U.S. patent application number 17/187399 was filed with the patent office on 2022-01-06 for auto/allo-immune defense receptors for the selective targeting of activated pathogenic t cells and nk cells.
The applicant listed for this patent is Baylor College of Medicine. Invention is credited to Malcolm K. Brenner, Maksim Mamonkin, Feiyan Mo.
Application Number | 20220000920 17/187399 |
Document ID | / |
Family ID | |
Filed Date | 2022-01-06 |
United States Patent
Application |
20220000920 |
Kind Code |
A1 |
Mamonkin; Maksim ; et
al. |
January 6, 2022 |
AUTO/ALLO-IMMUNE DEFENSE RECEPTORS FOR THE SELECTIVE TARGETING OF
ACTIVATED PATHOGENIC T CELLS AND NK CELLS
Abstract
Embodiments of the disclosure concern engineered
auto/allo-immune defense receptor (ADR)-expressing T cells that
selectively target activated T cells, including pathogenic T cells,
to incapacitate them. The chimeric receptors comprise moieties for
targeting 4-1BB, OX40, and CD40L, for example, whose expression is
indicative of activated T cells. In particular embodiments, there
are methods of preventing or treating conditions associated with
activated T cells using adoptive T-cell transfer of cells encoding
the ADRs.
Inventors: |
Mamonkin; Maksim; (Houston,
TX) ; Brenner; Malcolm K.; (Houston, TX) ; Mo;
Feiyan; (Houston, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Baylor College of Medicine |
Houston |
TX |
US |
|
|
Appl. No.: |
17/187399 |
Filed: |
February 26, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17049561 |
Oct 21, 2020 |
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PCT/US2019/029163 |
Apr 25, 2019 |
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17187399 |
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62662817 |
Apr 26, 2018 |
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International
Class: |
A61K 35/17 20060101
A61K035/17; A61P 37/06 20060101 A61P037/06; C07K 14/705 20060101
C07K014/705 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under P50
CA126752 awarded by National Institutes of Health and the National
Cancer Institute. The government has certain rights in the
invention.
Claims
1. An isolated polynucleotide, comprising sequence encoding a
polypeptide, wherein said polypeptide comprises: (1) one or more of
an OX40-specific ligand, a 4-1BB-specific ligand, CD40L-specific
ligand, or functional derivatives thereof; that is operably linked
to (2) a signaling domain promoting T-cell activation.
2. (canceled)
3. (canceled)
4. (canceled)
5. The polynucleotide of claim 1, wherein the OX40-specific ligand
is OX40L, an antibody that targets OX40, an OX40L-Fc fusion, or a
combination thereof.
6. The polynucleotide of claim 1, wherein the 4-1BB-specific ligand
is 4-1BBL, an antibody that targets 4-1BB, a 4-1BBL-Fc fusion, or a
combination thereof.
7. The polynucleotide of claim 1, wherein the CD40L-specific ligand
is CD40, an antibody that targets CD40L, a CD40-Fc fusion, or any
other engineered protein capable of specific binding to CD40L.
8. (canceled)
9. The polynucleotide of claim 1, wherein the polynucleotide
further comprises sequence that encodes a spacer between (1) and
(2).
10. The polynucleotide of claim 9, wherein the spacer is between 10
and 220 amino acids in length.
11. The polynucleotide of claim 10, wherein the spacer has sequence
that facilitates surface detection with an antibody.
12. The polynucleotide of claim 11, wherein the spacer is
detectable with an anti-Fc Ab.
13. The polynucleotide of claim 12, wherein the spacer comprises
IgG Fc portion.
14. The polynucleotide of claim 1, wherein the polynucleotide
further encodes a chimeric antigen receptor, a T-cell receptor, or
both.
15. The polynucleotide of claim 14, wherein there is a 2A element
or IRES element on the polynucleotide between sequence that encodes
the (a) a polypeptide that comprises (1) one or more of an
OX40-specific ligand, a 4-1BB-specific ligand, CD40L-specific
ligand, or functional derivatives thereof; that is operably linked
to (2) a signaling domain promoting T-cell activation and (b) the
polynucleotide that encodes the chimeric antigen receptor, a T-cell
receptor, or both.
16. (canceled)
17. (canceled)
18. (canceled)
19. (canceled)
20. The polynucleotide of claim 1, wherein the polynucleotide is
present in a cell.
21. (canceled)
22. The polynucleotide of claim 20, wherein the cell is a T cell
that comprises one or more chimeric antigen receptors or one or
more engineered T cell receptors (TCRs).
23. (canceled)
24. (canceled)
25. (canceled)
26. (canceled)
27. (canceled)
28. A polypeptide expressed by a polynucleotide of claim 1.
29. A polypeptide, comprising: (1) one or more of an OX40-specific
ligand, a 4-1BB-specific ligand, and CD40; that is operably linked
to (2) a signaling domain promoting T-cell activation.
30. (canceled)
31. (canceled)
32. A chimeric receptor-expressing cell, comprising the
polynucleotide of claim 1.
33. The cell of claim 32, wherein the cell is cell a CAR-transduced
T cell or a T cell receptor (TCR)-transduced T cell.
34. (canceled)
35. (canceled)
36. (canceled)
37. (canceled)
38. The cell of claim 32, wherein the cell is engineered to lack
endogenous expression of one or more genes.
39. The cell of claim 38, wherein the cell is engineered to lack
endogenous expression of 4-1BB, OX40 and/or CD40L.
40. (canceled)
41. (canceled)
42. A method of preparing cells for cell therapy, comprising the
step of transfecting immune effector cells with the polynucleotide
of claim 1.
43. (canceled)
44. The method of claim 42, further comprising the step of
modifying the cells to express one or more chimeric antigen
receptors and/or one or more recombinant T-cell receptors.
45. The method of claim 42, further comprising the step of
providing an effective amount of the cells to an individual in need
thereof.
46. (canceled)
47. A method of treating an individual for a medical condition with
allogeneic therapeutic cells, comprising the step of providing to
the individual a therapeutically effective amount of allogeneic
cells, wherein said cells express a polypeptide comprising (1) one
or more of an OX40-specific ligand, a 4-1BB-specific ligand,
CD40L-specific ligand, or functional derivatives thereof; that is
operably linked to (2) a signaling domain promoting T-cell
activation.
48. (canceled)
49. The method of claim 47, wherein the cells express one or more
chimeric antigen receptors and/or one or more recombinant T-cell
receptors.
50. A method of avoiding rejection of allogeneic cells, tissue, or
organs in an individual, comprising the step of delivering to the
individual an effective amount of allogeneic immune cells
expressing an engineered chimeric receptor that comprises an
extracellular domain that targets compounds that are selectively
present on activated T cells and that comprises CD3 zeta, wherein
the delivering step results in the following in the individual: (1)
inhibition of endogenous alloreactive T cells in the individual;
and/or (2) suppression of NK cell activation in the individual.
51. The method of claim 50, wherein the allogeneic cells are the
allogeneic immune cells expressing the chimeric receptor.
52. The method of claim 50, wherein the allogeneic cells express a
chimeric antigen receptor or an engineered T cell receptor.
53. The method of claim 50, wherein the allogeneic immune cells are
delivered to the individual before, during, and/or after tissue
and/or organ transplantation in the individual.
54. The method of claim 50, wherein the activated T cells are
pathogenic T cells.
55. A method of selectively targeting activated T cells in an
individual, comprising the step of providing to the individual an
effective amount of immune cells expressing an engineered chimeric
receptor, said chimeric receptor comprising: (1) an extracellular
domain that targets compounds that are selectively present on
activated T cells; and (2) a signaling domain promoting T-cell
activation.
56. (canceled)
57. The method of claim 55, wherein the activated T cells are
pathogenic T cells.
58. A method of preventing or treating a medical condition related
to activated T cells in an individual, comprising the step of
delivering to the individual an effective amount of immune cells
expressing an engineered chimeric receptor that selectively targets
said activated T cells, said chimeric receptor comprising: (1) an
extracellular domain that targets compounds that are selectively
present on activated T cells; and (2) a signaling domain promoting
T-cell activation.
59. (canceled)
60. The method of claim 58, wherein the medical condition is an
autoimmune disorder.
61. The method of claim 58, wherein the medical condition comprises
graft rejection, graft-versus-host disease, type I diabetes,
multiple sclerosis, autoimmune colitis, or a combination
thereof.
62. A method of avoiding NK cell-mediated host rejection of
allogeneic T cells, tissues, or organs in an individual, comprising
the step of providing to the individual an effective amount of
immune cells expressing an engineered chimeric receptor that
comprises an extracellular domain that targets compounds that are
selectively present on activated T cells and that also comprises a
signaling domain promoting T-cell activation.
63. The method of claim 62, wherein the immune cells expressing the
engineered chimeric receptor are the allogeneic T cells.
64. The method of claim 62, wherein the immune cells express a
chimeric antigen receptor or an engineered T cell receptor.
65. (canceled)
66. (canceled)
67. (canceled)
68. (canceled)
Description
[0001] This application is a continuation of U.S. Non-Provisional
application Ser. No. 17/049,561 filed Oct. 21, 2020 which is a
national phase application under 35 U.S.C. .sctn. 371 that claims
priority to International Application No. PCT/US2019/029163 filed
Apr. 25, 2019, which claims priority to U.S. Provisional Patent
Application 62/662,817, filed Apr. 26, 2018, all of which are
incorporated by reference herein in their entirety.
INCORPORATION OF SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing, named
"Seq_Listing.txt" (18,804 bytes), created Feb. 26, 2021, which has
been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety.
TECHNICAL FIELD
[0004] Embodiments of the disclosure include at least the fields of
immunology, cell biology, molecular biology, and medicine.
BACKGROUND
[0005] Unwanted activation of T- and NK-cells often promotes
life-threatening allo-immune reactions in patients receiving
transplants or third party-derived therapeutic cells, leading to
rejection of a transplanted organ/tissue or development of
graft-versus-host disease (GvHD). Likewise, unwanted activation of
autoreactive T-cells can lead to devastating autoimmune conditions,
such as diabetes mellitus, autoimmune colitis, and multiple
sclerosis. Currently, most of these diseases are not curable
because of the inability to selectively eliminate pathogenic T
cells. Instead, the patients are often treated with
immunosuppressive drugs that render them immunodeficient and
therefore susceptible to infections and malignant
transformations.
[0006] The present disclosure provides solutions for a long felt
need in the art of safe and effective tissue transplantation and
adoptive cell transfer, including utilizing off-the-shelf cells, by
enhancing their ability of the transferred cells to control
pathogenic conditions because of unwanted activation of the immune
system.
BRIEF SUMMARY
[0007] The present disclosure is directed to compositions and
methods related to cells utilized for adoptive transfer to control
pathogenic conditions due to immune activation. The composition and
methods apply to autologous and allogeneic cells. Although some
steps may be taken to reduce the reactivity of allogeneic cells in
the recipient individual, such cells would still be targeted by the
immune system of the recipient (primarily T- and NK-cells), which
would recognize them as foreign leading to rejection and limiting
therapeutic benefit.
[0008] The present disclosure overcomes this problem by modifying
adoptive therapy cells to target activated pathogenic T, NK-T, and
NK cells to prevent or treat medical conditions associated with
their presence. In particular embodiments, the compositions and
methods utilize adoptive T-cell transfer with cells that express
receptors that selectively target pathogenic T cells while sparing
resting T cells. In specific embodiments, the adoptive T-cells for
transfer are engineered to express chimeric molecules that target
pathogenic T cells that express certain target molecules whose
presence on T cells is indicative of pathogenic T cells. In
particular embodiments the disclosure concerns auto/allo-immune
defense receptors (ADRs) for the selective targeting of pathogenic
T-cells.
[0009] Particular embodiments of the disclosure include methods of
protecting engineered allogeneic T cells from elimination in a host
individual by providing to the individual cells armed with ADRs.
Embodiments also include methods that avoid allo-immune reactions
in individuals receiving tissue or organ transplants, for
example.
[0010] In particular embodiments, cells encompassed by the
disclosure have been modified or can be modified to allow them to
survive in a recipient, including an allogeneic recipient. In
specific cases, cells for adoptive cell therapy (including T cells,
NKT cells, and so forth) are suitable for being utilized
"Off-the-shelf", which herein refers to cells kept in a repository,
or bank, that may be provided (with or without further
modification) to an individual in need thereof for a specific
purpose. The individual in many cases is not the individual from
which the cells were originally derived. The cells utilized in such
a manner may be pre-manufactured to express an ADR, although in
some cases the cells are obtained from a bank and afterwards are
modified to express an ADR. The banked cells may or may not also
express a CAR or recombinant TCR, or the cells obtained from the
bank may afterwards be modified to express a CAR or recombinant
TCR. Such practices allow for ease of use of third party-derived
therapeutic cells without immune rejection by a host and without
having to manufacture a patient-specific produce every time one is
needed.
[0011] In particular embodiments, there is an isolated
polynucleotide, comprising sequence encoding: (1) one or more of an
OX40-specific ligand, a 4-1BB-specific ligand, CD40L-specific
ligand, or functional derivatives thereof; that is operably linked
to (2) a signaling domain promoting T-cell activation. The
polynucleotide may comprise an OX40-specific ligand, a
4-1BB-specific ligand, or a CD40L-specific ligand. The
OX40-specific ligand may be OX40L, an antibody that targets OX40,
an OX40L-Fc fusion, or a combination thereof, or any other
engineered protein capable of specific binding to OX40. The
4-1BB-specific ligand may be 4-1BBL, an antibody that targets
4-1BB, a 4-1BBL-Fc fusion, or a combination thereof, or any other
engineered protein capable of specific binding to 4-1BB. The
CD40L-specific ligand may be CD40, an antibody that targets CD40L,
a CD40-Fc fusion, or any other engineered protein capable of
specific binding to CD40L or combination thereof. In at least
certain cases, the polynucleotide further comprises sequence that
encodes a spacer between (1) and (2), such as between 10 and 220
amino acids in length, for example. The spacer may have sequence
that facilitates surface detection with an antibody, such as the
spacer being detectable with an anti-Fc Ab. The spacer may comprise
an IgG Fc portion.
