U.S. patent application number 15/038997 was filed with the patent office on 2016-12-29 for csgp4 - specific chimeric antigen receptor for cancer.
The applicant listed for this patent is BAYLOR COLLEGE OF MEDICINE. Invention is credited to Gianpietro Dotti, Soldano Ferrone.
Application Number | 20160376375 15/038997 |
Document ID | / |
Family ID | 52101600 |
Filed Date | 2016-12-29 |
View All Diagrams
United States Patent
Application |
20160376375 |
Kind Code |
A1 |
Dotti; Gianpietro ; et
al. |
December 29, 2016 |
CSGP4 - Specific Chimeric Antigen Receptor for Cancer
Abstract
Embodiments of the disclosure include methods and compositions
related to chimeric antigen receptors (CAR) that target chondroitin
sulfate proteoglycan-4 (CSPG4). T cells transduced with a
CSPG4-specific CAR are effective for inhibition of particular
cancer cells that express CSPG4. In certain embodiments, the cancer
is melanoma, breast cancer, head and neck cancer, mesothelioma,
glioblastoma, or renal cancer.
Inventors: |
Dotti; Gianpietro; (Chapel
Hill, NC) ; Ferrone; Soldano; (Boston, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BAYLOR COLLEGE OF MEDICINE |
Houston |
TX |
US |
|
|
Family ID: |
52101600 |
Appl. No.: |
15/038997 |
Filed: |
November 21, 2014 |
PCT Filed: |
November 21, 2014 |
PCT NO: |
PCT/US14/66953 |
371 Date: |
May 24, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61909788 |
Nov 27, 2013 |
|
|
|
Current U.S.
Class: |
424/134.1 |
Current CPC
Class: |
A61P 35/00 20180101;
C07K 2319/30 20130101; C07K 2319/03 20130101; C07K 14/70517
20130101; C07K 16/3076 20130101; C07K 14/7051 20130101; C07K
14/70521 20130101; C07K 14/70578 20130101; A61K 35/17 20130101;
C07K 2319/01 20130101; C07K 2317/622 20130101; C07K 16/30 20130101;
C07K 14/70596 20130101; C07K 14/70575 20130101; A61K 2039/505
20130101; C07K 2317/53 20130101 |
International
Class: |
C07K 16/30 20060101
C07K016/30; A61K 35/17 20060101 A61K035/17; C07K 14/705 20060101
C07K014/705 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under P30
CA125123 awarded by the National Cancer Institute and under CA
138188 awarded by the National Institutes of Health. The government
has certain rights in the invention.
Claims
1. A method of inhibiting proliferation of cancer cells, comprising
the step of contacting the cancer cells with a therapeutically
effective amount of immune cells that express a chimeric antigen
receptor (CAR) that targets chondroitin sulfate proteoglycan-4
(CSPG4), wherein the cancer is not melanoma.
2. The method of claim 1, wherein the cancer is head and neck
cancer, mesothelioma, breast cancer, glioblastoma, or renal
cancer
3. The method of claim 1, wherein the cancer is a sarcoma.
4. The method of claim 1, wherein said contacting is performed in
vitro.
5. The method of claim 1, wherein said contacting is performed in
cell culture.
6. The method of claim 1, wherein said contacting is performed in
vivo, and said immune cells are cells in an individual.
7. The method of claim 1, wherein said contacting is performed in
vivo, and said immune cells are T cells in an individual.
8. The method of claim 5, wherein said immune cells are autologous
to the individual.
9. The method of claim 5, wherein said immune cells are allogeneic
to the individual.
10. The method of claim 1, wherein said immune cells are T cells,
NK cells, dendritic cells, or a mixture thereof.
11. The method of claim 1, wherein said immune cells are T
cells.
12. The method of claim 10, wherein said T cells are CD4+ T
cells.
13. The method of claim 10, wherein said T cells are CD8+ T
cells.
14. The method of claim 10, wherein said T cells are Treg
cells.
15. The method of claim 1, wherein the CAR comprises a
transmembrane domain selected from the group consisting of
CD3-zeta, CD28, CD8.alpha., CD4, or a combination thereof.
16. The method of claim 1, wherein the CAR comprises a
co-stimulatory molecule endodomain selected from the group
consisting of CD28, CD27, 4-1BB, OX40 ICOS, and a combination
thereof.
17. The method of claim 4, wherein the individual has received, is
receiving, or will receive an additional cancer treatment.
18. The method of claim 15, wherein the additional cancer treatment
comprises chemotherapy, immunotherapy, radiation, surgery, hormone
therapy, or a combination thereof.
