cultured according
to experimental conditions 1, 2, 3, 12, 13, 14, 15, 16, 17, 18, 19
or 20, as determined by ELISPOT analysis. Frequencies shown are
subtracted for background signal obtained in control conditions
performed in the absence of EBV peptide stimulation. Donor 2 was
strongly responsive to LMP2.
[0370] FIG. 16 shows the total number of virus-specific T cells and
the proportion of IFN.gamma.+CD8+ CTLs obtained following
stimulation of autologous CD14- PBMCs derived from Donor 2 for 9
days with EBV peptide-pulsed mature DCs cultured under different
experimental conditions. Condition numbers 18, 19 and 20 (`PolyIC
MPLA wo`, `PolyIC MPLA PGE` and `MPLA IFN.gamma.`) were identified
as promising candidates for further evaluation as they were shown
to perform better than condition 2 (`Standard PGE`).
[0371] The inventors then undertook further investigation of the
four most promising DC maturation conditions 5, 18, 19 and 20 (see
Table 4), extending the characterization to cells derived from
three different donors.
[0372] FIGS. 17A and 17B show representative CD83, CD80, CCR7 and
PD-L1 expression by monocyte-derived DCs derived from three
different donors following culture according to the experimental
conditions 1, 2, 5, 18, 19 and 20. FIG. 17C shows the proportions
of CD80+CD83- DCs, FIG. 17D shows the proportions of CD80+CD83+ DCs
and 17E shows the proportions of PD-L1+ DCs.
[0373] FIG. 18 shows the total number of virus-specific T cells
obtained following stimulation of autologous CD14- PBMCs derived
from the three different donors for 9 days with EBV peptide-pulsed
mature DCs cultured according to the experimental conditions 2, 5,
18, 19 and 20. Mature DCs expanded under the different conditions
had similar overall levels of expansion.
[0374] FIG. 19 shows the proportions of IFN.gamma.+CD8+ CTLs and
IFN.gamma.+CD4+ Th cells obtained following stimulation of
autologous CD14- PBMCs derived from the different donors for 9 days
with EBV peptide-pulsed mature DCs cultured according to the
experimental conditions 2, 5, 18, 19 and 20, as determined by
ELISPOT analysis. Frequencies shown are subtracted for background
signal obtained in control conditions performed in the absence of
EBV peptide stimulation.
[0375] FIG. 20 shows the proportions of EBV antigen-reactive
IFN.gamma.+CD8+ CTLs and IFN.gamma.+CD4+ Th cells obtained
following stimulation of autologous CD14- PBMCs derived from the
different donors for 9 days with EBV peptide-pulsed mature DCs
cultured according to the experimental conditions 2, 5, 18, 19 and
20 as determined by ELISPOT analysis. Frequencies shown are
subtracted for background signal obtained in control conditions
performed in the absence of EBV peptide stimulation.
[0376] FIG. 21 shows the scaled total number of virus-specific T
cells and the scaled frequency of IFN.gamma.+ T cells obtained
following stimulation of autologous CD14- PBMCs derived from the
three donors for 9 days with EBV peptide-pulsed mature DCs cultured
under different experimental conditions.
[0377] Overall, the results suggested that the best condition for
DC maturation is culture in the presence of GM-CSF, IL-4,
IL-1.beta., IL-6, TNF.alpha. and CD40L. Another good condition is
culture in the presence of GM-CSF, IL-4, MPLA and IFN.gamma..
Example 5
Further Optimization of DC Maturation
[0378] Blood samples are collected from EBV-reactive donors in
heparin vacutainers and PBMCs are isolated from the blood samples
by Ficoll density gradient centrifugation. CD14+ microbeads are
then used to isolate CD14+ cells from within the PBMCs by positive
selection. The number of cells is counted, and the CD14- cell
population is frozen and stored.