[0012] In particular embodiments, polynucleotides of the disclosure
may further encode a chimeric antigen receptor, a T-cell receptor,
or both. The polynucleotide may be in any form including present on
a vector, such as a viral vector (retroviral vector, lentiviral
vector, adenoviral vector, or adeno-associated viral vector) or
non-viral vector (plasmid, transposon, etc.). In particular cases,
the polynucleotide is present in a cell, including a eukaryotic
cell or a bacterial cell. The cell may be an immune cell, such as a
T cell. The cell may be engineered. The cell may comprise one or
more chimeric antigen receptors (CARs) and/or one or more
engineered T cell receptors (TCRs).
[0013] Polypeptides expressed by any polynucleotide encompassed by
the disclosure are included as part of the disclosure. In
particular embodiments there is a polypeptide, comprising: (1) one
or more of an OX40-specific ligand, a 4-1BB-specific ligand, and
CD40; that is operably linked to (2) a signaling domain promoting
T-cell activation. The signaling domain promoting T-cell activation
may be from CD3 zeta subunit, DAP12, an Fc receptor, or a
combination thereof.
[0014] Any cell encompassed by the disclosure is part of the
disclosure. In specific embodiments, any chimeric
receptor-expressing cell is part of the disclosure, including
cells, comprising any polynucleotide contemplated herein and/or any
polypeptides contemplated herein. The cell may be an engineered
cell. The cell may be an immune cell, such as a T cell, including a
CAR-transduced T cell and/or a T cell receptor (TCR)-transduced T
cell. In specific embodiments, the cell is engineered to lack
endogenous expression of one or more genes, such as lack one or
more of 4-1BB, OX40 and/or CD40L. The cell may be engineered using
CRISPR/Cas9, zinc finger nucleases, TALE nucleases, or
meganucleases. Alternatively, the cell may be engineered to prevent
surface expression of ADR ligands, for example, by trapping the ADR
ligand with a specific antibody or a receptor anchored in the
endoplasmic reticulum or another intracellular compartment.
[0015] In one embodiment there is a method of avoiding rejection of
allogeneic cells, tissue, or organs in an individual, comprising
the step of delivering to the individual an effective amount of
allogeneic immune cells expressing an engineered chimeric receptor
that comprises an extracellular domain that targets compounds that
are selectively present on activated T cells and that comprises CD3
zeta, wherein the delivering step results in the following in the
individual: (1) inhibition of endogenous alloreactive T cells in
the individual; and/or (2) suppression of NK cell activation in the
individual. In specific embodiments, the allogeneic cells are the
allogeneic immune cells expressing the chimeric receptor. The
allogeneic cells may express a chimeric antigen receptor and/or an
engineered T cell receptor. The allogeneic immune cells may be
delivered to the individual before, during, and/or after tissue
and/or organ transplantation in the individual. In specific cases,
the activated T cells are pathogenic T cells.
[0016] In one embodiment, there is a method of selectively
targeting activated T cells in an individual, comprising the step
of providing to the individual an effective amount of cells
expressing an engineered chimeric receptor, said chimeric receptor
comprising: (1) an extracellular domain that targets compounds that
are selectively present on activated T cells; and (2) a signaling
domain promoting T-cell activation. The signaling domain promoting
T-cell activation may be derived from CD3 zeta subunit, DAP12, an
Fc receptor, any ITAM-comprising sequence, or a combination
thereof. In specific cases, the activated T cells are pathogenic T
cells.
[0017] In a certain embodiment, there is a method of preventing or
treating a medical condition related to activated T cells in an
individual, comprising the step of delivering to the individual an
effective amount of immune cells expressing an engineered chimeric
receptor that selectively targets said activated T cells, said
chimeric receptor comprising: (1) an extracellular domain that
targets compounds that are selectively present on activated T
cells; and (2) a signaling domain promoting T-cell activation. The
medical condition may be an autoimmune disorder, such as graft
rejection, graft-versus-host disease, type I diabetes, multiple
sclerosis, autoimmune colitis, or a combination thereof, for
example.
[0018] In one embodiment, there is a method of avoiding NK
cell-mediated host rejection of allogeneic T cells, tissues, or
organs in an individual, comprising the step of providing to the
individual an effective amount of immune cells expressing an
engineered chimeric receptor that comprises an extracellular domain
that targets compounds that are selectively present on activated T
cells and that also comprises a signaling domain promoting T-cell
activation. The immune cells expressing the engineered chimeric
receptor are the allogeneic T cells, in certain cases. The immune
cells may express a chimeric antigen receptor and/or an engineered
T cell receptor. The amount of immune cells expressing the
engineered chimeric receptor that are provided to the individual
may be in a range of 10.sup.2-10.sup.12 per m.sup.2. The cells
expressing the chimeric receptor may be provided to the individual
systemically or locally. The immune cells may be T cells. The
immune cells may be delivered to the individual once or more than
once.
[0019] The foregoing has outlined rather broadly the features and
technical advantages of the present disclosure in order that the
detailed description that follows may be better understood.
Additional features and advantages will be described hereinafter
which form the subject of the claims herein. It should be
appreciated by those skilled in the art that the conception and
specific embodiments disclosed may be readily utilized as a basis
for modifying or designing other structures for carrying out the
same purposes of the present designs. It should also be realized by
those skilled in the art that such equivalent constructions do not
depart from the spirit and scope as set forth in the appended
claims. The novel features which are believed to be characteristic
of the designs disclosed herein, both as to the 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
disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] For a more complete understanding of the present disclosure,
reference is now made to the following descriptions taken in
conjunction with the accompanying drawing, in which:
[0021] FIGS. 1A-1E. ADRs can be expressed on cell surface of immune
cells and promote cytotoxicity against respective targets. (FIG.
1A) Schematic of ADR. GFP is optional (FIG. 1B) Expression of ADR
on the cell surface (FIG. 1C) Expansion of ADR T cells after
transduction (FIG. 1D) Cytotoxicity of ADR T cells against target
cells expressing ADR ligands (FIG. 1E) Expansion of wild-type vs
4-1BB KO T cells expressing 4-1BB ADR and their cytotoxicity
against 4-1BB+ targets showing that knocking out ADR ligand on T
cells can further enhance expansion and cytotoxicity and
demonstrating co-expression of ADR and its ligand on T cells is not
required for ADR-T cell expansion or function
[0022] FIGS. 2A-2E. Selective expression of ADR ligands on
activated T cells enables their selective elimination by ADR T
cells. (FIG. 2A-FIG. 2C) Expression of ADR ligands on resting vs
activated T cells after TCR stimulation. (FIG. 2D) Absence of
cytotoxicity of ADR T cells against resting CD4+ and CD8+ T cells
(FIG. 2E) Elimination of activated CD4+ and CD8+ T cells by ADR T
cells after a 48 h coculture.
[0023] FIGS. 3A-3F. Expression of 4-1BB ADR protects T cells from
immune rejection in an MLR model (FIG. 3A) Representative dot plots
showing TCRKO T cells xo-expressing ADR are protected from
rejection after coculture with allogeneic PBMC at a 1:10 ADR T:PBMC
ratio. (FIG. 3B-FIG. 3C) Absolute counts of donor T cells and
allogeneic T cells in the PBMC during coculture (FIG. 3D-FIG. 3F)
same for virus-specific ADR T cells.
[0024] FIGS. 4A-4C. Expression of ADR protects allogeneic
virus-specific T cells from immune rejection in a mixed lymphocyte
reaction in vitro (FIG. 4A) Representative dot plots showing ADR
VST are protected from immune rejection by recipient allogeneic
PBMC (FIG. 4B-FIG. 4C) Absolute counts of recipient T cells and
donor VST at various time points during MLR.
[0025] FIG. 5. ADR VSTs retain anti-viral function. ADR VSTs were
cocultured with viral pepmix-pulsed monocytes, and monocyte counts
indicated that they eliminated viral infected cells equally well
compared to unmodified VSTs.
[0026] FIGS. 6A-6H. Activated NK cells upregulate ADR ligands and
can be selectively targeted by ADR T cells (FIG. 6A-FIG. 6B)
Expression of 4-1BB on resting vs activated NK cells (FIG. 6C)
Residual counts of resting vs activated NK cells after 24 hr
coculture with of 4-1BB ADR T cells (FIG. 6D) ADR T cells lacking
MHC are protected from immune rejection by allogeneic PBMC by
controlling the expansion of NK cells (FIG. 6E) Absolute counts of
donor T cells and allogeneic NK cells during coculture. (FIG. 6F)
ADR T cells lacking MHC resist immune rejection by NK cells upon 48
h coculture at a 1:1 E:T ratio. (FIG. 6G-FIG. 6H) ADR T cells
control the expansion of alloreactive NK cells during MLR with
PBMC, with absolute counts of NK cells plotted in H.
[0027] FIGS. 7A-7E. ADR expression protects allogeneic T cells from
immune rejection in vivo (FIG. 7A) Schematic of the mouse model of
immune rejection where mice were given T cells from an HLA-A2+
donor after a sublethal irradiation, followed by administration of
allogeneic HLA-A2- T cells 4 days later. (FIG. 7B-FIG. 7C) Control
T cells from the HLA-A2-donor were rejected by Day 18 while
ADR-expressing cells were protected (FIG. 7C) Absolute counts of T
cells from HLA-A2+ and HLA-A2- donors at various time points. (FIG.
7D) Modified in vivo model where instead of allogeneic T cells mice
received whole PBMC (containing both T- and NK-cells) from donor 1.
(FIG. 7E) Representative flow plots showing ADR T cells were
protected from immune rejection and also protected mice from rapid
onset of fatal GvHD.
[0028] FIGS. 8A-8E. Coexpression of CAR and ADR preserves functions
of both receptors (FIG. 8A) Schematic representation of an immune
cell co-expressing ADR and a CAR (FIG. 8B) Coexpression of CAR and
ADR on the cell surface (FIG. 8C) Cytotoxicity of CAR-ADR T cells
against NALM-6 (CD19+ CAR target) (FIG. 8D) Cytotoxicity of CAR-ADR
T cells against activated T cells (ADR target) (FIG. 8E) Cytotoxic
activity of CAR-ADR T cells against both targets upon simultaneous
co-culture with both cell targets.
[0029] FIGS. 9A-9E. CAR-ADR T cells are protected from immune
rejection and exert potent anti-tumor activity (FIG. 9A) Schematic
of the mouse model. Mice received allogeneic T cells from Donor 1
and b2mKO NALM6 24 hr apart, followed by a single dose of CAR-ADR T
cells from Donor 2. (FIG. 9B) Kinetics of T cells from Donor 2 in
peripheral blood (FIG. 9C) Kinetics of Donor 1 T cells in the
experimental groups (FIG. 9D) Leukemia burden in mice (FIG. 9E)
overall survival of mice.
[0030] FIGS. 10A-10C. CAR-ADR T cells are protected from immune
rejection and exert potent anti-tumor activity in a solid tumor
model. (FIG. 10A) Schematic of the mouse model. Mice received
allogeneic T cells from Donor 1 and b2mKO neuroblastoma cell line
CHLA255 24 hr apart, followed by a single dose of CAR-ADR T cells
from Donor 2. (FIG. 10B) Donor 2 GD2 CAR T cells were rejected by
D18, whereas CAR-ADR T cells resisted allogeneic rejection and
persisted in peripheral blood. (FIG. 10C) Tumor burden in mice, *
indicates xenogeneic-GvHD associated deaths in ATC+GD2 CAR T
group.
[0031] FIGS. 11A-11E. TCR-knockout CAR-ADR T cells are protected
from immune rejection and exert potent anti-tumor activity (FIG.
11A) Schematic of the mouse model. Mice received allogeneic T cells
from Donor 1 and b2mKO NALM6 24 hr apart, followed by a single dose
of TCR-edited CAR-ADR T cells from Donor 2. (FIG. 11B) Kinetics of
T cells from Donor 2 in peripheral blood (FIG. 11C) Kinetics of
Donor 1 T cells in the experimental groups (FIG. 11D) Leukemia
burden in mice (FIG. 11E) overall survival of mice.
[0032] FIGS. 12A-12D. ADR T cells protect mice against fatal
xenogeneic GvHD (FIG. 12A) Schematic of the model (FIG. 12B)
Expansion of FFLuc-labeled ADR T cells in vivo (FIG. 12C) Kinetics
of weight gain/loss in mice (FIG. 12D) Overall survival of
mice.
[0033] FIGS. 13A-13G. 2nd generation ADR with CD28 intracellular
signaling domain (ADR.28zeta). (FIG. 13A) Structure of ADR.28zeta.
(FIG. 13B-FIG. 13C) in vitro cytotoxicity of ADR.28zeta against
target-expressing cell lines. (FIG. 13D-FIG. 13G) ADR.28zeta
protected mice from xeno-GvHD. (FIG. 13D) Schematic of the model
(FIG. 13E) Expansion of FFLuc-labeled ADR.28zeta T cells in vivo
(FIG. 13F) Kinetics of weight gain/loss in mice (FIG. 13G) Overall
survival of mice.