19. The method of claim 1, wherein the immune cells harbor a
polynucleotide that encodes the CAR.
20. The method of claim 17, wherein the polynucleotide further
comprises a suicide gene.
21. A method of inhibiting proliferation of cancer cells,
comprising the step of contacting the cancer cells with a
therapeutically effective amount of immune cells that express a
chimeric antigen receptor (CAR) that targets chondroitin sulfate
proteoglycan-4 (CSPG4), wherein the CAR comprises a scFv antibody
that is not derived from mAb 225.28S.
22. A method of inhibiting proliferation of cancer cells,
comprising the step of contacting the cancer cells with a
therapeutically effective amount of immune cells that express a
chimeric antigen receptor (CAR) that targets chondroitin sulfate
proteoglycan-4 (CSPG4), wherein the CAR comprises a scFv 763.74
antibody.
23. A method of inhibiting proliferation of cancer cells,
comprising the step of contacting the cancer cells with a
therapeutically effective amount of immune cells that express a
chimeric antigen receptor (CAR) that targets chondroitin sulfate
proteoglycan-4 (CSPG4), wherein the CAR comprises part or all of
the IgG1 hinge.
24. The method of claim 23, wherein the CAR further comprises the
IgG1 C.sub.H2 C.sub.H3 domain.
25. The method of claim 23, wherein the CAR comprises the CD28
transmembrane domain.
26. The method of claim 23, wherein the CAR comprises CD28
endodomain or 4-1BB endodomain.
27. The method of claim 23, wherein the CAR does not comprise the
IgG1 C.sub.H2 C.sub.H3 domain.
28. The method of claim 23, wherein the CAR comprises the hinge of
IgG1, CD8 alpha transmembrane domain, and one of CD28 endodomain or
4-1BB endodomain.
29. The method of claim 23, wherein the CAR comprises the hinge of
IgG1, CD8 alpha transmembrane domain, CD28 endodomain, and 4-1BB
endodomain.
30. The method of claim 23, wherein the cancer is not melanoma or
is not ovarian cancer or is not triple negative breast cancer.
31. The method of claim 23, wherein the cancer is head and neck
cancer, mesothelioma, glioblastoma, or renal cancer.
32. A method of inhibiting proliferation of cancer cells,
comprising the step of contacting the cancer cells with a
therapeutically effective amount of immune cells that express a
chimeric antigen receptor (CAR) that targets chondroitin sulfate
proteoglycan-4 (CSPG4), wherein the CAR comprises the entire CD8a
alpha stalk, CD8a hinge, CD8a transmembrane domain, and one of CD28
endodomain or 4-1BB endodomain.
33. The method of claim 30, wherein the CAR comprises the entire
CD8a alpha stalk, CD8a hinge, CD8a transmembrane domain, CD28
endodomain, and 4-1BB endodomain.
34. The method of claim 23, wherein the cancer is not melanoma or
is not ovarian cancer or is not triple negative breast cancer.
35. The method of claim 23, wherein the cancer is head and neck
cancer, mesothelioma, glioblastoma, or renal cancer.
Description
[0001] This application claims priority to U.S. Provisional Patent
Application Ser. No. 61/909,788, filed Nov. 27, 2013, which is
incorporated by reference herein in its entirety.
TECHNICAL FIELD
[0003] The fields of embodiments of the disclosure include at least
cell biology, molecular biology, immunology, and medicine,
including cancer medicine.
BACKGROUND
[0004] Chondroitin sulfate proteoglycan-4 (CSPG4), also known as
high molecular weight-melanoma associated antigen (HMW) and
melanoma-associated chondroitin sulfate proteoglycan (MCSP), is a
well characterized cell surface proteoglycan first identified on
human melanoma cells. Subsequent studies showed it to be highly
expressed on other solid tumors such as mesothelioma and triple
negative breast carcinoma, all of which often show an aggressive
clinical course. In contrast, CSPG4 has a restricted distribution
in normal tissues. CSPG4 participates in tumor migration, invasion,
angiogenesis, and metastasis. It interacts with .alpha.4.beta.1
integrins to directly modulate cell adhesion, motility and
metastasis, as demonstrated by its ectopic expression in tumor
cells. Given its restricted expression in normal tissues, high
expression on various types of solid tumors and its role in the
biology of tumor cells, CSPG4 is an attractive target for
immunotherapy.
