U.S. patent application number 15/293011 was filed with the patent office on 2017-02-02 for immunotherapy of cancer using genetically engineered gd2-specific t cells.
The applicant listed for this patent is Baylor College of Medicine. Invention is credited to Nabil M. Ahmed, Malcolm Brenner, Gianpietro Dotti, Stephen M. G. Gottschalk, Zakaria Grada, Claudia Rossig.
Application Number | 20170027988 15/293011 |
Document ID | / |
Family ID | 45810967 |
Filed Date | 2017-02-02 |
United States Patent
Application |
20170027988 |
Kind Code |
A1 |
Brenner; Malcolm ; et
al. |
February 2, 2017 |
IMMUNOTHERAPY OF CANCER USING GENETICALLY ENGINEERED GD2-SPECIFIC T
CELLS
Abstract
The present invention concerns immunotherapy for cancers having
cells that comprise the ganglioside GD2 antigen. In specific
embodiment, T cells having a chimeric receptor that targets GD2 is
employed. In particular cases, the chimeric receptor comprises
antibody, cytoplasmic signaling domain from the T cell receptor,
and/or costimulatory molecule(s).
Inventors: |
Brenner; Malcolm; (Bellaire,
TX) ; Dotti; Gianpietro; (Chapel Hill, NC) ;
Ahmed; Nabil M.; (Houston, TX) ; Rossig; Claudia;
(Heidelberg, DE) ; Gottschalk; Stephen M. G.;
(Houston, TX) ; Grada; Zakaria; (Houston,
TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Baylor College of Medicine |
Houston |
TX |
US |
|
|
Family ID: |
45810967 |
Appl. No.: |
15/293011 |
Filed: |
October 13, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13820931 |
Apr 3, 2013 |
9493740 |
|
|
PCT/US2011/050780 |
Sep 8, 2011 |
|
|
|
15293011 |
|
|
|
|
61380761 |
Sep 8, 2010 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 5/0638 20130101;
A61K 2039/5156 20130101; C07K 2317/622 20130101; A61K 39/001171
20180801; A61K 35/17 20130101; C07K 16/18 20130101; C07K 16/3084
20130101; A61K 39/0011 20130101; A61K 2039/5158 20130101 |
International
Class: |
A61K 35/17 20060101
A61K035/17; A61K 39/00 20060101 A61K039/00; C12N 5/0783 20060101
C12N005/0783; C07K 16/30 20060101 C07K016/30 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under PO1
CA94237 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A method of targeting a cancer cell having a GD2 antigen,
comprising the steps of providing to the cell a cytotoxic T
lymphocyte with a chimeric receptor that recognizes the GD2
antigen.
2. The method of claim 1, wherein the cancer cell is in vitro or in
vivo.
3. The method of claim 1, wherein the chimeric receptor comprises
antibody that binds GD2
4. The method of claim 2, wherein the antibody is a scFv
antibody.
5. The method of claim 2, wherein the antibody is the 14g2a scFv
antibody.
6. The method of claim 1, wherein the chimeric receptor comprises
the effector domain of the T-cell receptor zeta chain or related
signal transduction endodomains derived from the T cell
receptor.
7. The method of claim 1, wherein the chimeric receptor comprises
one or more costimulatory molecules.
8. The method of claim 6, wherein the costimulatory molecules
comprise CD28, OX40, 4-1BB, or a combination thereof.
Description
[0001] This application is a divisional of U.S. patent application
Ser. No. 13/820,931 filed Apr. 3, 2013 which is a national phase
application under 35 U.S.C. .sctn.371 that claims priority to
International Application No. PCT/US2011/050780 filed Sep. 8, 2011,
which claims priority to U.S. Provisional Application Ser. No.
61/380,761 filed Sep. 8, 2010, all of which are incorporated by
reference herein in their entirety.
TECHNICAL FIELD
[0003] The present invention generally concerns the fields of cell
biology, molecular biology, and medicine. In particular, the field
of the invention concerns immunotherapy of cancer.
BACKGROUND OF THE INVENTION
[0004] The rising incidence of cutaneous melanoma and the failure
to significantly improve outcomes in metastatic disease have led to
increasing interest in immunotherapeutic approaches, because these
can be remarkably effective (Jemal et al., 2008; Bajetta et al.,
2002; Rosenberg et al., 2008). Several investigators have focused
on targeting tumor-associated antigens that fall into the cancer
testis antigen group, including MAGE, BAGE, GAGE, and NY-ESO-1, or
the melanocyte differentiation protein group, including gp100,
Melan-A/MART-1, and tyrosinase, which are widely present on
melanoma cells. These studies have used cytotoxic T cell lines
(Mackensen et al., 2006; Butler et al., 2007), clones with native
(Rosenberg et al., 2004) or transgenic .alpha..beta. T cell
receptors (Morgan et al., 2006) specific for cancer testis
antigen-derived peptides that are recognized in association with
human leukocyte antigen (HLA) class I antigens on the tumor cell
surface. It is clear, however, that the heterogeneity of protein
antigen expression and presentation in melanoma is a characteristic
that helps limit the proportion of patients who are able to respond
to such targeted strategies (Ohnmacht and Marincola, 2000). One
means of increasing the effectiveness of targeted T cell therapy of
melanoma, therefore, may be to use artificial chimeric receptors
derived, for example, from the antigen binding domain of a
monoclonal antibody (Pule et al., 2003). When coupled to
appropriate intracellular signaling domains, T cells expressing
these chimeric antigen receptors (CAR) can kill tumor cell targets
(Haynes et al., 2002). They have the advantage of acting in a MHC
unrestricted manner, allowing them to target tumor cells in which
antigen processing or presentation pathways are disrupted.
Moreover, they can be directed to nonpeptide antigens on the cell
surface, broadening the range of target structures that can be
recognized on malignant cells. Hence, CAR-expressing T cells could
complement MHC restricted cytotoxic T cells, and increase the
overall effectiveness of this cellular immunotherapy.
[0005] Many melanoma cells express a range of gangliosides,
including GD2, GM2, GM3, and GD3, that may be a good choice of
target for CAR-T cells, because their expression is highly
tissue-restricted (Yun et al., 2000; Livingston, 1998). Although
these carbohydrate antigens are expressed by both normal
melanocytes and melanoma cells, expression is significantly
up-regulated after malignant transformation of melanocytes
(Tsuchida et al., 1989; Albino et al., 1992), and is associated
with changes in the proliferation, migration, and metastatic
potential of the tumor cells (Ravindranath et al., 2008). Moreover,
natural or vaccine-induced antibodies to gangliosides in melanoma
patients have been correlated with improved disease relapse-free
survival (Livingston et al., 2008; Ragupathi et al., 2003).
BRIEF SUMMARY OF THE INVENTION
[0006] The present invention concerns methods and compositions for
the treatment of cancer, including treatment of cancer employing
immunotherapy. In particular cases, the immunotherapy includes T
lymphocytes engineered to target certain cancers. Although any
cancers may be targeted using the inventive therapy (including
brain, breast, pancreatic, liver, kidney, lung, spleen, gall
bladder, anal, testicular, ovarian, cervical, skin, bone, blood, or
colon, for example), in particular cases the cancer is melanoma or
lung cancer, including non small cell lung cancer. In specific
embodiments, the cancer being treated has cancer cells with GD2 as
an antigen on the surface of the cancer cells. In particular cases,
the cytotoxic T lymphocytes (CTLs) employed to target GD2 on the
surface of cancer cells comprise a receptor for GD2 and, in
specific cases, the receptor on the CTLs is chimeric, non-natural
and engineered at least in part by the hand of man. In particular
cases, the engineered chimeric antigen receptor (CAR) has one, two,
three, four, or more components, and in some embodiments the one or
more components facilitate targeting or binding of the T lymphocyte
to the GD2 antigen-comprising cancer cell, although in some cases
one or more components are useful to promote T cell growth and
maturity.
