U.S. patent application number 16/743987 was filed with the patent office on 2020-05-07 for immunosuppressive tgf-b signal converter.
The applicant listed for this patent is Baylor College of Medicine. Invention is credited to Ann Marie Leen, Cliona M. Rooney, Juan Fernando Vera Valdes, Norihiro Watanabe.
Application Number | 20200140520 16/743987 |
Document ID | / |
Family ID | 50877664 |
Filed Date | 2020-05-07 |
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United States Patent
Application |
20200140520 |
Kind Code |
A1 |
Vera Valdes; Juan Fernando ;
et al. |
May 7, 2020 |
IMMUNOSUPPRESSIVE TGF-B SIGNAL CONVERTER
Abstract
Embodiments of the disclosure concern cell therapy methods and
compositions utilizing cells expressing at least a chimeric
TGF.beta. receptor including the exodomain of a TGF.beta.II
receptor and an endodomain that is not from TGF.beta. receptor,
thereby converting the negative signal of TGF.beta. for T cell
proliferation into a T cell activation signal. In at least certain
aspects, cells harboring the chimeric TGF.beta. receptor also
harbor one or more chimeric antigen receptors.
Inventors: |
Vera Valdes; Juan Fernando;
(Bellaire, TX) ; Rooney; Cliona M.; (Bellaire,
TX) ; Leen; Ann Marie; (Houston, TX) ;
Watanabe; Norihiro; (Houston, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Baylor College of Medicine |
Houston |
TX |
US |
|
|
Family ID: |
50877664 |
Appl. No.: |
16/743987 |
Filed: |
January 15, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14785248 |
Oct 16, 2015 |
10584158 |
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PCT/US2014/034570 |
Apr 17, 2014 |
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16743987 |
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61812917 |
Apr 17, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 38/1774 20130101; A61K 38/179 20130101; C07K 2319/00 20130101;
A61N 5/10 20130101; A61K 45/06 20130101; A61P 35/02 20180101; C07K
14/70596 20130101; C07K 14/71 20130101 |
International
Class: |
C07K 14/71 20060101
C07K014/71; C07K 14/705 20060101 C07K014/705; A61N 5/10 20060101
A61N005/10; A61K 45/06 20060101 A61K045/06; A61K 38/17 20060101
A61K038/17 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under
W81XWH-11-1-0625 awarded by the Department of Defense. The
government has certain rights in the invention.
Claims
1. A chimeric TGF.beta. receptor polypeptide, wherein the receptor
comprises an exodomain of TGF.beta. receptor and an endodomain of
TLR4, wherein said polypeptide is comprised in a T cell.
2. The polypeptide of claim 1, wherein the cell comprises a
chimeric antigen receptor (CAR).
3. The polypeptide of claim 2, wherein the CAR is specific for an
antigen selected from the group consisting of PSCA, HER2, CD19,
CD20, CD22, Kappa or light chain, CD30, CD33, CD123, CD38, ROR1,
ErbB3/4, EGFR, EGFRvIII, EphA2, FAP, carcinoembryonic antigen,
EGP2, EGP40, mesothelin, TAG72, PSMA, NKG2D ligands, B7-H6, IL-13
receptor .alpha.2, IL-11 receptor Ra, MUC1, MUC16, CA9, GD2, GD3,
HMW-MAA, CD171, Lewis Y, G250/CAIX, HLA-AI MAGE A1, HLA-A2
NY-ESO-1, PSC1, folate receptor-.alpha., CD44v7/8, 8H9, NCAM, VEGF
receptors, 5T4, Fetal AchR, NKG2D ligands, CD44v6, TEM1, TEM8 and a
combination thereof.
Description
[0001] This application is continuation of U.S. patent application
Ser. No. 14/785,248 filed Oct. 16, 2015, which is a national phase
application under 35 U.S.C. .sctn. 371 that claims priority to
International Application No. PCT/US2014/034570 filed Apr. 17,
2014, which claims priority to U.S. Provisional Patent Application
Ser. No. 61/812,917, filed Apr. 17, 2013, all of which are
incorporated by reference herein in their entirety.
TECHNICAL FIELD
[0003] Embodiments of the disclosure encompass at least the fields
of immunology, cell biology, molecular biology, and medicine,
including cancer medicine.
BACKGROUND
[0004] Transforming growth factor beta (TGF.beta.) has been
demonstrated to play an important role in the regulation of the
immune response, primarily through its suppressive function towards
cells of the immune system. TGF.beta. is a suppressor of
antigen-specific T cell proliferation at least through reduction of
the cell-cycle rate, as opposed to induction of apoptosis. In
particular, TGF.beta. acts on cytotoxic T lymphocytes (CTLs) to
specifically inhibit the expression of at least five cytolytic gene
products: perforin, granzyme A, granzyme B, Fas ligand, and
interferon gamma that are important for CTL-mediated tumor
cytotoxicity (Thomas and Massague, 2005).
BRIEF SUMMARY
[0005] Embodiments of the disclosure include methods and
compositions for cancer therapy. In particular embodiments, there
are methods and compositions related to cell therapy for cancer.
Although particular embodiments may be useful for any cancer, in
specific embodiments the methods and compositions are particularly
effective against cancers that secrete TGF.beta. or that reside in
a tumor microenvironment (including tumor stroma) that releases
TGF.beta.. Exemplary cancers that are suitable for treatment with
the compositions and methods disclosed herein include but are not
limited to cancers of the prostate, breast, melanoma, pancreatic,
lung, brain, colon, esophageal, liver, kidney, testicular, ovarian,
cervical, gall bladder, thyroid, anal, endometrial, bladder,
pituitary gland, leukemia, lymphoma, stomach, and spleen.
[0006] Aspects of the disclosure utilize cells that are modified to
express one or more non-native molecules that render the cells
effective against cancers that secrete TGF.beta.. The cells may be
utilized for adoptive cell therapy, in specific embodiments. In
particular aspects, the cells are T cells, NK cells, NKT cells,
cytotoxic T lymphocytes, antigen-specific T cells, including tumor-
or pathogen (such as viral or bacterial)-specific T cells, T cells
having .alpha..beta. T cell receptors (TCR), T cells that comprise
at least one chimeric antigen receptor, and so forth. Cells may be
genetically engineered to be effective in the presence of
TGF.beta., and in addition may or may not have other non-naturally
occurring genetic modifications. In addition to the chimeric
TGF.beta. receptor, the cells may express other non-native
molecules. In particular embodiments, the non-native molecules are
cell surface receptors. The molecules may be a chimeric antigen
receptor (CAR) specific for a tumor cell surface molecule or
.alpha..beta. TCRs, for example. In certain aspects, a cell may
employ one or more chimeric antigen receptors and a chimeric
TGF.beta. receptor.
[0007] In embodiments of the disclosure, the cells of the
disclosure convert the normally inhibitory signal from TGF.beta.
into an activation stimulus for T cells by utilizing a chimeric
TGF.beta. receptor that employs an extracellular receptor for
TGF.beta. on the cell surface linked to an endodomain from another
entity that is capable of an activation signal. In particular
embodiments, T cells modified with such chimeric receptors have
increased potency compared to cells that lack such chimeric
receptors. Specific embodiments of the disclosure employ a chimeric
TGF.beta. receptor expressing the exodomain of TGF.beta.RII linked
to an endodomain from another molecule. In specific embodiments the
exodomain and the endodomain are linked through a transmembrane
domain, and in certain cases the transmembrane domain is the
naturally occurring transmembrane domain for the selected
endodomain. Although the receptor for TGF.beta. can be part or all
of any one of TGF.beta. receptor 1, TGF.beta. receptor 2, or
TGF.beta. receptor 3, in specific embodiments it comprises the
TGF.beta. receptor 2 isoform of the receptor. In specific
embodiments, the endodomain comprises the endodomain of the
exemplary toll-like receptor (TLR) 4, although others such as TLR1,
TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, CD28, 4-1BB, OX40,
CD3zeta, CD40, CD27, or a combination thereof, may be employed, for
example. In specific embodiments, the chimeric cytokine receptor is
labeled, such as with GFP, mOrange, blue fluorescent protein, and
so forth.
[0008] In certain aspects of the disclosure, the cell that
comprises a chimeric TGF.beta. receptor also comprises a chimeric
antigen receptor. The chimeric antigen receptor (CAR) may be of any
kind, but in specific embodiments the CAR targets the cancer that
also secretes the TGF.beta. molecule. Exemplary CARs include at
least CARs specific for any one or more of the following: EphA2,
HER2, GD2, Glypican-3, 5T4, 8H9, .alpha..sub.v.beta..sub.6
integrin, B cell maturation antigen (BCMA) B7-H3, B7-H6, CAIX, CA9,
CD19, CD20, CD22, kappa light chain, CD30, CD33, CD38, CD44,
CD44v6, CD44v7/8, CD70, CD123, CD138, CD171, CEA, CSPG4, EGFR,
EGFRvIII, EGP2, EGP40, EPCAM, ERBB3, ERBB4, ErbB3/4, FAP, FAR, FBP,
fetal AchR, Folate Receptor u, GD2, GD3, HLA-AI MAGE A1, HLA-A2,
IL11Ra, IL13Ra2, KDR, Lambda, Lewis-Y, MCSP, Mesothelin, Muc1,
Muc16, NCAM, NKG2D ligands, NY-ESO-1, PRAME, PSCA, PSC1, PSMA,
ROR1, Sp17, SURVIVIN, TAG72, TEM1, TEM8, VEGRR2, carcinoembryonic
antigen, HMW-MAA, VEGF receptors, and/or other exemplary antigens
that are present with in the extracellular matrix of tumors, such
as oncofetal variants of fibronectin, tenascin, or necrotic regions
of tumors and other tumor-associated antigens or actionable
mutations that are identified through genomic analysis and or
differential expression studies of tumors, for example, or a
combination thereof. The cells of the disclosure may have more than
one CAR, and a CAR may have more than one scFv specific for
different antigens.
[0009] In certain aspects of the disclosure, the cell that
comprises (such as expresses) a chimeric TGF.beta. receptor also
comprises (such as expresses) an antigen-specific receptor. The
antigen may be a tumor antigen or a pathogen antigen, including
viral or bacterial, for example.
[0010] Exemplary tumor antigens include at least the following:
carcinoembryonic antigen (CEA) for bowel cancers; CA-125 for
ovarian cancer; MUC-1 or epithelial tumor antigen (ETA) or CA15-3
for breast cancer; tyrosinase or melanoma-associated antigen (MAGE)
for malignant melanoma; and abnormal products of ras, p53 for a
variety of types of tumors; alphafetoprotein for hepatoma, ovarian,
or testicular cancer; beta subunit of hCG for men with testicular
cancer; prostate specific antigen for prostate cancer; beta 2
microglobulin for multiple myelom and in some lymphomas; CA19-9 for
colorectal, bile duct, and pancreatic cancer; chromogranin A for
lung and prostate cancer; TA90 for melanoma, soft tissue sarcomas,
and breast, colon, and lung cancer. Examples of tumor antigens are
known in the art, for example in Cheever et al., 2009, which is
incorporated by reference herein in its entirety. Specific examples
of tumor antigens include at least CEA, gp100, mesothelin, TRP1,
CD40, EGFP, Her2, TCR alpha, trp2, MUC1, cdr2, ras, GITR, WT1,
MUC1, LMP2, HPV E6 E7, EGFRvIII, HER-2/neu, MAGE A3, p53 nonmutant,
NY-ESO-1, PSMA, GD2, Melan A/MART1, Ras mutant, gp 100, p53 mutant,
Proteinase3 (PR1), bcr-abl, Tyrosinase, Survivin, PSA, hTERT,
EphA2, PAP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2 ETS fusion gene),
NA17, PAX3, ALK, Androgen receptor, Cyclin B1, Polysialic acid,
MYCN, RhoC, TRP-2, GD3, Fucosyl GM1, Mesothelin, PSCA, MAGE A1,
sLe(a), CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1,
RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OY-TES1, Sperm
protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, Legumain, Tie
2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-13, MAD-CT-2, and
Fos-related antigen 1, for example.
[0011] Specific viruses for which an antigen receptor T cell may be
directed includes one selected from the group consisting of EBV,
CMV, Adenovirus, BK virus, HHV6, RSV, Influenza, Parainfluenza,
Bocavirus, Coronavirus, LCMV, Mumps, Measles, Metapneumovirus,
Parvovirus B, Rotavirus, West Nile Virus, JC, HHV7, and a
combination thereof. In some cases, the virus is EBV and the
antigen is selected from the group consisting of EBNA1, LMP2, and
BZLF; the virus may be CMV and the antigen may be selected from the
group consisting of IE1 and pp65; the virus may be Adv and the
antigen may be selected from the group consisting of Hexon and
penton; the virus may be BK virus and the antigen may be selected
from the group consisting of LT and VP-1; the virus may be HHV6 and
the antigen may be selected from the group consisting of U14, U11,
U71, U54, and U90; the virus may be RSV and the antigen may be
selected from the group consisting of N and F; the virus may be
Influenza and the antigen may be selected from the group consisting
of MP1 and NP1.
[0012] In embodiments of the disclosure, there is transgenic
expression of a novel immunosuppressive signal converter on T
cells. The signal converter changes the normally inhibitory action
of TGF.beta. on an immune effector cell into a stimulatory signal
for the cell through the use of the chimeric TGF.beta. receptor
that has the TGF.beta. exodomain linked to an endodomain that is
not from TGF.beta. receptor and provides an activation signal.
[0013] In embodiments of the disclosure, there is a polynucleotide
comprising a nucleotide sequence encoding a chimeric TGF.beta.
receptor, wherein the receptor comprises an exodomain of TGF.beta.
receptor and a non-TGF.beta. receptor endodomain. In specific
embodiments, the endodomain comprises an endodomain of an immune
stimulant protein, or functional fragment thereof, wherein the
endodomain or fragment thereof is from toll-like receptor (TLR) 1,
TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, CD28, 41BB, 41BB,
OX40, CD40, CD27, or a combination thereof. In specific cases, the
endodomain is from toll-like receptor (TLR) 4. In particular
embodiments, the polynucleotide further comprises a polynucleotide
sequence that encodes a chimeric antigen receptor (CAR) or an
.alpha..beta. cell receptor (TCR). In some embodiments, the
expression of the chimeric TGF.beta. receptor and the expression of
the CAR and/or (p TCR are under control of the same or different
regulatory element or elements.
[0014] In embodiments wherein one or more polynucleotides are
utilized, the polynucleotide may be further defined as an
expression construct or vector, and the vector may be a viral
vector (such as a retroviral vector, lentiviral vector, adenoviral
vector, or adeno-associated viral vector) or a non-viral vector,
such as plasmid or RNA, including mRNA.
[0015] In certain embodiments, provided herein are one or more
cells that comprise a polynucleotide as provided herein. Such cells
may be, for example, T cells, natural killer (NK) cells, natural
killer T (NKT) cells, antigen-specific T cells, or T cells
comprising an .alpha..beta.TCR, or CTLs. In a specific embodiment,
the antigen-specific T cells are further defined as tumor-specific
T cells or pathogen-specific T cells. In specific cases, the
TGF.beta. receptor comprises part or all of TGF.beta. receptor II.
In some cases, the antigen-specificity of the antigen-specific T
cells is natural, whereas in certain cases, the antigen-specificity
of the antigen-specific T cells is recombinantly generated. In
particular cases, the cell is an antigen-specific T cell, and/or
comprises an (p TCR, which may be native to the T cell or is an
engineered molecule. In particular aspects, the antigen-specific T
cell is further defined as a tumor-specific T cell or
pathogen-specific T cell. In some cases, the antigen-specificity of
the antigen-specific T cell is natural, whereas in certain cases,
the antigen-specificity of the antigen-specific T cell is
recombinantly generated. In some cases, the (p TCR on the cell is
natural, whereas in other cases the .alpha..beta. TCR on the cell
is recombinantly generated; the .alpha..beta. TCR may be selected
for, such as from a library.