[0005] CSPG4 has been targeted with monoclonal antibodies (mAbs) in
models of melanoma, mesothelioma, and breast carcinoma, resulting
in the inhibition of tumor growth and survival in addition to
thwarting the metastatic capability of tumor cells. Recent advances
in potentiating the antitumor effects of a specific mAb rely on
coupling its antigen-binding specificity with the effector function
and long-term persistence of T lymphocytes to generate specific
chimeric antigen receptors (CARs). These molecules are obtained by
fusing the extracellular antigen-binding domain of the mAb with the
intracellular signaling domains derived from the CD3-.zeta. chain
of the T-cell receptor, in tandem to costimulatory endodomains to
support survival and proliferative signals. Because CAR-modified T
cells function independently of a patient's MHC and can readily be
generated for clinical use, the targeting of CSPG4 with a CAR
based-approach is useful.
[0006] The present embodiments provide a solution to a long-felt
need in the art to provide effective methods and/or compositions
for the treatment of particular cancers.
BRIEF SUMMARY
[0007] The present embodiments are directed to methods and/or
compositions for the treatment of cancer. In particular cases, the
disclosure concerns methods and/or compositions for the treatment
of cancers in which the cancer cells comprise CSPG4 as a tumor
antigen. Although in certain aspects the cancer may be of any kind,
in particular cases the cancer is melanoma, breast cancer, head and
neck cancer, mesothelioma, glioblastoma, or renal cancer. In
specific embodiments, the cancer comprises solid tumors. In at
least some cases, the cancer is not melanoma. In specific
embodiments, the cancer is not breast cancer, such as not being
triple negative breast cancer, for example. In a certain
embodiment, the cancer is not ovarian cancer.
[0008] Embodiments of the disclosure encompass immune cells that
express a CSPG4-targeting chimeric antigen receptor (CAR). In
certain aspects, the CAR comprises a scFv specific for CSPG4. In
specific embodiments, the scFv is not derived from the murine mAb
225.28S. In particular cases, the antibody is scFv 763.74 or any
other commercially-available or otherwise available anti-CSPG4
antibodies. In certain embodiments, the antibody is not scFv 763.74
or is not derived from its respective monoclonal antibody. In
particular embodiments, the CAR utilizes an scFv specific for CSPG4
that is known in the art, although in other embodiments, the CAR
does not utilize an scFv specific for CSPG4 that is known in the
art. In certain embodiments, the CAR utilizes an scFv specific for
CSPG4 that is derived from a monoclonal antibody known in the art,
whereas in other cases the CAR utilizes an scFv specific for CSPG4
that is not derived from a monoclonal antibody known in the
art.
[0009] The CAR may include one or more costimulatory endodomains,
such as CD28, CD27, 4-1BB, OX40, ICOS, or a combination thereof.
The CAR may include one or more transmembrane domains, such as one
selected from the group consisting of CD3-zeta, CD28, CD8.alpha.,
CD4, or a combination thereof. In some embodiments, the immune
cells are one of T cells, NK cells, dendritic cells, or a mixture
thereof. In certain aspects, T cells redirected against CSPG4
control the growth of CSPG4-expressing cancers, either in vitro or
in vivo, e.g., in an individual having a cancer comprising tumor
cells that express CSPG4. The cells are effective against multiple
solid tumors, in particular embodiments.
[0010] As described in detail herein, the expression of CSPG4 was
validated in an extensive panel of tumor arrays and normal tissues
and in gene expression profiling datasets. A CSPG4-specific CAR
(referred to as CAR.CSPG4) was generated that showed that when it
was expressed by T cells, melanoma was effectively targeted in
vitro, and antitumor activity was observed in vitro and in vivo
against many solid tumors including breast carcinoma, head and neck
squamous cell carcinoma (HNSCC) and mesothelioma, for example.
Redirecting T cells to CSPG4 using CARs thus represents a robust
platform to target multiple types of solid tumors.
[0011] Because CSPG4 protein is expressed by several solid tumors
including melanoma, breast cancer, HNSCC and mesothelioma, and
because CSPG4 mRNA is detected in other tumors such as
glioblastoma, renal cell carcinoma and at least some type of
sarcomas, this antigen is an optimal target for adoptive T-cell
immunotherapy based on CSPG4-CAR-redirected T cells. In addition,
the lack of significant expression of CSPG4 in normal tissues
further highlights its relevance for immunotherapy.
[0012] In one embodiment, there is a method of inhibiting
proliferation of cancer cells, comprising the step of contacting
the cancer cells with a therapeutically effective amount of immune
cells that express a chimeric antigen receptor (CAR) that targets
chondroitin sulfate proteoglycan-4 (CSPG4), wherein the cancer is
not melanoma. In specific embodiments, the cancer is head and neck
cancer, mesothelioma, breast cancer, glioblastoma, or renal cancer.