[0007] In certain embodiments, the present invention includes T
lymphocytes engineered to comprise a chimeric receptor having an
antibody for GD2, part or all of a cytoplasmic signaling domain,
and/or part or all of one or more costimulatory molecules, for
example endodomains of costimulatory molecules. In specific
embodiments, the antibody for GD2 is a single-chain variable
fragment (scFv), although in certain aspects the antibody is
directed at other target antigens on the cell surface, such as HER2
or CD19, 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 would include,
but are not limited to, endodmains from co-stimulatory molecules
such as CD28, 4-1BB, and OX40 or the signalling components of
cytokine receptors such as IL7 and IL15. In particular embodiments,
costimulatory molecules are employed to enhance the activation,
proliferation, and cytotoxicity of T cells produced by the CAR
after antigen engagement. In specific embodiments, the
costimulatory molecules are CD28, OX40, and 4-1BB and cytokine and
the cytokine receptors are IL7 and IL15.
[0008] Genetic engineering of human T lymphocytes to express
tumor-directed chimeric antigen receptors (CAR) can produce
antitumor effector cells that bypass tumor immune escape mechanisms
that are due to abnormalities in protein-antigen processing and
presentation. Moreover, these transgenic receptors can be directed
to tumor-associated antigens that are not protein-derived, such as
the ganglioside GD2, which is expressed in a high proportion of
melanoma cells.
[0009] In certain embodiments, the present invention provides
chimeric T cells specific for the ganglioside GD2 by joining an
extracellular antigen-binding domain derived from the GD2-specific
antibody sc14.G2a to cytoplasmic signaling domains derived from the
T-cell receptor .zeta.-chain, with the endodomains of the exemplary
costimulatory molecules CD28 and OX40, for examples. This CAR was
expressed in human T cells and the targeting of GD2-positive
melanoma tumors was assessed in vitro and in a murine xenograft,
for example.
[0010] As described herein, upon coincubation with GD2-expressing
melanoma cells, CAR-GD2 T lymphocytes incorporating the CD28 and
OX40 endodomains secreted significant levels of cytokines in a
pattern comparable with the cytokine response obtained by
engagement of the native CD3 receptor. These CAR-T cells had
antimelanoma activity in vitro and in an exemplary xenograft model,
increasing the survival of tumor-bearing animals. Thus, redirecting
human T lymphocytes to the tumor-associated ganglioside GD2
generates effector cells with antimelanoma activity that is useful
in subjects with disease.
[0011] In some embodiments, there is a method of targeting a cancer
cell having a GD2 antigen, comprising the steps of providing to the
cell a cytotoxic T lymphocyte with a chimeric receptor that
recognizes the GD2 antigen. In specific embodiments, the cancer
cell is in vitro or in vivo. In certain embodiments, the chimeric
receptor comprises antibody that binds GD2, such as a scFv
antibody, for example the 14g2a scFv antibody.
[0012] In particular embodiments, the chimeric receptor comprises
the effector domain of the T-cell receptor zeta chain or related
signal transduction endodomains derived from the T cell receptor.
In specific cases, the chimeric receptor comprises one or more
costimulatory molecules, such as CD28, OX40, 4-1BB, or a
combination thereof, for example. In specific embodiments, the
cancer cell is in an individual with melanoma or non small cell
lung cancer.
[0013] In one embodiment of the present invention, there is a
method of treating melanoma or non small cell lung cancer in an
individual, comprising the steps of administering to an individual
cytotoxic T lymphocytes having a chimeric receptor that recognizes
a GD2 antigen on the surface of cancer cells. In certain
embodiments, the chimeric receptor comprises antibody that binds
GD2, for example a scFv antibody, such as the 14g2a scFv antibody,
as one instance.
[0014] In particular cases the chimeric receptor comprises the
effector domain of the T-cell receptor zeta chain. In a specific
embodiment, the chimeric receptor comprises one or more
costimulatory molecules, such as CD28, OX40, 4-1BB, or a
combination thereof, for example. In specific aspects, the
individual has had and/or is having an additional cancer therapy
for the respective melanoma or non small cell lung cancer.
[0015] The foregoing has outlined some of the features and
technical advantages of the present invention in order that the
detailed description of the invention that follows may be better
understood. Additional features and advantages of the invention
will be described hereinafter which form the subject of the claims
of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 shows expression of GD2 antigen in human melanoma
cell lines. The expression of GD2 was evaluated by FACS analysis in
11 melanoma cell lines. Six (45%) and three (27%) tumor cell lines
showed GD2 expression at intermediate/high (++/+++) or low levels
(+), respectively. In two tumor cell lines (18%) GD2 was
undetectable. A GD2-normal skin fibroblast line was used as a
negative control for GD2 expression. Open histograms, isotype
control of the GD2 staining (grey histograms).
[0017] FIGS. 2A and 2B demonstrate that T lymphocytes can be
genetically modified to express CARs targeting GD2. Activated T
lymphocytes were genetically modified to express CAR-GD2. FIG. 2A
shows expression of CAR-GD2 as assessed by FACS analysis using a
specific 14.g2a anti-idiotype antibody (1A7). Graph is a
representative expression of CAR-GD2 from four different transduced
T cell lines. FIG. 2B shows that both CD4-positive and CD8-positive
T lymphocytes expressed the CAR-GD2 after gene transfer.
[0018] FIG. 3 demonstrates lymphocytes redirected to express
CAR-GD2 kill GD2-positive melanoma cell lines. A .sup.51Cr release
assay was used to evaluate the cytotoxic activity of T lymphocytes
expressing CAR-GD2 and nontransduced (NT) T cells. Target cells
were melanoma lines with absent GD2 (4405 M) or low (CBL),
intermediate (SEMMA), or high (P1143) GD2 expression. Left and
right graphs, mean and SD of .sup.51Cr release from four T cell
lines after 6 and 18 h incubation, respectively.
[0019] FIG. 4 shows T lymphocytes expressing GD2-CAR produce Th1
and Th2 cytokines in response to GD2-positive melanoma cell lines.
T lymphocytes transduced with CAR-GD2 or nontransduced (NT) T cells
were cocultured (ratio T lymphocytes:tumor cells of 20:1) with four
different melanoma cell lines either negative for GD2 (4405 M) or
expressing dim (CBL), intermediate (SEMMA), or high (P1143) levels
of GD2. Culture supernatant was collected 24 h later and the
production of IL-2, IL-5, IFN-.gamma., and TNF-.alpha. measured
using a CBA assay. Neither IL-4 nor IL-10 was detected in the 24-h
supernatants. The results of four experiments are presented.
[0020] FIGS. 5A and 5B provide T-lymphocytes redirected to express
CAR-GD2 eliminate GD2-positive melanoma cell lines in vitro. To
evaluate the capacity of T lymphocytes expressing CAR-GD2 to
eliminate melanoma cells, nontransduced (NT) or CAR-GD2 transduced
T lymphocytes were cultured with melanoma cell lines that were
GD2-negative (4405 M) or expressed dim (CBL), intermediate (SEMMA),
or high (P1143) levels of the target antigen. T lymphocytes and
melanoma cell lines were plated at 20:1 ratio and cultured for 5 d
without adding IL-2 to the culture. Residual melanoma cells were
enumerated by FACS analysis. In FIG. 5A, there are mean and SD of
surviving cells expressing GFP for four T cell lines. In FIG. 5B,
there is phenotypic analysis of coculture experiments.