[0016] In embodiments of the disclosure, there are one or more
cells, comprising a first polynucleotide that encodes a chimeric
TGF.beta. receptor, wherein the chimeric TGF.beta. receptor
comprises an exodomain of TGF.beta. receptor and a non-TGF.beta.
receptor endodomain; and a second polynucleotide that encodes a CAR
and/or a .alpha..beta. TCR. In some cases, the first and second
expression construct are the same or different molecule. The
antigen-specific T cell may be a tumor-specific T cell receptor or
a pathogen-specific T cell receptor. The CAR may be specific for an
antigen such as one of the following: PSCA, HER2, CD19, CD20, CD22,
Kappa or light chain, CD30, CD33, CD123, CD38, ROR1, ErbB3/4, EGFR,
EGFRvIII, EphA2, FAP, carcinoembryonic antigen, EGP2, EGP40,
mesothelin, TAG72, PSMA, NKG2D ligands, B7-H6, IL-13 receptor
.alpha.2, IL-11 receptor R .alpha., MUC1, MUC16, CA9, GD2, GD3,
HMW-MAA, CD171, Lewis Y, G250/CAIX, HLA-AI MAGE A1, HLA-A2
NY-ESO-1, PSC1, folate receptor-.alpha., CD44v7/8, 8H9, NCAM, VEGF
receptors, 5T4, Fetal AchR, NKG2D ligands, CD44v6, TEM1, TEM8 or a
combination thereof.
[0017] In some embodiments, provided herein are one or more cells,
defined as: 1) being an antigen-specific T cell, and/or a cell
comprising a CAR, and/or a cell comprising an .alpha..beta. T cell
receptor; and 2) comprising a chimeric TGF.beta. receptor, wherein
the receptor comprises an exodomain of TGF.beta. receptor and a
non-TGF.beta. receptor endodomain. In specific embodiments, the
cell is autologous with respect to a recipient of said cell. In
some cases, the cell is allogenic with respect to a recipient of
said cell.
[0018] In embodiments of the disclosure, provided herein is a
method of treating and/or preventing cancer in an individual,
comprising the step of delivering a therapeutically effective
amount of a plurality of cells as provided herein to the
individual, wherein the individual has a cancer that has cells that
secrete TGF.beta. and/or the cancer has a microenvironment that
produces TGF.beta.. Although in some cases the cancer may be of any
kind, in specific cases, the cancer is prostate, breast, melanoma,
pancreatic, lung, brain, colon, esophageal, liver, kidney,
testicular, ovarian, cervical, gall bladder, thyroid, anal,
endometrial, bladder, pituitary gland, leukemia, lymphoma, stomach,
spleen, or myeloma. In specific embodiments, the cells are
delivered intravenously, intraperitoneally, intratumorally,
intrathecally, and/or transrectally. In some embodiments, exposure
of TGF.beta. to the cell protects or enhances the anti-tumor
activity of the cell. In particular aspects, the individual is
provided with another cancer therapy, such as surgery,
chemotherapy, immunotherapy, hormone therapy, radiation, or a
combination thereof. In certain embodiments, the cells are
allogenic or autologous to said individual. In some aspects, the
methods of the disclosure further comprise the step of obtaining
peripheral blood mononuclear cells (PBMCs) from the individual or
from another individual. In specific embodiments, the method
comprises the step of obtaining T cells from the PBMCs and
modifying the T cells to comprise a polynucleotide comprising a
nucleotide sequence encoding a chimeric TGF.beta. receptor, wherein
the receptor comprises an exodomain of TGF.beta. receptor and a
non-TGF.beta. receptor endodomain. In embodiments of the
disclosure, the cells are provided to the individual more than
once. In specific cases, when the cells are provided more than once
and the cells comprise a CAR specific for a particular tumor
antigen, upon one or more subsequent deliveries the cells comprise
a CAR specific for a different tumor antigen.
[0019] In an embodiment of the disclosure, there is a method of
converting a T cell-inhibitory cytokine signal into a T
cell-stimulatory signal, comprising the step of exposing the
inhibitory cytokine to a receptor on a T cell, said receptor having
an exodomain and an endodomain that is not naturally linked to the
exodomain, wherein the exodomain is capable of binding the
inhibitory cytokine and the endodomain provides a stimulatory
signal to the T cell, wherein the exodomain and the endodomain pair
as homodimers. In specific embodiment, the cytokine is TGF.beta. or
VEGF.
[0020] In an embodiment of the disclosure, there is a method of
converting a T cell-inhibitory cytokine signal into a T
cell-stimulatory signal, comprising the step of exposing the
inhibitory cytokine to a receptor on a T cell, said receptor having
an exodomain and an endodomain that is not naturally linked to the
exodomain, wherein the exodomain is capable of binding the
inhibitory cytokine and the endodomain provides a stimulatory
signal to the T cell, wherein the inhibitory cytokine is not IL4 or
IL7 or both. In specific embodiments, the cytokine is TGF.beta. or
VEGF.
[0021] Embodiments of the disclosure provide improvements in
activity of cells for adoptive transfer therapy in vivo in the
presence of cancer cells that secrete the inhibitor TGF3.
[0022] In an embodiment of the disclosure, there is a kit
comprising a polynucleotides or polypeptides as described herein
and/or a cell or cells as described herein.
[0023] The foregoing has outlined the features and technical
advantages of the present disclosure in order that the detailed
description of the disclosure that follows may be better
understood. Additional features and advantages of the disclosure
will be described hereinafter which form the subject of the claims
of the disclosure. It should be appreciated by those skilled in the
art that the conception and specific embodiment disclosed may be
readily utilized as a basis for modifying or designing other
structures for carrying out the same purposes of the present
disclosure. It should also be realized by those skilled in the art
that such equivalent constructions do not depart from the spirit
and scope of the disclosure as set forth in the appended claims.
The novel features which are believed to be characteristic of the
disclosure, both as to its organization and method of operation,
together with further objects and advantages will be better
understood from the following description when considered in
connection with the accompanying figures. It is to be expressly
understood, however, that each of the figures is provided for the
purpose of illustration and description only and is not intended as
a definition of the limits of the present disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] 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:
[0025] FIG. 1 illustrates an exemplary embodiment of arming T cells
to withstand the inhibitory tumor microenvironment.
[0026] FIG. 2 provides exemplary retroviral vector maps of the
control DNRII, RIID2 and RIID4.
[0027] FIG. 3 shows generation of DNRII, RIID2 and RIID4 transgenic
T cells;
[0028] FIGS. 4A-4B demonstrate determination of co-expression of
DNRII, RIID2 and RIID4 with CAR-PSCA.2G.
[0029] FIG. 5 demonstrates whether RIID2 and RIID4 protect
CAR-modified T cells exposed to TGF.beta..
[0030] FIGS. 6A-6C show whether in suppressive conditions RIID2-
and RIID4-modified T cells are protected.
[0031] FIG. 7 shows administration of TGF.beta.1 selects RIID2 and
RIID4-transducted T cells.
[0032] FIG. 8 demonstrates that CARPSCA.2G/RIID2 and/RIID4 T cells
require antigen stimulation for their expansion.
[0033] FIG. 9 provides that RIID2 and RIID4 enhance anti-tumor
effect of CARPSCA.2G against DU145 cells.
[0034] FIG. 10 shows the impact of TGF.beta. exposure on exemplary
CAR T cells. A) signaling, B) cytolytic function, C) expansion, D)
viability, and E) PD1 expression.
[0035] FIG. 11. illustrates A) wild type TGF.beta.R, and B) RIID4
signaling.
[0036] FIG. 12 illustrates A) the retroviral vector for an example
of a RIID4 construct, B) expression of RIID4 on transduced cells,
C) microscopic photographs of T cell cultures in presence or
absence of TGF.beta., and D) expression of PD1 in T cells as
measured by flow.
[0037] FIG. 13 illustrates exemplary chimeric cytokine retroviral
receptors.
[0038] FIG. 14 demonstrates that TGF.beta.1 does not induce pSmad
2/3 in RIID4-modified T cells.
[0039] FIG. 15 shows protection by RIID4 of T cells from
TGF.beta.1-induced apoptosis.
[0040] FIG. 16 demonstrates that RIID4 prevents PD1
upregulation.
[0041] FIG. 17 demonstrates that RIID4 prevents TGF.beta.1-induced
inhibition of cytolytic function.
[0042] FIG. 18 shows that RIID4+ cells are selected by TGF.beta.1
exposure.
[0043] FIG. 19 demonstrates that withdrawal of antigen and
TGF.beta.1 leads to culture failure.
[0044] FIG. 20 illustrates an example of a co-culture experimental
set-up.
[0045] FIG. 21 provides evidence that 2G.CAR-PSCA/RIID4 cells
eliminated tumors in the presence of TGF.beta. 1.
[0046] FIG. 22 shows that 2G.CAR-PSCA T cells did not proliferate
in the presence of TGF.beta.1.
[0047] FIG. 23 demonstrates that 2G.CAR-PSCA/RIID4 cells were able
to expand and that the cells depend on antigen existence.
[0048] FIG. 24 illustrates that TGF.beta.1 administration promotes
proliferation of 2G.CAR-PSCA/RIID4 T cells.
DETAILED DESCRIPTION
[0049] 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 disclosure
may consist of or consist essentially of one or more elements,
method steps, and/or methods of the disclosure. It is contemplated
that any method or composition described herein can be implemented
with respect to any other method or composition described herein
embodiments which are disclosed and still obtain a like or similar
result without departing from the spirit and scope of the
disclosure.
[0050] Embodiments of the present disclosure encompass cells for
adoptive cell transfer that have been modified to switch an
inhibitory signal that suppresses immune cell proliferation to a
stimulatory signal that enhances immune cell proliferation and, at
least in some cases, anti-tumor activity. In particular
embodiments, immune cells of the disclosure are equipped with
receptors that are designed to bind to inhibitory molecules but
transmit a positive signal rather than a suppressive one. That is,
the cells transform a "brake" signal (such as TGF.beta.) into an
"accelerator" signal to improve the anti-tumor effects of the
respective immune cells.
[0051] The cells, in certain embodiments, comprise a chimeric
cytokine receptor that encompasses a chimera of a TGF.beta.
exodomain (for example) with an endodomain that is from another
molecule such that binding of TGF.beta. to the exodomain causes the
endodomain to stimulate activity of the T cell. The cells may or
may not have other traits, natural and/or genetically engineered by
man, including receptors for other molecules, including at least
.alpha..beta.TCR, antigen-specific receptors (including
tumor-specific receptors), and/or chimeric antigen receptors, for
example.
[0052] I. Chimeric Cytokine Receptors
[0053] In embodiments of the disclosure, there are compositions
that comprise chimeric cytokine receptors that bind to a Th2 or
immunosuppressive cytokine (TGF.beta. is an example) but induce
immune stimulatory signaling instead of immunosuppression,
resulting in the maintenance of a Th1 (effector) phenotype,
proliferation and cytotoxic profile. The receptors as polypeptides
and nucleotides that encode them are encompassed in the
disclosure.
[0054] Embodiments of the disclosure include polypeptides (and the
polynucleotides that encode them) that comprise an exodomain of a
receptor for an immunosuppressive cytokine fused with an endodomain
of an immunostimulatory molecule. In specific embodiments, the
exodomain of TGF.beta.R is fused with one or more stimulatory
endodomains including for example TLR1, TLR2, TLR3, TLR4, TLR5,
TLR6, TLR7, TLR8, TLR9, CD28, 4-1BB, OX40, or combinations of any
of the foregoing. Cells harboring such chimeric cytokine receptors
protect tumor-reactive immune cells from immunosuppressive
cytokines such as TGF.beta..
[0055] In certain embodiments, provided herein are chimeric
TGF.beta. receptors that include only the exodomain of TGF.beta.
receptor or a TGF.beta.-binding fragment thereof.
[0056] The chimeric TGF.beta. receptor, in certain embodiments,
comprises the exodomain of the TGF.beta. receptor. Any isoform of
the natural TGF.beta. receptor may be used for its exodomain,
including TGF.beta. receptor 1, TGF.beta. receptor 2, and TGF.beta.
receptor 3. In particular embodiments, the exodomain for TGF.beta.
receptor 2 is employed in compositions (including expression
constructs and cells, for example) of the disclosure. Exemplary
nucleotide sequences encoding TGF.beta. receptor 2 include
GenBank.RTM. Accession Nos. NM_001024847.2 (SEQ ID NO:3)
(polypeptide is NP_001020018; SEQ ID NO:4) or NM_003242.5
(polypeptide is NM_003242). An example of an endodomain is TLR4,
and an exemplary sequence for TLR4 is at GenBank.RTM. Accession No.
U88880 (SEQ ID NO:5) (polypeptide is AAC34135; SEQ ID NO:6).
[0057] Polynucleotides for the receptor may include a nucleotide
sequence encoding the signal peptide of TGF.beta. receptor, in
specific embodiments. In a certain embodiment, the signal peptide
is as follows (amino acid position 1-22 from TGF.beta. _receptor
2): MGRGLLRGLWPLHIVLWTRIAS (SEQ ID NO: 1). In a specific
embodiment, the exodomain (which may be referred to as the
extracellular domain) includes amino acid position 23-166 from
TGF.beta. receptor 2 as shown in SEQ ID NO:2:
TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQE
VCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSD
ECNDNIIFSEEYNTSNPDLLLVIFQ (SEQ ID NO:2). In other specific
embodiments, the exodomain is from TGF.beta. receptor 1, TGF.beta.
receptor 3, or from any other TGF.beta. receptor or variant
thereof.
[0058] The disclosure encompasses polynucleotides and polypeptides
for the chimeric TGF.beta. receptors and cells that harbor them.
The disclosure includes use of the chimeric TGF.beta. receptors in
methods of treating cancers that secrete TGF.beta. or that are in a
tumor environment that provides TGF.beta.. The chimeric TGF.beta.
receptors may be used in conjunction on cells that also include one
or more CARs.
[0059] In particular embodiments, the chimeric TGF.beta. receptor
of the disclosure comprises an endodomain that is not from the
endogenous TGF.beta. receptor. The endodomain may be of any kind so
long as it imparts an activation signal to the immune effector cell
in which it resides upon binding of TGF.beta. to the chimeric
TGF.beta. receptor exodomain, e.g., causes T cells that express the
chimeric TGF.beta. receptor to proliferate in the presence of
TGF.beta.. Exemplary endodomains include, for example, an immune
stimulant endodomain or fragment thereof from one or more of TLR1,
TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, CD28, 4-1BB, OX40,
CD3zeta, CD40, CD27, or a combination thereof.
[0060] In embodiments of the disclosure, the chimeric TGF.beta.
receptor may also include a transmembrane domain between the
exodomain and endodomain. The transmembrane domain may come from
any source, although in specific embodiments it is from the same
molecule as the endodomain. In particular embodiments, the chimeric
receptor comprises the exodomain of the TGF.beta. receptor, an
endodomain of an immunostimulatory molecule and a transmembrane
domain; in particular aspects the transmembrane domain is from the
same molecule as the immunostimulatory molecule. In specific
embodiments, the chimeric TGF.beta. 3 receptor comprises a
transmembrane domain and one or more additional amino acids (such
as one, two, three, four, five, or more additional amino acids
(aa)) from the same molecule as the transmembrane domain (TM). An
exemplary pattern for the chimeric TGF.beta. receptor is as
follows:
[0061] TGF.beta.R exodomain-1, 2, 3, or more aa from
TM-TM-non-TGF.beta.R endodomain
[0062] In certain embodiments, the exodomain may not include the
entire exodomain but instead comprise an epitope and, in some
cases, a spacer connected to the endodomain.
[0063] II. Host Cells Comprising Chimeric Cytokine Receptors
[0064] In embodiments of the disclosure, cells are employed for
therapy. The cells encompassed in the disclosure may be immune
cells, such as immune effector cells that encompass a T cell,
including a cytotoxic T cell (also known as TC, Cytotoxic T
Lymphocyte, CTL, T-Killer cell, cytolytic T cell, CD8+ T-cells or
killer T cell); NK cells; NKT cells; and other immune cells that
can elicit an effector function. In specific embodiments, cells
that incorporate the receptors of the disclosure exhibit a
bystander effect at the tumor microenvironment at least by
depleting inhibitory cytokines from the tumor microenvironment, for
example.
[0065] As used herein, the terms "cell," "cell line," and "cell
culture" may be used interchangeably. All of these terms also
include their progeny, which is any and all subsequent generations.
