U.S. patent application number 17/266509 was filed with the patent office on 2021-10-07 for rejuvenation of car t cell.
The applicant listed for this patent is Purdue Research Foundation. Invention is credited to Philip Stewart Low et al., John V. Napoleon, Boning Zhang.
Application Number | 20210308267 17/266509 |
Document ID | / |
Family ID | 1000005694073 |
Filed Date | 2021-10-07 |
United States Patent
Application |
20210308267 |
Kind Code |
A1 |
Low et al.; Philip Stewart ;
et al. |
October 7, 2021 |
REJUVENATION OF CAR T CELL
Abstract
A payload of drug conjugated to a targeting ligand specifically
designed to deliver to exhausted CART cells to rejuvenate these CAR
T cells is provided herein. The targeted CAR T cells are modified
with a fusion receptor which can bind to the targeting ligand and
internalize the conjugated payload of drug to execute its
regulatory function to exhausted CAR T cell.
Inventors: |
Low et al.; Philip Stewart;
(West Lafayette, IN) ; Zhang; Boning; (West
Lafayette, IN) ; Napoleon; John V.; (West Lafayette,
IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Purdue Research Foundation |
West Lafayette |
IN |
US |
|
|
Family ID: |
1000005694073 |
Appl. No.: |
17/266509 |
Filed: |
July 21, 2019 |
PCT Filed: |
July 21, 2019 |
PCT NO: |
PCT/US19/42726 |
371 Date: |
February 5, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62715666 |
Aug 7, 2018 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 49/0043 20130101;
A61K 47/6835 20170801; A61K 49/0052 20130101; A61K 47/55 20170801;
A61K 47/62 20170801; A61K 31/4545 20130101; A61K 31/437 20130101;
A61K 31/7076 20130101; A61K 31/4535 20130101 |
International
Class: |
A61K 47/55 20060101
A61K047/55; A61K 49/00 20060101 A61K049/00; A61K 47/68 20060101
A61K047/68; A61K 47/62 20060101 A61K047/62; A61K 31/437 20060101
A61K031/437; A61K 31/4545 20060101 A61K031/4545; A61K 31/4535
20060101 A61K031/4535; A61K 31/7076 20060101 A61K031/7076 |
Claims
1. A system to rejuvenate an exhausted classical CAR T cell,
comprising at least two components: a first component is a
conjugate comprising a targeting ligand covalently linked to a
payload drug; and a second component is a targeting ligand binding
module linked to a membrane-anchoring module, wherein the targeting
ligand binding module of the second component recognizes the
targeting ligand in the first component with high affinity to form
a complex, the payload drug either blocks the inhibitory signaling
of the exhausted classical CAR T, or re-activates said CAR T
through an antigen independent pathway, and wherein the
membrane-anchoring module mediates internalization of the two
component complex into the exhausted CAR T cell.
2. The system according to claim 1, wherein the targeting ligand of
the first component is folate, FITC or FK506.
3. The system according to claim 1, wherein the targeting ligand
binding module of the second component comprises an anti-FITC
antibody fragment or FKBP or folate receptor.
4. The system according to claim 1, wherein the membrane anchoring
module is a folate receptor.
5. The system according to claim 1, wherein the first component
comprises a releasable linker between the targeting ligand and the
payload drug.
6. The system according to claim 1, wherein the first component
comprises a non-releasable linker between the targeting ligand and
the payload drug.
7. The system according to claim 1, wherein the binding affinity
between the targeting ligand and the ligand-binding module is in
sub-nanomolar range.
8. The system according to claim 1, wherein the payload of drug is
a Toll Like Receptor 7 (TLR7) agonist or Simulator of interferon
genes (STING) agonist.
9. The system according to claim 1, wherein the payload of drug is
an inhibitor to following proteins: SHP1/2, TC-PTP or DGK.alpha.,
TGF.beta..
10. The system according to claim 8, wherein the TLR7 agonist has
the structure of ##STR00026##
11. The system according to claim 1, wherein the first component is
a Fluorescein-TLR7 agonist having the structure of ##STR00027##
12. The system according to claim 1, wherein the first component is
a FK506-TLR7 agonist having the structure of ##STR00028##
13. The system according to claim 1, wherein the first component is
one of the following: ##STR00029##
14. The system according to claim 1, wherein the first component
comprising the payload drug selected from the group consisting of
following TC-PTP phosphatase inhibitors: ##STR00030##
15. The system according to claim 14, wherein the phosphatase
inhibitor is connected to the fluorescein or FK506 (tacrolimus) to
form the following structures: ##STR00031##
16. The system according to claim 1, wherein the payload drug in
the first component comprises a STING agonist of one of the
following structures. ##STR00032##
17. The system according to claim 1, wherein the first component
comprises a spacer between the targeting ligand and the payload
drug selected from the group consisting of the following
structures: ##STR00033##
18. A method to rejuvenate an exhausted CAR T cell, comprising: a.
providing said exhausted CAR T cell a first component comprising a
conjugate, wherein the conjugate comprises a targeting ligand
covalently linked to a payload of drug through a releasable or
non-releasable linker; b. providing said exhausted CAR T cell a
second component comprising a fusion receptor linked to the
exhausted CAR construct, wherein the fusion receptor comprises a
targeting ligand binding module and a membrane bound receptor
module; c. letting the targeting ligand binding module of the
second component bind to the targeting ligand in the first
component to form a complex, d. letting the membrane bound CAR
module mediate internalization of the complex into the exhausted
CAR T cell; e. letting the payload drug either block the inhibitory
signaling of the exhausted CAR T, or re-activate said CAR T through
an antigen independent pathway.
19. The method according to claim 18, wherein the payload drug
executes its function within the endosome of the exhausted CAR T,
and the targeting ligand and the payload drug are linked by a
nonreleasable linker.