The cancer may be a sarcoma. In some embodiments, the contacting is
performed in vitro, in cell culture, or in vivo. In particular
cases, the contacting is performed in vivo, and the immune cells
are cells in an individual, such as T cells. In particular cases,
the immune cells are autologous or allogeneic to the individual. In
particular embodiments, the immune cells are T cells, NK cells,
dendritic cells, or a mixture thereof. The T cells may be CD4+ T
cells or CD8+ T cells or Treg cells. The immune cells may harbor a
polynucleotide that encodes the CAR, and the polynucleotide may
further comprise a suicide gene.
[0013] In some embodiments, the CAR comprises a transmembrane
domain selected from the group consisting of CD3-zeta, CD28,
CD8.alpha., CD4, or a combination thereof. In particular
embodiments, the CAR comprises a co-stimulatory molecule endodomain
selected from the group consisting of CD28, CD27, 4-1BB, OX40 ICOS,
and a combination thereof.
[0014] In specific methods of the disclosure, an individual
subjected to the methods has received, is receiving, or will
receive an additional cancer treatment, such as chemotherapy,
immunotherapy, radiation, surgery, hormone therapy, or a
combination thereof.
[0015] In one embodiment, there is a method of inhibiting
proliferation of cancer cells, comprising the step of contacting
the cancer cells with a therapeutically effective amount of immune
cells that express a chimeric antigen receptor (CAR) that targets
chondroitin sulfate proteoglycan-4 (CSPG4), wherein the CAR
comprises a scFv antibody that is not derived from mAb 225.28S.
[0016] In a particular embodiment, there is a method of inhibiting
proliferation of cancer cells, comprising the step of contacting
the cancer cells with a therapeutically effective amount of immune
cells that express a chimeric antigen receptor (CAR) that targets
chondroitin sulfate proteoglycan-4 (CSPG4), wherein the CAR
comprises a scFv 763.74 antibody.
[0017] In embodiments, one or more structural components of a
CSPG4-specific CAR are contemplated herein. In specific
embodiments, one can alter the length of the hinge between the
V.sub.H and V.sub.L domains; short hinges and long hinges may be
utilized. In specific embodiments, the hinge is between about 10 to
25 amino acids. In specific embodiments, a full hinge is employed.
In certain embodiments, the hinge is rich in glycine, such as for
flexibility, and/or is rich in serine and/or threonine, such as for
solubility. In particular embodiments, the hinge can either connect
the N-terminus of the V.sub.H with the C-terminus of the V.sub.L,
or vice versa. In certain embodiments, part or all of the hinge of
IgG1 is employed. Optimization may occur in vitro and/or in vivo in
NSG tumor-bearing mice, for example.
[0018] In some embodiments, one can optimize the transmembrane
domain of the CAR. In specific embodiments, the transmembrane is
derived from that of CD28 or. 4-1BB or CD8 alpha, for example. In
other embodiment, one can optimize the number and kind of
co-stimulatory endodomains; in specific cases, CD28 co-stimulatory
endodomain, 4-1BB co-stimulatory endodomain, or both are
employed.
[0019] Examples of certain combinations of structural components of
a CSPG4-specific CAR are as follows: 1) full hinge of IgG1 and
IgG1.C.sub.H2 C.sub.H3 domain with CD28 transmembrane domain and
CD28 or 4-1BB endodomains; 2) only hinge of IgG1 (in the absence of
the IgG1C.sub.H2 C.sub.H3 domain) with CD28 transmembrane domain
and CD28 or 4-1BB endodomains; 3) only hinge of IgG1 (in the
absence of the IgG1CH2CH3 domain) with CD8a alpha transmembrane
domain and CD28 or 4-1BB endodomains; 4) full CD8a alpha stalk
including hinge and transmembrane domain and CD28 or 4-1BB
endodomains; and 5) all of these combinations plus CD28 and 4-1BB
endodomains to make third generation CARs.