[0034] FIG. 14. Cytotoxicity of ADR-expressing cells in cancer.
(left) Cytotoxicity of 4-1BB ADR-expressing T cells against HDLM-2
Hodgkin's lymphoma cells (right) Cytotoxicity of 4-1BB
ADR-expressing T cells against K562 chronic myeloid leukemia (CML)
cells. Absolute counts of tumor cells upon a 48 h coculture at a
1:1 effector-to-target ratio is shown.
DETAILED DESCRIPTION
[0035] As used herein, 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.
[0036] Throughout this specification, unless the context requires
otherwise, the words "comprise", "comprises" and "comprising" will
be understood to imply the inclusion of a stated step or element or
group of steps or elements but not the exclusion of any other step
or element or group of steps or elements. By "consisting of" is
meant including, and limited to, whatever follows the phrase
"consisting of." Thus, the phrase "consisting of" indicates that
the listed elements are required or mandatory, and that no other
elements may be present. By "consisting essentially of" is meant
including any elements listed after the phrase, and limited to
other elements that do not interfere with or contribute to the
activity or action specified in the disclosure for the listed
elements. Thus, the phrase "consisting essentially of" indicates
that the listed elements are required or mandatory, but that no
other elements are optional and may or may not be present depending
upon whether or not they affect the activity or action of the
listed elements.
[0037] Reference throughout this specification to "one embodiment,"
"an embodiment," "a particular embodiment," "a related embodiment,"
"a certain embodiment," "an additional embodiment," or "a further
embodiment" or combinations thereof means that a particular
feature, structure or characteristic described in connection with
the embodiment is included in at least one embodiment of the
present invention. Thus, the appearances of the foregoing phrases
in various places throughout this specification are not necessarily
all referring to the same embodiment. Furthermore, the particular
features, structures, or characteristics may be combined in any
suitable manner in one or more embodiments.
[0038] The term "subject," as used herein, generally refers to an
individual in need of a therapy for a medical condition of any
kind. A subject can be an animal of any kind. The subject can be
any organism or animal subject that is an object of a method or
material, including mammals, e.g., humans, laboratory animals
(e.g., primates, rats, mice, rabbits), livestock (e.g., cows,
sheep, goats, pigs, turkeys, and chickens), household pets (e.g.,
dogs, cats, and rodents), horses, and transgenic non-human animals.
The subject can be a patient, e.g., have or be suspected of having
a disease (that may be referred to as a medical condition), such as
one or more infectious diseases, one or more genetic disorders, one
or more cancers, or any combination thereof. The disease may be
pathogenic. The subject may being undergoing or having undergone
antibiotic treatment. The subject may be asymptomatic. The subject
may be healthy individuals. The term "individual" may be used
interchangeably, in at least some cases. The "subject" or
"individual", as used herein, may or may not be housed in a medical
facility and may be treated as an outpatient of a medical facility.
The individual may be receiving one or more medical compositions
via the internet. An individual may comprise any age of a human or
non-human animal and therefore includes both adult and juveniles
(i.e., children) and infants and includes in utero individuals. The
individual may be of any race and gender. It is not intended that
the term connote a need for medical treatment, therefore, an
individual may voluntarily or involuntarily be part of
experimentation whether clinical or in support of basic science
studies.
[0039] The term "engineered" as used herein refers to a molecule
that is not present in nature and has been generated by the hand of
man, such as by genetic recombination techniques standard in the
art.
[0040] In the context of the present disclosure, an "effective
amount" or a "therapeutically effective amount" refers to the
amount of cells that, when administered to an individual, allows
for targeting of activated T cells and/or alleviates the signs and
or symptoms of a medical condition or prevents a medical condition.
The actual amount to be administered can be determined based on
studies done either in vitro or in vivo where the functional immune
cells exhibit pharmacological activity against a medical
condition.
[0041] I. Auto/Allo-Immune Defense Receptors and Compositions and
Uses Thereof
[0042] The present disclosure encompasses synthetic chimeric
receptor molecules that provide for selective targeting of
activated T cells, including pathogenic T cells. The engineered
molecules are synthetic and may be produced by recombinant
technology. The molecules may be referred to as auto/allo-immune
defense receptors that target activated T cells, including
activated pathogenic T cells, including with high specificity.
[0043] In particular embodiments, auto/allo-immune defense
receptors (ADRs) comprise an entity that targets one or more
compounds that are upregulated on activated T cells. Although the
compound that is upregulated on activated T cells may be any one or
combination thereof, in specific embodiments OX40, 4-1BB, and CD40L
are upregulated on activated T cells and are the subjects of which
the ADRs are targeting. The ADRs are present on allogeneic immune
cells that are allogeneic with respect to the individual receiving
the cells, in particular embodiments. In other instances, the ADRs
are expressed on autologous T cells, xenogeneic cells, and/or
synthetic cells.
[0044] A. Auto/Allo-Immune Defense Receptor (ADR) Molecules
[0045] ADR molecules are synthetic, non-natural, and produced by
the hand of man and comprise at least (1) an extracellular domain
that targets compounds that are selectively present on activated T
cells (and in specific embodiments, the extracellular domain is a
protein or functional fragment or derivative thereof that targets
one or more compounds that are upregulated on activated T cells);
that is operably linked to (2) a signaling domain promoting T-cell
activation, including those derived from CD3 zeta subunit, DAP12,
and Fc receptors, or another ITAM-comprising sequence, for example.
The ADR molecule may comprise or consist of or consist essentially
of elements (1) and (2). In at least certain cases, the ADR
comprises one or more components of a Type I transmembrane protein
and/or one or more components of a Type II transmembrane
protein.
[0046] In specific embodiments, in the ADR molecule the
extracellular domain comprises a protein that selectively binds an
associated protein on an activated T cell. For example, the ADR
extracellular domain may comprise a ligand for a receptor on an
activated T cell, or the ADR extracellular domain may comprise a
receptor for a ligand on an activated T cell.
[0047] In specific embodiments, in the ADR molecule the
extracellular domain comprises a ligand for OX40, a ligand for
4-1BB, and/or CD40. These particular examples have associated
proteins on activated T cells that are OX40, 4-1BB, and CD40L,
respectively. In alternative embodiments, other particular
compositions on the activated T cells are targeted. For example,
one may target other activation markers that are upregulated on the
cell surface of T cells (like CD69, CD25, CD71, etc.) can be
targeted using a similar approach. In such cases, the corresponding
ADR molecule would have a respective CD69, CD25, or CD71 ligand, or
an antibody-derived targeting moiety, instead of
4-1BB/OX40-specific ligands.
[0048] In some cases, activated T cells are targeted that have
upregulation of expression of OX40, in contrast to T cells that are
not activated. To target these activated T cells, a ligand of OX40
could be utilized in the ADR to be able to target the activated T
cells. In cases wherein a ligand for OX40 is utilized in the ADR,
the ligand for OX40 may be any suitable ligand for OX40 including
at least OX40L, an antibody (or functional fragment thereof) that
binds to OX40, a fusion of Fc with OX40L, or functional derivatives
or fragments thereof. OX40L may also be referred to as tumor
necrosis factor (ligand) superfamily, member 4
(tax-transcriptionally activated glycoprotein 1, 34 kDa), OX40L,
CD252, TNFSF4, TXGP1, OX-40L, or gp34.
[0049] In some cases, activated T cells are targeted that have
upregulation of expression of 4-1BB, in contrast to T cells that
are not activated. To target these activated T cells, a ligand of
4-1BB could be utilized in the ADR to be able to target the
activated T cells. In cases wherein a ligand for 4-1BB is utilized
in the ADR, the ligand for 4-1BB may be any suitable ligand for
4-1BB including 4-1BBL, an antibody (or functional fragment
thereof) that targets 4-1BB, a fusion of Fc with 4-1BBL, or
functional derivatives or fragments thereof.
[0050] In certain cases, activated T cells are targeted that have
upregulation of expression of CD40L, in contrast to T cells that
are not activated. To target these activated T cells, a receptor
for CD40L could be utilized in the ADR to be able to target the
activated T cells. In cases wherein a receptor for CD40L is
utilized in the ADR, the ADR may comprise CD40 (that may also be
referred to as Bp50, CDW40, TNFRSF5, or p50), an antibody (or
functional fragment thereof) that targets CD40L, or functional
derivatives or fragments thereof.
[0051] In some cases, the ADR molecule comprises two or more
extracellular domains to facilitate targeting of the activated T
cells. Such combinations may enhance targeting of activated T cells
generally or may allow for specific targeting of certain subsets of
activated T cells. For example, the ADR may comprise both OX40L and
4-1BBL as extracellular domains in the same ADR molecule to allow
for targeting of activated T cells that express either OX40 or
4-1BB. Such an example of a combination would selectively target
activated T cells that express either OX40 or 4-1BB regardless of
whether or not those activated T cells also express CD40L.
Analogously, ADRs may comprise both CD40 and OX40L to target
activated T cells that express either CD40L or OX40 regardless of
whether or not those activated T cells also express 4-1BB.
[0052] In the ADR molecule, the extracellular domain may be
operably linked to one or more components, including components
that are part of the ADR molecule. One such component may be a
protein that mediates downstream signaling during T cell
activation. In particular embodiments the ADR comprises CD3zeta
(also referred to as CD247, CD3-ZETA, CD3H, CD3Q, CD3Z, IMD25, T3Z,
or TCRZ) or a functional fragment or derivative thereof. CD3zeta
mediates downstream ITAM-derived signaling during T cell
activation. Other ITAM-containing signaling domains may include
those derived from DAP12, Fc receptors, other CD3 subunits, etc.
The signaling domains may be non-covalently linked to the ADR via
another domain.
[0053] In particular embodiments, the ADR comprises a spacer
between the CD3 zeta and the extracellular protein that targets one
or more compounds that are upregulated on activated T cells. In
other cases, a spacer is not utilized. The spacer may comprise
sequence that is inert or contributes substantially little or
nothing with respect to any function the ADR may have, whereas in
other cases the spacer comprises sequence that enhances a function
of the ADR and/or allows it to be detectable and/or able to be
targeted for inhibition, as examples. In specific embodiments, the
spacer comprises encoded protein sequence that facilitates
detection of cells that express the ADR. For example, the spacer
may encode Fc region or fragments thereof that would allow for
surface detection of the cells, such as using anti-Fc Abs. In
particular embodiments, the spacer provides separation between the
ligand binding domain and the membrane to avoid potential steric
hindrances, such as those caused by the splicing of Type II
transmembrane proteins (4-1BBL, OX40L) with the Type I ADR backbone
(TM, signaling domains). The spacer may be of any suitable length,
including about 10-220 amino acids as an example. The spacer length
may be in a range of 10-220, 10-200, 10-150, 10-100, 10-50, 25-200,
25-150, 25-100, 25-75, 25-50, 50-200, 50-150, 50-125, 50-100,
50-75, 75-200, 75-150, 75-100, 100-200, 100-175, 100-150, 100-125,
125-200, 125-175, 125-150, 150-200, 150-175, 175-200, and so forth.
The spacer may be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,
105, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, or 220
amino acids in length. In other cases, the spacer is less than 10
amino acids or more than 200 amino acids.
[0054] In some cases, ADRs comprise one, two, three, or more
costimulatory domains that enhance cytokine production from the
cells that express the ADR. The costimulatory domains may be
derived from the intracellular signaling domains of costimulatory
proteins including CD28, CD27, 4-1BB, OX40, ICOS, CD30, HVEM, CD40,
and so forth. As an example only, when the ADR comprises 4-1BBL,
the costimulatory domain of the ADR may or may not be from
4-1BB.
[0055] In some embodiments, the ADRs will comprise a transmembrane
domain that may be of any kind so long as it allows the CD3 zeta
component of the ADR to be located intracellularly and the
extracellular domain that targets one or more compounds that are
upregulated on activated T cells to be located extracellularly. In
other instances, ADRs are soluble proteins that can bind to the
respective ligand on activated T cells and promote cytotoxicity by
crosslinking TCR (e.g., ADR-CD3 T-cell engager protein). In a case
wherein the extracellular domain is from a surface protein having a
transmembrane domain, (CD40, for example), the ADR may comprise the
transmembrane domain from that corresponding endogenous molecule.
In some cases in which the ADR molecule comprises one or more
costimulatory domains, the transmembrane domain (TM) may be from
the same endogenous molecule that has the costimulatory domain.
Examples of TMs include those from CD3, CD8.alpha., CD27, CD28,
4-1BB, OX40, CD4, etc.
[0056] In an example of a ADR polypeptide, the components may be in
a particular N-terminal (N) to C-terminal (C) order. For a general
ADR, the receptor may comprise one of the following (as examples
only) and wherein the extracellular domain comprises the protein
that selectively binds an associated protein on an activated T
cell:
[0057] N-extracellular domain-signaling domain-C
[0058] N-extracellular domain-CD3zeta-C
[0059] N-extracellular domain-spacer-CD3zeta-C
[0060] N-extracellular domain-spacer-costimulatory
domain-CD3zeta-C
[0061] N-extracellular domain-spacer-two costimulatory
domains-CD3zeta-C
[0062] N-two extracellular domains-spacer-costimulatory
domain-CD3zeta-C
[0063] N-two extracellular domains-spacer-two costimulatory
domains-CD3zeta-C
[0064] In any case, the transmembrane domain may be C-terminal with
respect to the spacer. A signal peptide at the N-terminus may be
utilized to facilitate expression of Type II ligands (such as OX40L
and 4-1BBL) on a Type I transmembrane protein backbone (such as the
transmembrane domain, signaling domains, CD3 zeta).