20. The method according to claim 18, wherein the payload drug
executes its function as a free drug in the cytosol of the
exhausted CAR T, and the targeting ligand and the payload drug are
linked by a releasable linker.
21. The method according to claim 18, wherein the targeting ligand
of the first component is folate, FITC or FK506.
22. The method according to claim 18, wherein the targeting ligand
binding module of the second component anti-FITC, folate receptor,
or FKBP.
23. The method according to claim 18, wherein the ligand binding
module is Folate Receptor alpha (FRa).
24. The method according to claim 18, wherein the payload of drug
is a Toll Like Receptor 7 (TLR7) agonist or Simulator of interferon
genes (STING) agonist.
25. The method according to claim 18, wherein the payload of drug
is an inhibitor to following proteins: SHP1/2, TC-PTP or
DGK.alpha., TGF.beta..
26. The method according to claim 18, wherein the TLR7 agonist has
the structure of ##STR00034##
27. The method according to claim 18, wherein the first component
is a Fluorescein-TLR7 agonist having the structure of
##STR00035##
28. The method according to claim 18, wherein the first component
is a FK506-TLR7 agonist having the structure of ##STR00036##
29. The method according to claim 18, wherein the first component
is one of the following ##STR00037##
30. The method according to claim 18, wherein the first component
comprising the payload drug selected from the group consisting of
following TC-PTP phosphatase inhibitors: ##STR00038##
31. The method according to claim 30, wherein the Phosphatase
inhibitor is connected to the fluorescein or FK506 (tacrolimus) to
form the following structures: ##STR00039##
32. The method according to claim 18, wherein the payload drug in
the first component comprising a STING agonist of the following
structures. ##STR00040##
33. The method according to claim 18, wherein the first component
comprising a spacer between the targeting ligand and the payload
drug selected from the group consisting of the following
structures: ##STR00041##
Description
FIELD OF INVENTION
[0001] This disclosure provides a system to rejuvenate cancer
antigen exhausted chimeric antigen receptor T (CAR T) cells.
Specifically, the system comprising a fusion receptor in a
classical CAR construct, wherein the fusion receptor provides a
ligand binding module that recognizes a high affinity
ligand-payload drug conjugate to deliver payload of drugs that are
designed to either block the inhibitory signaling in the exhausted
CAR T, or to re-activate CAR T through an antigen independent
pathway.
BACKGROUND
[0002] The field of chimeric antigen receptor (CAR) T cell therapy
has made tremendous progress over the last two decades. The CAR
construct consists of four parts: (1) an extracellular binding
moiety against tumor specific antigen, (2) a hinge domain, (3) a
transmembrane domain and (4) a combination of various activation
domains, for example, CD28, 4-1BB and CD3.zeta. chain. The most
impressive successes have been seen in CAR T therapy against CD19
positive B cell leukemias, where more than 80% complete remission
rate has been achieved in several clinical trials
[0003] In contrast, the treatment of solid tumor by CAR T has so
far proven to be more challenging. Great successes have been
achieved in preclinical study using syngeneic mice model or
xenograft tumor model in immunodeficient mice. However, none of the
clinical trials involving solid tumor, for example, breast cancer,
ovarian cancer or lung cancer has shown improvement compare to
control. In general, it suffers from the same limitations that are
faced by other adoptive cell therapies, including (1) poor tumor
penetration; (2) hypoxic pressure; (3) an immunosuppressive tumor
microenviroment which includes tumor associated macrophages,
fibroblasts and suppressive cytokines.sup.1. CAR T cells, as same
as tumor infiltrated lymphocytes (TIL), can be re-educated by this
suppressive microenviroment and turn to a "hypofunctional" status,
which is characterized by overexpression of co-inhibitory molecules
(i.e., PD-1, Tim-3, LAG-3 etc.), decreased INF.gamma. secretion and
killing capability. Data from the patient samples of ovarian cancer
shows that the majority of PD-1.sup.+CD8.sup.+ T cell lacked
expression of CD127, which is known to be important for the
effector-to-memory transition in T cell. Overexpression of LAG-3
also negatively correlated with the effector function of TCR
specific CD8.sup.+ TIL. In addition, PD-1.sup.+ LAG-3.sup.+ double
positive T cell exhibited lower INF.gamma. production.sup.2.
Similarly, NY-ESO-1 TCR specific human T cell became hypofunctional
in mice solid tumor model, showing high expression of the
co-inhibitory molecules and less efficient anti-tumor effect, due
to both the microenvrioment and the constant activation of T cell
by the continuous exposure to antigen.sup.3. Therefore, a reversion
of the suppressive microenviroment, more importantly, a
rejuvenation of the exhausted CAR T cell is highly desired for a
better solid tumor treatment.
SUMMARY OF THE INVENTION
[0004] This disclosure provides system to rejuvenate an exhausted
classical CAR T cell. The system comprises at least two components:
a first component is a conjugate comprising a targeting ligand
covalently linked to a payload of drug; and a second component is a
targeting ligand binding module linked to membrane-anchoring
module. The targeting ligand binding module of the second component
recognizes the targeting ligand in the first component with high
affinity to form a complex, and the payload drug either blocks the
inhibitory signaling of the exhausted CAR T, or re-activates said
CAR T through an antigen independent pathway. The
membrane-anchoring module mediates internalization of the two
component complex into the exhausted CAR T cell.
[0005] In some preferred embodiment, the aforementioned targeting
ligand of the first component is folate, FITC or FK506.
[0006] In some preferred embodiment, the aforementioned targeting
ligand binding module of the second component comprises a folate
receptor, an anti-FITC antibody fragment or FKBP.
[0007] In some preferred embodiment, the aforementioned
membrane-anchoring module is a folate receptor.
[0008] In some preferred embodiment, the aforementioned first
component comprises a releasable linker between the targeting
ligand and the payload drug.