[0020] Additional features and advantages of the invention will be
described hereinafter which form the subject of the claims of the
invention. It should be appreciated by those skilled in the art
that the conception and specific embodiment disclosed may be
readily utilized as a basis for modifying or designing other
structures for carrying out the same purposes of the present
invention. It should also be realized by those skilled in the art
that such equivalent constructions do not depart from the spirit
and scope of the invention as set forth in the appended claims. The
novel features which are believed to be characteristic of the
invention, both as to its organization and method of operation,
together with further objects and advantages will be better
understood from the following description when considered in
connection with the accompanying figures. It is to be expressly
understood, however, that each of the figures is provided for the
purpose of illustration and description only and is not intended as
a definition of the limits of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] For a more complete understanding of the present invention,
reference is now made to the following descriptions taken in
conjunction with the accompanying drawing, in which:
[0022] FIG. 1. CSPG4 expression in primary solid tumors and
tumor-derived cell lines. Panel A. Representative
immunohistochemistry (IHC) and scoring of analyzed solid tumor
tissue arrays. Representative melanoma and breast carcinoma samples
are shown at 200.times. magnification. Panel B. Scoring summary of
a panel of solid tumors which includes melanoma, breast carcinoma
(Breast CA), HNSCC, neuroblastoma (NeuroB) and mesothelioma
(MesoT). Panel C. CSPG4 mRNA expression by TCGA in a variety of
solid tumors. Box plots show median, 25%/75% range, 5%/95% range,
and minimum/maximum. Panel D. CSPG4 mRNA expression analysis,
comparing tumor versus corresponding normal tissues, for
astrocytoma/glioblastoma (GBM), HNSCC, clear cell renal carcinoma,
and melanoma. Indicated P-values were calculated by t-test. Panel
E. CSPG4 expression in the indicated array of melanoma cell lines
as assessed by flow cytometry (FACS). Panel F. FACS analysis of
CSPG4 expression in the selected mesothelioma (MILL and PHI), HNSCC
(PCI-30), and breast cancer-derived (MDA-MB-231 and UACC-812) cells
lines. Dotted and bold lines indicate isotype and CSPG4 mAbs,
respectively.
[0023] FIG. 2. Expression and function of CAR.CSPG4 in T cells.
Panel A. Schematic representation of a retroviral vector encoding
an example of a CSPG4-specific CAR. The CAR incorporates the CD28
costimulatory endodomain. Panel B. Representative FACS analysis
showing the expression of the CAR in CD3, CD4 and CD8 T cells after
retroviral transduction. Panel C. Representative expression of the
CD62L, CD45RO, and CCR7 markers on control and CAR.CSPG4+ T cells
by flow cytometry on day 14 of culture. Numbers represent
percentages of cells per quadrant.
[0024] FIG. 3. Cytotoxic function of CAR.CSPG4+ T cells against
CSPG4+ tumors but not against epithelial cells from lung, kidney
and prostate. Panel A. Cytotoxic activity of control T cells and
CAR.CSPG4.sup.+ T cells evaluated in a 6 hour .sup.51Cr release
assay. Target cells used were the CSPG4.sup.+ tumor cell line
(SENMA), CSPG4- target cell line (P1143) and K562 to quantify
natural killer activity. Data show averages and SD results of T
cells from 4 donors. Panel B. Co-culture experiments of control and
CAR.CSPG4.sup.+ T cells with GFP+ tumor cell lines, assessed by
flow cytometry 72 hours later. The plots describe a representative
experiment of T cells co-cultured with SENMA (CSPG4.sup.+ target)
or P1143 (CSPG4.sup.- target). Numbers represent percentages of
cells per quadrant. Panel C. Summary of co-culture experiments of
control and CAR.CSPG4.sup.+ T cells against a panel of CSPG4.sup.+
tumor targets. Data represent averages.+-.SD of 4 donors.
*=p<0.05, and ***=p<0.001. Panel D. FACS analysis of CSPG4
expression in primary epithelial cells derived from normal small
airway, kidney and prostate. Dotted and bold lines indicate isotype
and CSPG4 mAbs, respectively Panel E. Cytotoxic activity of control
T cells and CAR.CSPG4.sup.+ T cells from a representative donor of
two independent experiments evaluated in a 5 hour .sup.51Cr release
assay against these normal epithelial cells.
[0025] FIG. 4. T lymphocytes transduced with CAR.CSPG4 proliferate
and release IL-2 and IFN.gamma. upon specific antigen engagement.
Panel A. Control and CAR.CSPG4.sup.+ T cells, labeled with CFSE,
were stimulated with irradiated CSPG4.sup.+ (SENMA) tumor target.
The panel illustrates the CFSE dilution in CD4 or CD8 gated T cells
after 96 hours of culture for a representative donor. Panel B.
Summary of 3 independent CFSE dilution assays. Data represents
mean.+-.SD. Panel C. IL-2 cytokine-release assessment using
specific ELISA by T lymphocytes transduced with CAR.CSPG4 and
control T cells 24 hours post co-culture (E:T ratio 5:1) with
either CSPG4.sup.+ tumors or CSPG4.sup.- target cells (P1143).
Results of 5 experiments are presented with mean.+-.SD. Panel D
illustrates the detection of IFN.gamma. in the same culture
supernatant. Results of 5 experiments with mean.+-.SD are shown.
*=P<0.05, **=p<0.01, and ***=p<0.001.