[0021] FIGS. 6A and 6B demonstrates that CAR-GD2 T lymphocytes
control tumor growth in vivo. SCID mice were infused i.v. with
2.times.10.sup.6 melanoma cells from the cell lines 4405 M (0% GD2
positive) or P1143 (95% GD2 positive) labeled with FFLuc gene.
Tumor growth and engraftment was monitored using an in vivo imaging
system (Xenogen-IVIS Imaging System). Four and 21 d after tumor
infusion, mice were treated with T lymphocytes CAR-GD2 or
nontransduced (NT) T cells (1.times.10.sup.7 cells/mouse). No
exogenous cytokines were injected into the mice. A, tumor growth
measured as light emission in a representative cohort of 5 mice
from each group of NT and CAR-GD2 T cell-treated animals. B,
survival curve of mice engrafted with the P1143 (95% GD2 positive)
tumor cells receiving either tumor alone, NT T cells or CAR-GD2 T
lymphocytes.
[0022] FIG. 7 shows cytolytic activity of T-lymphocytes redirected
to express GD2-CAR against normal cell lines. The inventors used a
.sup.51CR release assay to evaluate the cytotoxic activity of T
lymphocytes expressing CAR-GD2. Target cells were allogeneic
peripheral blood mononuclear cells (PBMCs) and GD2-skin
fibroblasts. The inventors also used a GD2+ mesenchymal stem cell
(MSC) line, which was susceptible to CAR-GD2 T cell killing. Data
illustrate the mean and SD of .sup.51Cr release from 4 T cell lines
after 6 and 18 hours incubation. Non-transduced T cells did not
kill any of the targets tested.
[0023] FIG. 8 shows T lymphocytes expressing CAR-GD2 and
co-expressing CD28-OX40 endodomains proliferate in response to GD2+
melanoma cell lines. CAR-GD2 T cells labeled with CFSE, to evaluate
T cell division, were co-cultured (Ratio T cells:tumor cells of
20:1) with three melanoma cell lines expressing dim (CLB),
intermediate (SENMA) or high (P1143) levels of GD2. Non transuced
(NT) T lymphocytes were used as negative controls. CFSE expression
by T cells was analyzed by FACS on day 4. Only T cells expressing
CAR-GD2 divided multiply in response to GD2+ melanoma cell lines.
Data are representative of repeat experiments.
[0024] FIGS. 9A and 9B show GD2 expression in lung cancer. FIG. 9A
shows expression in small cell lung cancer. FIG. 9B shows
expression in non-small cell lung cancer.
[0025] FIG. 10 shows flow cytometry for both small cell and non
small cell lung cancer.
[0026] FIG. 11 shows transduction rates with GD2 CAR using GD2
specific proteins.
[0027] FIGS. 12A and 12B demonstrate that GD2 CAR-transduced T
lymphocytes recognize and kill lung cancer cell lines. FIG. 12A
shows killing of small cell lung cancer, and FIG. 12B shows killing
of non-small cell lung cancer.
[0028] FIG. 13 demonstrates that GD2 CAR-transduced T lymphocytes
secrete immunostimulatory cytokines in coculture of GD2 positive
lung cancer cell lines.
DETAILED DESCRIPTION OF THE INVENTION
[0029] In the following description, certain details are set forth
such as specific quantities, sizes, etc. so as to provide a
thorough understanding of the present embodiments disclosed herein.
However, it will be obvious to those skilled in the art that the
present disclosure may be practiced without such specific details.
In many cases, details concerning such considerations and the like
have been omitted inasmuch as such details are not necessary to
obtain a complete understanding of the present disclosure and are
within the skills of persons of ordinary skill in the relevant
art.
[0030] In keeping with long-standing patent law convention, the
words "a" and "an" when used in the present specification in
concert with the word comprising, including the claims, denote "one
or more." Some embodiments of the invention may consist of or
consist essentially of one or more elements, method steps, and/or
methods of the invention. It is contemplated that any method or
composition described herein can be implemented with respect to any
other method or composition described herein.
I. DEFINITIONS
[0031] As used herein, the term "costimulatory molecule" refers to
a molecular component that promotes activation, proliferation and
effector function of a T cell after engagement of an antigen
specific receptor.
[0032] As used herein, the term "cytoplasmic signaling domain"
refers to the component of a co-stimulatory molecule or cytokine
receptor that exists inside the cell and is responsible for
transducing the external signal received to the internal metabolic
processes of the cell, thereby altering its phenotype and
function.
[0033] In embodiments of the present invention, the overexpression
of GD2 by human primary melanoma cells allows these cells to be
targeted in vitro and in vivo by GD2 CAR-expressing primary T
cells, and incorporation of endodomains from both CD28 and OX40
molecules (Pule et al., 2005) mediates costimulation of the T
lymphocytes, inducing T cell activation, proliferation, and
cytotoxicity against GD2-positive melanoma cells.
[0034] In particular embodiments of the invention, there are
methods for killing metastatic melanoma cells using genetically
manipulated T-cells that express a chimeric antigen receptor (CAR)
directed against the ganglioside antigen GD2. Engagement (antigen
binding) of this CAR leads to activation of the linked T-cell
receptor .zeta. chain and the costimulatory molecules CD28 and
OX40. The present invention leads to the suppression of metastatic
melanoma xenografts in vivo, and the skilled artisan recognizes
that such practices extrapolate to non small cell lung cancer, in
certain embodiments.
II. SCFV ANTIBODIES
[0035] In particular embodiments of the invention, the CAR receptor
comprises a single-chain variable fragment (scFv) that recognizes
GD2. The skilled artisan recognizes that scFv is a fusion protein
of the variable regions of the heavy (VH) and light chains (VL) of
immunoglobulins, connected with a short linker peptide of ten to
about 25 amino acids. The linker may be rich in glycine for
flexibility and/or it may have serine or threonine for solubility,
in certain cases. In a particular embodiment, the 14g2a scFv
antibody is used in the CAR. The scFv may be generated by methods
known in the art.
[0036] Other examples of ScFv made and successfully tested in
pre-clinical studies include, but are not limited to, CD20, CD19,
CD30, Her2, kappa light chain, and lambda light chain, and in
certain embodiments one or more of these are employed in the
invention.
[0037] In certain aspects, one can use cytokine exodomains or other
ligand/receptor molecules as exodomains to provide targeting to the
tumor cells.