[0065] In some cases, the ADR comprises one or more detectable
markers, such as markers that are colorimetric, fluorescent, and/or
radioactive, and so forth. Examples include green fluorescent
protein, blue fluorescent protein, and so forth.
[0066] The ADR may be in the form of a polynucleotide or
polypeptide expressed by a polynucleotide, although the ADR may be
synthetically generated as a protein. Recombinant technology to
produce ADR polynucleotides and polypeptides are known in the
art.
[0067] In certain cases, a ADR polynucleotide is in an expression
construct or is part of an expression construct present on a vector
that may be a viral vector or a non-viral vector. Examples of
non-viral vectors include plasmids. Examples of viral vectors
include lentiviral, retroviral, adenoviral, and adeno-associated
viral vectors. Any vector expressing the ADR will have appropriate
element(s) to allow expression in an eukaryotic cell, including an
immune cell, such as a T cell, NK cell, or NKT cell, for example.
Such appropriate elements include promoters and so forth.
TABLE-US-00001 4-1BB ADR (SEQ ID NO: 1)
MEFGLSWLFLVAILKGVQCGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDP
GLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSL
ALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLG
VHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEESKYGPPCP
PCPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
QKSLSLSPGKKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSRVKFSRS
ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG
LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ
ALPPRTSAAAGGGGSGGGGSGGGGSMVSKGEELFTGVVPILVELDGDVNG
HKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFTYGVQCFARY
PDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
LKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIKVNFKTRHNIEDG
SVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFV TAAGITLGMDELYK
OX40 ADR (SEQ ID NO: 2)
MEFGLSWLFLVAILKGVQCQVSHRYPRIQSIKVQFTEYKKEKGFILTSQK
EDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKK
VRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFC
VLESKYGPPCPPCPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD
IAVEWESNGQPENNYKTTPPVLDSDGSHLYSKLTVDKSRWQQGNVFSCSV
MHEALHNHYTQKSLSLSPGKKDPKFWVLVVVGGVLACYSLLVTVAFIIFW
VRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG
KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT
KDTYDALHMQALPPRTSAAAGGGGSGGGGSGGGGSMVSKGEELFTGVVPI
LVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTF
TYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIHKDDGNYKTRAEVKFE
GDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIKVNF
KTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKR
DHMVLLEFVTAAGITLGMDELYK CD40L ADR (SEQ ID NO: 3)
MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSD
CTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETD
TICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSDTICEPCPVGF
FSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRESKYGPP
CPPCPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG
QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH
YTQKSLSLSPGKKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSRVKFS
RSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ
EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH
MQALPPRTSAAAGGGGSGGGGSGGGGSMVSKGEELFTGVVPILVELDGDV
NGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFTYGVQCFA
RYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNR
IELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIKVNFKTRHNIE
DGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
FVTAAGITLGMDELYK
[0068] In certain embodiments, the extracellular domain that
targets activated T cells comprises an antibody or functional
fragment or derivative thereof. The term "antibody," as used
herein, refers to an immunoglobulin molecule that specifically
binds with an antigen. Antibodies can be intact immunoglobulins
derived from natural sources or from recombinant sources and can be
immunoreactive portions of intact immunoglobulins. Antibodies are
typically tetramers of immunoglobulin molecules. The antibodies in
the present invention may exist in a variety of forms including,
for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab
and F(ab).sub.2, as well as single chain antibodies and humanized
antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al.,
1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor,
N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA
85:5879-5883; Bird et al., 1988, Science 242:423-426).
[0069] In some cases, the extracellular domain of the ADR comprises
an antibody fragment. The term "antibody fragment" refers to a
portion of an intact antibody and refers to the antigenic
determining variable regions of an intact antibody. Examples of
antibody fragments include, but are not limited to, Fab, Fab',
F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and
multispecific antibodies formed from antibody fragments.
[0070] Synthetic antibodies may be used in the ADR. By the term
"synthetic antibody" as used herein, is meant an antibody which is
generated using recombinant DNA technology, such as, for example,
an antibody expressed by a bacteriophage as described herein. The
term should also be construed to mean an antibody that has been
generated by the synthesis of a DNA molecule encoding the antibody
and which DNA molecule expresses an antibody protein, or an amino
acid sequence specifying the antibody, wherein the DNA or amino
acid sequence has been obtained using synthetic DNA or amino acid
sequence technology that is available and well known in the
art.
[0071] B. Cells Expressing ADRs
[0072] Allogeneic cells used for adoptive transfer are prone to
having limited efficacy because of the immune reaction by the
recipient individual. Although in some cases, the cells may be
modified to remove endogenous TCR (such as with CRISPR), for
example to prevent graft-versus-host disease, alternatively one can
utilize virus-specific T cells (intact or CAR/TCR-modified) to
retain anti-viral activity, which is useful in certain pathologic
conditions. Although such VSTs have very limited graft-versus-host
activity because their TCRs are more restricted to viral antigens,
they are still susceptible to deleterious reaction by the
recipient.
[0073] Encompassed in the disclosure are cells that are improved
for allogeneic use by being modified to express a synthetic ADR
molecule. Thus, the disclosure includes cells harboring the ADR as
a polynucleotide and as an expressed ADR polypeptide on the surface
of the cells. In particular cases, the ADR-expressing cells are
produced for the purpose of being maintained in a repository for
off-the-shelf use. The cells may be housed in a repository already
being configured to express an ADR, or they may be housed in a bank
and configured to express an ADR following retrieval from the
repository. Certain cells, such as bacterial cells, may be utilized
to generate the ADR molecules, whereas other cells, such as
eukaryotic cells, harboring the ADR may be used for methods of the
disclosure including targeting activated T cells. As shown herein,
ADR-expressing immune cells selectively eliminate activated T cells
and ADR-expressing immune cells are protected against cytolysis by
alloreactive T cells.
[0074] Cells that express the ADR molecule may be of any kind, but
in specific embodiments they are immune cells, for example immune
effector cells, such as T cells, NK cells, NKT cells, or cell lines
derived from the said lineages or engineered to have cytotoxic
activity that have been modified to express the ADR and are
therefore not found in nature. Populations of the non-natural
ADR-expressing cells are contemplated, including populations that
are at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98,
99, or 100% of the population being ADR-expressing cells. The cells
may be generated by standard methods of transfection or
transduction of synthetic ADR polynucleotides, as an example.
[0075] In some cases, the ADR molecules that modified to express
the ADR molecule may already be engineered or subsequently are
engineered to have another engineered, non-natural molecule other
than the ADR. For example, cells that express chimeric antigen
receptors (CAR) or engineered T cell receptors (TCR) and in doing
so can protect such cells against host rejection and therefore
increase their therapeutic potency. Cells expressing one or more
CARs and/or one or more TCRs may be engineered to express one or
more ADRs, or cells that express one or more ADRs may be engineered
to express one or more CARs and/or one or more TCRs. Thus, in some
cases a ADR is expressed on a different vector than a CAR and/or
TCR, yet in other cases a ADR molecule is expressed on the same
vector as a CAR and/or TCR. In cases wherein a ADR is expressed on
the same vector as a CAR (as an example), the ADR and CAR
expression may be directed from the same or different regulatory
elements. In any case, the ADR and CAR may be expressed as a single
polypeptide with a cleavable element between them, such as 2A.
[0076] In cases wherein ADR-expressing cells also express a CAR or
TCR, the CAR or TCR may target any particular antigen. In cases
wherein CARs are employed, the CARs may be first generation, second
generation, third generation and so on. The CAR may be bispecific,
in specific cases.
[0077] In some cases, cells expressing the ADR molecules are
engineered, such as engineered to lack expression of one or more
endogenous molecules. In specific cases the cells are engineered to
lack expression of one or more endogenous genes that would
facilitate fratricide of the cells otherwise. In specific cases the
cells expressing the ADR molecules are engineered to lack
expression of 4-1BB or OX40, for example. Engineering of the cells
may occur by CRISPR/Cas9, merely as an example.
[0078] II. Methods of Using Auto/Allo-Immune Defense Receptors
[0079] Embodiments of the disclosure include methods of providing
an effective amount of ADR-expressing cells to an individual for
any purpose. Methods include providing selectively targeting
activated T cells in an individual for any purpose. The activated T
cells are targeted by exposing activated T cells to an effective
amount of ADR-expressing immune cells, such as ADR-expressing T
cells. Such exposure may have one or more resultant applications,
for example.
[0080] In some embodiments, the ADRs are used to selectively target
activated immune cells other than activated T cells, such as B
cells (that would be useful for controlling unwanted B-cell
responses such as with lupus, rheumatoid arthritis, etc.), as well
as targeting activation of innate immunity (such as macrophage
activation syndrome, and so on). In other embodiments, the ADRs are
utilized to specifically target malignant cells expressing their
corresponding target, including 4-1BB or OX40 or CD40L, for
example.
[0081] The regimen for providing to an individual an effective
amount of ADR-expressing cells may be known or determined by an
individual or individuals delivering the cells for therapy or
prevention, or regardless of the method of use. In preventative
cases, for example, the cells may be delivered prior to detection
of one or more symptoms, or the cells may be delivered following
detection of one or more symptoms but before further symptom(s)
develop and/or worsen. For treatment cases, the individual may be
provided the effective amount of cells after one, two, or more
symptoms develop and may be after clinical diagnosis.
[0082] In specific aspects of the methods, the individual may be
given a single dose of a therapeutically effective amount of cells,
or the individual may be given multiple deliveries of the
therapeutically effective amount of the cells, such as multiple
deliveries separated by 1, 2, 3, 4, 5, 6, 7 days, or 1, 2, 3, or 4
weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or 1, 2,
3, 4, 5, or more years, or any range there between, for example.
The time between doses may vary in a single regimen.
[0083] The administration of the ADR-expressing cells may be via
any suitable route to the individual, including locally or
systemically. In specific embodiments the ADR-expressing cells are
delivered intravenously, orally, rectally, topically,
intramuscularly, by infusion, enterically, nasally, by inhalation,
sublingually, bucally, transdermally, subcutaneously, and so forth.
The cells may or may not be delivered as a bolus. With multiple
administrations, the cells may or may not be provided to the
individual in different delivery routes. When the cells are
delivered to an individual, they may be delivered in a
pharmaceutically acceptable carrier or excipient. Particular
examples of doses for ADR-expressing cells include 10.sup.4
cells/m.sup.2, 10.sup.5 cells/m.sup.2, 10.sup.6 cells/m.sup.2,
10.sup.7 cells/m.sup.2, 10.sup.8 cells/m.sup.2, 10.sup.9
cells/m.sup.2, 10.sup.10 cells/m.sup.2, 10.sup.11 cells/m.sup.2, or
10.sup.12 cells/m.sup.2 and ranges there between.
[0084] A. Use for Off-the-Shelf Embodiments
[0085] The disclosure encompasses cells for adoptive transfer that
are able to be utilized off-the-shelf, including able to be
obtained from a repository for the purpose of use in an individual
other than the individual from which the cells were originally
obtained. The cells may already express the ADR prior to being
deposited in the repository, or the cells may be modified
afterwards to express the ADR. The cells may be any kind of immune
effector cells for adoptive transfer. The cells prior to deposit in
the repository or after obtaining them from the repository may be
modified to express a tumor-specific receptor, for example (e.g., a
CAR or a TCR).
[0086] As shown elsewhere herein, cells expressing ADRs selectively
target activated T- and NK-cells while sparing resting subsets. The
ADRs protect allogeneic T cells from immune rejection mediated by
T- and/or NK-cells in vitro in addition to protecting T-cells from
immune rejection in vivo. The ADRs in doing so do not interfere
with the function of an engineered anti-tumor receptor (CAR, as an
example) as T-cells co-expressing ADR and CAR can efficiently
eliminate both tumor and activated T cells in vitro. The disclosure
further provides anti-cancer activity in vivo in a mouse model
using "off-the-shelf" T cells co-expressing CAR and ADR while
retaining resistance to immune rejection from allogeneic T-cells
present in the same mice. As an example, in FIG. 14 it is shown
that 4-1BB ADR is effective against 4-1BB+ tumor cells such that
ADR can be used as a therapeutic modality against 4-1BB-expressing
malignancies.
[0087] In particular embodiments, "off-the-shelf" therapeutic cells
express ADR to resist immune rejection and either retain endogenous
TCR specificity (e.g., to viral or tumor antigens) or have
endogenous TCRs replaced with engineered anti-tumor receptors, such
as one or more CARs and/or one or more recombinant TCRs.
[0088] In specific embodiments, off-the-shelf cells are housed in a
repository and may be modified for a specific purpose either before
or after deposit in the repository. For example, ADR-expressing T
cells may be housed in a repository and ready for use, such as
after a tissue or organ transplant, to prevent graft rejection.
ADR-expressing T cells may be housed in a repository and may be
selected or engineered with a native or transgenic TCR, for example
against viral infection or cancer. ADR-expressing T cells may be
housed in a repository and may be transduced with a CAR directed to
cancer or pathogenic infection. ADR-expressing cells may be housed
in a repository and may be transduced with one or more CARs and/or
one or more TCRs directed to specific cancer-associated antigens or
neoantigens expressed by the patient's specific tumor.
[0089] In some cases, banked allogeneic cells are utilized for
prevention of rejection of solid organ grafts by destroying
rejecting host immune cells, particularly in cases when the
ADR-expressing cells were not themselves alloreactive.
[0090] Although the cells that are housed in the repository may be
allogeneic with respect to a recipient individual, in alternative
embodiments the cells housed in the repository are autologous with
respect to a recipient individual. For example, an individual with
cancer may have ADR-expressing T cells deposited in a repository
for subsequent use, such as in the event that the cancer comes out
of remission. In other cases, autologous ADR-expressing cells are
housed in a repository for treatment of autoimmune disorders.