[0379] The CD14+ cells are then differentiated to immature
dendritic cells (iDCs). Briefly, CD14+ cells are cultured in wells
of a 24-well plate at a concentration of 0.5.times.10.sup.6
cells/ml in DC cell culture medium containing 400 IU/ml IL-4 and
800 IU/ml GM-CSF, for 5 days.
[0380] The iDCs are then matured to mature DCs by culture for 24
hours in DC cell culture medium according to the conditions shown
in one of 1, 2, 21 or 25 in Table 5 below:
TABLE-US-00005 TABLE 5 DC Maturation Culture Conditions Condition
No. Conditions 1 800 U/ml GM-CSF, 400 U/ml IL-4 2 800 U/ml GM-CSF,
400 U/ml IL-4, 10 pg/ml IL-1.beta., 100 pg/ml IL-6, 10 pg/ml
TNF.alpha., 1 ng/ml PGE-1 21 800 U/ml GM-CSF, 100 ng/ml IFN.alpha.
22 800 U/ml GM-CSF, 400 U/ml IL-4, 100 ng/ml IFN.gamma., 1 .mu.g/ml
AmpB
[0381] The mature DCs are then analyzed by flow cytometry for
expression of CD80, CD83, CCR7 and PD-L1. The previously frozen
CD14- fraction is thawed and rested in cell culture medium for 24
hours.
[0382] Immature and mature DCs are then pulsed with individual EBV
antigen pepmixes (LMP1, LMP2, BRAF1 or EBNA1), and the
peptide-pulsed DCs are used to stimulate the autologous CD14- cells
by coculture for 3 days or 9 days in the presence of IL-7 (10
ng/ml) and IL-15 (100 ng/ml). The expanded T cell populations are
then analyzed by flow cytometry for expression of CCR7, CD45RO, EBV
peptide reactivity and intracellular staining for IFN.gamma..
[0383] DCs matured according to conditions 21 and 22 are expected
to be associated with advantageous properties as compared to DCs
matured according to condition 2.
Example 6
Characterization of Virus-Specific T Cells Expanded by Stimulation
Culture in the Presence of Different Costimulatory Cells
[0384] The inventors next investigated the use of K562cs cells and
HLA-negative LCLs (also referred to herein as universal LCLs
(ULCLs)) as costimulatory cells in stimulations in methods for
generating/expanding populations of virus-specific T cells.
Briefly, PBMCs were harvested, and pulsed with EBV pepmixes
corresponding to EBV antigens (EBNA-1, LMP-1, LMP-2 and BARF-1) in
the presence of IL-7 (10 ng/ml) and IL-15 (100 ng/ml) to stimulate
the expansion of EBV-specific T cells. On day 9, and every 7 days
thereafter, restimulation steps were performed either with (i) EBV
pepmix-pulsed, irradiated autologous ATCs, in the presence of IL-7
(10 ng/ml) and IL-15 (100 ng/ml), and in the presence of irradiated
K562-cs co-stimulatory cells, at a ratio of responder
cells:ATCs:K562-cs of 1:1:5, or (ii) EBV pepmix-pulsed, irradiated
autologous ATCs, in the presence of IL-7 (10 ng/ml) and IL-15 (100
ng/ml), and in the presence of irradiated ULCLs, at a ratio of
responder cells:ATCs:ULCLs of 1:1:5.
[0385] LCLs lacking surface expression of HLA class I and HLA class
II (i.e. HLA-negative LCLs) were obtained by targeted knockout of
genes encoding HLA class I and HLA class II molecules. The
HLA-negative LCLs were further modified to knockout genes necessary
for EBV replication. Three different ULCL clones (#4, #5 and #13)
were analyzed.
[0386] FIGS. 22A and 22B show the results of two separate
experiments, demonstrating that the overall fold expansion of the
cells in culture was higher for cells expanded using K562-cs cells
as costimulatory cells as compared to ULCLs. However, as shown in
FIGS. 23A and 23B, ULCL clone #5 was found to expand greater
numbers of virus-specific T cells, as determined by ELISPOT
analysis of the number of IFN.gamma. producing cells.
[0387] The inventors next analyzed the proportions of different
cell types expanded after the indicated number of stimulations
using K562-cs or ULCLs as costimulatory cells, by flow cytometry
using antibodies specific for cell surface markers. FIGS. 32A to
32C show scatterplots from a representative donor (n=7) showing the
proportions of CD3-CD56+ NK cells, CD4+ T cells, CD8+ T cells,
gamma delta T cells and alpha beta T cells in the expanded
populations. Similar proportions of CD3-CD56+ NK cells were found
in the expanded populations (FIG. 24A), whilst populations obtained
by stimulations using K562cs cells tended to have a reduced
proportion of CD4+ T cells in the expanded populations (FIG. 24B),
and populations obtained by stimulations using ULCLs tended to have
an increased proportion of alpha beta T cells in the expanded
populations (FIG. 24C). FIG. 25 shows representative results of
characterization of gamma delta TCR expression by ULCL clones #5
and #13, as compared to expression by K562cs cells.