III. COSTIMULATORY MOLECULES
[0038] The skilled artisan recognizes that T cells utilize
co-stimulatory signals that are antigen non-specific to become
fully activated. In particular cases they are provided by the
interaction between co-stimulatory molecules expressed on the
membrane of APC and the T cell. In specific embodiments, the one or
more costimulatory molecules in the chimeric receptor come from the
B7/CD28 family, TNF superfamily, or the signaling lymphocyte
activation molecule (SLAM) family. Exemplary costimulatory
molecules include one or more of the following: B7-1/CD80; CD28;
B7-2/CD86; CTLA-4; B7-H1/PD-L1; ICOS; B7-H2; PD-1; B7-H3; PD-L2;
B7-H4; PDCD6; BTLA; 4-1BB/TNFRSF9/CD137; CD40 Ligand/TNFSF5; 4-1BB
Ligand/TNFSF9; GITR/TNFRSF18; BAFF/BLyS/TNFSF13B; GITR
Ligand/TNFSF18; BAFF R/TNFRSF13C; HVEM/TNFRSF14; CD27/TNFRSF7;
LIGHT/TNFSF14; CD27 Ligand/TNFSF7; OX40/TNFRSF4; CD30/TNFRSF8; OX40
Ligand/TNFSF4; CD30 Ligand/TNFSF8; TACI/TNFRSF13B; CD40/TNFRSF5;
2B4/CD244/SLAMF4; CD84/SLAMF5; BLAME/SLAMF8; CD229/SLAMF3; CD2
CRACC/SLAMF7; CD2F-10/SLAMF9; NTB-A/SLAMF6; CD48/SLAMF2;
SLAM/CD150; CD58/LFA-3; CD2; Ikaros; CD53; Integrin alpha 4/CD49d;
CD82/Kai-1; Integrin alpha 4 beta 1; CD90/Thy1; Integrin alpha 4
beta 7/LPAM-1; CD96; LAG-3; CD160; LMIR1/CD300A; CRTAM; TCL1A;
DAP12; TIM-1/KIM-1/HAVCR; Dectin-1/CLEC7A; TIM-4; DPPIV/CD26; TSLP;
EphB6; TSLP R; and HLA-DR.
[0039] The CAR of the invention may employ one, two, three, four,
or more costimulatory molecules.
IV. EFFECTOR DOMAIN OF THE T-CELL RECEPTOR ZETA CHAIN
[0040] The effector domain is a signalling domain that tranduces
the event of receptor ligand binding to an intracellular signal
that partially activates the T lymphocyte. Absent appropriate
co-stimulatory signals, this event is insufficient for useful T
cell activation and proliferation.
V. COMBINATION THERAPY
[0041] In certain embodiments of the invention, methods of the
present invention 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).
[0042] 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 gene therapy. For
example, the herpes simplex-thymidine kinase (HS-tK) gene, when
delivered to brain tumors by a retroviral vector system,
successfully induced susceptibility to the antiviral agent
ganciclovir (Culver, et al., 1992). In the context of the present
invention, it is contemplated that cell therapy could be used
similarly in conjunction with chemotherapeutic, radiotherapeutic,
or immunotherapeutic intervention, in addition to other
pro-apoptotic or cell cycle regulating agents.
[0043] Alternatively, the present inventive therapy may precede or
follow the other agent treatment by intervals ranging from minutes
to weeks. In embodiments where the other agent and present
invention are applied separately to the individual, one would
generally ensure that a significant period of time did not expire
between the time of each delivery, such that the agent and
inventive therapy would still be able to exert an advantageously
combined effect on the cell. In such instances, it is contemplated
that one may contact the cell with both modalities within about
12-24 h of each other and, more preferably, within about 6-12 h of
each other. In some situations, it may be desirable to extend the
time period for treatment significantly, however, where several 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.
[0044] Various combinations may be employed, present invention 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
[0045] 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.
[0046] A. Chemotherapy
[0047] Cancer therapies also include a variety of combination
therapies with both chemical and radiation based treatments.
Combination chemotherapies include, for example, abraxane,
altretamine, docetaxel, herceptin, methotrexate, novantrone,
zoladex, cisplatin (CDDP), carboplatin, procarbazine,
mechlorethamine, cyclophosphamide, camptothecin, ifosfamide,
melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin,
daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin,
etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding
agents, taxol, gemcitabien, navelbine, farnesyl-protein tansferase
inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin
and methotrexate, or any analog or derivative variant of the
foregoing.
[0048] In specific embodiments, chemotherapy for melanoma is
employed in conjunction with the invention, for example before,
during and/or after administration of the invention. Exemplary
chemotherapeutic agents for melanoma include at least dacarbazine
(also termed DTIC), temozolimide, paclitaxel, cisplatin,
carmustine, fotemustine, vindesine, vincristine, or bleomycin.
[0049] B. Radiotherapy
[0050] 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.
[0051] 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.
[0052] C. Immunotherapy
[0053] Immunotherapeutics generally rely on the use of immune
effector cells and molecules to target and destroy cancer cells.
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.
[0054] Immunotherapy 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 invention. 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.
[0055] Immunotherapy for melanoma may include interleukin-2 (IL-2)
or interferon (IFN), for example.
[0056] D. Genes
[0057] 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 invention
clinical embodiments. A variety of expression products are
encompassed within the invention, including inducers of cellular
proliferation, inhibitors of cellular proliferation, or regulators
of programmed cell death.
[0058] E. Surgery
[0059] 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 invention,
chemotherapy, radiotherapy, hormonal therapy, gene therapy,
immunotherapy and/or alternative therapies.
[0060] 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 invention may be used in conjunction
with removal of superficial cancers, precancers, or incidental
amounts of normal tissue.
[0061] 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.
[0062] F. Other Agents
[0063] It is contemplated that other agents may be used in
combination with the present invention 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-1beta,
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 invention 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 invention to improve the anti-hyerproliferative
efficacy of the treatments. Inhibitors of cell adhesion are
contemplated to improve the efficacy of the present invention.
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 invention to improve the treatment
efficacy.
EXAMPLES
[0064] The following examples are presented in order to more fully
illustrate the preferred embodiments of the invention. They should
in no way, however, be construed as limiting the broad scope of the
invention.
Example 1
Exemplary Materials and Methods
[0065] Establishment of cell lines. After informed consent, tumor
biopsies (from metastatic skin lesions) were obtained from five
patients with stage III or IV melanoma. The tumor tissue was minced
and the fragments resuspended in 30 mL of digestion medium
containing DNAse at 30 U/mL, hyaluronidase at 0.1 mg/mL, and
collagenase at 1 mg/mL (all from Sigma-Aldrich), in complete medium
prepared as follows: DMEM (Cambrex) supplemented with 10% of heat
inactivated FCS (HyClone), 200 IU/mL penicillin, 200 mg/mL
streptomycin, 100 mg/mL gentamicin (Invitrogen), and 2 mmol/L
GlutaMAX (Invitrogen). After 4 h incubation at 37.degree. C. in 5%
CO.sub.2, the cell suspension supernatant (free of tissue debris)
was collected, transferred to a new tube, and then centrifuged at
400.times.g for 5 min. Cells were resuspended in a 6-well plate in
fresh complete medium containing 1 mmol/L sodium pyruvate
(Invitrogen), and cultured at 37.degree. C. in 5% CO.sub.2. Culture
medium was renewed every 72 h. At day 6, the antibiotics present in
the complete medium were reduced to 100 IU/mL penicillin and 100
mg/mL streptomycin.
[0066] When tumor cells reached confluence, they were transferred
to a T25 flask for further amplification. The established tumor
cell lines (CLB, SENMA, Plaode, RR-371953, and P1143) were
characterized by fluorescence activated cell sorting (FACS)
analysis (MCSP and GD2) and immunofluorescence (gp100, MAGE-1, and
MART-1). Low passage number (<20) of the primary melanoma cell
lines was used in in vitro and in vivo experiments.