It is understood that all progeny may not be identical due to
deliberate or inadvertent mutations. In the context of expressing a
heterologous nucleic acid sequence, "host cell" refers to a
eukaryotic cell that is capable of replicating a vector and/or
expressing a heterologous gene encoded by a vector. A host cell
can, and has been, used as a recipient for vectors. A host cell may
be "transfected" or "transformed," which refers to a process by
which exogenous nucleic acid is transferred or introduced into the
host cell. A transformed cell includes the primary subject cell and
its progeny. As used herein, the terms "engineered" and
"recombinant" cells or host cells are intended to refer to a cell
into which an exogenous nucleic acid sequence, such as, for
example, a vector, has been introduced. Therefore, recombinant
cells are distinguishable from naturally occurring cells which do
not contain an introduced recombinant nucleic acid.
[0066] In certain embodiments, it is contemplated that RNAs or
proteinaceous sequences may be co-expressed with other selected
RNAs or proteinaceous sequences in the same cell, such as the same
CTL. Co-expression may be achieved by co transfecting or
co-transducing the CTL with two or more distinct recombinant
vectors. Alternatively, a single recombinant vector may be
constructed to include multiple distinct coding regions for RNAs,
which could then be expressed in CTLs transfected or transduced
with the single vector.
[0067] Some vectors may employ control sequences that allow it to
be replicated and/or expressed in both prokaryotic and eukaryotic
cells. One of skill in the art would further understand the
conditions under which to incubate all of the above described host
cells to maintain them and to permit replication of a vector. Also
understood and known are techniques and conditions that would allow
large-scale production of vectors, as well as production of the
nucleic acids encoded by vectors and their cognate polypeptides,
proteins, or peptides.
[0068] The cells can be autologous cells, syngeneic cells,
allogenic cells and even in some cases, xenogeneic cells, in
relation to the individual that receives them.
[0069] In many situations one may wish to be able to kill the
modified cells, where one wishes to terminate the treatment, the
cells become neoplastic, in research where the absence of the cells
after their presence is of interest, or other event. For this
purpose one can provide for the expression of certain gene products
in which one can kill the modified cells under controlled
conditions, such as inducible suicide genes.
[0070] A. Additional Cellular Characteristics
[0071] In addition to the cell comprising a chimeric cytokine
receptor, the cell may have one or more other characteristics that
are useful for cellular immunotherapy. Such additional
characteristics may be inherent to the cell or may be a part of the
cell following genetic manipulation by man. There may be more than
one characteristic in addition to the chimeric TGF.beta. receptor.
In specific embodiments, in addition to the cell having a chimeric
TGF.beta. receptor, the cell may have a chimeric antigen receptor
(CAR), an .alpha..beta. T cell receptor, and/or an antigen-specific
receptor, such as a tumor-specific receptor.
[0072] In one embodiment, the host cell is a T-cell comprising one
or more chimeric TGF.beta. receptors and comprising one or more of
an engineered .alpha..beta.TCR receptor, a native receptor specific
for a tumor antigen, or a CAR; in each case, the additional
modification may target a tumor antigen of choice.
[0073] Naturally occurring T-cell receptors comprise two subunits,
an .alpha.-subunit and a .beta.-subunit, each of which is a unique
protein produced by recombination event in each T-cell's genome,
and libraries of TCRs may be screened for their selectivity to
particular target antigens, including tumor antigens. An
"engineered TCR" refers to a natural TCR, which has a high-avidity
and reactivity toward target antigens that is selected, cloned,
and/or subsequently introduced into a population of T-cells used
for adoptive immunotherapy. In contrast to engineered TCRs, CARs
are engineered to bind target antigens in an MHC-independent
manner.
[0074] In particular embodiments, a CAR comprises an extracellular
binding domain including, but not limited to, an antibody or
antigen binding fragment thereof; a transmembrane domain; one or
more intracellular costimulatory signaling domains and a primary
signaling domain.
[0075] In embodiments wherein T cell receptors are generated in
cells that express or will express a chimeric cytokine receptor, in
specific embodiments the methods use exposure of peripheral
mononuclear blood cells with libraries of mixture of peptide from a
tumor antigen (see PCT/US2013/025342, which is incorporated by
reference herein in its entirety).
[0076] In certain embodiments of the disclosure, there are immune
effector cells that include the chimeric TGF.beta. receptor and
also that are modified to comprise at least one CAR that allows
bypass of tumor immune escape mechanisms that are due to
abnormalities in protein-antigen processing and presentation.
[0077] In particular cases, the cells include a CAR that is
chimeric, non-natural and engineered at least in part by the hand
of man. In particular cases, the engineered CAR has one, two,
three, four, or more components, and in some embodiments the one or
more components facilitate targeting or binding of the cell (such
as a T lymphocyte) to the tumor antigen-comprising cancer cell. In
specific embodiments, the CAR comprises a part of an antibody for
the tumor antigen, part or all of a cytoplasmic signaling domain,
and/or part or all of one or more co-stimulatory molecules, for
example endodomains of co-stimulatory molecules. In specific
embodiments, the antibody is a single-chain variable fragment
(scFv). In certain aspects the antibody is directed at target
antigens on the cell surface of cancer cells that secrete
TGF.beta., for example. In certain embodiments, a cytoplasmic
signaling domain, such as those derived from the T cell receptor
zeta-chain, is employed as at least part of the chimeric receptor
in order to produce stimulatory signals for T lymphocyte
proliferation and effector function following engagement of the
chimeric receptor with the target antigen. Examples include, but
are not limited to, endodomains from co-stimulatory molecules such
as CD27, CD28, 4-1BB, and OX40. In particular embodiments,
co-stimulatory molecules are employed to enhance the activation,
proliferation, and cytotoxicity of T cells produced by the CAR
after antigen engagement. In specific embodiments, the
co-stimulatory molecules are CD28, OX40, and 4-1BB.
[0078] The CAR may be first generation, second generation, or third
generation (CAR in which signaling is provided by CD3.zeta.
together with co-stimulation provided by CD28 and a tumor necrosis
factor receptor (TNFr), such as 4-1BB or OX40), for example. The
CAR may be specific for PSCA, HER2, CD19, CD20, CD22, Kappa or
light chain, CD30, CD33, CD123, CD38, ROR1, ErbB2, ErbB3/4, EGFR,
EGFRvIII, EphA2, FAP, carcinoembryonic antigen, EGP2, EGP40,
mesothelin, TAG72, PSMA, NKG2D ligands, B7-H6, IL-13 receptor
.alpha.2, IL-11 receptor .alpha., MUC1, MUC16, CA9, GD2, GD3,
HMW-MAA, CD171, Lewis Y, G250/CAIX, HLA-AI MAGE A1, HLA-A2
NY-ESO-1, PSC1, folate receptor-.alpha., CD44v7/8, 8H9, NCAM, VEGF
receptors, 5T4, Fetal AchR, NKG2D ligands, CD44v6, TEM1, TEM8,
viral-associated antigens expressed by the tumor, or other
tumor-associated antigens that are identified through genomic
analysis and or differential expression studies of tumors. A single
cell may have multiple CARs, including CARs that target different
tumor antigens.
[0079] In particular embodiments the CAR is encoded on an
expression vector that may or may not also encode the chimeric
TGF.beta. receptor. The vector may be bicistronic, in particular
embodiments. When present on the same expression construct, the CAR
coding sequence may be configured 5' or 3' to the chimeric
TGF.beta. receptor coding sequence. The expression of the CAR and
the chimeric TGF.beta. receptor may be under the direction of the
same or different regulatory sequences.
[0080] B. Introduction of Constructs into Host Cells
[0081] Expression vectors that encode the chimeric cytokine
receptor and optionally comprise at least one CAR,
.alpha..beta.TCR, and/or antigen-specific receptor can be
introduced as one or more DNA molecules or constructs, where there
may be at least one marker that will allow for selection of host
cells that contain the construct(s). The constructs can be prepared
in conventional ways, where the genes and regulatory regions may be
isolated, as appropriate, ligated, cloned in an appropriate cloning
host, analyzed by restriction or sequencing, or other convenient
means. Particularly, using PCR, individual fragments including all
or portions of a functional unit may be isolated, where one or more
mutations may be introduced using "primer repair", ligation, in
vitro mutagenesis, etc., as appropriate. The construct(s) once
completed and demonstrated to have the appropriate sequences may
then be introduced into the CTL by any convenient means. The
construct(s) may be integrated and packaged into non-replicating,
defective viral genomes like Adenovirus, Adeno-associated virus
(AAV), or Herpes simplex virus (HSV) or others, including
retroviral vectors, for infection or transduction into cells. The
constructs may include viral sequences for transfection, if
desired. Alternatively, the construct may be introduced by fusion,
electroporation, biolistics, transfection, lipofection, or the
like. The host cells may be grown and expanded in culture before
introduction of the construct(s), followed by the appropriate
treatment for introduction of the construct(s) and integration of
the construct(s). The cells are then expanded and screened by
virtue of a marker present in the construct. Various markers that
may be used successfully include hprt, neomycin resistance,
thymidine kinase, hygromycin resistance, etc.
[0082] In some instances, 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 O-vectors. See, for
example, Thomas and Capecchi, Cell (1987) 51, 503-512; Mansour, et
al., Nature (1988) 336, 348-352; and Joyner, et al., Nature (1989)
338, 153-156.
[0083] The constructs may be introduced as a single DNA molecule
encoding at least the chimeric TGF.beta. receptor and optional
CAR(s), or different DNA molecules having one or more genes. The
constructs may be introduced simultaneously or consecutively, each
with the same or different markers.
[0084] Vectors containing useful elements such as bacterial or
yeast origins of replication, selectable and/or amplifiable
markers, promoter/enhancer elements for expression in prokaryotes
or eukaryotes, etc. that may be used to prepare stocks of construct
DNAs and for carrying out transfections are well known in the art,
and many are commercially available.
[0085] III. Polynucleotides Encoding Chimeric Cytokine
Receptors
[0086] The present disclosure also encompasses a composition
comprising a nucleic acid sequence encoding a chimeric cytokine
receptor as defined above and cells harboring the nucleic acid
sequence. The nucleic acid molecule is a recombinant nucleic acid
molecule, in particular aspects, and it may be synthetic. It may
comprise DNA, RNA as well as PNA (peptide nucleic acid) and it may
be a hybrid thereof.
[0087] It is evident to the person skilled in the art that one or
more regulatory sequences may be added to the nucleic acid molecule
comprised in the composition of the disclosure. For example,
promoters, transcriptional enhancers and/or sequences that allow
for induced expression of the polynucleotide of the disclosure may
be employed. A suitable inducible system is for example
tetracycline-regulated gene expression as described, e.g., by
Gossen and Bujard (Proc. Natl. Acad. Sci. USA 89 (1992), 5547-5551)
and Gossen et al. (Trends Biotech. 12 (1994), 58-62), or a
dexamethasone-inducible gene expression system as described, e.g.
by Crook (1989) EMBO J. 8, 513-519.
[0088] In certain embodiments, the chimeric TGF.beta. receptor is
expressed constitutively by the cell or vector. In other
embodiments, expression of the chimeric TGF.beta. 3 receptor is
under the control of an inducible promoter, e.g., a promoter that
is inducible by TGF.beta. 3 or by another molecule present in the
tumor microenvironment.
[0089] Furthermore, it is envisaged for further purposes that
nucleic acid molecules may contain, for example, thioester bonds
and/or nucleotide analogues. The modifications may be useful for
the stabilization of the nucleic acid molecule against endo- and/or
exonucleases in the cell. The nucleic acid molecules may be
transcribed by an appropriate vector comprising a chimeric gene
that allows for the transcription of said nucleic acid molecule in
the cell. In this respect, it is also to be understood that such
polynucleotides can be used for "gene targeting" or "gene
therapeutic" approaches. In another embodiment the nucleic acid
molecules are labeled. Methods for the detection of nucleic acids
are well known in the art, e.g., Southern and Northern blotting,
PCR or primer extension. This embodiment may be useful for
screening methods for verifying successful introduction of the
nucleic acid molecules described above during gene therapy
approaches.
[0090] The nucleic acid molecule(s) may be a recombinantly produced
chimeric nucleic acid molecule comprising any of the aforementioned
nucleic acid molecules either alone or in combination. In specific
aspects, the nucleic acid molecule is part of a vector.
[0091] The present disclosure therefore also relates to a
composition comprising a vector comprising the nucleic acid
molecule described in the present disclosure.
[0092] Many suitable vectors are known to those skilled in
molecular biology, the choice of which would depend on the function
desired and include plasmids, cosmids, viruses, bacteriophages and
other vectors used conventionally in genetic engineering. Methods
that are well known to those skilled in the art can be used to
construct various plasmids and vectors; see, for example, the
techniques described in Sambrook et al. (1989) and Ausubel, Current
Protocols in Molecular Biology, Green Publishing Associates and
Wiley Interscience, N.Y. (1989), (1994).
[0093] Alternatively, the polynucleotides and vectors of the
disclosure can be reconstituted into liposomes for delivery to
target cells. A cloning vector may be used to isolate individual
sequences of DNA. Relevant sequences can be transferred into
expression vectors where expression of a particular polypeptide is
required. Typical cloning vectors include pBluescript SK, pGEM,
pUC9, pBR322 and pGBT9. Typical expression vectors include pTRE,
pCAL-n-EK, pESP-1, pOP13CAT.
[0094] In specific embodiments, there is a vector that comprises a
nucleic acid sequence that is a regulatory sequence operably linked
to the nucleic acid sequence encoding a chimeric cytokine receptor
construct defined herein. Such regulatory sequences (control
elements) are known to the artisan and may include a promoter, a
splice cassette, translation initiation codon, translation and
insertion site for introducing an insert into the vector. In
specific embodiments, the nucleic acid molecule is operatively
linked to said expression control sequences allowing expression in
eukaryotic or prokaryotic cells.
[0095] It is envisaged that a vector is an expression vector
comprising the nucleic acid molecule encoding a chimeric cytokine
receptor construct defined herein. In specific aspects, the vector
is a viral vector, such as a lentiviral vector. Lentiviral vectors
are commercially available, including from Clontech (Mountain View,
Calif.) or GeneCopoeia (Rockville, Md.), for example.
[0096] The term "regulatory sequence" refers to DNA sequences that
are necessary to effect the expression of coding sequences to which
they are ligated. The nature of such control sequences differs
depending upon the host organism. In prokaryotes, control sequences
generally include promoters, ribosomal binding sites, and
terminators. In eukaryotes generally control sequences include
promoters, terminators and, in some instances, enhancers,
transactivators or transcription factors. The term "control
sequence" is intended to include, at a minimum, all components the
presence of which are necessary for expression, and may also
include additional advantageous components.
[0097] The term "operably linked" refers to a juxtaposition wherein
the components so described are in a relationship permitting them
to function in their intended manner. A control sequence "operably
linked" to a coding sequence is ligated in such a way that
expression of the coding sequence is achieved under conditions
compatible with the control sequences. In case the control sequence
is a promoter, it is obvious for a skilled person that
double-stranded nucleic acid is preferably used.
[0098] Thus, the recited vector is an expression vector, in certain
embodiments. An "expression vector" is a construct that can be used
to transform a selected host and provides for expression of a
coding sequence in the selected host. Expression vectors can for
instance be cloning vectors, binary vectors or integrating vectors.
Expression comprises transcription of the nucleic acid molecule
preferably into a translatable mRNA. Regulatory elements ensuring
expression in prokaryotes and/or eukaryotic cells are well known to
those skilled in the art. In the case of eukaryotic cells they
comprise normally promoters ensuring initiation of transcription
and optionally poly-A signals ensuring termination of transcription
and stabilization of the transcript. Possible regulatory elements
permitting expression in prokaryotic host cells comprise, e.g., the
P.sub.L, lac, trp or tac promoter in E. coli, and examples of
regulatory elements permitting expression in eukaryotic host cells
are the AOX1 or GAL1 promoter in yeast or the CMV-, SV40-,
RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a
globin intron in mammalian and other animal cells.