[0388] The inventors next analyzed the frequency of
antigen-specific T-cells in the populations of cells expanded by
stimulations using ULCL clone #5 or #13, or K562cs cells by ELISPOT
analysis of IFN.gamma. production at the end of the second and
third stimulations. The results obtained from two different donors
are shown in FIGS. 26A and 26B. More virus-specific cells were
detected where cells had been expanded by stimulation using ULCL
cells as costimulatory cells. FIGS. 27A to 27D show the number of
cells per 100,000 cells specific for the indicated EBV antigens for
four different donors after the indicated number of stimulations
using K562cs cells, ULCL clone #5 or LCLs as costimulatory cells,
as determined by ELISPOT analysis.
[0389] The inventors extended the analysis of virus-specific T cell
expansion to four stimulations, and found that stimulations
employing ULCLs as costimulatory cells expanded populations
containing higher proportions of virus-specific cells as compared
to stimulations performed using K562cs cells (FIGS. 28A to 28D).
ULCL clone #4 cells were found not to differ significantly from
cells of the parental ULCL clone in relation to ability to expand
IFN.gamma. producing virus-specific T cells (FIGS. 29A and
29B).
[0390] In further experiments using PBMCs obtained from two
different donors, stimulations using ULCL clone #5 yielded
populations of cells comprising IFN.gamma. producing virus-specific
T cells at a higher frequency as compared to the frequency in
populations expanded using K562cs cells in stimulations (FIGS. 30A
and 30B), with similar fold expansion (FIGS. 31A and 31B). FIGS. 32
and 33 show that the proportions of CD3+ and CD56+ cells in the
expanded populations did not differ significantly in populations
expanded using ULCL clone #5 as compared to populations expanded
using K562cs cells, and FIGS. 34 and 35 show that populations
expanded using K562cs cells expanded slightly greater proportion of
central memory cells.
[0391] FIGS. 36A and 36B show results from experiments obtained
using PBMCs obtained from two further donors, demonstrating that
stimulations using ULCL clone #5 yielded populations of cells
comprising IFN.gamma. producing virus-specific T cells at a higher
frequency as compared to the frequency in populations expanded
using K562cs cells in stimulations. In these experiments overall
fold expansion was higher when K562cs cells were used in
stimulations (FIGS. 37A and 37B). FIGS. 38 and 39 show that the
proportions of CD3+ and CD56+ cells in the expanded populations did
not differ significantly in populations expanded using ULCL clone
#5 as compared to populations expanded using K562cs cells, and
FIGS. 40 and 41 show that populations expanded using K562cs cells
expanded slightly greater proportion of central memory cells.
Example 7
Further Characterization of HPV-Specific T Cells Expanded by
Stimulation Culture in the Presence of Different Combinations of
Cytokines and with Different Costimulatory Cells
[0392] HPV stimulated T-cells (HPVST) were first activated by HPV
E6/E7 peptide-pulsed autologous dendritic cells (DCs) at 10-20:1
PBMC:DC ratio, and cultured for 8 days in culture medium containing
IL-6 (100 ng/ml), IL-7 (10 ng/ml), IL-12 (10 ng/ml), IL-15 (10
ng/ml) (e.g. per the first stimulation step described by Ramos et
al., (J Immunother 2013; 36:66-76)).