[0009] In some preferred embodiment, the aforementioned first
component comprises a non-releasable linker between the targeting
ligand and the payload drug.
[0010] In some preferred embodiment, the binding affinity between
aforementioned targeting ligand and the targeting ligand-binding
module is in sub-nanomolar range.
[0011] In some preferred embodiment, the aforementioned payload of
drug is a Toll Like Receptor 7 (TLR7) agonist or Simulator of
interferon genes (STING) agonist.
[0012] In some preferred embodiment, the aforementioned payload of
drug is an inhibitor to following proteins: SHP1/2, TC-PTP or
DGK.alpha., TGF.beta..
[0013] In some preferred embodiment, the aforementioned TLR7
agonist has the structure of or
##STR00001##
[0014] In some preferred embodiment, the aforementioned first
component is a Fluorescein-TLR7 agonist having the structure of
##STR00002##
[0015] In some preferred embodiment, the aforementioned first
component is a FK506-TLR7 agonist having the structure of
##STR00003##
[0016] In some preferred embodiment, the aforementioned first
component is one of the following:
##STR00004##
[0017] In some preferred embodiment, the aforementioned first
component comprising the payload drug selected from the group
consisting of following TC-PTP phosphatase inhibitors:
##STR00005##
[0018] In some preferred embodiment, the aforementioned phosphatase
inhibitor is connected to the fluorescein or FK506 (tacrolimus) to
form the following structures:
##STR00006##
[0019] In some preferred embodiment, the aforementioned payload
drug in the first component comprises a STING agonist of one of the
following structures.
##STR00007##
[0020] In some preferred embodiment, the aforementioned first
component comprises a spacer between the targeting ligand and the
payload drug selected from the group consisting of the following
structures:
##STR00008##
[0021] This disclosure further provides a method to rejuvenate an
exhausted CAR T cell. The method comprises the steps of: [0022] a.
providing the exhausted CAR T cell a first component comprising a
conjugate, wherein the conjugate comprises a targeting ligand
covalently linked to a payload of drug through a releasable or
non-releasable linker; [0023] b. providing said exhausted CAR T
cell a second component comprising a fusion receptor linked to the
exhausted CAR construct, wherein the fusion receptor comprises a
targeting ligand binding module and a membrane-anchoring module;
[0024] c. letting the targeting ligand binding module of the second
component bind to the targeting ligand in the first component to
form a complex, [0025] d. letting the membrane-anchoring module
mediate internalization of the complex into the exhausted CAR T
cell; [0026] e. letting the payload drug either block the
inhibitory signaling of the exhausted CAR T, or re-activate said
CAR T through an antigen independent pathway.
[0027] In some preferred embodiment, the aforementioned method
carries a payload drug executing its function within the endosome
of the exhausted CAR T, and the targeting ligand and the payload
drug are linked by a nonreleasable linker.
[0028] In some preferred embodiment, the aforementioned method
carries a payload drug executing its function as a free drug in the
cytosol of the exhausted CAR T, and the targeting ligand and the
payload drug are linked by a releasable linker.
[0029] In some preferred embodiment, the targeting ligand of the
first component is folate, FITC or FK506 in aforementioned
method.
[0030] In some preferred embodiment, the targeting ligand-binding
module of the second component in aforementioned method is
anti-FITC, folate receptor, or FKBP.
[0031] In some preferred embodiment, the targeting ligand-binding
module of the second component in aforementioned method is Folate
Receptor alpha (FRa).
[0032] In some preferred embodiment, the payload of drug of the
first component in aforementioned method is a Toll Like Receptor 7
(TLR7) agonist or Simulator of interferon genes (STING)
agonist.
[0033] In some preferred embodiment the payload of drug the first
component in aforementioned method is an inhibitor to following
proteins: SHP1/2, TC-PTP or DGK.alpha., TGF.beta..
[0034] In some preferred embodiment the TLR7 agonist in
aforementioned method has the structure of
##STR00009##
[0035] In some preferred embodiment, the first component in
aforementioned method is a Fluorescein-TLR7 agonist having the
structure of
##STR00010##
[0036] In some preferred embodiment, the first component in
aforementioned method is a FK506-TLR7 agonist having the structure
of
##STR00011##
[0037] In some preferred embodiment, the first component in
aforementioned method is
##STR00012##
[0038] In some preferred embodiment, the first component in
aforementioned method comprising payload drug selected from the
group consisting of following TC-PTP phosphatase inhibitors:
##STR00013##
[0039] In some preferred embodiment, the first component in
aforementioned method comprising the Phosphatase inhibitor
connected to the fluorescein or FK506 (tacrolimus) to form the
following structures:
##STR00014##
[0040] In some preferred embodiment, the first component in
aforementioned method comprising a payload drug of a STING agonist
of the following structures.
##STR00015##
[0041] In some preferred embodiment, the first component in
aforementioned method comprising a spacer between the targeting
ligand and the payload drug that is selected from the group
consisting of the following structures:
##STR00016##
[0042] These and other features, aspects and advantages of the
present invention will become better understood with reference to
the following figures, associated descriptions and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] FIG. 1. la, Graph illustration of the exhaustion model. 1b,
antiCD19 CAR T cell became exhausted after 3 times of stimulation
of fresh Raji cell in vitro as shown by decreased killing
effect.
[0044] FIG. 2. Graph illustration of the secret passageway. FKBP or
antiFITC is linked to FRa as a fusion receptor, which constantly
internalizes and delivers FK506 or FITC linked payloads into the
cell.
[0045] FIG. 3. Binding affinity of FK506 and FITC to the
corresponding fusion receptors. FK506-Rhodamine shows Kd=3.39 nM
towards FKBP-FR fusion receptor, while FITC-AF647 shows Kd=8.03 nM
towards antiFITC-FR.