[0091] B. Use in Autoimmune Disorders
[0092] Unwanted activation of endogenous autoreactive T cells in an
individual can lead to devastating autoimmune diseases for the
individual, such as diabetes mellitus, autoimmune colitis, and
multiple sclerosis. In particular embodiments, one or more
autoimmune disorders are prevented or treated using ADR-expressing
immune cells in the individual that impacts the autoimmune disorder
(or its potential development) by inhibiting endogenous
autoreactive T cells in the individual. In specific embodiments,
such use of the ADR-expressing cells spares resting non-pathogenic
naive and memory T cells in the individual. Thus, certain methods
of the disclosure utilize particular cells modified to express ADRs
that are provided to an individual in a sufficient amount to target
activated T cells, including pathogenic T cells, thereby initiating
destruction of the activated T cells.
[0093] In vivo activation of T cells with unwanted specificity may
cause pathogenicity, and in particular embodiments cells expressing
one or more ADRs target the activated T cells. In some cases,
ADR-expressing cells target pathogenic cells that are a subset of
activated T cells.
[0094] In particular cases, ADR-expressing T cells can be used to
prevent or reverse life-threatening and debilitating conditions
driven by activated T cells (organ rejection, graft-versus-host
disease, type I diabetes, multiple sclerosis, autoimmune colitis,
lupus, rheumatoid arthritis, as examples) using adoptive T-cell
transfer with the ADR-expressing T cells.
[0095] In some cases, the ADR-expressing T cells also comprise one
or more compositions other than the ADR that facilitate treatment
or prevention of one or more autoimmune disorders.
[0096] In some cases, an individual being provided the
ADR-expressing T cells is given one or more additional therapies to
prevent or treat one or more autoimmune disorders. The individual
may or may not be given one or more immunosuppressive drugs, for
example, such as glucocorticoids, cytostatics, antibodies, and/or
drugs acting on immunophilins. In addition or alternatively, the
individual may be given one or more appropriate vaccines.
[0097] In some cases, an individual is at risk for an autoimmune
disorder and is provided an effective amount of ADR-expressing
cells to prevent onset of the autoimmune disorder or to delay onset
and/or lessen one or more symptoms, including in severity and/or
duration, for example. An individual at risk for an autoimmune
disorder, for example, is one having a personal or family history,
being a female of certain ethnicity, and so forth. The individual
may have one autoimmune disorder and desires to prevent or reduce
the severity and/or duration or delay the onset of another
autoimmune disorder(s), in some cases.
[0098] Examples of autoimmune disorders that may be prevented or
treated with ADR-expressing cells include at least the following:
Achalasia; Addison's disease; Adult Still's disease;
Agammaglobulinemia; Alopecia areata; Amyloidosis; Ankylosing
spondylitis; Anti-GBM/Anti-TBM nephritis; Antiphospholipid
syndrome; Autoimmune angioedema; Autoimmune dysautonomia;
Autoimmune encephalomyelitis; Autoimmune hepatitis; Autoimmune
inner ear disease (AIED); Autoimmune myocarditis; Autoimmune
oophoritis; Autoimmune orchitis; Autoimmune pancreatitis;
Autoimmune retinopathy; Autoimmune urticarial; Axonal &
neuronal neuropathy (AMAN); Bal disease; Behcet's disease; Benign
mucosal pemphigoid; Bullous pemphigoid; Castleman disease (CD);
Celiac disease; Chagas disease; Chronic inflammatory demyelinating
polyneuropathy (CIDP); Chronic recurrent multifocal osteomyelitis
(CRMO); Churg-Strauss Syndrome (CSS) or Eosinophilic Granulomatosis
(EGPA); Cicatricial pemphigoid; Cogan's syndrome; Cold agglutinin
disease; Congenital heart block; Coxsackie myocarditis; CREST
syndrome; Crohn's disease; Dermatitis herpetiformis;
Dermatomyositis; Devic's disease (neuromyelitis optica); Discoid
lupus; Dressler's syndrome; Endometriosis; Eosinophilic esophagitis
(EoE); Eosinophilic fasciitis; Erythema nodosum; Essential mixed
cryoglobulinemia; Evans syndrome; Fibromyalgia; Fibrosing
alveolitis; Giant cell arteritis (temporal arteritis); Giant cell
myocarditis; Glomerulonephritis; Goodpasture's syndrome;
Granulomatosis with Polyangiitis; Graves' disease; Guillain-Barre
syndrome; Hashimoto's thyroiditis; Hemolytic anemia;
Henoch-Schonlein purpura (HSP); Herpes gestationis or pemphigoid
gestationis (PG); Hidradenitis Suppurativa (HS) (Acne Inversa);
Hypogammalglobulinemia; IgA Nephropathy; IgG4-related sclerosing
disease; Immune thrombocytopenic purpura (ITP); Inclusion body
myositis (IBM); Interstitial cystitis (IC); Juvenile arthritis;
Juvenile diabetes (Type 1 diabetes); Juvenile myositis (JM);
Kawasaki disease; Lambert-Eaton syndrome; Leukocytoclastic
vasculitis; Lichen planus; Lichen sclerosus; Ligneous
conjunctivitis; Linear IgA disease (LAD); Lupus; Lyme disease
chronic; Meniere's disease; Microscopic polyangiitis (MPA); Mixed
connective tissue disease (MCTD); Mooren's ulcer; Mucha-Habermann
disease; Multifocal Motor Neuropathy (MMN) or MMNCB; Multiple
sclerosis; Myasthenia gravis; Myositis; Narcolepsy; Neonatal Lupus;
Neuromyelitis optica; Neutropenia; Ocular cicatricial pemphigoid;
Optic neuritis; Palindromic rheumatism (PR); PANDAS; Paraneoplastic
cerebellar degeneration (PCD); Paroxysmal nocturnal hemoglobinuria
(PNH); Parry Romberg syndrome; Pars planitis (peripheral uveitis);
Parsonnage-Turner syndrome; Pemphigus; Peripheral neuropathy;
Perivenous encephalomyelitis; Pernicious anemia (PA); POEMS
syndrome; Polyarteritis nodosa; Polyglandular syndromes type I, II,
III; Polymyalgia rheumatic; Polymyositis; Postmyocardial infarction
syndrome; Postpericardiotomy syndrome; Primary biliary cirrhosis;
Primary sclerosing cholangitis; Progesterone dermatitis; Psoriasis;
Psoriatic arthritis; Pure red cell aplasia (PRCA); Pyoderma
gangrenosum; Raynaud's phenomenon; Reactive Arthritis; Reflex
sympathetic dystrophy; Relapsing polychondritis; Restless legs
syndrome (RLS); Retroperitoneal fibrosis; Rheumatic fever;
Rheumatoid arthritis; Sarcoidosis; Schmidt syndrome; Scleritis;
Scleroderma; Sjogren's syndrome; Sperm & testicular
autoimmunity; Stiff person syndrome (SPS); Subacute bacterial
endocarditis (SBE); Susac's syndrome; Sympathetic ophthalmia (SO);
Takayasu's arteritis; Temporal arteritis/Giant cell arteritis;
Thrombocytopenic purpura (TTP); Tolosa-Hunt syndrome (THS);
Transverse myelitis; Type 1 diabetes; Ulcerative colitis (UC);
Undifferentiated connective tissue disease (UCTD); Uveitis;
Vasculitis; Vitiligo; Vogt-Koyanagi-Harada Disease; and Wegener's
granulomatosis (or Granulomatosis with Polyangiitis (GPA)).
[0099] C. Use to Eliminate NK Cells
[0100] ADR-expressing immune cells may be utilized to eliminate NK
cells in cases wherein it is desirable to do so. As demonstrated
herein, the presence of ADRs on certain immune cells provides a
specific cytotoxic activity against NK cells that are involved with
mediating rapid rejection of HLA.sup.low or HLA-mismatched cells.
Thus, in situations where it is desirable to maintain HLA.sup.low
or HLA-mismatched cells, for example to be able to utilize adoptive
transfer of allogeneic cells into certain individuals, use of
ADR-expressing cells avoids NK cell activation and rejection of the
HLA-mismatched cells. Specifically, as shown herein, co-culture of
T cells expressing ADRs leads to elimination of NK cells and thus
offsets the NK cell-mediated host rejection of ADR-expressing
allogeneic T cells.
[0101] D. Use to Facilitate the Engraftment of Allogeneic
Cell/Tissue/Organ Transplant
[0102] In a specific aspect of avoiding activation of alloreactive
T cells, one may avoid rejection of allogeneic cells, tissues, or
organs in individuals receiving transplants, which is mainly
mediated by a population of alloreactive T cells from the
recipient. Activation of recipient's alloreactive T cells would
lead to graft failure, for example, if steps were not taken to
avoid such rejection. Therefore, in particular embodiments, methods
of transplanting cells, tissue, or organs into an individual
utilize delivery of an effective amount of ADR-expressing cells
before, during, and/or after the respective transplants of cells,
tissue(s), or organ(s). In some cases, the ADR-expressing cells are
not themselves part of the cells, tissue(s), or organ(s) that are
the subject of the transplant, whereas in other cases the
ADR-expressing cells are part of the respective cells, tissue(s),
or organ(s).
[0103] Tissue for transplantation may be of any kind including at
least skin, cornea, bone, tendons, heart valves, veins, or
arteries, for example. Organ for transplantation may be of any
kind, including heart, kidneys, liver, lungs, pancreas, intestine,
and thymus, for example.
[0104] In particular embodiments, ADR-expressing cells enhance
allogeneic cell use in an individual as a two-pronged approach: (1)
they inhibit the endogenous alloreactive T-cells in the individual;
and (2) they suppress NK cell-mediated rejection in the individual.
As such, the ADR molecules can enhance the persistence and activity
of any type of third party-derived therapeutic cells in the
individual including, for example, allogeneic therapeutic cells,
including T-cells, NK cells, NK-T cells, mucosal associated
invariant T cells (MATT) and other cytotoxic cells, including those
expressing engineered constructs such as chimeric antigen receptor
(CAR), transgenic TCR, etc.
[0105] E. Use in Prophylaxis or Treatment of Graft-Versus-Host
Disease (GvHD) During Allogeneic Cell/Tissue/Organ Transplant
[0106] In another specific aspect of avoiding activation of
alloreactive T cells, one may avoid life-threatening allo-immune
reactions in individuals receiving transplants of allogeneic cells,
tissues, or organs. Such transplants would contain donor
alloreactive T cells that would elicit development of
graft-versus-host disease (GvHD), for example, if steps were not
taken to avoid their activation. Therefore, in particular
embodiments, methods of transplanting cells, tissue, or organs into
an individual utilize delivery of an effective amount of
ADR-expressing cells before, during, and/or after the respective
transplants of cells, tissue(s), or organ(s). In some cases, the
ADR-expressing cells are not themselves part of the cells,
tissue(s), or organ(s) that are the subject of the transplant,
whereas in other cases the ADR-expressing cells are part of the
respective cells, tissue(s), or organ(s).
[0107] Tissue for transplantation may be of any kind including at
least skin, cornea, bone, tendons, heart valves, veins, or
arteries, for example. Organ for transplantation may be of any
kind, including heart, kidneys, liver, lungs, pancreas, intestine,
and thymus, for example.
[0108] III. Production of ADR-Expressing Cells
[0109] Cells expressing the ADR molecules may be produced in a
variety of ways, all of which may be routine in the art. The
production methods may include obtaining the cells to be modified
to express the ADR molecule and also include generation of the ADR
molecules.
[0110] A. Sources of T Cells
[0111] Prior to expansion and genetic modification of the
ADR-expressing T cells of the disclosure, a source of T cells may
be obtained from a subject. Such a step of obtaining may or may not
be part of the method. In some cases, obtaining T cells to be
modified and their manipulation may be performed by a party other
than the party that provides the ADR expressing-T cells to an
individual. T cells can be obtained from a number of sources,
including peripheral blood mononuclear cells, bone marrow, lymph
node tissue, cord blood, thymus tissue, tissue from a site of
infection, ascites, pleural effusion, spleen tissue, and tumors. In
certain embodiments of the present disclosure, any number of T cell
lines available in the art may be used. In certain embodiments, T
cells can be obtained from a unit of blood collected from a subject
using any number of techniques known to the skilled artisan, such
as Ficoll.TM. separation. In one embodiment, cells from the
circulating blood of an individual are obtained by apheresis. The
apheresis product typically contains lymphocytes, including T
cells, monocytes, granulocytes, B cells, other nucleated white
blood cells, red blood cells, and platelets, for example. In one
embodiment, the cells collected by apheresis may be washed to
remove the plasma fraction and to place the cells in an appropriate
buffer or media for subsequent processing steps. In one embodiment,
the cells are washed with phosphate buffered saline (PBS). In an
alternative embodiment, the wash solution lacks calcium and may
lack magnesium or may lack many if not all divalent cations. As
those of ordinary skill in the art would readily appreciate a
washing step may be accomplished by methods known to those in the
art, such as by using a semi-automated "flow-through" centrifuge
(for example, the Cobe 2991 cell processor, the Baxter CytoMate, or
the Haemonetics Cell Saver 5) according to the manufacturer's
instructions. After washing, the cells may be resuspended in a
variety of biocompatible buffers, such as, for example,
Ca.sup.2+-free, Mg.sup.2+-free PBS, PlasmaLyte A, or other saline
solution with or without buffer. Alternatively, the undesirable
components of the apheresis sample may be removed and the cells
directly re-suspended in culture media.