[0393] Subsequent weekly restimulation steps were then carried out
as shown in Table 6, four a total of four stimulations:
TABLE-US-00006 TABLE 6 Second and subsequent stimulations Condition
Conditions K562cs 7/15 HPV E6/E7 peptide-pulsed autologous T-APC at
1:1 ratio, in the presence of equal number of irradiated allogeneic
K562-cs co-stimulatory cells, in cell culture media containing IL-7
(10 ng/ml) and IL-15 (100 ng/ml). ULCL 7/15 HPV E6/E7
peptide-pulsed autologous T-APC at 1:1 ratio, in the presence of
equal number of irradiated HLA- negative LCLs, in cell culture
media containing IL-7 (10 ng/ml) and IL-15 (100 ng/ml). K562cs HPV
E6/E7 peptide-pulsed autologous T-APC at 1:1 ratio, 6/7/12/15 in
the presence of equal number of irradiated allogeneic K562-cs
co-stimulatory cells, in cell culture media containing IL-6 (100
ng/ml), IL-7 (10 ng/ml), IL-12 (10 ng/ml) and IL-15 (10 ng/ml).
ULCL HPV E6/E7 peptide-pulsed autologous T-APC at 1:1 ratio,
6/7/12/15 in the presence of equal number of irradiated HLA-
negative LCLs, in cell culture media containing IL-6 (100 ng/ml),
IL-7 (10 ng/ml), IL-12 (10 ng/ml) and IL-15 (10 ng/ml).
[0394] At the end of each stimulation the cells were counted, and
analysed by flow cytometry to determine the proportions of
different cell types in the expanded populations, and analyzed by
ELISPOT to determine the frequency IFN.gamma. producing,
virus-specific cells in the expanded populations.
[0395] FIG. 42 shows that HPV-specific T cells were present at a
higher frequency in populations of cells expanded by methods
employing stimulations in the presence of IL-6, IL-7, IL-12 and
IL-15 as compared to stimulations in the presence of IL-7 and IL-15
only. HPV-specific T cells were also present at a higher frequency
in populations of cells expanded by methods using ULCLs as
costimulatory cells as compared to methods using K562cs cells as
costimulatory cells.
[0396] FIG. 43 shows the overall fold expansion of cells expanded
according to the different protocols. Methods comprising
stimulation in the presence of IL-7 and IL-15 only were found to
expand more HPV-specific T cells as compared to methods comprising
stimulation in the presence of IL-6, IL-7, IL-12 and IL-15. Methods
comprising stimulations using ULCLs as costimulatory cells were
shown to expand more HPV-specific T cells as compared methods
comprising stimulations using K562cs cells as costimulatory
cells.
[0397] FIG. 44 shows the expression of CD3 and CD56 by cells
stimulated according to the different protocols, after the
indicated number of stimulations. Cells expanded by stimulation in
the presence of IL-7 and IL-15 only were shown to have a greater
proportion of CD3+CD56- cells as compared to cells expanded by
stimulation in the presence of IL-6, IL-7, IL-12 and IL-15. Cells
expanded by stimulations using HLA-negative LCLs as costimulatory
cells were shown have a greater proportion of CD3+CD56- cells as
compared to cells expanded by stimulations using K562cs as
costimulatory cells.
[0398] FIG. 45 shows the expression of CD4 and CD8 by cells
stimulated according to the different protocols, after the
indicated number of stimulations. Cells expanded by stimulation in
the presence of IL-6, IL-7, IL-12 and IL-15 were shown to have a
greater proportion of CD8+ cells as compared to cells expanded by
stimulation in the presence of IL-7 and IL-15 only.
[0399] FIG. 46 shows the expression of CD45RO and CCR7 by cells
stimulated according to the different protocols, after the
indicated number of stimulations. Cells expanded by stimulation in
the presence of IL-7 and IL-15 only were shown to have a greater
proportion of central memory (i.e. CD45RO+CCR7+) cells as compared
to cells expanded by stimulation in the presence of IL-6, IL-7,
IL-12 and IL-15.
Example 8
Generation of Virus-Specific T Cells from PBMCs Depleted of CD45RA+
Cells
[0400] The inventors investigated modifications to the methods for
expanding virus-specific T cells to increase the frequency of
viral-specific antigen-specific T-cells and to reduce the frequency
of NK cells in expanded populations.
[0401] FIG. 47 provides a schematic representation of steps of the
method used to generate virus-specific T-cells in this Example.