[0046] FIG. 4. 4a. Structure of the TLR7 agonist. 4b. rejuvenation
effect of TLR7 agonist on exhausted antiCD19 CART cell, shown as
the increased killing effect, INFr level and decreased
co-inhibitory molecule level.
[0047] FIG. 5. Chemical Structure of TLR7 agonists.
[0048] FIG. 6. in vitro model for the induction of CAR T cell
exhaustion. (A) CD19.sup.+ Raji and anti-CD19 CART cells were
co-cultured at 1:1 ratio with fresh Raji cells added every 12 h.
(B) Lysis effect of CAR T cells gradually decreased as the number
of stimulus (number of Raji cell addition) increases.
CD19.sup.-K562 cells were used as control. (C) Expression level
change of co-inhibitory molecules, PD-1, LAG3 and Tim3 for stim1
and stim3 in CD4 and CD8 positive CAR T cells. FIG. 7. Evaluation
of TLR7 agonist and PTP1b inhibitor effect on rejuvenation of
exhausted CART cells. (A) Chemical structure of TLR7 agonist and
PTP1b inhibitor. (B-C) Exhausted CAR T cells were incubated with
different concentrations of TLR7 agonist and PTP1b inhibitor
monitored by lysis effect and INF.gamma. (B) and expression level
of PD-1 LAG-3 and Tim3 after incubation (C). * denotes a
p-value<0.05, **<0.01, ns=not significant.
[0049] FIG. 8. Evaluation of potential derivatization sites of the
TLR7 agonist for non-releasable ligand targeted delivery. (A)
Chemical structure of the TLR7 agonist analogs. (B) Exhausted CART
cells were incubated with different concentration of TLR7 analogs
and lysis effect was measured and compared to non-treated
group.
[0050] FIG. 9. Design and evaluation of releasable and
non-releasable targeted delivery of TLR7 agonist using FITC as a
targeting ligand. (A) Illustration of chemical structure of
FITC-TLR7 agonist. (B) Exhausted CAR T cells were incubated with
different concentration of releasable FITC-TLR7 and non-releasable
FITC-JTLR7 agonists and lysis effect was measured and compared to
non-treated group. * denotes a p-value<0.05, **<0.01, ns=not
significant. (C) Left, crystal structure of FITC (green) binding
with FITC scFv (grey) (PDB: 1X9Q, left), the distance between FITC
to the edge of FTIC scFv is measured to be around 18 .ANG.. Right,
similarly, crystal structure of R-848 (red) binding with TLR7
(grey) (PDB:5GMF) is shown with distance between R-848 and the edge
of TLR7 around 24 .ANG.. (D) Diagram illustrating the two possible
working mechanisms, "Reaching" or "Jumping" Mode, for the
non-releasable FITC-TLR7 agonists.
[0051] FIG. 10. Chemical linkers of variable rigidity and
hydrophobicity available for usage in the design of targeting
ligand-payload conjugates.
DETAILED DESCRIPTION
[0052] While the concepts of the present disclosure are illustrated
and described in detail in the figures and the description herein,
results in the figures and their description are to be considered
as exemplary and not restrictive in character; it being understood
that only the illustrative embodiments are shown and described and
that all changes and modifications that come within the spirit of
the disclosure are desired to be protected.
[0053] Unless defined otherwise, the scientific and technology
nomenclatures have the same meaning as commonly understood by a
person in the ordinary skill in the art pertaining to this
disclosure.
[0054] Chimeric antigen receptor (CAR) T cell therapies have
recently experienced substantial success in the treatment of
several types of hematopoietic cancers. In the meantime, one should
also recognize that some of the lymphoma and most solid tumor cases
still have a very low response rate or a high relapse rate with CAR
T cell therapies. This mainly result from one or combinations of
the following three reasons: 1. Emergence of antigen negative
cancer cell colonies under the selection pressure of CAR T cells,
as seen in the case of CD19 negative ALL relapse treated with
anti-CD19 CART cells; 2. Hindered initial homing and proliferation
of CART cells in solid tumor due to the aberrant tumor vasculature,
dense stromal barrier and suppressive microenvironment; 3. Gradual
exhaustion and lowered lysis effect of CART cells after continuous
tumor antigen exposure. Assuming loss of antigen is not present for
a given solid tumor patient (validated by biopsy sampling), the
causes of a potential failure of a CAR T cell therapy are most
likely to result from the latter two reasons. Therefore, to
increase CAR T cells efficacy in solid tumors, practical methods
for in vivo evaluation and rejuvenation of CAR T cells are highly
desired.
[0055] Here we describe the novel design of a private passageway
fusion receptor in CAR T cells as a universal platform to achieve
both objectives. This FITC-FR fusion receptor is composed of two
parts, scFv against FITC as the ligand binding domain at the N
terminal and FRa as the GPI anchoring and internalizing domain at
the C terminal. When independently expressed on CAR T cells, the
FITC-FR fusion receptor can be specifically targeted by a
FITC-immuno agonist to overcome the exhaustion status of CAR T
cells in the suppressive tumor microenvironment. These
immuno-agonists normally cause strong autoimmunity side effects,
and can now be systemically dosed in a FITC targeted form, and
safely delivered to FITC-FR positive CAR T cells. In the last few
decades, great advances have been made in this field regarding the
cell types, delivery methods and suitable diseases models. In terms
of cell types, current cell therapies can be roughly categorized as
chimeric antigen receptors (CARs), cell for tumor model and stem
cell based regenerative medicine.