[0099] Beside elements that are responsible for the initiation of
transcription such regulatory elements may also comprise
transcription termination signals, such as the SV40-poly-A site or
the tk-poly-A site, downstream of the polynucleotide. Furthermore,
depending on the expression system used leader sequences capable of
directing the polypeptide to a cellular compartment or secreting it
into the medium may be added to the coding sequence of the recited
nucleic acid sequence and are well known in the art. The leader
sequence(s) is (are) assembled in appropriate phase with
translation, initiation and termination sequences, and preferably,
a leader sequence capable of directing secretion of translated
protein, or a portion thereof, into the periplasmic space or
extracellular medium. Optionally, the heterologous sequence can
encode a fusion protein including an N-terminal identification
peptide imparting desired characteristics, e.g., stabilization or
simplified purification of expressed recombinant product; see
supra. In this context, suitable expression vectors are known in
the art such as Okayama-Berg cDNA expression vector pcDV1
(Pharmacia), pEF-Neo, pCDM8, pRc/CMV, pcDNA1, pcDNA3 (Invitrogen),
pEF-DHFR and pEF-ADA, (Raum et al. Cancer Immunol Immunother (2001)
50.beta.), 141-150) or pSPORT1 (GIBCO BRL).
[0100] In some embodiments, the expression control sequences are
eukaryotic promoter systems in vectors capable of transforming of
transfecting eukaryotic host cells, but control sequences for
prokaryotic hosts may also be used. Once the vector has been
incorporated into the appropriate host, the host is maintained
under conditions suitable for high level expression of the
nucleotide sequences, and as desired, the collection and
purification of the polypeptide of the disclosure may follow.
[0101] Additional regulatory elements may include transcriptional
as well as translational enhancers. Advantageously, the
above-described vectors of the disclosure comprises a selectable
and/or scorable marker. Selectable marker genes useful for the
selection of transformed cells are well known to those skilled in
the art and comprise, for example, antimetabolite resistance as the
basis of selection for dhfr, which confers resistance to
methotrexate (Reiss, Plant Physiol. (Life-Sci. Adv.) 13 (1994),
143-149); npt, which confers resistance to the aminoglycosides
neomycin, kanamycin and paromycin (Herrera-Estrella, EMBO J. 2
(1983), 987-995) and hygro, which confers resistance to hygromycin
(Marsh, Gene 32 (1984), 481-485). Additional selectable genes have
been described, namely trpB, which allows cells to utilize indole
in place of tryptophan; hisD, which allows cells to utilize
histinol in place of histidine (Hartman, Proc. Natl. Acad. Sci. USA
85 (1988), 8047); mannose-6-phosphate isomerase which allows cells
to utilize mannose (WO 94/20627) and ODC (ornithine decarboxylase)
which confers resistance to the ornithine decarboxylase inhibitor,
2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue, 1987, In:
Current Communications in Molecular Biology, Cold Spring Harbor
Laboratory ed.) or deaminase from Aspergillus terreus which confers
resistance to Blasticidin S (Tamura, Biosci. Biotechnol. Biochem.
59 (1995), 2336-2338).
[0102] Useful scorable markers are also known to those skilled in
the art and are commercially available. Advantageously, said marker
is a gene encoding luciferase (Giacomin, P1. Sci. 116 (1996),
59-72; Scikantha, J. Bact. 178 (1996), 121), green fluorescent
protein (Gerdes, FEBS Lett. 389 (1996), 44-47) or
.beta.-glucuronidase (Jefferson, EMBO J. 6 (1987), 3901-3907). This
embodiment is particularly useful for simple and rapid screening of
cells, tissues and organisms containing a recited vector.
[0103] As described above, the recited nucleic acid molecule can be
used in a cell, alone, or as part of a vector to express the
encoded polypeptide in cells. The nucleic acid molecules or vectors
containing the DNA sequence(s) encoding any one of the above
described chimeric cytokine receptor constructs is introduced into
the cells that in turn produce the polypeptide of interest. The
recited nucleic acid molecules and vectors may be designed for
direct introduction or for introduction via liposomes, or viral
vectors (e.g., adenoviral, retroviral) into a cell. In certain
embodiments, the cells are T-cells, CAR T-cells, NK cells,
NKT-cells, MSCs, neuronal stem cells, or hematopoietic stem cells,
for example.
[0104] In accordance with the above, the present disclosure relates
to methods to derive vectors, particularly plasmids, cosmids,
viruses and bacteriophages used conventionally in genetic
engineering that comprise a nucleic acid molecule encoding the
polypeptide sequence of a chimeric cytokine receptor construct
defined herein. In particular embodiments, the vector is an
expression vector and/or a gene transfer or targeting vector.
Expression vectors derived from viruses such as retroviruses,
vaccinia virus, adeno-associated virus, herpes viruses, or bovine
papilloma virus, may be used for delivery of the recited
polynucleotides or vector into targeted cell populations. Methods
which are well known to those skilled in the art can be used to
construct recombinant vectors; see, for example, the techniques
described in Sambrook et al. (loc cit.), Ausubel (1989, loc cit.)
or other standard text books. Alternatively, the recited nucleic
acid molecules and vectors can be reconstituted into liposomes for
delivery to target cells. The vectors containing the nucleic acid
molecules of the disclosure can be transferred into the host cell
by well-known methods, which vary depending on the type of cellular
host. For example, calcium chloride transfection is commonly
utilized for prokaryotic cells, whereas calcium phosphate treatment
or electroporation may be used for other cellular hosts; see
Sambrook, supra.
[0105] IV. Pharmaceutical Compositions
[0106] In accordance with this disclosure, the term "pharmaceutical
composition" relates to a composition for administration to an
individual and encompasses compositions of cells for immunotherapy.
In specific embodiments, the cells for immunotherapy are engineered
to express at least a chimeric cytokine receptor. In certain
embodiments, the cells comprise one or more additional
modifications, such as one or more receptors, including receptors
for tumor antigens.
[0107] In a particular embodiment, the pharmaceutical composition
comprises a composition for parenteral, transdermal, intraluminal,
intra-arterial, intrathecal or intravenous administration or for
direct injection into a cancer. It is in particular envisaged that
said pharmaceutical composition is administered to the individual
via infusion or injection.
[0108] Administration of the suitable compositions may be effected
by different ways, e.g., by intravenous, subcutaneous,
intraperitoneal, intramuscular, topical or intradermal
administration.
[0109] The pharmaceutical composition of the present disclosure may
further comprise a pharmaceutically acceptable carrier. Examples of
suitable pharmaceutical carriers are well known in the art and
include phosphate buffered saline solutions, water, emulsions, such
as oil/water emulsions, various types of wetting agents, sterile
solutions, etc. Compositions comprising such carriers can be
formulated by well-known conventional methods. These pharmaceutical
compositions can be administered to the subject at a suitable
dose.
[0110] The dosage regimen will be determined by the attending
physician and clinical factors. As is well known in the medical
arts, dosages for any one patient depends upon many factors,
including the patient's size, body surface area, age, the
particular compound to be administered, sex, time and route of
administration, general health, and other drugs being administered
concurrently. A particular dosage for administration might be in
the range of between 5.times.10.sup.6 per m2 and 3.times.10.sup.8
per m2.
[0111] The compositions of the disclosure may be administered
locally or systemically. The compositions provided herein, e.g.,
cells expressing the constructs provided herein, may, in certain
embodiments, be administered parenterally, e.g., intravenous,
intraarterial, intrathecal, subdermal or intramuscular
administration. In certain other embodiments, DNA encoding the
constructs provided herein may be administered directly to the
target site, e.g., by biolistic delivery to an internal or external
target site or by catheter to a site in an artery. In a preferred
embodiment, the pharmaceutical composition is administered
subcutaneously and in an even more preferred embodiment
intravenously. Preparations for parenteral administration include
sterile aqueous or non-aqueous solutions, suspensions, and
emulsions. Examples of non-aqueous solvents are propylene glycol,
polyethylene glycol, vegetable oils such as olive oil, and
injectable organic esters such as ethyl oleate. Aqueous carriers
include water, alcoholic/aqueous solutions, emulsions or
suspensions, including saline and buffered media. Parenteral
vehicles include sodium chloride solution, Ringer's dextrose,
dextrose and sodium chloride, lactated Ringer's, or fixed oils.
Intravenous vehicles include fluid and nutrient replenishes,
electrolyte replenishers (such as those based on Ringer's
dextrose), and the like. Preservatives and other additives may also
be present such as, for example, antimicrobials, anti-oxidants,
chelating agents, and inert gases and the like. In addition, the
pharmaceutical composition of the present disclosure might comprise
proteinaceous carriers, like, e.g., serum albumin or
immunoglobulin, preferably of human origin. It is envisaged that
the pharmaceutical composition of the disclosure might comprise, in
addition to the proteinaceous chimeric cytokine receptor constructs
or nucleic acid molecules or vectors encoding the same (as
described in this disclosure), further biologically active agents,
depending on the intended use of the pharmaceutical composition. In
specific embodiments, the cells are infused intravenously in 10%
DMSO, 40% saline and 50% human serum albumin.
[0112] V. Therapeutic Uses of Chimeric Cytokine Receptors and Host
T-cells Comprising Chimeric Cytokine Receptors
[0113] In various embodiments chimeric cytokine receptor
constructs, nucleic acid sequences, vectors, and/or host cells, as
contemplated herein and/or pharmaceutical compositions comprising
the same are used for the prevention, treatment or amelioration of
a cancerous disease, such as a tumorous disease. In particular
embodiments, the pharmaceutical composition of the present
disclosure may be particularly useful in preventing, ameliorating
and/or treating cancer, including solid tumors, for example. In
certain cases, the cancer has a tumor antigen.
[0114] As used herein "treatment" or "treating," includes any
beneficial or desirable effect on the symptoms or pathology of a
disease or pathological condition, and may include even minimal
reductions in one or more measurable markers of the disease or
condition being treated, e.g., cancer. Treatment can involve
optionally either the reduction or amelioration of symptoms of the
disease or condition, or the delaying of the progression of the
disease or condition. "Treatment" does not necessarily indicate
complete eradication or cure of the disease or condition, or
associated symptoms thereof.
[0115] As used herein, "prevent," and similar words such as
"prevented," "preventing" etc., indicate an approach for
preventing, inhibiting, or reducing the likelihood of the
occurrence or recurrence of, a disease or condition, e.g., cancer.
It also refers to delaying the onset or recurrence of a disease or
condition or delaying the occurrence or recurrence of the symptoms
of a disease or condition. As used herein, "prevention" and similar
words also includes reducing the intensity, effect, symptoms and/or
burden of a disease or condition prior to onset or recurrence of
the disease or condition.
[0116] In particular embodiments, the present invention
contemplates, in part, cells, chimeric cytokine receptor construct,
nucleic acid molecules and vectors that can administered either
alone or in any combination using standard vectors and/or gene
delivery systems, and in at least some aspects, together with a
pharmaceutically acceptable carrier or excipient. In certain
embodiments, subsequent to administration, said nucleic acid
molecules or vectors may be stably integrated into the genome of
the subject.
[0117] In specific embodiments, viral vectors may be used that are
specific for certain cells or tissues and persist in said cells.
Suitable pharmaceutical carriers and excipients are well known in
the art. The compositions prepared according to the disclosure can
be used for the prevention or treatment or delaying the above
identified diseases.
[0118] Furthermore, the disclosure relates to a method for the
prevention, treatment or amelioration of a tumorous disease
comprising the step of administering to a subject in need thereof
an effective amount of cells harboring the chimeric cytokine
receptor molecule, a nucleic acid sequence, a vector, as
contemplated herein and/or produced by a process as contemplated
herein.
[0119] Possible indications for administration of the
composition(s) of the exemplary chimeric cytokine
receptor-comprising cells are cancerous diseases, including
tumorous diseases, including breast, prostate, lung, and colon
cancers or epithelial cancers/carcinomas such as breast cancer,
colon cancer, prostate cancer, head and neck cancer, skin cancer,
cancers of the genito-urinary tract, e.g. ovarian cancer,
endometrial cancer, cervix cancer and kidney cancer, lung cancer,
gastric cancer, cancer of the small intestine, liver cancer,
pancreas cancer, gall bladder cancer, cancers of the bile duct,
esophagus cancer, cancer of the salivary glands and cancer of the
thyroid gland. The administration of the composition(s) of the
disclosure is useful for all stages and types of cancer, including
for minimal residual disease, early cancer, advanced cancer, and/or
metastatic cancer and/or refractory cancer, for example.
[0120] The disclosure further encompasses co-administration
protocols with other compounds, e.g. bispecific antibody
constructs, targeted toxins or other compounds, which act via
immune cells. The clinical regimen for co-administration of the
inventive compound(s) may encompass co-administration at the same
time, before or after the administration of the other component.
Particular combination therapies include the use of a
chemotherapeutic agent (e.g., a chemotherapeutic agent listed in
Section VII.A. below), radiation, surgery, hormone therapy, or
other types of immunotherapy, in combination with the chimeric
TGF.beta. receptor-expressing cells provided herein.
[0121] Embodiments relate to a kit comprising a chimeric cytokine
receptor construct as defined above, a nucleic acid sequence as
defined above, a vector as defined above and/or a host as defined
above. It is also contemplated that the kit of this disclosure
comprises a pharmaceutical composition as described herein above,
either alone or in combination with further medicaments to be
administered to an individual in need of medical treatment or
intervention.
[0122] By way of illustration, cancer patients or patients
susceptible to cancer or suspected of having cancer may be treated
as follows. Cells modified as described herein may be administered
to the individual and retained for extended periods of time. The
individual may receive one or more administrations of the cells,
and the timing of separations of administrations may be on the
order of days, weeks, months, or years. In specific embodiments,
multiple administrations occur within weeks or months of each
other, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks
or months. In some embodiments, the genetically modified cells are
encapsulated to inhibit immune recognition and placed at the site
of the tumor. In cases where cells are provided to the individual
following tumor recurrence after initially treating with cells of
the disclosure, the cells may be altered such that they recognize a
different target tumor antigen. For example, when initial rounds
include cells that harbor the chimeric cytokine receptor and
another receptor specific for a particular antigen, upon subsequent
rounds (including upon tumor recurrence, if it occurs) may utilize
a receptor for a different particular antigen.
[0123] In particular cases the individual is provided with
effective amounts of the therapeutic cells that encompass a
chimeric TGF.beta. receptor and, optionally, 2) a CAR, (p TCR,
and/or antigen-specific receptor. The cells may be delivered at the
same time or at different times from one or more other cancer
therapies. The cells and the other cancer therapy may be delivered
in the same or separate formulations. The cells and the other
cancer therapy may be provided to the individual in separate
delivery routes. The cells and/or the other cancer therapy may be
delivered by injection at a tumor site or intravenously or orally,
for example. Routine delivery routes for such compositions are
known in the art.
[0124] The cells that have been modified with the construct(s) are
then 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 modified host cells have been identified, they may then be used
as planned, e.g., expanded in culture or introduced into a host
organism.
[0125] 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
hone to the cancer or are modified to hone to the cancer. 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.
[0126] 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.
[0127] The cells may be administered as desired. In certain
embodiments, the regimen parameters may be modulated using various
protocols. In specific embodiments, the route or number or timing
of administration, the life of the cells, and/or the number of
cells present, may be varied. The number of administrations may
depend upon the factors described above, for example, at least in
part.
[0128] It should be appreciated that the system is subject to many
variables, such as the cellular response to the ligand, the
efficiency of expression and, as appropriate, the level of
secretion, the activity of the expression product, the particular
need of the individual, which may vary with time and circumstances,
the rate of loss of the cellular activity as a result of loss of
cells or expression activity of individual cells, and the like.
Therefore, it is expected that for each individual patient, even if
there were universal cells that could be administered to the
population at large, each patient would be monitored for the proper
dosage for the individual, and such practices of monitoring a
patient are routine in the art.
[0129] In specific embodiments, there is a screening assay employed
as part of, or not as part of, methods of the disclosure. For
example, in certain embodiments, a biopsy is taken and the level of
TGF.beta. production is assessed so as to determine whether the
construct would work in the individual from which the biopsy is
taken. In specific embodiments, the assay method identified cancers
that detectably express TGF.beta. or a certain level of
TGF.beta..
[0130] VI. Kits of the Invention
[0131] Any of the compositions described herein may be comprised in
a kit. In a non-limiting example, chimeric cytokine
receptor-expressing cells for use in cell therapy and/or the
reagents to generate one or more cells for use in cell therapy that
harbors recombinant expression vectors may be comprised in a kit.
The kit components are provided in suitable container means.