[0067] Normal mesenchymal stem cells and normal skin fibroblasts
were generated in laboratory as previously described (Yvon et al.,
2003; Gottschalk et al., 2003), and the K562 cell line was obtained
from the American Type Culture Collection. All cell lines were
maintained in RPMI (Hyclone) supplemented with 10% heat inactivated
FCS, 100 IU/mL penicillin, 100 mg/mL streptomycin, 1 mmol/L sodium
pyruvate (Invitrogen), and 2 mmol/L GlutaMAX. Six established
melanoma cell lines, isolated from surgical specimens at Istituto
Nazionale Tumori, Milan, were also used to screen GD2
expression.
[0068] Mononuclear cells. Peripheral blood, obtained after informed
consent from normal donors, was processed over Ficoll gradients,
and the resulting peripheral blood mononuclear cells (PBMC) were
cultured in complete T-cell medium containing 45% RPMI and 45%
Click's medium supplemented with 10% heat inactivated FCS, 100
IU/mL penicillin, 100 mg/mL streptomycin, and 2 mmol/L
GlutaMAX.
[0069] Retroviral constructs. The 14g2a scFv sequence was cloned in
the SFG retroviral backbone in frame with the human IgG1-CH2CH3
domain, followed by the CD28 and OX40 endodomains and the
.zeta.-chain of the T-cell receptor/CD3 complex, to form the
14g2a-CD28-OX40-.zeta. (CAR-GD2) construct as previously described
(Pule et al., 2005). Vectors encoding the Firefly Luciferase gene
(FF-Luc.) or the eGFP protein were also used to track cell survival
and proliferation in vivo, as previously described (Savoldo et al.,
2000). The RD114 retrovirus envelope (RDF plasmid) and the MoMLV
gag-pol (PegPam3-e plasmid) were used to engineer the retroviral
vectors.
[0070] Retrovirus production and transduction. Transient retroviral
supernatants were produced by cotransfection of 293T cells with the
PegPam-e, RDF, and the desired SFG vectors (CAR-GD2, eGFP, or
FF-Luc) using the Fugene6 transfection reagent (Roche), and used to
transduce OKT3 (Ortho Biotech) activated PBMCs, as previously
described (Vera et al., 2006).
[0071] The 4405M, CLB, SENMA, and P1143 melanoma cell lines were
transfected with retroviral vectors encoding either eGFP or FF-Luc.
The inventors plated 1.times.10.sup.5 tumor cells in 1 well of a
6-well plate and the cells were grown to 60% to 70% confluency.
Culture medium was replaced by the appropriate retroviral
supernatant (1.5 mL/well), and 1 .mu.g of polybrene was added. When
the tumor cells reached confluency, they were trypsinized and
plated in a T25 flask. The FF-Luc-transduced cells were then
selected with puromycin (Sigma-Aldrich) at 1 .mu.g/mL. The
eGFPtransduced tumor cell lines did not require selection as
>98% of the cells were eGFP-positive postretroviral
transduction.
[0072] Flow cytometry. FITC-, phycoerythrin (PE)-, or periodin
chlorophyll protein (perCP)-conjugated anti-CD4, -CD8, -CD80 and
-CD86 monoclonal antibodies (all from Becton-Dickinson) were used
to label lymphocytes, whereas anti-MCSP-PE (Miltenyi-Biotech Inc.)
and a purified anti-GD2 monoclonal antibody (Becton-Dickinson
Pharmingen) were used to stain the melanoma cells. A secondary
antibody (RAM-IgG2a+b-PE; Becton-Dickinson) was added to detect the
anti-GD2 (IgG2a) antibody by indirect immunofluorescence. CAR
expression by transduced T lymphocytes was detected using a
monoclonal anti-idiotype, 1A7 (TriGem, Titan), followed by staining
with the secondary antibody RAM-IgG1-PE (Becton-Dickinson; Rossig
et al., 2001). The proliferation of nontransduced and transduced T
cells, in the presence or absence of tumor cells, was evaluated by
FACS analysis after labeling T cells with CFSE (Invitrogen)
according to the manufacturer's instructions.
[0073] Cytotoxicity assays. The cytotoxic activity of the
nontransduced and CAR-GD2 T lymphocytes was evaluated in a standard
.sup.51Cr release assay, as previously described (Pule et al.,
2005; Vera et al., 2006). Isotope release was evaluated at 6 and 18
h in cultures with effector-to-target (E:T) ratios of 40:1, 20:1,
10:1, and 5:1, using a gamma counter (Perkin-Elmer).
[0074] Coculture experiments. Seven days after transduction,
nontransduced and CAR-GD2 cells were collected, counted, and plated
at 5.times.10.sup.5 cells/well in a 24-well plate at 20:1 ratio
with eGFP-expressing (>98%+) tumor cells. Cytokine release after
24 h of culture was measured using the CBAarray (BD Bioscience) and
the percent of CD3-positive T cells and eGFP-positive tumor cells
was evaluated by FACS analysis at day 5 of coculture, after
treatment with 0.5% trypsin EDTA (Invitrogen) to detach adherent
cells.
[0075] Xenogeneic SCIDmo use model of melanoma. To assess the in
vivo antitumor activity of the CAR-GD2 T lymphocytes, an exemplary
SCID mouse model was used and the P1143 or 4405 M melanoma line
expressing FF-Luciferase. SCID mice (8 to 9 weeks old) were
sublethally irradiated (250 rad) and injected i.v. with
2.times.10.sup.6 tumor cells. Tumor cell engraftment was monitored
using the IVIS 100 imaging system (Caliper Lifesciences), and on
days 4 and 21, 1.times.10.sup.7 nontransduced or CAR-GD2 T
lymphocytes were injected i.v. The animals were imaged weekly to
evaluate tumor growth, and photon emission from
luciferase-expressing cells was quantified using the "Living Image"
software provided with the IVIS system (Caliper Lifesciences).
Briefly, after drawing a region of interest over the tumor region,
the intensity of the signal measured was expressed as total
photons/s/cm2 (p/s/cm.sup.2/sr).
[0076] Statistical analysis. For cytotoxicity and cytokine
production, results were presented as mean.+-.SD and paired
Student's t test was used to determine statistical significance.
For the bioluminescence results, the signal intensity was
log-transformed and summarized using mean.+-.SD at baseline and
multiple subsequent time points for each group of mice. Changes in
intensity of signal from baseline at each time point were
calculated and compared using paired t-tests or Wilcoxon
signed-ranks test. P<0.05 was considered statistically
significant.
Example 2
Expression of GD2 by Primary Melanoma Cells
[0077] To characterize GD2 as a target for CAR-directed T cell
therapy, primary melanoma cells were dissociated from five patients
after biopsy of cutaneous metastatic melanoma and an additional six
established cell lines were used in a study. Cells from the five
patients and four of the established lines expressed GD2 on
immunofluorescence staining, and between 17% and 95% of the cells
were positive, with variable intensity of expression (FIG. 1).
Expression of GD2 was used on a normal skin fibroblast cell line
and it was confirmed that it did not express the ganglioside. To
confirm the absence of nonneoplastic cells in the primary culture,
expression of MCSP on the cells (Table 1) was examined, and to
compare the frequency of expression of GD2 with that of other known
melanoma tumor associated antigens, theexpression of gp100, MART1,
and MAGE-1 was also measured. The percentage range of GD2-positive
cells was comparable with the range of malignant cells expressing
these three other melanoma-associated antigen cells (Table 1). All
cell populations studied were negative for expression of the
costimulatory molecules CD80 or CD86.
TABLE-US-00002 TABLE 1 Characterization of the melanoma cell lines.