[0112] In another embodiment, T cells are isolated from peripheral
blood lymphocytes by lysing the red blood cells and depleting the
monocytes, for example, by centrifugation through a PERCOLL.TM.
gradient or by counterflow centrifugal elutriation. A specific
subpopulation of T cells, such as CD3.sup.+, CD28.sup.+, CD4.sup.+,
CD8.sup.+, CD45RA.sup.+, and CD45RO.sup.+ T cells, can be further
isolated by positive or negative selection techniques.
[0113] Enrichment of a T cell population by negative selection can
be accomplished with a combination of antibodies directed to
surface markers unique to the negatively selected cells. One method
is cell sorting and/or selection via negative magnetic
immunoadherence or flow cytometry that uses a cocktail of
monoclonal antibodies directed to cell surface markers present on
the cells negatively selected. For example, to enrich for CD4.sup.+
cells by negative selection, a monoclonal antibody cocktail
typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR,
and CD8. In certain embodiments, it may be desirable to enrich for
or positively select for regulatory T cells which typically express
CD4.sup.+, CD25.sup.+, CD62.sup.hi, GITR.sup.+, and FoxP3.sup.+.
Alternatively, in certain embodiments, T regulatory cells are
depleted by anti-C25 conjugated beads or other similar method of
selection.
[0114] For isolation of a desired population of cells by positive
or negative selection, the concentration of cells and surface
(e.g., particles such as beads) can be varied. In certain
embodiments, it may be desirable to significantly decrease the
volume in which beads and cells are mixed together (i.e., increase
the concentration of cells), to ensure maximum contact of cells and
beads. For example, in one embodiment, a concentration of 2 billion
cells/ml is used. In one embodiment, a concentration of 1 billion
cells/ml is used. In a further embodiment, greater than 100 million
cells/ml is used. In a further embodiment, a concentration of cells
of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used.
In yet another embodiment, a concentration of cells from 75, 80,
85, 90, 95, or 100 million cells/ml is used. In further
embodiments, concentrations of 125 or 150 million cells/ml can be
used. Using high concentrations can result in increased cell yield,
cell activation, and cell expansion. In another embodiment, it may
be desirable to use lower concentrations of cells. By significantly
diluting the mixture of T cells and surface (e.g., particles such
as beads), interactions between the particles and cells is
minimized
[0115] T cells for stimulation can also be frozen after a washing
step. Wishing not to be bound by theory, the freeze and subsequent
thaw step provides a more uniform product by removing granulocytes
and to some extent monocytes in the cell population. After the
washing step that removes plasma and platelets, the cells may be
suspended in a freezing solution. Many freezing solutions and
parameters are known in the art. In certain embodiments,
cryopreserved cells are thawed and washed as described herein and
allowed to rest for one hour at room temperature prior to
activation using the methods of the present invention.
[0116] Also contemplated in the context of the disclosure is the
collection of blood samples or apheresis product from a subject at
a time period prior to when the expanded cells as described herein
might be needed. As such, the source of the cells to be expanded
can be collected at any time point necessary, and desired cells,
such as T cells, isolated and frozen for later use in T cell
therapy for any number of diseases or conditions that would benefit
from T cell therapy, such as those described herein. In one
embodiment a blood sample or an apheresis is taken from a generally
healthy subject. In certain embodiments, a blood sample or an
apheresis is taken from a generally healthy subject who is at risk
of developing a disease, but who has not yet developed a disease,
and the cells of interest are isolated and frozen for later use. In
certain embodiments, the T cells may be expanded, frozen, and used
at a later time. In certain embodiments, samples are collected from
a patient shortly after diagnosis of a particular disease as
described herein but prior to any treatments. In a further
embodiment, the cells are isolated from a blood sample or an
apheresis from a subject prior to any number of relevant treatment
modalities, including but not limited to treatment with agents such
as natalizumab, efalizumab, antiviral agents, chemotherapy,
radiation, immunosuppressive agents, such as cyclosporin,
azathioprine, methotrexate, mycophenolate, and FK506, antibodies,
or other immunoablative agents such as CAMPATH, anti-CD3
antibodies, cytoxan, fludarabine, cyclosporin, FK506, rapamycin,
mycophenolic acid, steroids, FR901228, and irradiation. These drugs
inhibit either the calcium dependent phosphatase calcineurin
(cyclosporine and FK506) or inhibit the p70S6 kinase that is
important for growth factor induced signaling (rapamycin) (Liu et
al., Cell 66:807-815, 1991; Henderson et al., Immun 73:316-321,
1991; Bierer et al., Curr. Opin. Immun 5:763-773, 1993). In a
further embodiment, the cells are isolated for a patient and frozen
for later use in conjunction with (e.g., before, simultaneously or
following) bone marrow or stem cell transplantation, T cell
ablative therapy using either chemotherapy agents such as,
fludarabine, external-beam radiation therapy (XRT),
cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another
embodiment, the cells are isolated prior to and can be frozen for
later use for treatment following B-cell ablative therapy such as
agents that react with CD20, e.g., Rituxan.
[0117] B. Activation and Expansion of T Cells
[0118] Whether prior to or after genetic modification of the T
cells to express the ADR, the T cells can be activated and expanded
generally using methods as described, for example, in U.S. Pat.
Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358;
6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566;
7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S.
Patent Application Publication No. 20060121005. Generally, the T
cells of the disclosure are expanded by contact with a surface
having attached thereto an agent that stimulates a CD3/TCR complex
associated signal and a ligand that stimulates a co-stimulatory
molecule on the surface of the T cells. Such processes are known in
the art. In other instances, T cells can be modified to express ADR
without prior activation.
[0119] C. Generation of ADR Molecules
[0120] Turning generally to polynucleotides that encode the ADR,
the nucleic acid sequences coding for the ADR molecules can be
obtained using recombinant methods known in the art, such as, for
example by screening libraries from cells expressing the gene, by
deriving the gene from a vector known to include the same, or by
isolating directly from cells and tissues containing the same,
using standard techniques. Alternatively, the ADR polynucleotide of
interest can be produced synthetically, rather than cloned.
[0121] In brief summary, the expression of synthetic
polynucleotides encoding ADRs is typically achieved by operably
linking a nucleic acid encoding the ADR polypeptide or portions
thereof to a promoter, and incorporating the construct into an
expression vector. The vectors can be suitable for replication and
integration eukaryotes. Typical cloning vectors contain
transcription and translation terminators, initiation sequences,
and promoters useful for regulation of the expression of the
desired nucleic acid sequence.
[0122] The ADR polynucleotide can be cloned into a number of types
of vectors. For example, the nucleic acid can be cloned into a
vector including, but not limited to a plasmid, a phagemid, a phage
derivative, an animal virus, and a cosmid. Vectors of particular
interest include expression vectors, replication vectors, probe
generation vectors, and sequencing vectors.
[0123] Further, the expression vector may be provided to a cell in
the form of a viral vector. Viral vector technology is well known
in the art and is described, for example, in Sambrook et al. (2001,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, New York), and in other virology and molecular biology
manuals. Viruses, which are useful as vectors include, but are not
limited to, retroviruses, adenoviruses, adeno-associated viruses,
herpes viruses, and lentiviruses. In general, a suitable vector
contains an origin of replication functional in at least one
organism, a promoter sequence, convenient restriction endonuclease
sites, and one or more selectable markers, (e.g., WO 01/96584; WO
01/29058; and U.S. Pat. No. 6,326,193).
[0124] A number of viral-based systems have been developed for gene
transfer into mammalian cells. For example, retroviruses provide a
convenient platform for gene delivery systems. A selected gene can
be inserted into a vector and packaged in retroviral particles
using techniques known in the art. The recombinant virus can then
be isolated and delivered to cells of the subject either in vivo or
ex vivo. A number of retroviral systems are known in the art. In
some embodiments, adenovirus vectors are used. A number of
adenovirus vectors are known in the art. In one embodiment,
lentivirus vectors are used.
[0125] Additional promoter elements, e.g., enhancers, regulate the
frequency of transcriptional initiation. Typically, these are
located in the region 30-110 bp upstream of the start site,
although a number of promoters have recently been shown to contain
functional elements downstream of the start site as well. The
spacing between promoter elements frequently is flexible, so that
promoter function is preserved when elements are inverted or moved
relative to one another. In the thymidine kinase (tk) promoter, the
spacing between promoter elements can be increased to 50 bp apart
before activity begins to decline. Depending on the promoter, it
appears that individual elements can function either cooperatively
or independently to activate transcription.
[0126] One example of a suitable promoter is the immediate early
cytomegalovirus (CMV) promoter sequence. This promoter sequence is
a strong constitutive promoter sequence capable of driving high
levels of expression of any polynucleotide sequence operatively
linked thereto. Another example of a suitable promoter is
Elongation Growth Factor-1 alpha (EF-1alpha). However, other
constitutive promoter sequences may also be used, including, but
not limited to the simian virus 40 (SV40) early promoter, mouse
mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long
terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia
virus promoter, an Epstein-Barr virus immediate early promoter, a
Rous sarcoma virus promoter, as well as human gene promoters such
as, but not limited to, the actin promoter, the myosin promoter,
the hemoglobin promoter, and the creatine kinase promoter. Further,
the invention should not be limited to the use of constitutive
promoters. Inducible promoters are also contemplated as part of the
invention. The use of an inducible promoter provides a molecular
switch capable of turning on expression of the polynucleotide
sequence which it is operatively linked when such expression is
desired, or turning off the expression when expression is not
desired. Examples of inducible promoters include, but are not
limited to a metallothionine promoter, a glucocorticoid promoter, a
progesterone promoter, and a tetracycline promoter.
[0127] In order to assess the expression of an ADR polypeptide or
portions thereof, the expression vector to be introduced into a
cell can also contain either a selectable marker gene or a reporter
gene or both to facilitate identification and selection of
expressing cells from the population of cells sought to be
transfected or infected through viral vectors. In other aspects,
the selectable marker may be carried on a separate piece of DNA and
used in a co-transfection procedure. Both selectable markers and
reporter genes may be flanked with appropriate regulatory sequences
to enable expression in the host cells. Useful selectable markers
include, for example, antibiotic-resistance genes, such as neo and
the like.
[0128] Reporter genes are used for identifying potentially
transfected cells and for evaluating the functionality of
regulatory sequences. In general, a reporter gene is a gene that is
not present in or expressed by the recipient organism or tissue and
that encodes a polypeptide whose expression is manifested by some
easily detectable property, e.g., enzymatic activity. Expression of
the reporter gene is assayed at a suitable time after the DNA has
been introduced into the recipient cells. Suitable reporter genes
may include genes encoding luciferase, beta-galactosidase,
chloramphenicol acetyl transferase, secreted alkaline phosphatase,
or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000
FEBS Letters 479: 79-82). Suitable expression systems are well
known and may be prepared using known techniques or obtained
commercially. In general, the construct with the minimal 5'
flanking region showing the highest level of expression of reporter
gene is identified as the promoter. Such promoter regions may be
linked to a reporter gene and used to evaluate agents for the
ability to modulate promoter-driven transcription.
[0129] Methods of introducing and expressing ADR polynucleotides
into a cell are known in the art. In the context of an expression
vector, the vector can be readily introduced into a host cell,
e.g., mammalian, bacterial, yeast, or insect cell by any method in
the art. For example, the expression vector can be transferred into
a host cell by physical, chemical, or biological means.
[0130] Physical methods for introducing a ADR polynucleotide into a
host cell include calcium phosphate precipitation, lipofection,
particle bombardment, microinjection, electroporation, and the
like. Methods for producing cells comprising vectors and/or
exogenous nucleic acids are well-known in the art. See, for
example, Sambrook et al. (2001, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory, New York). One method for
the introduction of a polynucleotide into a host cell is calcium
phosphate transfection.
[0131] Biological methods for introducing a ADR polynucleotide of
interest into a host cell include the use of DNA and RNA vectors.
Viral vectors, and especially retroviral vectors, have become the
most widely used method for inserting genes into mammalian, e.g.,
human cells. Other viral vectors can be derived from lentivirus,
poxviruses, herpes simplex virus I, adenoviruses and
adeno-associated viruses, and the like. See, for example, U.S. Pat.
Nos. 5,350,674 and 5,585,362.
[0132] Chemical means for introducing a polynucleotide into a host
cell include colloidal dispersion systems, such as macromolecule
complexes, nanocapsules, microspheres, beads, and lipid-based
systems including oil-in-water emulsions, micelles, mixed micelles,
and liposomes. An exemplary colloidal system for use as a delivery
vehicle in vitro and in vivo is a liposome (e.g., an artificial
membrane vesicle).
[0133] In the case where a non-viral delivery system is utilized,
an exemplary delivery vehicle is a liposome. The use of lipid
formulations is contemplated for the introduction of the nucleic
acids into a host cell (in vitro, ex vivo or in vivo). In another
aspect, the nucleic acid may be associated with a lipid. The
nucleic acid associated with a lipid may be encapsulated in the
aqueous interior of a liposome, interspersed within the lipid
bilayer of a liposome, attached to a liposome via a linking
molecule that is associated with both the liposome and the
oligonucleotide, entrapped in a liposome, complexed with a
liposome, dispersed in a solution containing a lipid, mixed with a
lipid, combined with a lipid, contained as a suspension in a lipid,
contained or complexed with a micelle, or otherwise associated with
a lipid. Lipid, lipid/DNA or lipid/expression vector associated
compositions are not limited to any particular structure in
solution. For example, they may be present in a bilayer structure,
as micelles, or with a "collapsed" structure. They may also simply
be interspersed in a solution, possibly forming aggregates that are
not uniform in size or shape. Lipids are fatty substances which may
be naturally occurring or synthetic lipids. For example, lipids
include the fatty droplets that naturally occur in the cytoplasm as
well as the class of compounds which contain long-chain aliphatic
hydrocarbons and their derivatives, such as fatty acids, alcohols,
amines, amino alcohols, and aldehydes.