[0132] Some components of the kits may be packaged either in
aqueous media or in lyophilized form. The container means of the
kits will generally include at least one vial, test tube, flask,
bottle, syringe or other container means, into which a component
may be placed, and preferably, suitably aliquoted. Where there are
more than one component in the kit, the kit also will generally
contain a second, third or other additional container into which
the additional components may be separately placed. However,
various combinations of components may be comprised in a vial. The
kits of the present disclosure also will typically include a means
for containing the components in close confinement for commercial
sale. Such containers may include injection or blow molded plastic
containers into which the desired vials are retained.
[0133] When the components of the kit are provided in one and/or
more liquid solutions, the liquid solution is an aqueous solution,
with a sterile aqueous solution being particularly ueful. In some
cases, the container means may itself be a syringe, pipette, and/or
other such like apparatus, from which the formulation may be
applied to an infected area of the body, injected into an animal,
and/or even applied to and/or mixed with the other components of
the kit.
[0134] However, the components of the kit may be provided as dried
powder(s). When reagents and/or components are provided as a dry
powder, the powder can be reconstituted by the addition of a
suitable solvent. It is envisioned that the solvent may also be
provided in another container means. The kits may also comprise a
second container means for containing a sterile, pharmaceutically
acceptable buffer and/or other diluent.
[0135] In particular embodiments of the disclosure, cells that are
to be used for cell therapy are provided in a kit, and in some
cases the cells are essentially the sole component of the kit. The
kit may comprise reagents and materials to make the desired cell.
In specific embodiments, the reagents and materials include primers
for amplifying desired sequences, nucleotides, suitable buffers or
buffer reagents, salt, and so forth, and in some cases the reagents
include vectors and/or DNA that encodes a chimeric cytokine
receptor as described herein and/or regulatory elements
therefor.
[0136] In particular embodiments, there are one or more apparatuses
in the kit suitable for extracting one or more samples from an
individual and/or for delivering cells to an individual. The
apparatus may be a syringe, scalpel, and so forth.
[0137] In some cases of the disclosure, the kit, in addition to
cell therapy embodiments, also includes a second cancer therapy,
such as chemotherapy, hormone therapy, and/or another
immunotherapy, for example. The kit(s) may be tailored to a
particular cancer for an individual and comprise respective second
cancer therapies for the individual. VII. Combination Therapy
[0138] In certain embodiments of the disclosure, methods of the
present disclosure for clinical aspects are combined with other
agents effective in the treatment of hyperproliferative disease,
such as anti-cancer agents. An "anti-cancer" agent is capable of
negatively affecting cancer in a subject, for example, by killing
cancer cells, inducing apoptosis in cancer cells, reducing the
growth rate of cancer cells, reducing the incidence or number of
metastases, reducing tumor size, inhibiting tumor growth, reducing
the blood supply to a tumor or cancer cells, promoting an immune
response against cancer cells or a tumor, preventing or inhibiting
the progression of cancer, or increasing the lifespan of a subject
with cancer. More generally, these other compositions would be
provided in a combined amount effective to kill or inhibit
proliferation of the cell. This process may involve contacting the
cancer cells with the expression construct and the agent(s) or
multiple factor(s) at the same time. This may be achieved by
contacting the cell with a single composition or pharmacological
formulation that includes both agents, or by contacting the cell
with two distinct compositions or formulations, at the same time,
wherein one composition includes the expression construct and the
other includes the second agent(s).
[0139] Tumor cell resistance to chemotherapy and radiotherapy
agents represents a major problem in clinical oncology. One goal of
current cancer research is to find ways to improve the efficacy of
chemo- and radiotherapy by combining it with other therapy. In the
context of the present disclosure, it is contemplated that the cell
therapy could be used similarly in conjunction with
chemotherapeutic, radiotherapeutic, and/or immunotherapeutic
intervention.
[0140] Alternatively, the present inventive cell therapy may
precede or follow the other agent treatment by intervals ranging
from minutes to weeks. In embodiments where the other agent and
present disclosure are applied separately to the individual, one
would generally ensure that a significant period of time did not
expire between the time of each delivery, such that the agent and
inventive therapy would still be able to exert an advantageously
combined effect on the cell. In such instances, it is contemplated
that one may contact the cell with both modalities within about
12-24 h of each other and, more preferably, within about 6-12 h of
each other. In some situations, it may be desirable to extend the
time period for treatment significantly, however, where several d
(2, 3, 4, 5, 6 or 7) to several wk (1, 2, 3, 4, 5, 6, 7 or 8) lapse
between the respective administrations.
[0141] Various combinations may be employed, present disclosure is
"A" and the secondary agent, such as radio- or chemotherapy, is
"B":
TABLE-US-00001 A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B
B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B
A/A/A/B B/A/A/A A/B/A/A A/A/B/A
[0142] It is expected that the treatment cycles would be repeated
as necessary. It also is contemplated that various standard
therapies, as well as surgical intervention, may be applied in
combination with the inventive cell therapy.
[0143] A. Chemotherapy
[0144] Cancer therapies also include a variety of combination
therapies with both chemical and radiation based treatments.
Combination anti-cancer agents include, for example, acivicin;
aclarubicin; acodazole hydrochloride; acronine; adozelesin;
aldesleukin; altretamine; ambomycin; ametantrone acetate;
amsacrine; anastrozole; anthramycin; asparaginase; asperlin;
azacitidine; azetepa; azotomycin; batimastat; benzodepa;
bicalutamide; bisantrene hydrochloride; bisnafide dimesylate;
bizelesin; bleomycin sulfate; brequinar sodium; bropirimine;
busulfan; cactinomycin; calusterone; caracemide; carbetimer;
carboplatin; carmustine; carubicin hydrochloride; carzelesin;
cedefingol; celecoxib (COX-2 inhibitor); chlorambucil; cirolemycin;
cisplatin; cladribine; crisnatol mesylate; cyclophosphamide;
cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride;
decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate;
diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride;
droloxifene; droloxifene citrate; dromostanolone propionate;
duazomycin; edatrexate; eflomithine hydrochloride; elsamitrucin;
enloplatin; enpromate; epipropidine; epirubicin hydrochloride;
erbulozole; esorubicin hydrochloride; estrarustine; estramustine
phosphate sodium; etanidazole; etoposide; etoposide phosphate;
etoprine; fadrozole hydrochloride; fazarabine; fenretinide;
floxuridine; fludarabine phosphate; fluorouracil; fluorocitabine;
fosquidone; fostriecin sodium; gemcitabine; gemcitabine
hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide;
ilmofosine; iproplatin; irinotecan; irinotecan hydrochloride;
lanreotide acetate; letrozole; leuprolide acetate; liarozole
hydrochloride; lometrexol sodium; lomustine; losoxantrone
hydrochloride; masoprocol; maytansine; mechlorethamine
hydrochloride; megestrol acetate; melengestrol acetate; melphalan;
menogaril; mercaptopurine; methotrexate; methotrexate sodium;
metoprine; meturedepa; mitindomide; mitocarcin; mitocromin;
mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone
hydrochloride; mycophenolic acid; nocodazole; nogalamycin;
ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin;
pentamustine; peplomycin sulfate; perfosfamide; pipobroman;
piposulfan; piroxantrone hydrochloride; plicamycin; plomestane;
porfimer sodium; porfiromycin; prednimustine; procarbazine
hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin;
riboprine; safingol; safingol hydrochloride; semustine; simtrazene;
sparfosate sodium; sparsomycin; spirogermanium hydrochloride;
spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur;
talisomycin; tecogalan sodium; taxotere; tegafur; teloxantrone
hydrochloride; temoporfin; teniposide; teroxirone; testolactone;
thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine;
toremifene citrate; trestolone acetate; triciribine phosphate;
trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole
hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin;
vinblastine sulfate; vincristine sulfate; vindesine; vindesine
sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine
sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine
sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride;
20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone;
aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin;
ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine;
aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole;
andrographolide; angiogenesis inhibitors; antagonist D; antagonist
G; antarelix; anti-dorsalizing morphogenetic protein-1;
antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston;
antisense oligonucleotides; aphidicolin glycinate; apoptosis gene
modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA;
arginine deaminase; asulacrine; atamestane; atrimustine;
axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin;
azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL
antagonists; benzochlorins; benzoylstaurosporine; beta lactam
derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF
inhibitor; bicalutamide; bisantrene; bisaziridinylspermine;
bisnafide; bistratene A; bizelesin; breflate; bropirimine;
budotitane; buthionine sulfoximine; calcipotriol; calphostin C;
camptothecin derivatives; capecitabine; carboxamide-amino-triazole;
carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived
inhibitor; carzelesin; casein kinase inhibitors (ICOS);
castanospermine; cecropin B; cetrorelix; chlorlns;
chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin;
cladribine; clomifene analogues; clotrimazole; collismycin A;
collismycin B; combretastatin A4; combretastatin analogue;
conagenin; crambescidin 816; crisnatol; cryptophycin 8;
cryptophycin A derivatives; curacin A; cyclopentanthraquinones;
cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;
cytostatin; dacliximab; decitabine; dehydrodidenmin B; deslorelin;
dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;
diaziquone: didemnin B; didox; diethylnorspermine;
dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl
spiromustine; docetaxel; docosanol; dolasetron; doxifluridine;
doxorubicin; droloxifene; dronabinol; duocarmycin SA; ebselen;
ecomustine; edelfosine; edrecolomab; eflornithine; elemene;
emitefur; epirubicin; epristeride; estramustine analogue; estrogen
agonists; estrogen antagonists; etanidazole; etoposide phosphate;
exemestane; fadrozole; fazarabine; fenretinide; filgrastim;
finasteride; flavopiridol; flezelastine; fluasterone; fludarabine;
fluorodaunorunicin hydrochloride; forfenimex; formestane;
fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate;
galocitabine; ganirelix; gelatinase inhibitors; gemcitabine;
glutathione inhibitors; hepsulfam; heregulin; hexamethylene
bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene;
idramantone; ilmofosine; ilomastat; imatinib (e.g., GLEEVEC.RTM.),
imiquimod; immunostimulant peptides; insulin-like growth factor-1
receptor inhibitor; interferon agonists; interferons; interleukins;
iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine;
isobengazole; isohomohalicondrin B; itasetron; jasplakinolide;
kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin;
lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia
inhibiting factor; leukocyte alpha interferon;
leuprolide+estrogen+progesterone; leuprorelin; levamisole;
liarozole; linear polyamine analogue; lipophilic disaccharide
peptide; lipophilic platinum compounds; lissoclinamide 7;
lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone;
loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic
peptides; maitansine; mannostatin A; marimastat; masoprocol;
maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors;
menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF
inhibitor; mifepristone; miltefosine; mirimostim; mitoguazone;
mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast
growth factor-saporin; mitoxantrone; mofarotene; molgramostim;
Erbitux, human chorionic gonadotrophin; monophosphoryl lipid
A+myobacterium cell wall sk; mopidamol; mustard anticancer agent;
mycaperoxide B; mycobacterial cell wall extract; myriaporone;
N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;
naloxone+pentazocine; napavin; naphterpin; nartograstim;
nedaplatin; nemorubicin: neridronic acid; nilutamide; nisamycin;
nitric oxide modulators; nitroxide antioxidant; nitrullyn;
oblimersen (GENASENSE.RTM.); O.sup.6-benzylguanine; octreotide;
okicenone; oligonucleotides; onapristone; ondansetron; ondansetron;
oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin;
oxaunomycin; paclitaxel; paclitaxel analogues; paclitaxel
derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;
panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;
peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;
perflubron; perfosfamide; perillyl alcohol; phenazinomycin;
phenylacetate; phosphatase inhibitors; picibanil; pilocarpine
hydrochloride; pirarubicin; piritrexim; placetin A; placetin B;
plasminogen activator inhibitor; platinum complex; platinum
compounds; platinum-triamine complex; porfimer sodium;
porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2;
proteasome inhibitors; protein A-based immune modulator; protein
kinase C inhibitor; protein kinase C inhibitors, microalgal;
protein tyrosine phosphatase inhibitors; purine nucleoside
phosphorylase inhibitors; purpurins; pyrazoloacridine;
pyridoxylated hemoglobin polyoxyethylene conjugate; raf
antagonists; raltitrexed; ramosetron; ras farnesyl protein
transferase inhibitors; ras inhibitors; ras-GAP inhibitor;
retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin;
ribozymes; RII retinamide; rohitukine; romurtide; roquinimex;
rubiginone B1; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A;
sargramostim; Sdi 1 mimetics; semustine; senescence derived
inhibitor 1; sense oligonucleotides; signal transduction
inhibitors; sizofuran; sobuzoxane; sodium borocaptate; sodium
phenylacetate; solverol; somatomedin binding protein; sonermin;
sparfosic acid; spicamycin D; spiromustine; splenopentin;
spongistatin 1; squalamine; stipiamide; stromelysin inhibitors;
sulfinosine; superactive vasoactive intestinal peptide antagonist;
suradista; suramin; swainsonine; tallimustine; tamoxifen
methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur;
tellurapyrylium; telomerase inhibitors; temoporfin; teniposide;
tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline;
thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin
receptor agonist; thymotrinan; thyroid stimulating hormone; tin
ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin;
toremifene; translation inhibitors; tretinoin; triacetyluridine;
triciribine; trimetrexate; triptorelin; tropisetron; turosteride;
tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex;
urogenital sinus-derived growth inhibitory factor; urokinase
receptor antagonists; vapreotide; variolin B; velaresol; veramine;
verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole;
zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer, or
any analog or derivative variant of the foregoing and also
combinations thereof.
[0145] In specific embodiments, chemotherapy for the individual is
employed in conjunction with the disclosure, for example before,
during and/or after administration of the disclosure.
[0146] B. Radiotherapy
[0147] Other factors that cause DNA damage and have been used
extensively include what are commonly known as .gamma.-rays,
X-rays, and/or the directed delivery of radioisotopes to tumor
cells. Other forms of DNA damaging factors are also contemplated
such as microwaves and UV-irradiation. It is most likely that all
of these factors effect a broad range of damage on DNA, on the
precursors of DNA, on the replication and repair of DNA, and on the
assembly and maintenance of chromosomes. Dosage ranges for X-rays
range from daily doses of 50 to 200 roentgens for prolonged periods
of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.
Dosage ranges for radioisotopes vary widely, and depend on the
half-life of the isotope, the strength and type of radiation
emitted, and the uptake by the neoplastic cells.
[0148] The terms "contacted" and "exposed," when applied to a cell,
are used herein to describe the process by which a therapeutic
construct and a chemotherapeutic or radiotherapeutic agent are
delivered to a target cell or are placed in direct juxtaposition
with the target cell. To achieve cell killing or stasis, both
agents are delivered to a cell in a combined amount effective to
kill the cell or prevent it from dividing.
[0149] C. Immunotherapy
[0150] Immunotherapeutics generally rely on the use of immune
effector cells and molecules to target and destroy cancer cells,
and immunotherapeutics other than the chimeric cytokine-expressing
cells of the disclosure may be used, in certain embodiments. The
immune effector may be, for example, an antibody specific for some
marker on the surface of a tumor cell. The antibody alone may serve
as an effector of therapy or it may recruit other cells to actually
effect cell killing. The antibody also may be conjugated to a drug
or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera
toxin, pertussis toxin, etc.) and serve merely as a targeting
agent. Alternatively, the effector may be a lymphocyte carrying a
surface molecule that interacts, either directly or indirectly,
with a tumor cell target. Various effector cells include cytotoxic
T cells and NK cells.
[0151] Immunotherapy other than the inventive therapy described
herein could thus be used as part of a combined therapy, in
conjunction with the present cell therapy. The general approach for
combined therapy is discussed below. Generally, the tumor cell must
bear some marker that is amenable to targeting, i.e., is not
present on the majority of other cells. Many tumor markers exist
and any of these may be suitable for targeting in the context of
the present disclosure. Common tumor markers include
carcinoembryonic antigen, prostate specific antigen, urinary tumor
associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72,
HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor,
laminin receptor, erb B and p155.
[0152] In certain other embodiments, the immunotherapy comprises
use of an antibody against DLL4, Notch, or a Wnt pathway protein,
for example.
[0153] D. Genes
[0154] In yet another embodiment, the secondary treatment is a gene
therapy in which a therapeutic polynucleotide is administered
before, after, or at the same time as the present disclosure
clinical embodiments. A variety of expression products are
encompassed within the disclosure, including inducers of cellular
proliferation, inhibitors of cellular proliferation, or regulators
of programmed cell death.