[0026] FIG. 5. CAR.CSPG4+ T lymphocytes control tumor growth in
vivo. Panels show tumor growth, assessed by caliper measurement, of
NSG mice engrafted subcutaneously with melanoma (SENMA) (Panel A),
HNSCC (PCI-30), (Panel B) or breast carcinoma (UACC-812) (Panel C)
cell lines and infused i.v. with either CAR.CSPG4.sup.+ (closed
squares) or control (closed circles) T lymphocytes. Arrows indicate
T-cell infusions. Shown are mean.+-.SD from 15 mice per group (3
independent experiments) for the melanoma model and 10 mice per
group (2 independent experiments) for the HNSCC model and breast
carcinoma models. ***=p<0.001.
[0027] FIG. 6. Immunohistochemistry of normal tissue arrays.
Representative immunohistochemistry of normal tissue arrays.
Endometrium, lung, skeletal muscle, nerve, skin, and a lung
carcinoma are shown. 200.times. magnification.
[0028] FIG. 7. CSPG4 mRNA expression levels in normal tissues. Top
panel, CSPG4 expression across normal human tissues represented in
the Novartis GeneAtlas dataset (http://biogps.org). Values
represented are relative and the average of Affymetrix probe sets
214297_at and 204736_s_at. Highlighted are normal tissues for which
corresponding normal adjacent tissue data were available in TCGA
datasets. Bottom panel, CSPG4 mRNA expression by TCGA in a variety
of solid tumors (featured in main FIG. 1D) and normal adjacent
tissues (the normal tissues being also represented in GeneAtlas),
with the cancers showing higher CSPG4 levels over that of the
normal samples. Note shown is log 2 scale.
[0029] FIG. 8. Comparison of CAR.CSPG4+ and CAR.CD19+ T
lymphocytes. Panel A. Expression of CAR.CD19 and CAR.CSPG4 in T
lymphocytes after retroviral transduction, as assessed by FACS.
Panel B. Co-culture experiments in which CAR.CSPG4+ T cells,
CAR.CD19+ T cells, and control (non-transduced) cells were cultured
with GFP+ tumor cell lines. Pictures illustrate the elimination of
CSPG4+eGFP+ target (SENMA) by CAR.CSPG4+ T cells and not by
CAR.CD19.sup.+ T cells or control T cells after 72 hours of
co-culture. The CSPG4= cell lines P1143 was not targeted by either
CAR.CSPG4+ T cells or CAR.CD19.sup.+ T cells. Panel C.
Proliferation of control (non-transduced), CAR.CD19.sup.+, and
CAR.CSPG4.sup.+ T cells upon stimulation with CSPG4+ target in a 96
hour CFSE dilution assay. Data are representative of three
independent experiments using irradiated SENMA tumor cells. Panel
D. Control (non-transduced), CAR.CD19.sup.+, and CAR.CSPG4.sup.+ T
cells were evaluated in a 6 hour cytotoxicity assay against SENMA
(CSPG4.sup.+), K562 (natural killer cell target), and P1143
(CSPG4.sup.-). Data represents the average of three experiments
with mean.+-.SD.
[0030] FIG. 9. Expansion and antitumor activity of T cells
expressing a second versus a third generation CAR.CSPG4. Panel A
illustrates the expansion of control, CAR.CSPG4 (2nd gen, CD28
costimulation), and CAR.CSPG4 (3rd gen, CD28/4-1BB costimulation)
within 14 days of culture. Data represents the average of 4 T-cell
lines with mean.+-.SD. Panel B. Summary of co-culture experiments
of anti-tumor activity of control, CAR.CSPG4.sup.+ T cells either
2nd or 3rd generation against a panel of CSPG4.sup.+ tumor targets.
Data represent averages of 3 donors with mean.+-.SD. *=p<0.05,
and ***=p<0.001.
[0031] FIG. 10. Immunohistochemistry, flow cytometry and survival
curve analysis of the melanoma xenograft model. Panel A illustrates
the expression of CSPG4 in tumor samples (SENMA) isolated 30 days
after infusion of either CAR.CSPG4.sup.+ T cells or control T
cells. Panel B. FACs analysis to detect tumor cells and CD3+ T
cells in tumor biopsy, blood and spleen collected from mice at day
30 post tumor (SENMA) injection. Panel C. Survival curve of the
melanoma xenograft model. Data are representative of 15 control and
treated mice. Log-rank test: p<0.0001.