[0134] Lipids suitable for use can be obtained from commercial
sources. For example, dimyristyl phosphatidylcholine ("DMPC") can
be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate ("DCP")
can be obtained from K & K Laboratories (Plainview, N.Y.);
cholesterol ("Choi") can be obtained from Calbiochem-Behring;
dimyristyl phosphatidylglycerol ("DMPG") and other lipids may be
obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock
solutions of lipids in chloroform or chloroform/methanol can be
stored at about -20.degree. C. Chloroform is used as the only
solvent since it is more readily evaporated than methanol.
"Liposome" is a generic term encompassing a variety of single and
multilamellar lipid vehicles formed by the generation of enclosed
lipid bilayers or aggregates. Liposomes can be characterized as
having vesicular structures with a phospholipid bilayer membrane
and an inner aqueous medium. Multilamellar liposomes have multiple
lipid layers separated by aqueous medium. They form spontaneously
when phospholipids are suspended in an excess of aqueous solution.
The lipid components undergo self-rearrangement before the
formation of closed structures and entrap water and dissolved
solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology
5: 505-10). However, compositions that have different structures in
solution than the normal vesicular structure are also encompassed.
For example, the lipids may assume a micellar structure or merely
exist as nonuniform aggregates of lipid molecules. Also
contemplated are lipofectamine-nucleic acid complexes.
[0135] In some instances, the ADR molecules may be integrated into
an endogenous nucleic acid of the cells. One may have a target site
for homologous recombination, where it is desired that a construct
be integrated at a particular locus. For example,) can knock-out an
endogenous gene and replace it (at the same locus or elsewhere)
with the gene encoded for by the construct using materials and
methods as are known in the art for homologous recombination. For
homologous recombination, one may use either OMEGA or 0-vectors.
CRISPR/Cas9, zinc finger nucleases, TALE nucleases, meganucleases,
and other site directed nucleases may be used to target and cleave
a specific site in the genome to promote homologous
recombination.
[0136] The exemplary T cells that have been engineered to include
the ADR-expressing construct(s) may be grown in culture under
selective conditions and cells that are selected as having the
construct may then be expanded and further analyzed, using, for
example; the polymerase chain reaction for determining the presence
of the construct in the host cells. Once the engineered host cells
have been identified, they may then be used as planned, e.g.
expanded in culture or introduced into a host organism.
[0137] Depending upon the nature of the cells, the cells may be
introduced into a host organism, e.g. a mammal, in a wide variety
of ways. The cells may be introduced at the site of the tumor, in
specific embodiments, although in alternative embodiments the cells
home to the cancer or are modified to home to the infected tissue.
The number of cells that are employed will depend upon a number of
circumstances, the purpose for the introduction, the lifetime of
the cells, the protocol to be used, for example, the number of
administrations, the ability of the cells to multiply, the
stability of the recombinant construct, and the like. The cells may
be applied as a dispersion, generally being injected at or near the
site of interest. The cells may be in a physiologically-acceptable
medium.
[0138] The DNA introduction need not result in integration in every
case. In some situations, transient maintenance of the DNA
introduced may be sufficient. In this way, one could have a short
term effect, where cells could be introduced into the host and then
turned on after a predetermined time, for example, after the cells
have been able to home to a particular site.
EXAMPLES
[0139] The following examples are presented in order to more fully
illustrate particular embodiments of the disclosure. They should in
no way, however, be construed as limiting the broad scope of the
disclosure.
Example 1
Auto/Allo-Immune Defense Receptors for the Selective Targeting of
Pathogenic T Cells
[0140] Disclosed herein is a novel approach to specifically target
pathogenic T cells using auto/allo-immune defense receptors (ADRs)
expressed on normal T cells. ADR-expressing T cells find and
eliminate only activated T cells and spare resting non-pathogenic
naive and memory T cells, which constitute the majority of
circulating lymphocytes
[0141] The concept of the ADR-mediated targeting is based on the
observation that within 24 h of activation, T cells transiently
upregulate costimulatory genes 4-1BB, OX40, and/or CD40L on their
cell surface. The expression of 4-1BB, OX40, and/or CD40L is only
maintained when the T-cells are actively cytotoxic and is gradually
downregulated within 4-5 days, when TCR signaling has stopped.
Notably, activated CD8.sup.+ T cells showed higher magnitude of
4-1BB expression whereas CD4.sup.+ T cells preferentially expressed
OX40 and/or CD40L. Apart from activated T cells, ADR ligands may
only be expressed on activated NK cells and on some other
non-critical and replenishable subsets of cells. The pattern of
expression of 4-1BB, OX40, and CD40L makes these genes attractive
targets to target activated cells with high specificity while
avoiding permanently damaging critical immune and non-immune
tissues.
[0142] To explore the feasibility of targeting these activated T
cells, auto/allo-immune defense receptors (ADRs) were designed to
comprise of a 4-1BB- or OX40-specific ligand, or a CD40L-specific
receptor directly connected via a spacer to a CD3.zeta. chain
encoded in a gammaretroviral vector SFG. The spacer region was
incorporated a) to enable integration of type II proteins 4-1BBL
and OX40L into the Type I backbone of the ADR and b) to facilitate
detection of ADR on the cell surface by FACS staining. Transduction
of T cells with this construct efficiently forced ADR expression on
the cell surface. These ADR T cells had potent and robust
cytotoxicity against 4-1BB-, OX40-, and CD40L-expressing cells,
eliminating 90-99% of the target cells within 48 h. These results
demonstrate the feasibility of generating functional 4-1BB-, OX40-,
and CD40L-specific ADR T cells.
[0143] Because ADR signaling in T cells in turn upregulates 4-1BB,
OX40, and CD40L, thus promoting fratricide and impeding effector
cell expansion, the effects of CRISPR/Cas9 genomic disruption of
ADR target genes in the effector T cells were explored. The
inventors had previously shown that this CRISPR/Cas9 approach can
prevent fratricide of primary human T cells expressing a
CD7-specific CAR. In this context, with CRISPR/Cas9 the inventors
were able to knock out 4-1BB expression in .about.70% of ADR T
cells that consistently increased ADR T cell expansion >2-fold
at 48 h following coculture with 4-1BB.sup.+ target cells without
affecting the cytotoxicity.
[0144] Next, the ability of ADR T cells was tested to selectively
eliminate activated T cells. 4-1BB, OX40, and CD40L ADR T cells
were co-cultured with fluorescently labeled resting or CD3/CD28
activated T cells. Residual live CD4.sup.+ and CD8.sup.+ T cells
were quantified by flow cytometry with counting beads. There was no
reactivity against resting autologous T cells after 72h of
co-culture with T cells expressing 4-1BB-, OX40-, or CD40L-specific
ADR (FIG. 2B). In contrast, co-culture of 4-1BB ADR T cells with
CD3/CD28-activated T cells eliminated most CD8.sup.+ and some
CD4.sup.+ T cells within 48 h. Incubation with OX40 ADR T cells
resulted in a reciprocal high-level depletion of activated
CD4.sup.+ T cells and modest depletion of activated CD8+ T cells.
CD40L ADR T cells produced moderate cytotoxic effect on activated
CD4.sup.+ T cells yet no effect was seen on CD8.sup.+ T cells. The
differential targeting profiles of OX40, CD40L and 4-1BB ADR T
cells against activated CD4.sup.+ and CD8.sup.+ T cells correlates
with observed differences in the magnitude and kinetics of OX40,
CD40L and 4-1BB expression on each T cell subset. This property of
ADRs can be utilized to preferentially target either or both
subsets of allo- or auto-reactive T cells according to need.
Therefore, ADR expression enables T cells to specifically target
activated (pathogenic) T cells but spare resting cells, suggesting
their clinical use.
[0145] It was assessed whether virus-specific T cells (VST)
expressing ADRs can resist allogeneic rejection in an in vitro
mixed lymphocyte reaction (MLR) assay. CMV-specific T cells were
generated from an HLA-A2-negative donor and mixed control
non-transduced or ADR-transduced VST with alloreactive HLA-A2.sup.+
PBMC at a 1:2 cell-to-cell ratio. The inventors then cultured the
cells for 12 days. At the end of co-culture, control VSTs were
almost completely eliminated by HLA-A2.sup.+ PBMC whereas VSTs
expressing either 4-1BB ADR or OX40 ADR resisted rejection. Taken
together, these results demonstrate the feasibility and selectivity
of targeting activated T cells using the newly developed ADR
platform embodiment.
Example 2
Auto/Allo-Immune Defense Receptors for the Selective Targeting of
NK Cells
[0146] The ADRs demonstrate a specific cytotoxic activity against
NK cells, a key cell population mediating rapid rejection of
HLA.sup.low or HLA-mismatched cells.
[0147] NK cells are capable of recognizing HLA-mismatched cells or
cells with low HLA expression, as a part of the anti-tumor and
anti-viral immune surveillance. Adoptive transfer of allogeneic
cells into immunoreplete patients would thus result in NK-cell
activation and rejection of the HLA-mismatched cells. Here, it is
shown that co-culture of T cells expressing 4-1BB- and
OX40-specific auto/allo-immune defense receptors (ADRs) leads to
elimination of NK cells and thus would offset the NK cell-mediated
host rejection of ADR-armed allogeneic T cells. Therefore, ADRs do
not only inhibit the alloreactive T-cell response but also suppress
NK cell mediated rejection, further supporting the application of
ADRs to enhance the persistence and activity of "off-the-shelf"
therapeutic T cells.
Example 3
ADR-Expressing T Cells Eliminate Target Cells
[0148] ADRs can be expressed on cell surface of immune cells and
promote cytotoxicity against respective targets. FIG. 1A
illustrates one example of a schematic of ADR (a label such as GFP
is optional). The expression of ADR on the surface of T cells was
confirmed (FIG. 1B) and the cells were expanded commensurate with
controls (FIG. 1C). (FIG. 1D) The ADR-expressing T cells were
cytotoxicity against target cells expressing corresponding ADR
ligands (FIG. 1D). FIG. 1E demonstrates expansion of wild-type vs
4-1BB KO T cells expressing 4-1BB ADR and their cytotoxicity
against 4-1BB+ targets. Knocking out the ADR ligand on T cells can
further enhance expansion and cytotoxicity, and co-expression of
ADR and its ligand on T cells is not required for ADR-T cell
expansion or function (FIG. 1E).
[0149] Selective expression of ADR ligands on activated T cells
enables their selective elimination by ADR T cells. Expression of
ADR ligands on resting vs activated T cells after TCR stimulation
is determined (FIG. 2A-FIG. 2C). There was no cytotoxicity of ADR T
cells against resting CD4+ and CD8+ T cells (FIG. 2D), yet there
was elimination of activated CD4+ and CD8+ T cells by ADR T cells
after a 48 h co-culture (FIG. 2E).
[0150] As one example, expression of 4-1BB ADR protects T cells
from immune rejection in an MLR model. Representative dot plots
showing TCRKO T cells co-expressing ADR are protected from
rejection after co-culture with allogeneic PBMC at a 1:10 ADR
T:PBMC ratio (FIG. 3A). Absolute counts of donor T cells and
allogeneic T cells in the PBMC during co-culture (FIG. 3B-FIG. 3C)
are the same for virus-specific ADR T cells (FIG. 3D-FIG. 3F).
[0151] Expression of ADR protects allogeneic virus-specific T cells
from immune rejection in a mixed lymphocyte reaction in vitro.
Representative dot plots showing ADR VST are protected from immune
rejection by recipient allogeneic PBMC (FIG. 4A). The absolute
counts of recipient T cells and donor VST at various time points
during MLR are provided in FIG. 4B and FIG. 4C.
[0152] In FIG. 5, ADR VSTs retain anti-viral function. ADR VSTs
were co-cultured with viral pepmix-pulsed monocytes, and monocyte
counts indicated that they eliminated viral infected cells equally
well compared to unmodified VSTs.
[0153] Activated NK cells upregulate ADR ligands and can be
selectively targeted by ADR T cells. Expression of 4-1BB on resting
vs activated NK cells is confirmed (FIG. 6A-FIG. 6B). Residual
counts of resting vs activated NK cells after 24 hr co-culture with
4-1BB ADR T cells are determined (FIG. 6C). In FIG. 6D, ADR T cells
lacking MHC are protected from immune rejection by allogeneic PBMC
by controlling the expansion of NK cells. Absolute counts of donor
T cells and allogeneic NK cells during co-culture are determined in
FIG. 6E. ADR T cells lacking MHC resist immune rejection by NK
cells upon 48 h co-culture at a 1:1 E:T ratio (FIG. 6F). ADR T
cells control the expansion of alloreactive NK cells during MLR
with PBMC (FIG. 6G), with absolute counts of NK cells plotted in
FIG. 6H.
[0154] ADR expression protects allogeneic T cells from immune
rejection in vivo. In FIG. 7A, one example is shown of a mouse
model of immune rejection where mice were given T cells from an
HLA-A2+ donor after a sublethal irradiation, followed by
administration of allogeneic HLA-A2- T cells 4 days later. Control
T cells from the HLA-A2- donor were rejected by Day 18 while
ADR-expressing cells were protected (FIG. 7B). Absolute counts of T
cells from HLA-A2+ and HLA-A2- donors at various time points were
determined (FIG. 7C). A modified in vivo model in FIG. 7D depicts
where, instead of allogeneic T cells mice, received whole PBMC
(containing both T- and NK-cells) from donor 1. Representative flow
plots in FIG. 7E show that ADR T cells were protected from immune
rejection and also protected mice from rapid onset of fatal
GvHD.