Expression of GD2, MCSP, gp100, MART1 and Mage-1 is presented. MCSP
and GD2 expression was evaluated by FACS analysis and fluorescence
used to determine the expression of gp100, MART1 and Mage-1. GD2
MCSP gp100 MART1 Mage-1 (%/MFI) (%) (%) (%) (%) CLB 19/182 100 99
11 46 SENMA 45/884 100 92 80 98 PLAODE 22/219 100 11 22 3 RR-371952
17/775 100 47 99 1 P1143 95/838 10 100 92 10 18588M 85/162 100 1 3
80 18732M 93/150 100 99 76 26 4405M 0/NA 100 26 9 1 10538P 80/142
100 57 14 62 879M 0/NA 100 59 71 55 8959M 77/71 100 99 32 82
[0078] T cells expressing a GD2-specific chimeric receptor kill
GD2-positive melanoma cell lines. T cells from four healthy donors
were transduced with a vector encoding the 14g2a single-chain
antibody linked to .zeta. and to the endodomains of the two
costimulatory molecules CD28 and OX40, which enhance the
activation, proliferation, and cytotoxicity of T cells produced by
the CAR after antigen engagement (Pule et al., 2005). Five days
after transduction, the expression of GD2-specific CAR was measured
by flow cytometry using the anti-14g2a idiotypic antibody 1A7, and
it was found that 95% of cells transduced with the
14g2a-CD28-OX40-.zeta. retroviral vector were CAR positive (range,
93-97%; FIG. 2A). The CAR-GD2 construct transduced CD4-positve and
CD8-positive T cell populations with equal efficiency (FIG.
2B).
[0079] To mimic the range of GD2 expression seen on primary
melanoma cells, the ability was measured of CAR-GD2 T cells to kill
three melanoma cell lines with different GD2 expression. P1143 was
a high expressor [95% positive, mean fluorescence intensity
(MFI)=838]; SENMA was intermediate (45% positive, MFI=884); CLB was
low (19% positive, MFI=182), and finally, the melanoma cell line
4405 M was used as a GD2-negative tumor cell control. At 6 hours
and 18 hours, .sup.51Cr release assays showed that the antitumor
activity was proportional to the level of GD2 antigen expression
(FIG. 3). As anticipated, the CAR-GD2 T cells had little activity
against the GD2-negative tumor cell line (4405 M), the GD2-NK-cell
target line K562, or against normal skin fibroblasts or PBMCs that
are also GD2 negative (FIG. 7).
[0080] CAR-GD2 T cells were, however, able to kill a mesenchymal
stem cell line positive for GD2 (95% positive, MFI=799; FIG. 7). Of
note, such cross-reactivity with GD2-positive normal mesenchymal
stem cells has not produced discernible adverse effects in any
clinical trial of GD2 monoclonal antibodies in patients with
neuroectodermal tumors (Saleh et al., 1995; Murray et al., 1995) or
in a phase I study of CAR-GD2 T cells in patients with
neuroblastoma (Pule et al., 2008).
[0081] CAR-GD2-expressing T cells secrete cytokines upon
stimulation with GD2-expressing melanoma cells. Functional
activation of CAR-GD2-expressing T cells following their exposure
to GD2-positive melanoma cells was measured by cytokine release
assay. As described above, GD2-positive target cells were killed by
T cells expressing the CAR, and significant interleukin 2 (IL-2),
IL-5, IFN-.gamma., and tumor necrosis factor .alpha. (TNF-.alpha.)
release occurred during coculture with the three melanoma
lines.
[0082] As FIG. 4 shows, the quantity of IL-2, IL-5, IFN-.gamma.,
and TNF-.alpha. secreted by the CAR-GD2 T cells after 24 hours of
culture correlated with the level of GD2 expression on the target
cells, and was highest for P1143 and lowest for CLB. Neither IL-4
nor IL-10 was detected in the supernatants of stimulated cells.
[0083] CAR-GD2 induces sustained killing and clonal expansion in
coculture experiments. It was next determined whether the killing
and cytokine release mediated by CAR-GD2 T cells could lead to
CAR-T cell proliferation and tumor cell eradication in vitro in a
5-day coculture experiment. CFSE-labeled control or CAR-GD2 T cells
were used to determine whether CAR stimulation by
CAR-GD2-expressing T cells induces effector T cell
proliferation.
[0084] Nontransduced T cells proliferated only in the presence of
exogenous IL-2 (100 U/mL), whereas proliferation of CAR-expressing
T cells increased in response to all three GD2-expressing tumor
cell lines, irrespective of whether these tumor cells expressed
high, intermediate, or low levels of GD2 (FIG. 8). To discover if
these expanded CAR-GD2 T cells were functional, tumor cells were
labeled with eGFP and were cocultured at the CAR-T cell:tumor cell
ratio of 20:1 in the absence of IL-2. After 5 days of culture,
viable GFP-positive cells were enumerated by flow cytometric
analysis. FIG. 5 shows that viable tumor cells were eradicated in
cocultures with T cells expressing CARGD2 but not in cocultures
with nontransduced T cells. Hence CAR-GD2 T cells proliferate in
vitro in response to the GD2 antigen and eradicate melanoma cells
that express the antigen. As expected, the GD2-negative cell line
4405 M was not killed in the 5-day coculture experiment, showing
GD2 antigen recognition is essential for the activity of
CAR-GD2-expressing T cells.
[0085] Adoptive transfer of GD2-specific T cells provide antitumor
effect in a xenogeneic SCIDmodel. The antitumor activity was
measured of CAR-GD2 T cells in vivo. To monitor tumor cells in
vivo, the firefly luciferase (FFLuc) gene was expressed in 4405 M
and P1143 cells, together with the puromycinresistance gene. After
puromycin selection, 2.times.10.sup.6 FFLuc-P1143 or 4405 M tumor
cells were injected i.v. into SCID mice.
[0086] After 4 days, FFLuc expression was evaluated by
bioluminescence imaging and the mice were divided into three groups
that received nontransduced T cells or T cells expressing CAR-GD2
at 1.times.10.sup.7 i.v. and finally a group that received tumor
cells alone. A second injection of nontransduced or CAR-GD2 T cells
was given at day 21 and luciferase signal was measured every week
in the 10 mice of each of the groups. FIG. 6A shows five
representative mice from the nontransduced and CAR-GD2 T cells
group, and shows that tumor grew rapidly in the lungs of mice
receiving nontransduced T cells. By contrast, the tumors in mice
receiving T cells expressing CAR-GD2 diminished within 48 to 72
hours of injection, and luciferase derived remained largely absent
in the group receiving CARGD2 T cells. Although the survival of the
mice receiving the tumor cells alone or tumor cells plus
nontransduced T cells was 68.+-.6 days and 72.+-.12 days,
respectively (P=0.03), 80% of the mice from the group receiving
CAR-GD2 T cells were still alive at day 100 and showed a
significant survival advantage when receiving CAR-GD2-specific T
cells (P=0.006; FIG. 6B). Finally, no tumor regression was reserved
when CAR-GD2 T cells were infused in mice bearing GD2-4405 M tumor
cells (FIG. 6A).
Example 3
Significance of Certain Embodiments of the Invention
[0087] In particular aspects of the invention, the ganglioside
antigen GD2 is expressed on the majority of primary melanoma cell
lines, and T cells engineered to express a CAR directed to this
antigen are able to recognize and lyse GD2-positive melanoma target
cells in vitro and in a SCID mouse model in vivo. The transgenic
receptor construct included the signaling endodomains of the CD28
and OX40 costimulatory molecules, and redirected T cells showed
activation, proliferation, and cytokine release after T-cell
receptor engagement by GD2.