[0155] E. Surgery
[0156] Approximately 60% of persons with cancer will undergo
surgery of some type, which includes preventative, diagnostic or
staging, curative and palliative surgery. Curative surgery is a
cancer treatment that may be used in conjunction with other
therapies, such as the treatment of the present disclosure,
chemotherapy, radiotherapy, hormonal therapy, gene therapy,
immunotherapy and/or alternative therapies.
[0157] Curative surgery includes resection in which all or part of
cancerous tissue is physically removed, excised, and/or destroyed.
Tumor resection refers to physical removal of at least part of a
tumor. In addition to tumor resection, treatment by surgery
includes laser surgery, cryosurgery, electrosurgery, and
miscopically controlled surgery (Mohs' surgery). It is further
contemplated that the present disclosure may be used in conjunction
with removal of superficial cancers, precancers, or incidental
amounts of normal tissue.
[0158] Upon excision of part of all of cancerous cells, tissue, or
tumor, a cavity may be formed in the body. Treatment may be
accomplished by perfusion, direct injection or local application of
the area with an additional anti-cancer therapy. Such treatment may
be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or
every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, or 12 months. These treatments may be of varying dosages as
well.
[0159] F. Other Agents
[0160] It is contemplated that other agents may be used in
combination with the present disclosure to improve the therapeutic
efficacy of treatment. These additional agents include
immunomodulatory agents, agents that affect the upregulation of
cell surface receptors and GAP junctions, cytostatic and
differentiation agents, inhibitors of cell adhesion, or agents that
increase the sensitivity of the hyperproliferative cells to
apoptotic inducers. Immunomodulatory agents include tumor necrosis
factor; interferon alpha, beta, and gamma; IL-2 and other
cytokines; F42K and other cytokine analogs; or MIP-1, MIP-lbeta,
MCP-1, RANTES, and other chemokines. It is further contemplated
that the upregulation of cell surface receptors or their ligands
such as Fas/Fas ligand, DR4 or DR5/TRAIL would potentiate the
apoptotic inducing abililties of the present disclosure by
establishment of an autocrine or paracrine effect on
hyperproliferative cells. Increases intercellular signaling by
elevating the number of GAP junctions would increase the
anti-hyperproliferative effects on the neighboring
hyperproliferative cell population. In other embodiments,
cytostatic or differentiation agents can be used in combination
with the present disclosure to improve the anti-hyerproliferative
efficacy of the treatments. Inhibitors of cell adhesion are
contemplated to improve the efficacy of the present disclosure.
Examples of cell adhesion inhibitors are focal adhesion kinase
(FAKs) inhibitors and Lovastatin. It is further contemplated that
other agents that increase the sensitivity of a hyperproliferative
cell to apoptosis, such as the antibody c225, could be used in
combination with the present disclosure to improve the treatment
efficacy.
EXAMPLES
[0161] The following examples are included to demonstrate preferred
embodiments of the disclosure. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples that
follow represent techniques discovered by the inventor to function
well in the practice of the disclosure, and thus can be considered
to constitute preferred modes for its practice. However, those of
skill in the art should, in light of the present disclosure,
appreciate that many changes can be made in the specific
embodiments which are disclosed and still obtain a like or similar
result without departing from the spirit and scope of the
disclosure.
Example 1
Exemplary Embodiments of the Disclosure
[0162] FIG. 1 illustrates an exemplary method to arm T cells to
overcome the inhibitory tumor microenvironment. The method permits
conversion of an inhibitory signal from TGF.beta. into a positive
signal for proliferation of tumor-specific CTLs (for example). The
conversion location occurs at a receptor for TGFb wherein the
receptor employs the cytokine receptor exodomain with an endodomain
that transmits the signal instead as a positive one. FIG. 2
provides exemplary vectors suitable for the disclosure by encoding
chimeric TGF.beta. receptors, including one that employs a TLR2
endodomain (RIID2) or one that employs a TLR4 endodomain (referred
to herein as RIID4). A control is shown that is a dominant negative
receptor including the TGF.beta. receptor exodomain with no
endodomain. Although any type of vector may include the expression
construct, retroviral vectors were employed.
[0163] FIG. 3 demonstrates generation of the DNRII control and
exemplary chimeric TGF.beta. receptors of RIID2 and RIID4. The flow
cytometry images demonstrate the percentage of cells expressing the
particular chimeric receptors (FIG. 3).
[0164] FIG. 4A demonstrates co-expression of the dominant negative
control DNRII and the exemplary chimeric TGF.beta. receptors of
RIID2 (labeled with mOrange) and RIID4 (labeled with GFP) with an
exemplary second generation chimeric antigen receptor (CAR)
specific for PSCA. FIG. 4B further demonstrates that RIID4 can be
expressed on second generation CAR-PSCA T cells.
[0165] FIG. 5 shows that RIID2 and RIID4 protect CAR-modified T
cells exposed to TGF.beta.. T cells that are in a healthy
environment and proliferating are visualized under a microscope as
clusters of cells (weekly antigen stimulation and administration of
5 ng/ml of TGF.beta.1; no IL2 administration). FIG. 5 shows that
although control DNRII and RIID2 show some clustering of the cells
in the presence of TGF.beta.1, the RIID4 cells are nearly
indistinguishable when comparing the absence and presence of
TGF.beta. 1.
[0166] FIGS. 6A through 6C show that in suppressive conditions
RIID2- and RIID4-modified T cells are protected in the presence of
TGF.beta.. FIG. 6A demonstrates that in control cells harboring
only a CAR, T cell proliferation decreases, particularly fast in
the presence of TGF.beta.1. In the presence of the dominant
negative receptor DNR, a decrease in proliferation occurs at a
later time. In FIG. 6B, the presence of the chimeric TGF.beta.
receptor RIID2 enhances proliferation, even in the presence of
TGF.beta.. Even after almost 100 days, the cells proliferate in the
presence of TGF.beta.. FIG. 6C also shows that in the presence of
the chimeric TGF.beta. receptor RIID4 enhances proliferation, even
in the presence of TGF.beta..
[0167] FIG. 7 demonstrates that cells harboring the exemplary
chimeric TGF.beta. receptors RIID2 and RIID4 are selected for in
the presence of TGF.beta.1, and FIG. 8 demonstrates that the cells
harboring RIID2 and RIID4 require antigen stimulation for their
expansion. Therefore, in vivo when the cells are successful in
reducing tumor load, their proliferation will decrease, in specific
embodiments.
[0168] FIG. 9 shows that RIID2 and RIID4 enhance the anti-tumor
effect of the exemplary PSCA CAR against the exemplary DU145
prostate cancer cells.
Example 2
Immunosuppressive TGF-.beta. Signal Converter
[0169] Chimeric antigen receptor (CAR)-transduced T cells are
promising tools for the treatment of cancers. To extend this
therapeutic modality to prostate cancer, the inventors generated a
2.sup.nd generation CAR targeting the tumor antigen PSCA
(2G.CAR-PSCA), which provides cells with the ability to kill
PSCA+prostate tumor cells (51.7.+-.1.2% specific lysis of Du145
cells at 20:1 E:T). However, many tumors, including prostate
cancer, secrete TGF.beta., which inhibits in vivo T cell
proliferation, activation and function. The present disclosure
addresses this need in the art to overcome this in vivo limitation
of T cells.
[0170] It has previously been demonstrated that
adoptively-transferred T cells can be protected from the inhibitory
effects of TGF.beta. through the transgenic expression of a
truncated, dominant-negative receptor (DNRII), which blocks
transmission of TGF.beta. signal. The inventors herein have
extended this strategy by converting the inhibitory signal from
TGF.beta. into an activation stimulus for T cells. The inventors
generated a chimeric cytokine receptor expressing the exodomain of
TGF.beta.RII linked to the endodomain of the exemplary toll-like
receptor (TLR) 4 and GFP (RIID4).
[0171] The inventors generated a chimeric cytokine receptor
expressing the exodomain of TGF.beta.RII linked to the endodomain
of the exemplary toll-like receptor (TLR) 4 and GFP (RIID4). The
inventors transduced primary T cells with RIID4 and obtained
69.3.+-.6.0% transduction that was stable for >60 days of
culture.
[0172] To address whether transgenic expression of RIID4 protected
against TGF.beta., the inventors modified 2G.CAR-PSCA T cells to
co-express either the dominant negative DNRII or RIID4 receptors.
These T cells were then stimulated weekly with PSCA+tumor cells
(K562-PSCA) with or without exogenous TGF.beta.1 (5 ng/mL). In the
absence of TGF.beta.1, 2G.CAR-PSCA, 2G.CAR-PSCA(DNRII) or
2G.CAR-PSCA(RIID4) T cells proliferated at similar levels for 30
days (6.4.times.10.sup.2, 2.5.times.10.sup.3, 5.9.times.10.sup.3
fold, respectively). But, in the presence of TGF.beta.1,
2G.CAR-PSCA T cells did not expand, and cultures failed within 2
weeks. In contrast, transgenic expression of DNRII or RIID4
protected the cells from the inhibitory impact of this cytokine
(7.4 and 21 fold at 2 weeks of culture, respectively). To determine
whether there were long term differences between DNRII- and
RIID4-modified cells, the inventors monitored cell expansion and
found that only RIID4-modified T cells were able to expand for
>60 days in the presence of TGF.beta.1 (3.0.times.10.sup.5 fold)
while DNRII cells began to contract after 30 days in culture (0.72
fold).
[0173] Administration of TGF.beta.1 also selected
2G.CAR-PSCA(RIID4) T cells, leading to an enrichment in this
transgenic cell population over time (from 63.6% to 93.3%). This
modification is safe, because the administration of TGF.beta.1
alone was insufficient to drive transgenic T cell proliferation
(0.04 fold), and the withdrawal of antigenic stimulation resulted
in T cell contraction (0.02 fold). Finally, to address whether this
modification could improve the anti-tumor activity of CAR-T cells,
the inventors co-cultured 1.times.10.sup.6 firefly-luciferase-Du145
cells, which express PSCA and produce TGF.beta.1, with
1.times.10.sup.5 2G.CAR-PSCA, 2G.CAR-PSCA(DNRII) or
2G.CAR-PSCA(RIID4) T cells. After 6 days there was superior control
of tumor growth by RIID4-expressing T cells compared with DNRII or
CAR alone conditions (Total Flux; 9.+-.0.1.times.10.sup.9,
10.+-.1.times.10.sup.9, 20.+-.1.times.10.sup.9 p/s, respectively).
Therefore, RIID4 not only protects cells from the inhibitory
effects TGF.beta. but converts this cytokine signal into one that
is stimulant.
Example 3
Chimeric Cytokine Receptors in the Tumor Microenvironment
[0174] TGF.beta. has been detected at high levels in patients with
cancer, including at least prostate cancer, where it acts by
suppressing effector T cell function while promoting Treg
development. Under wild type conditions, TGF.beta. engagement
results in phosphorylation of Smad2/3, which triggers multiply
inhibitory pathways (FIG. 10A). To evaluate the influence of
TGF.beta. on tumor-specific T cells, CAR-PSCA T cells were cultured
with TGF.beta. (5 ng/ml) and administered twice weekly. As
expected, this resulted in phosphorylation of Smad2/3 and decreased
cytolytic function in CAR-PSCA T cells (FIGS. 10A and 10B). In
addition, TGF.beta. exposure resulted in a decrease in the
expansion of CAR T cells, in part due to higher cell death (FIGS.
10C and 10D). Interestingly, exposure of CAR-PSCA T cells to
TGF.beta. also resulted in the upregulation of PD1, suggesting T
cell exhaustion (FIG. 10E). Interestingly, TGF.beta.1 does not
induce pSmad2/3 in RIID4-modified T cells (FIG. 14).
[0175] Arming CAR-PSCA T Cells Against TGF.beta.:
[0176] To determine whether Chimeric Cytokine Receptors could be
utilized to protect tumor-specific T cells from the
immunosuppressive effects of TGF.beta., the TGF.beta. endodomain
was substituted for that of an immunostimulatory molecule. One
example of an endodomain derives from the Toll-like Receptor (TLR)
family, which constitutes an important component of the innate
immune response. In particular, TLR4 signaling in T cells has been
demonstrated to improve T cell activation while decreasing the need
for co-stimulation. Therefore, with the purpose of transforming the
inhibitory signal of TGF.beta. into an immunostimulatory one, the
TGF.beta.R endodomain was substituted for the TLR4 endodomain
(referred to herein as "RIID4") (FIGS. 11B and 12A).
[0177] RIID4 Expression Protects CAR T Cells from TGF.beta..
[0178] To evaluate whether a Chimeric Cytokine Receptor could
protect CAR T cells from TGF.beta., a retroviral vector was
generated expressing the extracellular component of TGF.beta.R and
the intracellular signaling domain of TLR4 (RIID4). This sequence
was then linked with GFP using an IRES, allowing detection of the
transgenic T cell population (FIG. 12A). Activated T cells were
then transduced with the retroviral vector encoding for RIID4/GFP,
as shown in FIG. 12B. RIID4 expression on T cells was stable as
illustrated by a direct correlation of receptor expression with
GFP. To evaluate the protective properties of the construct, there
was co-expression of RIID4 on CAR-PSCA T cells and culturing of
them in the presence of TGF.beta.. Importantly, only the CAR T
cells expressing RIID4 were able to expand when co-cultured with
TGF.beta., as shown by the culture photographs in FIG. 12C. As
expected, administering TGF.beta. to CAR T cells induced
upregulation of PD1 expression. In contrast, T cells modified with
the RIID4 construct did not express PD1, indicating the lack of
exhaustion in these T cells (FIG. 12D).
[0179] Embodiments of the disclosure extend the target range of
chimeric cytokine receptors. Specific embodiments include an array
of different permutations of chimeric cytokine receptors expressing
the exodomain of TGF.beta. receptor with the endodomain of
different co-stimulatory molecules as shown by example in FIG. 13.
Next, one can identify which of these constructs is the most
efficient in enhancing T cell function in the presence of TGF.beta.
and determine whether the incorporation of such a modification
could provide a positive bystander effect by decreasing the
function of suppressive TGF.beta.-producing Tregs.
[0180] Vector Generation and Assessment:
[0181] A variety of different TGF.beta./immunostimulatory
endodomain constructs (TGF.beta.R/Th1) may be generated by standard
recombinant means in the art. An example of a retroviral vector
expressing the exodomain of TGF.beta. receptor and the endodomain
of TLR4, RIID4, is shown in FIG. 12A. Using this vector as an
example of a template, one can subclone additional configurations
by substituting the transmembrane domain and endodomain of TLR4
with OX40, CD28, 41BB, and CD28/41BB, for example. In specific
embodiments the vectors may also co-express a marker, such as GFP.
These different vectors may then be compared for expression and
function in T cells.
[0182] Construct Assessment:
[0183] To characterize the different constructs for suitability to
reverse the inhibitory signaling of TGF.beta., one can generate
viral supernatant and transduce T cells. Protein expression can be
assessed by flow cytometric analysis by correlating TGF.beta.
receptor with GFP expression as shown in FIG. 12B. Transgenic
function can be assessed by comparing T cell (i) expansion, (ii)
cytokine production, (iii) phenotypic profile including expression
of effector, memory and exhaustion markers and (iv) cytolytic
function in the presence or absence of TGF.beta., for example.
[0184] In specific embodiments, the different TGF.beta. cytokine
receptor constructs can achieve a transduction efficiency of
>60%. In specific embodiments, the constructs are stably
expressed on the surface of T cells as detected by flow cytometry
analysis. In general embodiments, permutations of the
TGF.beta.R/Th1 constructs exhibit superior T cell expansion,
cytokine production, phenotypic profile and cytolytic function in
the presence of TGF.beta. 3. However because of the difference in
the co-stimulatory endodomains in certain embodiments the T cells
modified with the CD28/41BB construct have a greater potential for
expansion than cells expressing receptors containing a single
endodomain. In certain aspects, this enhanced proliferative
capacity leads to over-activation and cell exhaustion as measured
by upregulation of PD1, TIM3 and LAG3, for example.