[0056] CAR T also known as chimeric T cell receptors, chimeric
immunoreceptors or artificial T cell receptors, enable immune
effector cells (usually T cells or NK cells) to recognize target
cells with corresponding antigen and exercise their cytotoxic
activity. The emergence and development of CAR-T technology
provides promises to certain types of cancers, which turns CAR-T
into a superstar in the field of both biomedical research and
clinical studies. Some traditional and improved CAR T cell design
are disclosed in U.S. application Ser. No. 15/296,666, the content
of which is incorporated herein entirely. In '666 application, a
CAR system is produced by providing a cytotoxic lymphocytes
expressing CARs that target a moiety that is not produced or
expressed by cells of the subject being treated. This CAR system
thus allows for focused targeting of the cytotoxic lymphocytes to
target cells, such as cancer cells. The targeted moiety is part of
a small conjugate molecule (SCM) that also comprises a ligand of a
tumor cell receptor. Administration of a SCM along with the
CAR-expressing cytotoxic lymphocytes results in the tarting of the
cytotoxic lymphocyte response to only those cells expressing the
tumor receptor to which the SCM is bound.
[0057] Despite the rapid progress of CAR T cell therapy in both
research and clinical use field, there are concerns accompanied
with CAR T therapy. One lethal side effect is cytokine storm
generated from the fast lysis of tumor cells as well as it kills
normal cells bearing CAR. In order to address such side effects,
targeted delivery of CAR T cell with specific payload of drug to
the target tumor cells to control such side effect is developed in
PCT/US2018/018557, the content of which is incorporated herein
entirely. Briefly, an engineered protein is coupled with a high
affinity targeting ligand, wherein the targeting ligand carries at
least one payload of drug to be internalized by the CAR T cell
through the engineered protein to regulate transplanted cell
therapy effects.
[0058] Another limitation of CAR T therapy is their tendency to get
exhausted after repeated stimulation of cancer antigen. The
reversibility of the exhausted phenotype of T cell is proven as T
cells isolated from the solid tumor tissue show higher INF.gamma.
secretion and killing effect if kept away from antigens ("rested")
overnight before re-stimulation.sup.4. However, it will be more
appealing if rejuvenation can be achieved in a more clinical
relevant way using drugs: either to block the inhibitory signaling
or to activate the T cell through other pathways. Antibodies
targeting checkpoint inhibitors (i.e. PD-1, CTLA-4, etc.) have
shown some success in solid tumors in clinic, however, two or more
targets in combination are often found to be necessary.sup.5 6.
Moreover, antibody therapy also suffers from poor penetration in
solid tumor. Therefore, less reports have been seen for the
combination therapy of CAR T and antibody for checkpoint inhibitors
in solid tumor. Inhibiting the phosphatases, such as SHP1/2.sup.7
and TC-PTP.sup.8, that mediates TCR deactivating, is another way to
block the inhibitory pathways. Both knockout experiments and small
molecules inhibitors of these phosphatases have shown potent effect
on lowering TCR threshold and increasing T cell activity, but none
of them have been used in CAR T therapy. DGK.alpha. is another
physiological inhibitor of TCR signaling and it's overexpressed in
exhausted TIL. DGK.alpha. catabolize DAG to PA thereby reducing DAG
levels, which results in attenuation of Ras and MARK ERK signaling.
Inhibitor of DGK.alpha. recovers the degranulation and increases
the killing effect of TIL and CAR T.sup.3 9. Another approach to
rejuvenate the T cell is to activate it through an antigen
independent pathway. It has been known that certain pathogen
pattern recognition (PPR) receptors, including Toll like receptors
(TLR), do express on non-myeloid cell populations, including T
cells, and can be activated in a similar way. Research has also
shown that TLR2.sup.10, 4.sup.11 and 7/8.sup.12 13 agonists can
activate CD8 T cells and increase INF.gamma. secretion. However,
due to the strong side effects of systemic dosing of TLR
agonists.sup.14, none of these agonists have been used in CAR T
therapy to re-activate the T cell or change the immunosuppressive
microenviroment. It's also hindered by the controversial effect of
TLR agonists on tumor cell itself.sup.15. Stimulator of interferon
genes (STING) is a cytosolic DNA sensor (CDS) that widely expressed
in hematopoietic cells in peripheral lymphoid tissues, including T
cell, myeloid cells and monocytes. STING agonists have been used as
an immune stimulator for many immunotherapies, and may also have a
profound effect in CAR T therapy. However, although the mentioned
inhibitors and agonists may have a profound rejuvenation effect on
CAR T cell, it's also highly possible that it may induce severe
side effects if systematically dosed due to their highly potent
pro-inflammatory functions. Therefore, a targeted delivery of the
potential payloads to the CAR T cell is highly desired.
[0059] To solve the specific delivery problem, we designed a secret
passageway platform, which can be expressed in T cell together with
the CAR construct, so that certain payloads can be systemically
dosed and specifically accumulated within the CAR T cell only,
rendering other cells untouched. The system consists a fusion
receptor and a classical CAR construct, linked through a T2A
self-cleavable sequence. The fusion receptor contains two parts:
(1) a ligand binding module, which can recognize a high affinity
ligand-payload conjugate; (2) a membrane bound receptor module,
which can mediate the internalization of the receptor/conjugate
complex into the cell. Two protein/ligand pairs have been chosen
for part 1, FKBP/FK506 and scFv against FITC (4M5.3)/FITC, for the
following reasons: (1) the absence of FKBP or 4M5.3 on natural cell
membrane guarantees the specific delivery of payload to fusion
receptor positive CAR T cell, thus reduces the side effects to
other cells; (2) the sub-nanomolar binding affinity between
protein/ligand pairs promote sufficient payload accumulation inside
the targeted cells. For the membrane bound receptor in part 2,
Folate Receptor alpha (FRa) was chosen for its constitutive
internalization properties regardless of Folate Acid
binding.sup.16. We also designed a target-payload linkage system,
where payload can be linked to targeting ligands with either
non-releasable linker or a disulfide releasable linker, depending
on the target. More specifically, since TLR7 locates in the
endosome, secret passageway delivered TLR7 agonist can exert its
function as soon as it enters the endosome through receptor
mediated internalization. Therefore, releasable linker is not
necessary in this case. While for other targets that are located in
the cytosol instead of the endosome, such as SHP1/2, TC-PTP, DGK,
TGF.beta. and STING, release of the free drug from the
target-payload conjugate is necessary for its escape from the
endosome. Together, this secret passageway system provides a
versatile platform for specific in vivo delivery of numerous
payloads to the exhausted CAR T cell.