[0088] Melanoma has long been a target of cellular immunotherapies
directed to the tumor-associated antigens expressed by the
malignant cells. Although earlier clinical research focused on
reinfusion of expanded tumor infiltrating lymphocytes, efforts have
recently been directed against the cancer testis series of antigens
such as MAGE and the melanocyte differentiation proteins such as
MART-1, by generation of T cells expressing conventional
.alpha..beta.-T-cell receptors specific for these antigens. These
receptors recognize peptide fragments in association with MHC class
I molecules, and are therefore restricted in their patient range to
individuals with the appropriate MHC polymorphism. Moreover, they
are unable to recognize tumor subclones in which the antigen
processing machinery is deficient. T cells that express synthetic
or chimeric receptors that recognize unprocessed structures on the
cell surface may thus have an advantage over T cells whose tumor
reactivity is mediated through their native receptor.
[0089] It has been known for some time that the ganglioside GD2 is
expressed by tumors derived from neuroectoderm, including
neuroblastoma, sarcoma, and small lung cancer. This
tumor-associated carbohydrate antigen is also expressed by many
melanoma cells (Cheresh et al., 1984), in which it is involved in
cell adhesion and may contribute to metastasis (Hakomori, 2001).
Although GD2 is present on the surface of many melanoma cells, it
is absent on most normal tissue, with only limited expression in
brain and on peripheral nerves, making this ganglioside an
attractive target for adoptive cell therapy in metastatic melanoma
(Hersey et al., 1998).
[0090] GD2 monoclonal antibodies have already been used with
benefit in patients with other GD2-positive malignancies, such as
neuroblastoma, but melanoma cells have more variable (and usually
lower) expression of the antigen. Hence, the benefits of GD2
antibody infusion in melanoma have been limited (Saleh et al.,
1992; Murray et al., 1994). The level of GD2 expression on melanoma
cells, however, is evidently sufficient to produce a cytotoxic
response from T cells expressing the same monoclonal antibody
binding site in the form of a chimeric T-cell receptor. There was
complete killing of the tumor cells even when GD2 expression was
low, consistent with previous observations that even tumor cells
with dim expression of the targeted antigen can be eliminated by
CAR-modified T cells (Vera et al., 2006). The killing of cells that
are resistant to antibodies of the same specificity may be related
to the improved avidity of multiple antibody-derived binding
domains when they are arrayed on a cell surface rather than
existing as bivalent molecules in solution, or it may reflect a
superior cytolytic activity of T effector cells compared with
antibody (Weijtens et al., 2007).
[0091] Hence, tumor cells with dim antigen expression can be
completely eliminated after coculture experiments, even when short
term assays based on .sup.51Cr release assay may produce lower
immediate cytotoxicity than tumor cells with high antigen
expression Like most malignancies, melanoma cells lack expression
of the T-cell costimulatory molecules required for complete
activation of T lymphocytes that engage tumor-associated antigens
through their native or chimeric receptors. Hence, to optimize T
cell triggering and effector function, the chimeric receptor was
coupled to co-stimulatory endodomains to increase T cell survival
and expansion. Following CAR engagement, endodomains from single
co-stimulatory molecules, such as CD28, 4-1BB, or OX40, into the
CAR may be sufficient to activate the cellular components of the
killing machinery and to produce IL-2 release and T cell
proliferation (Willemsen et al., 2005; Imai et al., 2004). It has
been previously shown, however, that the simultaneous expression in
cis of two endodomains such as CD28 and OX40 within a GD2-CAR
produces superior T cell proliferation and effector function than
expression of a single costimulatory endodomain (Pule et al.,
2005). In certain embodiments, this benefit occurs because both
CD28 and OX40 signals are both functional, and produce greater
activation of NF-.kappa.B than either endodomain alone, because
they act through two independent pathways (Pule et al., 2005).
[0092] If adoptive transfer of CAR-modified T cells in melanoma is
to be of clinical value, it will be essential to be able to treat
metastatic disease. Accordingly, the effects of the CAR-GD2 T cells
were studied in an exemplary xenograft lung metastatic model. Human
T cells expressing the 14g2a-CD28-OX40-.zeta. CAR produced
significant antitumor activity in this model, but were unable to
completely eradicate the disease. This incomplete benefit may
reflect the difficulties of sustaining human T cell function and
trafficking in a xenogeneic environment, or it may also represent
the limitations of even the combination of CD28 and OX40
endodomains, which on their own cannot completely recapitulate the
temporo-spatial features of the costimulatory events required to
sustain T cell activation physiologically (Pule et al., 2008;
Heemskerk et al., 2004).
[0093] Thus, in aspects of the invention, GD2 on melanoma cells is
a useful target for CAR-T cells. Administration of such cells will
usefully complement other cellular immunotherapies and biotherapies
for this disease, in at least some embodiments.
Example 4
Targeting the Disialo-Ganglioside GD2 in Lung Cancer Using Chimeric
Antigen Receptor (CAR) T Lymphocytes
[0094] FIGS. 9A-9B demonstrate GD2 expression in lung cancer: To
study the expression of GD in lung cancer cell the inventors
performed cytochemistry on a number of lung cancer cytospin
specimens and flow cytometry. FIG. 9A shows expression in small
cell lung cancer, and FIG. 9B shows expression in non-small cell
lung cancer.
[0095] These findings were validated using flow cytometry for both
small cell lung cancer and non-small cell lung cancer (FIG.
10).
[0096] Transduction of T lymphocytes was employed to express GD2
CD28.zeta CAR. The inventors used a retroviral system to transduce
human peripheral blood lymphocytes to express the GD2-specific
third generation CAR molecule with a CD28, OX40 and zeta signaling
domains (FIG. 11). More than 50% transduction rates was
consistently obtained with various donors.
[0097] In FIGS. 12A-12B, GD2 CAR-transduced T lymphocytes recognize
and kill lung cancer cell lines. The inventors used standard 4-6
hour .sup.51Cr release assays to assess the degree of cytolysis of
exemplary lung cancer cell lines at various tumor to T cell ratios.
The neuroblastoma cell line LAN1 was used as a positive control.
Lymphoblastoid cell lines (LCL) were used as the negative control.
FIG. 12A shows killing of small cell lung cancer, and FIG. 12B
shows killing of non-small cell lung cancer.
[0098] GD2 CAR-transduced T lymphocytes secrete immunostimulatory
cytokines in coculture of GD2 positive lung cancer cell lines (FIG.
13). ELISA was performed to detect the cytokine release (IFN.gamma.
and IL-2) 24 to 48 hours after co-culture initiation. GD2
expressing T cells secreted immunostimulatory cytokines upon
encounter of GD2 positive lung cancer cells above non-transduced
controls from the same donor.
REFERENCES
[0099] All patents and publications mentioned in this specification
are indicative of the level of those skilled in the art to which
the invention pertains. All patents and publications herein are
incorporated by reference to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference in their entirety. [0100] Albino A P,
Sozzi G, Nanus D M, Jhanwar S C, Houghton A N. Malignant
transformation of human melanocytes: induction of a complete
melanoma phenotype and genotype. Oncogene 1992; 7:2315-21. [0101]
Bajetta E, Del Vecchio M, Bernard-Marty C, et al. Metastatic
melanoma: chemotherapy. Semin Oncol 2002; 29:427-45. [0102] Butler
M O, Lee J S, Ansen S, et al. Long-lived antitumor CD8+ lymphocytes
for adoptive therapy generated using an artificial
antigen-presenting cell. Clin Cancer Res 2007; 13:1857-67. [0103]
Cheresh D A, Harper J R, Schulz G, Reisfeld R A. Localization of
the gangliosides GD2 and GD3 in adhesion plaques and on the surface
of the human melanoma cells. Proc Natl Acad Sci USA 1984;
81:5767-71. [0104] Gottschalk S, Edwards O L, Sili U, et al.