[0185] The in-vitro and in-vivo function of TGF.beta.R/Th1 in
dual-specific CTLs may be evaluated. These studies can show whether
TGF.beta.R/Th1 can be expressed by dual-specific CTLs, and whether
transgenic CTLs maintain their proliferative capacity and
anti-tumor activity even in the presence of TGF.beta.. One or more
of the TGF.beta.R/Th1 constructs may be included in a bicistronic
retroviral vector. In a specific example, the bicistronic
retroviral vector encodes CAR-PSCA and TGF.beta.R/Th1.
[0186] One can confirm that CTLs maintain their function and
anti-tumor activity in the presence of TGF.beta. by using
IFN.gamma. ELIspot with PSMA and PSCA pepmixes as stimulators, and
by measuring killing using TRPC tumor as a target in the presence
of TGF.beta. in short (4 hr Cr.sup.51 assay), long-term (4 day
co-culture) cytotoxicity assays and in TRPC tumor-bearing mice, for
example.
[0187] In particular embodiments, dual-specific CTLs expressing the
TGF.beta.R/Th1 retain the antigen specificity against their
respective targets (such as PSCA and PSMA).
[0188] However in specific embodiments, only TGF.beta.R/Th1
expressing T cells are functional in the presence of TGF.beta.
resulting in a more potent in-vitro and in-vivo anti-tumor effect
(Table 1).
TABLE-US-00002 TABLE 1 Expected Assessments of Dual-CTLs expressing
Chimeric Cytokine Receptors In-vitro assessment In-vivo asssessment
Cell Signaling CTL expansion Tumor Growth +IL2 +TGF.beta. +IL2
+TGF.beta. +IL2 +TGF.beta. Dual-CTL Th1 Th2 +++ -- (-/+) (++++)
Dual-CTL- Th1 Th1 +++ +++ (-/+) (-) TGF.beta.R/Th1
[0189] Assessing the Bystander Effect of TGF.beta.R/Th1
[0190] One can evaluate the ability of TGF.beta.R/Th1 to provide a
positive bystander effect on the recipient immune system by
depleting inhibitory cytokines from the tumor microenvironment. To
assess this one can recapitulate the immunosuppressive tumor
environment in an animal model by performing an autologous transfer
of TGF.beta.-producing Tregs, which will deliver TGF.beta. at the
tumor site.
[0191] Experimental groups may be subdivided into two: (i)
dual-CTLs-TGF.beta.R/Th1 or (ii) dual-CTLs as a control. Two weeks
after T cell treatment a cohort of mice can be sacrificed to
evaluate intra-tumor levels of TGF.beta. as well as the number and
function of the Tregs.
[0192] In specific embodiments, there is only a marginal anti-tumor
response from Dual-CTLs (alone) in presence of Tregs (Table 2). In
particular embodiments, and in contrast, treatment with dual-CTLs
expressing the TGF.beta.R/Th1 depletes the levels of TGF.beta. from
the tumor microenvironment affecting the function and persistence
of Tregs (Table 2), resulting in an overall enhanced anti-tumor
response.
TABLE-US-00003 TABLE 2 Example of Outcome with Dual-CTLs comprising
a Chimeric Cytokine Receptor Tumor levels of TGF.beta. Treg
Function Anti-tumor effect Dual-CTLs (++++) (++++) (+) Dual-CTLs-
(-/+) (+) (++++) TGF.beta.R/Th1
[0193] T cells harboring RIID4 are protected from
TGF.beta.1-induced apoptosis (FIG. 15); furthermore, RIID4 prevents
PD1 upregulation (FIG. 16) and TGF.beta.1-induced inhibition of
cytolytic function (FIG. 17).
[0194] FIG. 18 shows that RIID4+cells are selected by TGF.beta.1
exposure, whereas withdrawal of antigen and TGF.beta.1 leads to
culture failure (FIG. 19).
[0195] FIG. 20 illustrates an example of a co-culture experimental
set-up.
[0196] FIG. 21 demonstrates that 2G.CAR-PSCA/RIID4 cells eliminated
tumors in the presence of TGF.beta. 1.
[0197] FIG. 22 shows that 2G.CAR-PSCA T cells did not proliferate
in the presence of TGF.beta.1.
[0198] FIG. 23 demonstrates that 2G.CAR-PSCA/RIID4 cells were able
to expand dependent upon antigen existence. FIG. 24 illustrates
TGF.beta.1 administration promotes proliferation of
2G.CAR-PSCA/RIID4 T cells. Therefore, RIID4 modified T cells can
use TGF.beta.1 for their proliferation.
[0199] In specific embodiments, the TGF.beta.R/Th1 chimeric
cytokine receptor may be used with another chimeric cytokine
receptor. In specific embodiments, the TGF.beta.R/Th1 chimeric
cytokine receptor is used in the same cell as a IL4Ra/IL7R (4/7R)
chimeric cytokine receptor. In specific embodiment, the
TGF.beta.R/Th1 chimeric cytokine receptor and the additional
chimeric cytokine receptor results in cooperative signaling.
Therefore in specific embodiments, there are provided dual-CTLs
expressing the (i) 4/7R (ii) the TGF.beta.R/Th1 or (iii) 4/7R and
TGF.beta.R/Th1. In certain embodiments, the use of both 4/7R and
TGF.beta.R/Th1 chimeric cytokine receptors results in a synergistic
response.
[0200] 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 disclosure as defined by the
appended claims. Moreover, the scope of the present application is
not intended to be limited to the particular embodiments of the
process, machine, manufacture, composition of matter, means,
methods and steps described in the specification. As one of
ordinary skill in the art will readily appreciate from the
disclosure of the present 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
6122PRTHomo sapiens 1Met Gly Arg Gly Leu Leu Arg Gly Leu Trp Pro
Leu His Ile Val Leu1 5 10 15Trp Thr Arg Ile Ala Ser 202144PRTHomo
sapiens 2Thr Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met
Ile Val1 5 10 15Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys
Lys Phe Cys 20 25 30Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser
Cys Met Ser Asn 35 40 45Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln
Glu Val Cys Val Ala 50 55 60Val Trp Arg Lys Asn Asp Glu Asn Ile Thr
Leu Glu Thr Val Cys His65 70 75 80Asp Pro Lys Leu Pro Tyr His Asp
Phe Ile Leu Glu Asp Ala Ala Ser 85 90 95Pro Lys Cys Ile Met Lys Glu
Lys Lys Lys Pro Gly Glu Thr Phe Phe 100 105 110Met Cys Ser Cys Ser
Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser 115 120 125Glu Glu Tyr
Asn Thr Ser Asn Pro Asp Leu Leu Leu Val Ile Phe Gln 130 135
14034704DNAHomo sapiens 3ggagagggag aaggctctcg ggcggagaga
ggtcctgccc agctgttggc gaggagtttc 60ctgtttcccc cgcagcgctg agttgaagtt
gagtgagtca ctcgcgcgca cggagcgacg 120acacccccgc gcgtgcaccc
gctcgggaca ggagccggac tcctgtgcag cttccctcgg 180ccgccggggg
cctccccgcg cctcgccggc ctccaggccc cctcctggct ggcgagcggg
240cgccacatct ggcccgcaca tctgcgctgc cggcccggcg cggggtccgg
agagggcgcg 300gcgcggaggc gcagccaggg gtccgggaag gcgccgtccg
ctgcgctggg ggctcggtct 360atgacgagca gcggggtctg ccatgggtcg
ggggctgctc aggggcctgt ggccgctgca 420catcgtcctg tggacgcgta
tcgccagcac gatcccaccg cacgttcaga agtcggatgt 480ggaaatggag
gcccagaaag atgaaatcat ctgccccagc tgtaatagga ctgcccatcc
540actgagacat attaataacg acatgatagt cactgacaac aacggtgcag
tcaagtttcc 600acaactgtgt aaattttgtg atgtgagatt ttccacctgt
gacaaccaga aatcctgcat 660gagcaactgc agcatcacct ccatctgtga
gaagccacag gaagtctgtg tggctgtatg 720gagaaagaat gacgagaaca
taacactaga gacagtttgc catgacccca agctccccta 780ccatgacttt
attctggaag atgctgcttc tccaaagtgc attatgaagg aaaaaaaaaa
840gcctggtgag actttcttca tgtgttcctg tagctctgat gagtgcaatg
acaacatcat 900cttctcagaa gaatataaca ccagcaatcc tgacttgttg
ctagtcatat ttcaagtgac 960aggcatcagc ctcctgccac cactgggagt
tgccatatct gtcatcatca tcttctactg 1020ctaccgcgtt aaccggcagc
agaagctgag ttcaacctgg gaaaccggca agacgcggaa 1080gctcatggag
ttcagcgagc actgtgccat catcctggaa gatgaccgct ctgacatcag
1140ctccacgtgt gccaacaaca tcaaccacaa cacagagctg ctgcccattg
agctggacac 1200cctggtgggg aaaggtcgct ttgctgaggt ctataaggcc
aagctgaagc agaacacttc 1260agagcagttt gagacagtgg cagtcaagat
ctttccctat gaggagtatg cctcttggaa 1320gacagagaag gacatcttct
cagacatcaa tctgaagcat gagaacatac tccagttcct 1380gacggctgag
gagcggaaga cggagttggg gaaacaatac tggctgatca ccgccttcca
1440cgccaagggc aacctacagg agtacctgac gcggcatgtc atcagctggg
aggacctgcg 1500caagctgggc agctccctcg cccgggggat tgctcacctc
cacagtgatc acactccatg 1560tgggaggccc aagatgccca tcgtgcacag
ggacctcaag agctccaata tcctcgtgaa 1620gaacgaccta acctgctgcc
tgtgtgactt tgggctttcc ctgcgtctgg accctactct 1680gtctgtggat
gacctggcta acagtgggca ggtgggaact gcaagataca tggctccaga
1740agtcctagaa tccaggatga atttggagaa tgttgagtcc ttcaagcaga
ccgatgtcta 1800ctccatggct ctggtgctct gggaaatgac atctcgctgt
aatgcagtgg gagaagtaaa 1860agattatgag cctccatttg gttccaaggt
gcgggagcac ccctgtgtcg aaagcatgaa 1920ggacaacgtg ttgagagatc
gagggcgacc agaaattccc agcttctggc tcaaccacca 1980gggcatccag
atggtgtgtg agacgttgac tgagtgctgg gaccacgacc cagaggcccg
2040tctcacagcc cagtgtgtgg cagaacgctt cagtgagctg gagcatctgg
acaggctctc 2100ggggaggagc tgctcggagg agaagattcc tgaagacggc
tccctaaaca ctaccaaata 2160gctcttctgg ggcaggctgg gccatgtcca
aagaggctgc ccctctcacc aaagaacaga 2220ggcagcagga agctgcccct
gaactgatgc ttcctggaaa accaaggggg tcactcccct 2280ccctgtaagc
tgtggggata agcagaaaca acagcagcag ggagtgggtg acatagagca
2340ttctatgcct ttgacattgt cataggataa gctgtgttag cacttcctca
ggaaatgaga 2400ttgattttta caatagccaa taacatttgc actttattaa
tgcctgtata taaatatgaa 2460tagctatgtt ttatatatat atatatatat
ctatatatgt ctatagctct atatatatag 2520ccataccttg aaaagagaca
aggaaaaaca tcaaatattc ccaggaaatt ggttttattg 2580gagaactcca
gaaccaagca gagaaggaag ggacccatga cagcattagc atttgacaat
2640cacacatgca gtggttctct gactgtaaaa cagtgaactt tgcatgagga
aagaggctcc 2700atgtctcaca gccagctatg accacattgc acttgctttt
gcaaaataat cattccctgc 2760ctagcacttc tcttctggcc atggaactaa
gtacagtggc actgtttgag gaccagtgtt 2820cccggggttc ctgtgtgccc
ttatttctcc tggacttttc atttaagctc caagccccaa 2880atctgggggg
ctagtttaga aactctccct caacctagtt tagaaactct accccatctt
2940taataccttg aatgttttga accccacttt ttaccttcat gggttgcaga
aaaatcagaa 3000cagatgtccc catccatgcg attgccccac catctactaa
tgaaaaattg ttcttttttt 3060catctttccc ctgcacttat gttactattc
tctgctccca gccttcatcc ttttctaaaa 3120aggagcaaat tctcactcta
ggctttatcg tgtttacttt ttcattacac ttgacttgat 3180tttctagttt
tctatacaaa caccaatggg ttccatcttt ctgggctcct gattgctcaa
3240gcacagtttg gcctgatgaa gaggatttca actacacaat actatcattg
tcaggactat 3300gacctcaggc actctaaaca tatgttttgt ttggtcagca
cagcgtttca aaaagtgaag 3360ccactttata aatatttgga gattttgcag
gaaaatctgg atccccaggt aaggatagca 3420gatggttttc agttatctcc
agtccacgtt cacaaaatgt gaaggtgtgg agacacttac 3480aaagctgcct
cacttctcac tgtaaacatt agctctttcc actgcctacc tggaccccag
3540tctaggaatt aaatctgcac ctaaccaagg tcccttgtaa gaaatgtcca
ttcaagcagt 3600cattctctgg gtatataata tgattttgac taccttatct
ggtgttaaga tttgaagttg 3660gccttttatt ggactaaagg ggaactcctt
taagggtctc agttagccca agtttctttt 3720gcttatatgt taatagtttt
accctctgca ttggagagag gagtgcttta ctccaagaag 3780ctttcctcat
ggttaccgtt ctctccatca tgccagcctt ctcaaccttt gcagaaatta
3840ctagagagga tttgaatgtg ggacacaaag gtcccatttg cagttagaaa
atttgtgtcc 3900acaaggacaa gaacaaagta tgagctttaa aactccatag
gaaacttgtt aatcaacaaa 3960gaagtgttaa tgctgcaagt aatctctttt
ttaaaacttt ttgaagctac ttattttcag 4020ccaaatagga atattagaga
gggactggta gtgagaatat cagctctgtt tggatggtgg 4080aaggtctcat
tttattgaga tttttaagat acatgcaaag gtttggaaat agaacctcta
4140ggcaccctcc tcagtgtggg tgggctgaga gttaaagaca gtgtggctgc
agtagcatag 4200aggcgcctag aaattccact tgcaccgtag ggcatgctga
taccatccca atagctgttg 4260cccattgacc tctagtggtg agtttctaga
atactggtcc attcatgaga tattcaagat 4320tcaagagtat tctcacttct
gggttatcag cataaactgg aatgtagtgt cagaggatac 4380tgtggcttgt
tttgtttatg tttttttttc ttattcaaga aaaaagacca aggaataaca
4440ttctgtagtt cctaaaaata ctgacttttt tcactactat acataaaggg
aaagttttat 4500tcttttatgg aacacttcag ctgtactcat gtattaaaat