DETAILED DESCRIPTION
[0032] As used herein the specification, "a" or "an" may mean one
or more. As used herein in the claim(s), when used in conjunction
with the word "comprising", the words "a" or "an" may mean one or
more than one. As used herein "another" may mean at least a second
or more. In specific embodiments, aspects of the subject matter may
"consist essentially of" or "consist of" one or more elements or
steps of the subject matter, for example. Some embodiments of the
subject matter may consist of or consist essentially of one or more
elements, method steps, and/or methods of the subject matter. It is
contemplated that any method or composition described herein can be
implemented with respect to any other method or composition
described herein.
I. General Embodiments
[0033] Adoptive transfer of CAR-redirected T lymphocytes represents
a useful therapy for patients with malignancies. Here the
applicability of this strategy is extended to a broad array of
solid tumors by targeting the CSPG4 antigen; this antigen is
over-expressed by numerous tumor types while having negligible
expression in normal tissues. The disclosure provides evidence that
CSPG4-redirected T cells can control the growth of at least human
melanoma, HNSCC and breast cancer both ex vivo and in vivo in
xenograft models.
[0034] Particular aspects of the disclosure include methods of
treating CSPG4-expressing cancers. The cancers may be head and neck
cancer, mesothelioma, breast cancer, glioblastoma, or renal cancer,
for example, and in at least some cases the cancer is not melanoma.
In specific embodiments of the disclosure, a particular scFv for
CSPG4 is employed, and in particular cases the CSPG4 scFv is not
derived from the murine mAb 225.28S. In at least one specific case,
the methods are for treating CSPG4-expressing cancer that is not
melanoma and wherein the CSPG4 CAR does not employ a scFv derived
from the murine mAb 225.28S. In at least a specific case, the
methods are for treating CSPG4-expressing cancer that is not
melanoma and wherein the CSPG4 CAR employ scFv 763.74.
[0035] Indicia of successful treatment could be, e.g., detectable
reduction in the growth of a tumor (e.g., as seen by MRI or the
like), or reduction in one or more symptoms of a cancer that
expresses CSPG4, for example.
II. Chondroitin Sulfate Proteoglycan 4
[0036] Embodiments of the disclosure use methods and/or
compositions that include targeting of Chondroitin Sulfate
Proteoglycan 4 (CSPG4) on a cancer cell.
[0037] A skilled artisan recognizes that Chondroitin Sulfate
Proteoglycan 4 has multiple names, including at least
Melanoma-Associated Chondroitin Sulfate Proteoglycan; Chondroitin
Sulfate Proteoglycan 4 (Melanoma-Associated); Chondroitin Sulfate
Proteoglycan NG2; Melanoma Chondroitin Sulfate Proteoglycan; MCSP;
MCSPG; MSK16; NG2; High molecular weight-melanoma associated
antigen (HMW-MAA); MEL-CSPG; EC 2.7.8; and EC 3.6.3. An example of
a CSPG4 nucleic acid sequence is provided under the National Center
for Biotechnology Institute's GenBank.RTM. accession number
NM_001897, which is incorporated by reference herein in its
entirety. An example of a CSPG4 amino acid sequence is provided
under the National Center for Biotechnology Institute's
GenBank.RTM. accession number NP_001888, which is incorporated by
reference herein in its entirety.
III. Chimeric Antigen Receptors
[0038] 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. In
certain embodiments of the invention, there are immune cells that
are modified to comprise a CAR that targets CSPG4. In specific
embodiments, the immune cells are T cells, NKT cells, or NK
cells.
[0039] In particular cases, immune cells include a CAR receptor
that 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 immune cell to the
CSPG4-comprising cancer cell. In specific embodiments, the CAR
comprises an antibody for CSPG4, part or all of one or more
cytoplasmic signaling domains, 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).