Genearting CTLs against subdominant Epstein-Barr virus LMP1 antigen
for the adoptive immunotherapy of EBV-associated malignancies.
Blood 2003; 101:1905-12. [0105] Hakomori S. Tumor-associated
carbohydrate antigens defining tumor malignancy: basis for
development of anti-cancer vaccines. Adv Exp Med Biol 2001;
491:369-402. [0106] Haynes N M, Trapani J A, Teng M W, et al.
Single-chain antigen recognition receptors that costimulate potent
rejection of established experimental tumors. Blood 2002;
100:3155-63. [0107] Heemskerk M H M, Hoogeboom M, Hagedoorn R,
Kester M G D, Willemze R, Falkenburg F J H. Reprogramming of
virus-specific T cells into Leukemia-reactive T cells using T cell
receptor gene transfert. J Exp Med 2004; 199:885-94. [0108] Hersey
P, Jamal O, Henderson C, Zardawi I, D'Alessandro G. Expression of
the gangliosides GM3, GD3 and GD2 in tissue sections of normal
skin, naevi, primary and metastatic melanoma. Int J Cancer 1988;
41:336-43. [0109] Imai C, Mihara K, Andreansky M, et al. Chimeric
receptors with 4-1BB signaling capacity provoke potent cytotoxicity
against acute lymphoblastic leukemia. Leukemia 2004; 18:676-84.
[0110] Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2008.
CA Cancer J Clin 2008; 58:71-96. [0111] Livingston P., Ganglioside
vaccines with emphasis on GM2. Semin Oncol 1998; 25:636-45. [0112]
Livingston P O, Wong G Y, Adluri S, et al. Improved survival in
stage III melanoma patients with GM2 antibodies: a randomized trial
of adjuvant vaccination with GM2 ganglioside. J Clin Oncol 1994;
12:1036-44. [0113] Mackensen A, Meidenbauer N, Vogl S, Laumer M,
Berger J, Andreesen R. Phase I study of adoptive T-cell therapy
using antigen-specific CD8+ T cells for the treatment of patients
with metastatic melanoma. J Clin Oncol 2006; 24:5060-9. [0114]
Morgan R A, Dudley M E, Wunderlich J R, et al. Cancer regression in
patients after transfer of genetically engineered lymphocytes.
Science 2006; 314:126-9. [0115] Murray J L, Cunningham J E, Brewer
H, et al. Phase I trial of murine monoclonal antibody 14G2a
administered by prolonged intravenous infusion in patients with
neuroectodermal tumors. J Clin Oncol 1994; 12:184-93. [0116]
Ohnmacht G A, Marincola F M. Heterogeneity in expression of human
leukocyte antigens and melanoma-associated antigens in advanced
melanoma. J Cell Physiol 2000; 182:332-8. [0117] Pule M, Finney H,
Lawson A. Artificial T-cell receptors. Cytotherapy 2003; 5:211-26.
[0118] Pule M A, Savoldo B, Myers G D, et al. Virus-specific T
cells engineered to coexpress tumor-specific receptors: persistence
and antitumor activity in neuroblastoma patients. Nat Med 2008;
14:1264-70. [0119] Pule M A, Straathof K C, Dotti G, Heslop H E,
Rooney C M, Brenner M K. A chimeric T cell antigen receptor that
augments cytokine release and supports clonal expansion of primary
human T cells. Mol Ther 2005; 12:933-41. [0120] Ragupathi G,
Livingston P O, Hood C, et al. Consistent antibody response against
ganglioside GD2 induced in patients with melanoma by a GD2
lactone-keyhole limpet hemocyanin conjugate vaccine plus
immunological adjuvant QS-21. Clin Cancer Res 2003; 9:5214-20.
[0121] Ravindranath M H, Muthugounder S, Presser N. Ganglioside
signatures of primary and nodal metastatic melanoma cell lines from
the same patient. Melanoma Res 2008; 18:47-55. [0122] Rosenberg S
A, Dudley M E. Cancer regression in patients with metastatic
melanoma after the transfer of autologous antitumor lymphocytes.
Proc Natl Acad Sci USA 2004; 101:14639-45. [0123] Rosenberg S A,
Restifo N P, Yang J C, Morgan R A, Dudley M E. Adoptive cell
transfer: a clinical path to effective cancer immunotherapy. Nat
Rev Cancer 2008; 8:299-308. [0124] Rossig C, Bollard C M, Nuchtern
J G. Targeting of G(D2)-positive tumor cells by human T lymphocytes
engineered to express chimeric T-cell receptor genes. Int J Cancer
2001; 94:228-36. [0125] Saleh M N, Khazaeli M B, Wheeler R H, et
al. Phase I trial of the murine monoclonal anti-GD2 antibody 14G2a
in metastatic melanoma. Cancer Res 1992; 52:4342-7. [0126] Savoldo
B, Rooney C M, Di Stasi A, et al. Epstein Barr virus specific
cytotoxic T lymphocytes expressing the anti-CD30 artificial
chimeric T-cell receptor for immunotherapy of Hodgkin disease.
Blood 2007; 110:2620-30. [0127] Tsuchida T, Sazton R E, Morton D L,
Irie R F. Gangliosides of human melanoma. Cancer 1989; 63:1166-74.
[0128] Vera J, Savoldo B, Vigouroux S, et al. T lymphocytes
redirected against the .kappa. light chain of human immunoglobulin
efficiently kill mature B lymphocyte-derived malignant cells. Blood
2006; 108:3890-7. [0129] Weijtens M E, Hart E H, Bolhuis R L.
Functional balance between T cell chimeric receptor density and
tumor associated antigen density: CTL mediated cytolysis and
lymphokine production. Gene Ther 2000; 7:35-42. [0130] Willemsen R
A, Ronteltap C, Chames P, Debets R, Bolhuis R L H. T cell
retargeting with MHC class I-restricted antibodies: the CD28
costimulatory domain enhances antigen-specific cytotoxicity and
cytokine production. J Immunol 2005; 174:7853-8. [0131] Yun C O,
Nolan K F, Beecham E J, Reisfeld R A, Junghans P. Targeting of T
lymphocytes to melanoma cells through chimeric anti-GD3
immunoglobulin T-cell receptors. Neoplasia 2000; 2:449-59. [0132]
Yvon E S, Vigouroux S, Rousseau R F, et al. Overexpression of the
Notch ligand, Jagged-1, induces alloantigen-specific human
regulatory T cells. Blood 2003; 102:3815-21.
[0133] Although the present invention and its advantages have been
described in detail, it should be understood that various changes,
substitutions and alterations can be made herein without departing
from the invention as defined by the appended claims. Moreover, the
scope of the present application is not intended to be limited to
the particular embodiments of the process, machine, manufacture,
composition of matter, means, methods and steps described in the
specification. As one will readily appreciate from the 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. Accordingly, the appended claims are intended to
include within their scope such processes, machines, manufacture,
compositions of matter, means, methods, or steps.
* * * * *