[0060] These and other features, aspects and advantages of the
present invention will become better understood with the following
experiments examples.
Methods:
[0061] Exhaustion of the antiCD19 CAR T Cell and Drug
Treatment:
[0062] AntiCD19 CAR T cell were co-cultured with Raji at 1:1 ratio
in 6 well plate, while fresh Raji cell were added every 12 h to the
same well. Killing effect and co-inhibitory markers were quantified
by flow cytometry counting. For drug treatment, after 4 rounds of
stimulation with Raji cells, exhausted antiCD19 CAR T were further
incubated with drugs at different concentration for 12 h, and then
quantified similarly
Targeting Ligand Binding Assay
[0063] Fusion receptor positive cells were incubated with certain
ligand-dye molecule at different concentrations for 30 min at 4
degree. After incubation, cells were washed twice with PBS and then
submitted to flow cytometry. MFI or percentage of shift is used for
binding curve and calculation of Kd.
Materials
Cell Lines and Human T Cells
[0064] DMEM (Gibco) containing 10% heat-inactivated fetal bovine
serum and 1% penicillin-streptomycin was used for the culture of
MDAMB-231 and MDA-MB-231 CD19.sup.+ cells. Peripheral blood
mononuclear cells (PBMCs) were isolated by Ficoll density gradient
centrifugation (GE Healthcare Lifesciences, #17-5442-02) from human
whole blood obtained from healthy volunteers. Pure CD3+ T cells
were enriched from PBMCs using an EasySep.TM. Human T Cell
Isolation Kit (STEM CELL technologies, #17951).
Evaluation of Potential Payloads for In Vitro Rejuvenation of
Exhausted CAR T Cells
[0065] anti-CD19 CAR T cells were co-incubated with CD19.sup.+ Raji
cell at 1:1 ratio in 12-well plate at density of 2.times.10.sup.6
CAR T and 2.times.10.sup.6 Raji per well, new Raji cell were added
every 12 h for 3 times, Raji cell population, lysis effect and
co-inhibitory receptors were then tested to confirm the exhaustion
of the CART cells. Both flow cytometry and luciferase-based assays
were used to quantify the lysis effect. To test the rejuvenating
efficacy of the potential payloads, this cell mixture was then
transferred to 96-well plate, around 2.times.10.sup.5 cells per
well, and different concentration of drugs were added. After 12 h,
Raji cell population, lysis effect and co-inhibitory receptors were
tested again and compared to the PBS treatment group.
EXAMPLES
Example 1. Exhaustion of the antiCD19 CAR T Cell In Vitro
[0066] In this Example, we illustrated a model of exhausted CAR T
cell. Briefly, FIG. 1a shows Raji cells, a type of B-lymphoma cells
were co-cultured with FMC63 CAR T cells, a type of anti CD19 CAR T
cells as described in the method section, and fresh Raji cells were
added to the co-culture every 12 hours for consecutive three days.
FIG. 1b shows these CAR T cells became exhausted after 3 times of
stimulation by fresh Raji cells in vitro, indicated by decreased
killing effect. Co cultured K562 cells served as negative
control.
Example 2. Design of the Secret Passageway
[0067] In this Example, we show a graphic illustration of the
secret passageway of delivery payload drug to targeted cell types.
FKBP or antiFITC is linked to FRa as a fusion receptor, which is
able to engage a targeting ligand FK506 or FITC linked to a
specific payload of drug. Due to the nature of FRa, it constantly
internalizes and delivers FK506 or FITC linked payloads into the
CART cell. Therefore, when CAR T is engaged to its target cells
through CAR, in this example, anti-CD 19 molecule on the CAR T
surface engages CD-19 of the cancer cell, the delivered payload
drug inside of CAR T may execute its function, i.e. regulate CAR T
activity based on the payload function. For example, some payload
drugs may be directed to act on PD-1, CTLA4, or LAGS T cell
function regulatory molecules to rejuvenate CAR T when
necessary.
Example 3. Reversion of the Exhausted antiCD19 CAR T Cell by TLR7
Agonist
[0068] In this Example, we show that targeting ligand FK506 or FITC
successfully engages its respective fusion receptor (FKBP or
FITC-AF647 linked to Folate Receptor by G.sub.4S). In this example
the payload drug is an imaging agent Rhodamine to show the payload
distribution in fusion receptor transfected Jurkat cells. The
binding affinity of FK506 and FITC are calculated by competitive
binding and FK506-Rhodamine shows Kd=3.39 nM towards FKBP-FR fusion
receptor, while FITC-AF647 shows Kd=8.03 nM towards antiFITC-FR
Example 4. Design of Potential Releasable and Non-Releasable
FK506-TLR7 Agonist and FITC-TLR7 Agonist
[0069] In this Example, we provided targeting ligand FK506
conjugated to a Toll Like Receptor 7 agonist with the structure
below to treat exhausted CAR T cells. A dose dependent pattern of
increased killing effect is observed when TLR7 agonist payload drug
is targeted to exhausted CAR T cells. Accordingly, the indicator of
CAR T activity, IFN.gamma. expression level, increased in a dose
dependent manner relative to payload drug concentration; and the
expression level of a T cell effect inhibitory molecule, Tim 3, had
showed a reverse dose dependence manner, i.e. Tim 3 expression
decreased as the concentration of payload drug is increased.