[0155] Coexpression of CAR and ADR preserves functions of both
receptors. FIG. 8A illustrates an example of a representation of an
immune cell co-expressing ADR and a CAR Coexpression of a CAR and
an ADR on the cell surface were confirmed (FIG. 8B). In FIG. 8C,
cytotoxicity is shown of CAR-ADR T cells against NALM-6 (A CD19+
CAR target), as one example of a target. Cytotoxicity of the
CAR-ADR T cells against activated T cells (ADR target) were also
determined in FIG. 8D. Cytotoxic activity of CAR-ADR T cells
against both targets upon simultaneous co-culture with both cell
targets is demonstrated in FIG. 8E.
[0156] CAR-ADR T cells are protected from immune rejection and
exert potent anti-tumor activity. An example of a mouse model and a
regimen is depicted in FIG. 9A. Mice received allogeneic T cells
from Donor 1 and b2mKO NALM6 24 hr apart, followed by a single dose
of CAR-ADR T cells from Donor 2, as one example of a regimen.
Kinetics of T cells from Donor 2 in peripheral blood are provided
in FIG. 9B, and kinetics of Donor 1 T cells in the experimental
groups are provided in FIG. 9C. FIG. 9D shows leukemia burden in
the mice, with determination of overall survival of the mice (FIG.
9E).
[0157] FIGS. 13A-13C. CAR-ADR T cells are protected from immune
rejection and exert potent anti-tumor activity in a solid tumor
model. Schematic of an example of a mouse model and treatment is
shown in FIG. 10A, wherein mice received allogeneic T cells from
Donor 1 and b2mKO neuroblastoma cell line CHLA255 24 hr apart,
followed by a single dose of CAR-ADR T cells from Donor 2. Donor 2
GD2 CAR T cells were rejected by D18, whereas CAR-ADR T cells
resisted allogeneic rejection and persisted in peripheral blood
(FIG. 10B). Tumor burden in mice is shown in FIG. 13C, where *
indicates xenogeneic-GvHD associated deaths in ATC+GD2 CAR T
group.
[0158] TCR-knockout CAR-ADR T cells are protected from immune
rejection and exert potent anti-tumor activity. Schematic of the
mouse model is provided in which mice received allogeneic T cells
from Donor 1 and b2mKO NALM6 24 hr apart, followed by a single dose
of TCR-edited CAR-ADR T cells from Donor 2 (FIG. 11A). Kinetics of
T cells from Donor 2 in peripheral blood are provided (FIG. 11B).
Kinetics of Donor 1 T cells in the experimental groups are shown
(FIG. 11C). Leukemia burden in mice (FIG. 11D) and overall survival
of mice are provided (FIG. 11E).
[0159] ADR T cells protect mice against fatal xenogeneic GvHD.
Schematic of a model is provided in FIG. 12A, and expansion of
FFLuc-labeled ADR T cells in vivo is demonstrated (FIG. 12B).
Kinetics of weight gain/loss in mice were determined (FIG. 12C).
Overall survival of mice is depicted (FIG. 12D).
[0160] Second generation ADR with CD28 intracellular signaling
domain ("ADR.28zeta")(as one example) were utilized. One example of
a structure of ADR.28zeta is depicted (FIG. 13A). In vitro
cytotoxicity was determined of ADR.28zeta against target-expressing
cells ((FIG. 13B and FIG. 13C). ADR.28zeta protected mice from
xeno-GvHD lines (FIG. 13B). Schematic of the model (FIG. 13D) is
shown. Expansion of FFLuc-labeled ADR.28zeta T cells in vivo was
confirmed (FIG. 13E) Kinetics of weight gain/loss in mice (FIG.
13F), and the overall survival of mice was determined (FIG.
13G).
[0161] Although the present disclosure 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 design 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 present
disclosure, 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
disclosure. Accordingly, the appended claims are intended to
include within their scope such processes, machines, manufacture,
compositions of matter, means, methods, or steps.
Sequence CWU 1
1
31714PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val
Ala Ile Leu Lys Gly1 5 10 15Val Gln Cys Gly Leu Leu Asp Leu Arg Gln
Gly Met Phe Ala Gln Leu 20 25 30Val Ala Gln Asn Val Leu Leu Ile Asp
Gly Pro Leu Ser Trp Tyr Ser 35 40 45Asp Pro Gly Leu Ala Gly Val Ser
Leu Thr Gly Gly Leu Ser Tyr Lys 50 55 60Glu Asp Thr Lys Glu Leu Val
Val Ala Lys Ala Gly Val Tyr Tyr Val65 70 75 80Phe Phe Gln Leu Glu
Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly 85 90 95Ser Val Ser Leu
Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly 100 105 110Ala Ala
Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu 115 120
125Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser
130 135 140Ala Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg
Ala Arg145 150 155 160His Ala Trp Gln Leu Thr Gln Gly Ala Thr Val
Leu Gly Leu Phe Arg 165 170 175Val Thr Pro Glu Ile Pro Ala Gly Leu
Pro Ser Pro Arg Ser Glu Glu 180 185 190Ser Lys Tyr Gly Pro Pro Cys
Pro Pro Cys Pro Gly Gln Pro Arg Glu 195 200 205Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 210 215 220Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile225 230 235
240Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
245 250 255Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys 260 265 270Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys 275 280 285Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu 290 295 300Ser Leu Ser Pro Gly Lys Lys Asp
Pro Lys Phe Trp Val Leu Val Val305 310 315 320Val Gly Gly Val Leu
Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe 325 330 335Ile Ile Phe
Trp Val Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp 340 345 350Ala
Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 355 360
365Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg
370 375 380Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln
Glu Gly385 390 395 400Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala
Glu Ala Tyr Ser Glu 405 410 415Ile Gly Met Lys Gly Glu Arg Arg Arg
Gly Lys Gly His Asp Gly Leu 420 425 430Tyr Gln Gly Leu Ser Thr Ala
Thr Lys Asp Thr Tyr Asp Ala Leu His 435 440 445Met Gln Ala Leu Pro
Pro Arg Thr Ser Ala Ala Ala Gly Gly Gly Gly 450 455 460Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Met Val Ser Lys Gly465 470 475
480Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly
485 490 495Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu
Gly Asp 500 505 510Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys
Thr Thr Gly Lys 515 520 525Leu Pro Val Pro Trp Pro Thr Leu Val Thr
Thr Phe Thr Tyr Gly Val 530 535 540Gln Cys Phe Ala Arg Tyr Pro Asp
His Met Lys Gln His Asp Phe Phe545 550 555 560Lys Ser Ala Met Pro
Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe 565 570 575Lys Asp Asp
Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly 580 585 590Asp
Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu 595 600
605Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His
610 615 620Lys Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys
Val Asn625 630 635 640Phe Lys Thr Arg His Asn Ile Glu Asp Gly Ser
Val Gln Leu Ala Asp 645 650 655His Tyr Gln Gln Asn Thr Pro Ile Gly
Asp Gly Pro Val Leu Leu Pro 660 665 670Asp Asn His Tyr Leu Ser Thr
Gln Ser Lys Leu Ser Lys Asp Pro Asn 675 680 685Glu Lys Arg Asp His
Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly 690 695 700Ile Thr Leu
Gly Met Asp Glu Leu Tyr Lys705 7102675PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
2Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly1 5
10 15Val Gln Cys Gln Val Ser His Arg Tyr Pro Arg Ile Gln Ser Ile
Lys 20 25 30Val Gln Phe Thr Glu Tyr Lys Lys Glu Lys Gly Phe Ile Leu
Thr Ser 35 40 45Gln Lys Glu Asp Glu Ile Met Lys Val Gln Asn Asn Ser
Val Ile Ile 50 55 60Asn Cys Asp Gly Phe Tyr Leu Ile Ser Leu Lys Gly
Tyr Phe Ser Gln65 70 75 80Glu Val Asn Ile Ser Leu His Tyr Gln Lys
Asp Glu Glu Pro Leu Phe 85 90 95Gln Leu Lys Lys Val Arg Ser Val Asn
Ser Leu Met Val Ala Ser Leu 100 105 110Thr Tyr Lys Asp Lys Val Tyr
Leu Asn Val Thr Thr Asp Asn Thr Ser 115 120 125Leu Asp Asp Phe His
Val Asn Gly Gly Glu Leu Ile Leu Ile His Gln 130 135 140Asn Pro Gly
Glu Phe Cys Val Leu Glu Ser Lys Tyr Gly Pro Pro Cys145 150 155
160Pro Pro Cys Pro Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
165 170 175Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu 180 185 190Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn 195 200 205Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser 210 215 220Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg225 230 235 240Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu 245 250 255His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Lys 260 265 270Asp
Pro Lys Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys 275 280
285Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser
290 295 300Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
Gln Gly305 310 315 320Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly
Arg Arg Glu Glu Tyr 325 330 335Asp Val Leu Asp Lys Arg Arg Gly Arg
Asp Pro Glu Met Gly Gly Lys 340 345 350Pro Arg Arg Lys Asn Pro Gln
Glu Gly Leu Tyr Asn Glu Leu Gln Lys 355 360 365Asp Lys Met Ala Glu
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 370 375 380Arg Arg Gly
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala385 390 395
400Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
405 410 415Thr Ser Ala Ala Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly 420 425 430Gly Gly Gly Ser Met Val Ser Lys Gly Glu Glu Leu
Phe Thr Gly Val 435 440 445Val Pro Ile Leu Val Glu Leu Asp Gly Asp
Val Asn Gly His Lys Phe 450 455 460Ser Val Ser Gly Glu Gly Glu Gly
Asp Ala Thr Tyr Gly Lys Leu Thr465 470 475 480Leu Lys Phe Ile Cys
Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr 485 490 495Leu Val Thr
Thr Phe Thr Tyr Gly Val Gln Cys Phe Ala Arg Tyr Pro 500 505 510Asp
His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly 515 520
525Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys
530 535 540Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn
Arg Ile545 550 555 560Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly
Asn Ile Leu Gly His 565 570 575Lys Leu Glu Tyr Asn Tyr Asn Ser His
Lys Val Tyr Ile Thr Ala Asp 580 585 590Lys Gln Lys Asn Gly Ile Lys
Val Asn Phe Lys Thr Arg His Asn Ile 595 600 605Glu Asp Gly Ser Val
Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro 610 615 620Ile Gly Asp
Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr625 630 635
640Gln Ser Lys Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val
645 650 655Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met
Asp Glu 660 665 670Leu Tyr Lys 6753716PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
3Met Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr1 5
10 15Ala Val His Pro Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr
Leu 20 25 30Ile Asn Ser Gln Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys
Leu Val 35 40 45Ser Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro
Cys Gly Glu 50 55 60Ser Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr His
Cys His Gln His65 70 75 80Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg
Val Gln Gln Lys Gly Thr 85 90 95Ser Glu Thr Asp Thr Ile Cys Thr Cys
Glu Glu Gly Trp His Cys Thr 100 105 110Ser Glu Ala Cys Glu Ser Cys
Val Leu His Arg Ser Cys Ser Pro Gly 115 120 125Phe Gly Val Lys Gln
Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu 130 135 140Pro Cys Pro
Val Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys145 150 155
160Cys His Pro Trp Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln
165 170 175Ala Gly Thr Asn Lys Thr Asp Val Val Cys Gly Pro Gln Asp
Arg Leu 180 185 190Arg Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys
Pro Gly Gln Pro 195 200 205Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr 210 215 220Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser225 230 235 240Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 245 250 255Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 260 265 270Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 275 280
285Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
290 295 300Ser Leu Ser Leu Ser Pro Gly Lys Lys Asp Pro Lys Phe Trp
Val Leu305 310 315 320Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser
Leu Leu Val Thr Val 325 330 335Ala Phe Ile Ile Phe Trp Val Arg Ser
Arg Val Lys Phe Ser Arg Ser 340 345 350Ala Asp Ala Pro Ala Tyr Gln
Gln Gly Gln Asn Gln Leu Tyr Asn Glu 355 360 365Leu Asn Leu Gly Arg
Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg 370 375 380Gly Arg Asp
Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln385 390 395
400Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr
405 410 415Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
His Asp 420 425 430Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
Thr Tyr Asp Ala 435 440 445Leu His Met Gln Ala Leu Pro Pro Arg Thr
Ser Ala Ala Ala Gly Gly 450 455 460Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Met Val Ser465 470 475 480Lys Gly Glu Glu Leu
Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu 485 490 495Asp Gly Asp
Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu 500 505 510Gly
Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr 515 520
525Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe Thr Tyr
530 535 540Gly Val Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln
His Asp545 550 555 560Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val
Gln Glu Arg Thr Ile 565 570 575Phe Phe Lys Asp Asp Gly Asn Tyr Lys
Thr Arg Ala Glu Val Lys Phe 580 585 590Glu Gly Asp Thr Leu Val Asn
Arg Ile Glu Leu Lys Gly Ile Asp Phe 595 600 605Lys Glu Asp Gly Asn
Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn 610 615 620Ser His Lys
Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys625 630 635
640Val Asn Phe Lys Thr Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu
645 650 655Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro
Val Leu 660 665 670Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Lys
Leu Ser Lys Asp 675 680 685Pro Asn Glu Lys Arg Asp His Met Val Leu
Leu Glu Phe Val Thr Ala 690 695 700Ala Gly Ile Thr Leu Gly Met Asp
Glu Leu Tyr Lys705 710 715
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