aggaatgtga atgctatata 4560ctctttttat atcaaaagtc tcaagcactt
atttttattc tatgcattgt ttgtctttta 4620cataaataaa atgtttatta
gattgaataa agcaaaatac tcaggtgagc atcctgcctc 4680ctgttcccat
tcctagtagc taaa 47044592PRTHomo sapiens 4Met Gly Arg Gly Leu Leu
Arg Gly Leu Trp Pro Leu His Ile Val Leu1 5 10 15Trp Thr Arg Ile Ala
Ser Thr Ile Pro Pro His Val Gln Lys Ser Asp 20 25 30Val Glu Met Glu
Ala Gln Lys Asp Glu Ile Ile Cys Pro Ser Cys Asn 35 40 45Arg Thr Ala
His Pro Leu Arg His Ile Asn Asn Asp Met Ile Val Thr 50 55 60Asp Asn
Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp65 70 75
80Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys
85 90 95Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala
Val 100 105 110Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val
Cys His Asp 115 120 125Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu
Asp Ala Ala Ser Pro 130 135 140Lys Cys Ile Met Lys Glu Lys Lys Lys
Pro Gly Glu Thr Phe Phe Met145 150 155 160Cys Ser Cys Ser Ser Asp
Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 165 170 175Glu Tyr Asn Thr
Ser Asn Pro Asp Leu Leu Leu Val Ile Phe Gln Val 180 185 190Thr Gly
Ile Ser Leu Leu Pro Pro Leu Gly Val Ala Ile Ser Val Ile 195 200
205Ile Ile Phe Tyr Cys Tyr Arg Val Asn Arg Gln Gln Lys Leu Ser Ser
210 215 220Thr Trp Glu Thr Gly Lys Thr Arg Lys Leu Met Glu Phe Ser
Glu His225 230 235 240Cys Ala Ile Ile Leu Glu Asp Asp Arg Ser Asp
Ile Ser Ser Thr Cys 245 250 255Ala Asn Asn Ile Asn His Asn Thr Glu
Leu Leu Pro Ile Glu Leu Asp 260 265 270Thr Leu Val Gly Lys Gly Arg
Phe Ala Glu Val Tyr Lys Ala Lys Leu 275 280 285Lys Gln Asn Thr Ser
Glu Gln Phe Glu Thr Val Ala Val Lys Ile Phe 290 295 300Pro Tyr Glu
Glu Tyr Ala Ser Trp Lys Thr Glu Lys Asp Ile Phe Ser305 310 315
320Asp Ile Asn Leu Lys His Glu Asn Ile Leu Gln Phe Leu Thr Ala Glu
325 330 335Glu Arg Lys Thr Glu Leu Gly Lys Gln Tyr Trp Leu Ile Thr
Ala Phe 340 345 350His Ala Lys Gly Asn Leu Gln Glu Tyr Leu Thr Arg
His Val Ile Ser 355 360 365Trp Glu Asp Leu Arg Lys Leu Gly Ser Ser
Leu Ala Arg Gly Ile Ala 370 375 380His Leu His Ser Asp His Thr Pro
Cys Gly Arg Pro Lys Met Pro Ile385 390 395 400Val His Arg Asp Leu
Lys Ser Ser Asn Ile Leu Val Lys Asn Asp Leu 405 410 415Thr Cys Cys
Leu Cys Asp Phe Gly Leu Ser Leu Arg Leu Asp Pro Thr 420 425 430Leu
Ser Val Asp Asp Leu Ala Asn Ser Gly Gln Val Gly Thr Ala Arg 435 440
445Tyr Met Ala Pro Glu Val Leu Glu Ser Arg Met Asn Leu Glu Asn Val
450 455 460Glu Ser Phe Lys Gln Thr Asp Val Tyr Ser Met Ala Leu Val
Leu Trp465 470 475 480Glu Met Thr Ser Arg Cys Asn Ala Val Gly Glu
Val Lys Asp Tyr Glu 485 490 495Pro Pro Phe Gly Ser Lys Val Arg Glu
His Pro Cys Val Glu Ser Met 500 505 510Lys Asp Asn Val Leu Arg Asp
Arg Gly Arg Pro Glu Ile Pro Ser Phe 515 520 525Trp Leu Asn His Gln
Gly Ile Gln Met Val Cys Glu Thr Leu Thr Glu 530 535 540Cys Trp Asp
His Asp Pro Glu Ala Arg Leu Thr Ala Gln Cys Val Ala545 550 555
560Glu Arg Phe Ser Glu Leu Glu His Leu Asp Arg Leu Ser Gly Arg Ser
565 570 575Cys Ser Glu Glu Lys Ile Pro Glu Asp Gly Ser Leu Asn Thr
Thr Lys 580 585 59053811DNAHomo sapiens 5acagggccac tgctgctcac
agaagcagtg aggatgatgc caggatgatg tctgcctcgc 60gcctggctgg gactctgatc
ccagccatgg ccttcctctc ctgcgtgaga ccagaaagct 120gggagccctg
cgtggagact tggccctaaa ccacacagaa gagctggcat gaaacccaga
180gctttcagac tccggagcct cagcccttca ccccgattcc attgcttctt
gctaaatgct 240gccgttttat cacggaggtg gttcctaata ttacttatca
atgcatggag ctgaatttct 300acaaaatccc cgacaacctc cccttctcaa
ccaagaacct ggacctgagc tttaatcccc 360tgaggcattt aggcagctat
agcttcttca gtttcccaga actgcaggtg ctggatttat 420ccaggtgtga
aatccagaca attgaagatg gggcatatca gagcctaagc cacctctcta
480ccttaatatt gacaggaaac cccatccaga gtttagccct gggagccttt
tctggactat 540caagtttaca gaagctggtg gctgtggaga caaatctagc
atctctagag aacttcccca 600ttggacatct caaaactttg aaagaactta
atgtggctca caatcttatc caatctttca 660aattacctga gtatttttct
aatctgacca atctagagca cttggacctt tccagcaaca 720agattcaaag
tatttattgc acagacttgc gggttctaca tcaaatgccc ctactcaatc
780tctctttaga cctgtccctg aaccctatga actttatcca accaggtgca
tttaaagaaa 840ttaggcttca taagctgact ttaagaaata attttgatag
tttaaatgta atgaaaactt 900gtattcaagg tctggctggt ttagaagtcc
atcgtttggt tctgggagaa tttagaaatg 960aaggaaactt ggaaaagttt
gacaaatctg ctctagaggg cctgtgcaat ttgaccattg 1020aagaattccg
attagcatac ttagactact acctcgatga tattattgac ttatttaatt
1080gtttgacaaa tgtttcttca ttttccctgg tgagtgtgac tattgaaagg
gtaaaagact 1140tttcttataa tttcggatgg caacatttag aattagttaa
ctgtaaattt ggacagtttc 1200ccacattgaa actcaaatct ctcaaaaggc
ttactttcac ttccaacaaa ggtgggaatg 1260ctttttcaga agttgatcta
ccaagccttg agtttctaga tctcagtaga aatggcttga 1320gtttcaaagg
ttgctgttct caaagtgatt ttgggacaac cagcctaaag tatttagatc
1380tgagcttcaa tggtgttatt accatgagtt caaacttctt gggcttagaa
caactagaac 1440atctggattt ccagcattcc aatttgaaac aaatgagtga
gttttcagta ttcctatcac 1500tcagaaacct catttacctt gacatttctc
atactcacac cagagttgct ttcaatggca 1560tcttcaatgg cttgtccagt
ctcgaagtct tgaaaatggc tggcaattct ttccaggaaa 1620acttccttcc
agatatcttc acagagctga gaaacttgac cttcctggac ctctctcagt
1680gtcaactgga gcagttgtct ccaacagcat ttaactcact ctccagtctt
caggtactaa 1740atatgagcca caacaacttc ttttcattgg atacgtttcc
ttataagtgt ctgaactccc 1800tccaggttct tgattacagt ctcaatcaca
taatgacttc caaaaaacag gaactacagc 1860attttccaag tagtctagct
ttcttaaatc ttactcagaa tgactttgct tgtacttgtg 1920aacaccagag
tttcctgcaa tggatcaagg accagaggca gctcttggtg gaagttgaac
1980gaatggaatg tgcaacacct tcagataagc agggcatgcc tgtgctgagt
ttgaatatca 2040cctgtcagat gaataagacc atcattggtg tgtcggtcct
cagtgtgctt gtagtatctg 2100ttgtagcagt tctggtctat aagttctatt
ttcacctgat gcttcttgct ggctgcataa 2160agtatggtag aggtgaaaac
atctatgatg cctttgttat ctactcaagc caggatgagg 2220actgggtaag
gaatgagcta gtaaagaatt tagaagaagg ggtgcctcca tttcagctct
2280gccttcacta cagagacttt attcccggtg tggccattgc tgccaacatc
atccatgaag 2340gtttccataa aagccgaaag gtgattgttg tggtgtccca
gcacttcatc cagagccgct 2400ggtgtatctt tgaatatgag attgctcaga
cctggcagtt tctgagcagt cgtgctggta 2460tcatcttcat tgtcctgcag
aaggtggaga agaccctgct caggcagcag gtggagctgt 2520accgccttct
cagcaggaac acttacctgg agtgggagga cagtgtcctg gggcggcaca
2580tcttctggag acgactcaga aaagccctgc tggatggtaa atcatggaat
ccagaaggaa 2640cagtgggtac aggatgcaat tggcaggaag caacatctat
ctgaagagga aaaataaaaa 2700cctcctgagg catttcttgc ccagctgggt
ccaacacttg ttcagttaat aagtattaaa 2760tgctgccaca tgtcaggcct
tatgctaagg gtgagtaatt ccatggtgca ctagatatgc 2820agggctgcta
atctcaagga gcttccagtg cagagggaat aaatgctaga ctaaaataca
2880gagtcttcca ggtgggcatt tcaaccaact cagtcaagga acccatgaca
aagaaagtca 2940tttcaactct tacctcatca agttgaataa agacagagaa
aacagaaaga gacattgttc 3000ttttcctgag tcttttgaat ggaaattgta
ttatgttata gccatcataa aaccattttg 3060gtagttttga ctgaactggg
tgttcacttt ttcctttttg attgaataca atttaaattc 3120tacttgatga
ctgcagtcgt caaggggctc ctgatgcaag atgccccttc cattttaagt
3180ctgtctcctt acagaggtta aagtctaatg gctaattcct aaggaaacct
gattaacaca 3240tgctcacaac catcctggtc attctcgaac atgttctatt
ttttaactaa tcacccctga 3300tatattttta tttttatata tccagttttc
atttttttac gtcttgccta taagctaata 3360tcataaataa ggttgtttaa
gacgtgcttc aaatatccat attaaccact atttttcaag 3420gaagtatgga
aaagtacact ctgtcacttt gtcactcgat gtcattccaa agttattgcc
3480tactaagtaa tgactgtcat gaaagcagca ttgaaataat ttgtttaaag
ggggcactct 3540tttaaacggg aagaaaattt ccgcttcctg gtcttatcat
ggacaatttg ggctataggc 3600atgaaggaag tgggattacc tcaggaagtc
accttttctt gattccagaa acatatgggc 3660tgataaaccc ggggtgacct
catgaaatga gttgcagcag atgtttattt ttttcagaac 3720aagtgatgtt
tgatggacct atgaatctat ttagggagac acagatggct gggatccctc
3780ccctgtaccc ttctcactga caggagaact a 38116799PRTHomo sapiens 6Met
Glu Leu Asn Phe Tyr Lys Ile Pro Asp Asn Leu Pro Phe Ser Thr1 5 10
15Lys Asn Leu Asp Leu Ser Phe Asn Pro Leu Arg His Leu Gly Ser Tyr
20 25 30Ser Phe Phe Ser Phe Pro Glu Leu Gln Val Leu Asp Leu Ser Arg
Cys 35 40 45Glu Ile Gln Thr Ile Glu Asp Gly Ala Tyr Gln Ser Leu Ser
His Leu 50 55 60Ser Thr Leu Ile Leu Thr Gly Asn Pro Ile Gln Ser Leu
Ala Leu Gly65 70 75 80Ala Phe Ser Gly Leu Ser Ser Leu Gln Lys Leu
Val Ala Val Glu Thr 85 90 95Asn Leu Ala Ser Leu Glu Asn Phe Pro Ile
Gly His Leu Lys Thr Leu 100 105 110Lys Glu Leu Asn Val Ala His Asn
Leu Ile Gln Ser Phe Lys Leu Pro 115 120 125Glu Tyr Phe Ser Asn Leu
Thr Asn Leu Glu His Leu Asp Leu Ser Ser 130 135 140Asn Lys Ile Gln
Ser Ile Tyr Cys Thr Asp Leu Arg Val Leu His Gln145 150 155 160Met
Pro Leu Leu Asn Leu Ser Leu Asp Leu Ser Leu Asn Pro Met Asn 165 170
175Phe Ile Gln Pro Gly Ala Phe Lys Glu Ile Arg Leu His Lys Leu Thr
180 185 190Leu Arg Asn Asn Phe Asp Ser Leu Asn Val Met Lys Thr Cys
Ile Gln 195 200 205Gly Leu Ala Gly Leu Glu Val His Arg Leu Val Leu
Gly Glu Phe Arg 210 215 220Asn Glu Gly Asn Leu Glu Lys Phe Asp Lys
Ser Ala Leu Glu Gly Leu225 230 235 240Cys Asn Leu Thr Ile Glu Glu
Phe Arg Leu Ala Tyr Leu Asp Tyr Tyr 245 250 255Leu Asp Asp Ile Ile
Asp Leu Phe Asn Cys Leu Thr Asn Val Ser Ser 260 265 270Phe Ser Leu
Val Ser Val Thr Ile Glu Arg Val Lys Asp Phe Ser Tyr 275 280 285Asn
Phe Gly Trp Gln His Leu Glu Leu
Val Asn Cys Lys Phe Gly Gln 290 295 300Phe Pro Thr Leu Lys Leu Lys
Ser Leu Lys Arg Leu Thr Phe Thr Ser305 310 315 320Asn Lys Gly Gly
Asn Ala Phe Ser Glu Val Asp Leu Pro Ser Leu Glu 325 330 335Phe Leu
Asp Leu Ser Arg Asn Gly Leu Ser Phe Lys Gly Cys Cys Ser 340 345
350Gln Ser Asp Phe Gly Thr Thr Ser Leu Lys Tyr Leu Asp Leu Ser Phe
355 360 365Asn Gly Val Ile Thr Met Ser Ser Asn Phe Leu Gly Leu Glu
Gln Leu 370 375 380Glu His Leu Asp Phe Gln His Ser Asn Leu Lys Gln
Met Ser Glu Phe385 390 395 400Ser Val Phe Leu Ser Leu Arg Asn Leu
Ile Tyr Leu Asp Ile Ser His 405 410 415Thr His Thr Arg Val Ala Phe
Asn Gly Ile Phe Asn Gly Leu Ser Ser 420 425 430Leu Glu Val Leu Lys
Met Ala Gly Asn Ser Phe Gln Glu Asn Phe Leu 435 440 445Pro Asp Ile
Phe Thr Glu Leu Arg Asn Leu Thr Phe Leu Asp Leu Ser 450 455 460Gln
Cys Gln Leu Glu Gln Leu Ser Pro Thr Ala Phe Asn Ser Leu Ser465 470
475 480Ser Leu Gln Val Leu Asn Met Ser His Asn Asn Phe Phe Ser Leu
Asp 485 490 495Thr Phe Pro Tyr Lys Cys Leu Asn Ser Leu Gln Val Leu
Asp Tyr Ser 500 505 510Leu Asn His Ile Met Thr Ser Lys Lys Gln Glu
Leu Gln His Phe Pro 515 520 525Ser Ser Leu Ala Phe Leu Asn Leu Thr
Gln Asn Asp Phe Ala Cys Thr 530 535 540Cys Glu His Gln Ser Phe Leu
Gln Trp Ile Lys Asp Gln Arg Gln Leu545 550 555 560Leu Val Glu Val
Glu Arg Met Glu Cys Ala Thr Pro Ser Asp Lys Gln 565 570 575Gly Met
Pro Val Leu Ser Leu Asn Ile Thr Cys Gln Met Asn Lys Thr 580 585
590Ile Ile Gly Val Ser Val Leu Ser Val Leu Val Val Ser Val Val Ala
595 600 605Val Leu Val Tyr Lys Phe Tyr Phe His Leu Met Leu Leu Ala
Gly Cys 610 615 620Ile Lys Tyr Gly Arg Gly Glu Asn Ile Tyr Asp Ala
Phe Val Ile Tyr625 630 635 640Ser Ser Gln Asp Glu Asp Trp Val Arg
Asn Glu Leu Val Lys Asn Leu 645 650 655Glu Glu Gly Val Pro Pro Phe
Gln Leu Cys Leu His Tyr Arg Asp Phe 660 665 670Ile Pro Gly Val Ala
Ile Ala Ala Asn Ile Ile His Glu Gly Phe His 675 680 685Lys Ser Arg
Lys Val Ile Val Val Val Ser Gln His Phe Ile Gln Ser 690 695 700Arg
Trp Cys Ile Phe Glu Tyr Glu Ile Ala Gln Thr Trp Gln Phe Leu705 710
715 720Ser Ser Arg Ala Gly Ile Ile Phe Ile Val Leu Gln Lys Val Glu
Lys 725 730 735Thr Leu Leu Arg Gln Gln Val Glu Leu Tyr Arg Leu Leu
Ser Arg Asn 740 745 750Thr Tyr Leu Glu Trp Glu Asp Ser Val Leu Gly
Arg His Ile Phe Trp 755 760 765Arg Arg Leu Arg Lys Ala Leu Leu Asp
Gly Lys Ser Trp Asn Pro Glu 770 775 780Gly Thr Val Gly Thr Gly Cys
Asn Trp Gln Glu Ala Thr Ser Ile785 790 795
* * * * *