FK506-TLR7 agonist conjugate should have one of the structures
given below:
##STR00017##
Similarly, Fluorescein-TLR7 agonist conjugate should have one of
the structures given below and rejuvenate exhausted CAR T if the
functional CAR T has an anti-FITC fusion receptor.
##STR00018##
Example 5. Design of Other Potential Payloads for the Rejuvenation
of the Exhausted CAR T Cell
[0070] In this Example we provide a list of structures of other
potential payloads that may revert CAR T exhaustion with lower
nanomolar range potency.
[0071] 1) TC-PTP phosphatase inhibitor should have the following
structures,
##STR00019## [0072] Phosphatase inhibitor mentioned above may be
connected to the fluorescein or FK506 (tacrolimus) in the following
way.
##STR00020##
[0073] 2) The STING agonist should have the following
structure,
##STR00021##
Example 6. Design of Spacers Between Targeting Ligands and the
Potential Payloads
[0074] Below are a list of spacers that can be employed to link the
targeting ligands and any potential payloads.
##STR00022##
Example 7. Evaluation of the Ability of Phosphatase Inhibitors and
TLR7 Agonists to Rejuvenate Exhausted CAR T Cells
[0075] One major limitation of CAR T cell therapies in solid tumors
is their tendency to become exhausted after repeated stimulation
with cancer antigens. This phenomenon however, is not specific to
CAR T cells, but has been described in both chronic virus
infections.sup.4 and tumor infiltration lymphocytes.sup.5. The
reversibility of the exhausted phenotype of T cells has been proven
in studies where T cells isolated from the solid tumor tissue show
a higher INF.gamma. secretion and a killing effect if kept away
from antigens ("rested") overnight before re-stimulation.sup.57.
However, it would will be more appealing if rejuvenation could be
achieved in a more clinically relevant way using commercially
available therapeutics: either to block the inhibitory signaling or
to activate the T cells through other pathways. Antibodies
targeting checkpoint inhibitors (i.e. PD-1, CTLA-4, etc.) have
shown some success in solid tumors in-clinic.sup.151, however, two
or more targets in combination often have been found to be
necessary. Moreover, antibody therapy also suffers from poor
penetration in solid tumors and this may have led to less reports
for the combination therapy of CAR T cells and checkpoint blockades
(ICB) in solid tumors.
[0076] The inhibition of the phosphatases, such as SHP1/2 and
TC-PTP, that mediate TCR deactivation, is a potential way to block
tonic CAR T signaling. SHP1/2 phosphatase is responsible for
mediating the signal from PD-1 and other exhaustion markers. Data
has shown that SHP1/2 phosphatase inhibitor or silencing can
increase the activity of T cells and CAR T cells.sup.69. TC-PTP is
known to be an important player in T cell activity signaling. Mice
harboring a T cell specific TC-PTP deficiency have increased
susceptibility to inflammation and autoimmunity due to heightened
antigen-driven T cell activation. TC-PTP inactivates Src family
kinase downstream of the TCR, thereby contributing to the threshold
of TCR activation.sup.11. Although both knockout experiments and
small molecule inhibitors of these phosphatases have shown potent
effect on lowering TCR threshold and increasing T cell activity,
none of them have been used in CAR T therapy. A representative
SHP1/2 inhibitor has the structure of
##STR00023##
[0077] Another approach to rejuvenate the T cells is to augment
their activity through the engagement of antigen independent innate
immune receptors. It has been known that certain pathogen pattern
recognition (PPR) receptors, including toll like receptors (TLR),
do express on non-myeloid cell populations, including T cells, and
can be activated in a similar way. Research has also shown that
co-stimulation of TLR7/8 agonists and TCR signaling can activate
CD8 T cells and increase INF.gamma. secretion.sup.13. However, due
to the strong side effects of systemic dosing of TLR agonists, none
of these agonists have been used in CAR T therapy to reactivate the
T cell or change the immunosuppressive microenvironment. The
employment of TLR agonists for cancer immunotherapy is also
hindered by the controversial effect of TLR agonists on the tumor
cells. Therefore, a targeted delivery of the potential payloads to
the CAR T cell is highly desired. A potent TLR7 agonist was found
in the literature (FIG. 7A), which is around 40 fold stronger than
the FDA approved imiquimod.
[0078] To set up an in vitro screening model, as shown in FIG. 6,
anti-CD19 CART cells were exposed to 4 rounds of addition of CD19
positive Raji cells, and became exhausted as marked by gradual
decreased lysis activity as well as increased co-inhibitory markers
in an in vitro co-culture model. It's worth noticing that the
culture medium is important for the introduction of CART exhaustion
and needs to be kept the same without new replenish or change
during the whole process. It indicates that the soluble components
that are released by cancer cells and/or CAR T cells into the
medium, most likely immunosuppressive cytokines and modulators
(adenosine etc.), play a pivotal role in this process. It also
suggests that the exhaustion of CAR T cells generated by this in
vitro model is at a rather pliable than irreversible status.
[0079] Treatment of the TLR7 agonist and PTP1b (highly homologous
to TC-PTP.sup.26) inhibitor.sup.27 of choice with the already
exhausted CAR T cells was shown to be able to reactivate them
compared to the no treatment group (FIG. 7). No significant
changes, however, were observed in the expression level of
co-inhibitory markers except for Tim3. As shown by FIG. 7C, the
PTP1b inhibitor in general does not show as strong of a
reactivation effect as the TLR7 agonist. Without being limited by
any theory, this result could be due to the current in vitro
screening model where "Reversion" rather than "Prevention" of
exhaustion is studied, and the phosphatases are already "silenced"
at the exhausted status, therefore their inhibition will have
little to no effect. A modified screening model for a future study
will test the effects of phosphatase inhibitors and other drugs
with a focus on the "prevention" of exhaustion by adding the drugs
at the beginning of all cultures and keeping the rest of the
settings the same. In this way, it may be possible to see whether
the inhibition of phosphatase can lower the tonic signaling of CAR
T while still keeping a functional killing effect. We will mainly
focus on TLR7 agonist for the following study.