Briefly, on day 0 PBMCs (which were either depleted of CD45RA+
cells, or which were not depleted of CD45RA+ cells) were pulsed
with viral peptides in the presence of IL-7 (10 ng/ml) and IL-15
(100 ng/ml or 5 ng/ml), or in the presence of IL-4 and IL-7. At day
9, the cells were re-stimulated using peptide-pulsed irradiated
autologous antigen-presenting activated T-cells (ATCs) in the
presence of irradiated HLA-negative LCLs as costimulatory cells,
and in the presence of IL-7 (10 ng/ml) and IL-15 (100 ng/ml or 5
ng/ml), or in the presence of IL-4 and IL-7. At day 16 cells were
harvested and analyzed.
[0402] FIG. 48 shows results of ELISPOT analysis showing that
methods using IL-7 and IL-15 in stimulations expanded
virus-specific T cells at a higher frequency as compared to methods
using IL-4 and IL-7 in stimulations.
[0403] Methods comprising stimulation in the presence of IL-7 and
IL-15 were found to be able to expand viral antigen-specific T
cells from PBMCs obtained from lymphoma patients (FIG. 49). Using
IL-15 instead of IL-4 was found to increase the frequency of
antigen-specific T-cells in patient EBV-specific T cells.
[0404] Investigation of the optimal concentration of IL-15 to be
used in stimulations revealed that a higher dose of IL-15 (100
ng/ml) was best for increasing the frequency of virus-specific T
cells (100 ng per mL compared to standard dose of 5 ng per mL)--see
FIG. 50. FIG. 51 demonstrates that high doses of IL-15 increased
the proportion of central memory EBV-specific T cells in the
expanded cell population.
[0405] The inventors next investigated how to minimize NK cell
outgrowth in populations of expanded cells generated from healthy
donor and lymphoma patient PBMCs. Preferential outgrowth of NK
cells was found to be higher in methods using IL-15 in stimulations
(NK cell populations appear to be larger in populations obtained by
stimulations using IL-15 and IL-7). To address this, conditions
were developed to avoid excessive NK cell outgrowth. Depletion of
CD45RA+ cells from the PBMCs prior to stimulations was
investigated. CD45RA is a naive T-cell marker that is also
expressed on natural T-regulatory cells and NK cells, so it was
reasoned that depletion of CD45RA+ cells would remove the NK cells
from the starting PBMC population. Depletion of CD45RA+ cells also
removes 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 can be achieved by any suitable
method, e.g. 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.
[0406] The process used to generation of pepmix-activated
EBV-specific T cells from CD45RA- depleted PBMCs is illustrated
schematically in FIG. 53. As shown therein, whole PBMCs populations
were depleted of CD45RA (or CD45RO for comparative purposes), and
beginning day 0 or day 1 the first stimulation (S1) EBVpepmix was
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
were 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. 54 demonstrates
the results that CD45RA depletion (CD45RA+ PBMC from healthy donors
were depleted using Miltenyi.RTM. columns and GMP grade CD45RA-
conjugated beads) was found to decrease the frequency of
CD3-CD56+NK cells in the expanded population, which was associated
with increased proliferation of EBVSTs (FIG. 55). FIG. 56
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 was found to enhance
antigen specificity of EBVSTs at the end of a second stimulation
(for example, day 16) (see FIGS. 57 and 58, both showing data for
healthy donors). The increased antigen specificity of EBVSTs
generated by expansion from CD45RA+ cell depleted PBMCs was found
to be sustained after a third stimulation (FIG. 59).
[0407] The effects of CD45RA depletion was then characterized in
virus-specific T cells obtained by expansion from PBMCs obtain from
lymphoma patients, chosen either because their EBVSTs grown without
depletion showed high frequencies of NK cells or because they
failed to grow or show antigen-specificity. FIG. 60 shows the total
NK cell population at the end of a second stimulation step in five
lymphoma patients, demonstrating that CD45RA depletion decreased NK
cell population outgrowth in lymphoma patient EBVSTs, and that 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. 61). This experiment was performed in the
absence of dendritic cells for the first stimulation (i.e. CD45RA+
cell depleted PBMCs were contacted directly with EBVPepmix).
Similar to the results observed in methods using PBMCs from healthy
donors, CD45RA depletion was found to increase antigen specificity
in EBVSTs from lymphoma patients (FIG. 62). Proliferation of the
lymphoma patients' EBVSTs is demonstrated in FIG. 63. 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. 64).
[0408] 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.
* * * * *