[0080] Since TLR7 is one of the 4 TLR family members that resides
inside the endo some, it is speculated that a non-releasable linker
between the TLR7 agonist and our secret passageway targeting ligand
would preserve its TLR7 agonist function.sup.28. To achieve that,
several TLR7 agonist analogs were prepared and tested to find the
proper derivatization sites for linkage. As shown in FIG. 8, the
TLR7 agonist with a CH.sub.2OH extension at the piperidine ring has
an even higher activity compared to the parent drug. Therefore,
this derivative site will be used for a non-releasable conjugate. A
disulfide bond linked self-immolative form also has been
synthesized. In order to understand the distance needed for this
TLR7 agonist to reach its own target, three different lengths of
linker (PEG3, 6, 16) between the FITC and TLR7 agonist were made
for the non-releasable FITC-TLR7. As shown in FIG. 9 C, all of the
non-releasable forms had some effect, while the PEG6 compound
showed the best dose-dependent response. These results indicate
that TLR7 agonists may dock with TLR7 either by reaching out while
binding with FITC-FR or jumping between TLR and FITC-FR, under
which conditions the length of the linker in between is not a
crucial factor (FIG. 9 D-E). Since the non-releasable FITC-TLR7 is
trapped inside the endosome and the volume of each endosome is much
smaller than the cytosol, the intra-endosome TLR7 can get to its
functional concentration much faster and quicker, resulting in a
smaller IC.sub.50.
Example 8. Other Potential Payloads for Revert/Prevent the
Exhaustion of CAR T Cells
[0081] Other than TLR7 agonist, there are several other potential
payloads that may revert/prevent the exhaustion of CAR T cells as
described below. Some of the targets may not have agonists or
inhibitors with IC.sub.50 suitable for our targeted drug delivery
approach for now, but are still worth noticing and may be explored
through other inhibitory mechanisms, such as CRISPR or targeted
microRNA delivery approaches.
[0082] STING Agonist
The Simulator of IFN Genes (STING) is a master adaptor involved in
cytosolic DNA sensing and the following IFN-.beta. production.
STING associates weakly to sdDNA, but strongly binds the endogenous
cyclic dinucleotide GMP-AMP (cGAMP) synthesized by the cGMP-AMP
synthase (sGAS). It is predominantly expressed in macrophages, T
cells, a variety of DCs, endothelial cells, and select fibroblasts
and epithelial cells. Studies of STING have mainly focused on its
function in macrophages and dendritic cells, and recently some
groups have noticed the direct effect of STING activation in T
cells.sup.40. It is possible that a STING agonist will have a
similar pro-inflammatory effect on T cells. ADU-S100 is one of the
many STING agonists that has been pursued in clinics.
[0083] DGK-.alpha. Inhibitor
Diacylglycerol Kinase-a (DGK-a) converts diacylglycerol (DAG), a
second messenger in TCR signaling together with IP3, to
phosphatidic acid (PA). DGK is more highly expressed in CD8TIL than
in CD8-NIL, and its inhibition promotes ERK phosphorylation and
lytic degranulation.sup.41; it also restores lytic functions of CAR
TIL that are isolated from in vivo.sup.5. Some DGK inhibitor
structures are as following;
##STR00024##
[0084] TGF.beta.RI (ALK5) Inhibitor
TGF.beta. is known for its immunosuppressive function in many
immune cells, such as the T cell, B cell, and macrophages. The
blockage of TGF.beta. type I receptor (TGF.beta.RI, also called
ALK5) in T cells reverts the immunosuppressive environment of the
tumor.sup.42. Small molecule inhibitors have been pursued with
galunisertib (LY2157299 monohydrate) and EW-7197 tested in
clinics.sup.43-44.
[0085] A TGF.beta. inhibitor structure is as following:
##STR00025##
[0086] EZH2 Inhibitor
Enhancer of Zeste Homolog 2 (EZH2) is a histone H3K27
methyltransferase with a strong correv with the Treg function.
Genetic or pharmacological disruption of EZH2 drove acquisition of
proinflammatory function of tumor infiltrating Treg.sup.45. Since
exhausted CTL in chronic virus infections is also characterized by
unique epigenetic changes.sup.174, it is possible that EZH2
inhibitors will be able to reverse this exhaustion status. Several
small molecules of EZH2 inhibitors have been developed, including
CPI1205, EPZ6438 and GSK126.
[0087] Therefore, the FITC-FR fusion receptor and the corresponding
FITC targeted immune-agonists payloads provide a universal platform
for the monitor and control of CAR T cells homing and persistence
in solid tumor. This approach can be easily incorporated into CAR T
cells for any antigen since the FITC-FR fusion receptor is
independently expressed to the CAR construct. The modular design of
targeting ligand-payload conjugates also makes it easier for the
switching and modification. This approach combines the benefits of
cell therapy and small molecule-based targeted drug delivery and
may require extra characterization of both the engineered cells and
the corresponding ligands.
[0088] The success of CAR T cells in solid tumor most likely
requires the combination of multiple approaches targeting other
players within the microenvironment as well, such as breaking down
of the extracellular matrix by PI3K kinase inhibitors, reprograming
of anti-inflammatory M2 macrophages to a proinflammatory M1
phenotype, and upregulation of the decreased MHC molecules level on
cancer cells. Therefore, more and more combinational therapy
studies will be conducted both preclinically and clinically.
However, at the same time, a careful examination and control of the
CAR T cell itself cannot be neglected and should be optimized by
using simple but robust systems like FITC-FR fusion receptors in
preclinical research first before it reaches to humans.
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