[0040] In particular embodiments, the scFv is a particular scFv for
CSPG4. In specific cases, the scFv for CSPG4 is scFv 763.74. The
nucleotide sequence of scFv 763.74 is as follows:
TABLE-US-00001 (SEQ ID NO: 1)
ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGG
TGTCCAGTGCTCTAGAATGGCCCAGGTCAAACTGAAGGAGTCTGGAC
CTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCT
TCTGGTTATACCTTCACAGACTATTCAATGCACTGGGTGAAGAAGAC
TCCAGGAAAGGGTTTAAAGTGGCTGGGCTGGATAAACACTGCGACTG
GTGAGCCAACATATGCAGATGACTTCAAGGGACGGTTTGCCATCTCT
TTGGAAACCTCTGCCAGGACTGTCTATTTGCAGATCAATAATCTCAG
AAATGAGGACACGGCTACATATTTCTGTTTTAGTTACTACGACTACT
GGGGCCAAGGCACCACGGTCACCGTCTCCTCAGGTGGGGGCGGTTCA
GGCGGAGGTGGCTCTGGCGGTGGCGGATTGGACATCAAGCTCACTCA
GTCTCCATCCATCCTGTCTGTGACTCCAGGTGAAACAGTCAGTCTTT
CCTGTAGGGCCAGCCAGACTATTTACAAGAACCTACACTGGTATCAA
CAGAAATCACATCGGTCTCCAAGGCTTCTCATCAAGTATGGTTCTGA
TTCCATCTCTGGGATCCCCTCCAGGTTCACTGGCAGTGGATCAGGGA
CAGATTACACTCTCAATATCAACAGTGTGAAGCCCGAAGATGAAGGA
ATATATTACTGTCTTCAAGGTTACAGTACACCTTGGACGTTCGGTGG
AGGGACCAAGCTGGAAATAAAACGG
[0041] The amino acid sequence of scFv763.74 is as follows:
TABLE-US-00002 (SEQ ID NO: 2)
MEFGLSWLFLVAILKGVQCSRMAQVKLKESGPELKKPGETVKISCKA
SGYTFTDYSMHWVKKTPGKGLKWLGWINTATGEPTYADDFKGRFAIS
LETSARTVYLQINNLRNEDTATYFCFSYYDYWGQGTTVTVSSGGGGS
GGGGSGGGGLDIKLTQSPSILSVTPGETVSLSCRASQTIYKNLHWYQ
QKSHRSPRLLIKYGSDSISGIPSRFTGSGSGTDYTLNINSVKPEDEG
IYYCLQGYSTPWTFGGGTKLEIKR
[0042] In certain embodiments, the antibody is not scFv 763.74,
scFv 225.28; scFv 763.74; scFv VF1-TP41.2; scFv VT80.112; scFv
653.25; scFv TP61.5, scFv D2.8.5-C4B8 scFv T8-203, scFv C21 or is
not derived from their respective monoclonal antibodies. In
specific embodiments, the scFv is not derived from HMW-MAA-specific
mouse mAbs 149.53, 225.28, 763.74, TP61.5, VF1-TP34, or VF1-TP41.2.
In particular embodiments, the scFv is not derived from the
monoclonal antibody designated as TP109 American Type Culture
Collection (ATCC) Accession No. PTA-9582 or is not derived from the
monoclonal antibody designated as VF20-VT1.7 ATCC Accession No.
PTA-9583. In particular embodiments, the scFv is not derived from
the mouse monoclonal antibody 11BD-2E11-2 produced by the hybridoma
deposited with ATCC as accession number PTA-5643. In certain
embodiments, the scFv is not derived from the monoclonal antibodies
IND-1 or IND-2 and is not produced by the hybridoma XMMME-001 or
XMMME-002, deposited with ATCC as HB8759 and H88760, respectively.
In particular embodiments, the scFvs utilized in methods of the
disclosure comprise CDRs that differ from the heavy and light
chains described in U.S. Pat. No. 8,318,162, incorporated by
reference herein in its entirety. In specific embodiments, the
complementarity determining region (CDR) in the scFv utilized
herein differs from one in which residues 24-34, 26-32, 50-52,
50-56, 89-97, or 91-96 in the light chain variable domain of IND-1
or IND-2 are employed and also differs from one in which residues
26-32, 31-35, 50-65, 53-55, 95-102, or 96-101 in the heavy chain
variable domain of IND-1 or IND-2 are employed. In particular
embodiments, a scFv does not target the membrane-proximal domain of
CSPG4 (amino acids 1740-2221 of CSPG4). In specific embodiments,
the scFv utilized in methods of the disclosure allows for reduction
in interaction of CSPG4 on its cancer cell with P-selectin, such as
P-selectin present on platelets, for example.
[0043] 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,
endodomains from co-stimulatory molecules such as CD28, CD27,
4-1BB, ICOS, OX40, a combination thereof, or the signaling
components of cytokine receptors such as IL7 and IL15. In
particular embodiments, co-stimulatory molecules are employed to
enhance the activation, proliferation, and cytotoxicity of T cells
produced by the CSPG4-comprising CAR after antigen engagement. In
specific embodiments, the co-stimulatory molecules are CD28, OX40,
and 4-1BB, for example.
[0044] 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 CSPG4 and it may include other CARs, such
as those specific for CD19, CD20, CD22, CD138, Glypican-3, Kappa or
light chain, CD30, CD33, CD123, CD38, ROR1, ErbB2, ErbB3/4, EGFR
vIII, 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, or CD44v6, and other tumor-associated antigens
or actionable mutations that are identified through genomic
analysis and or differential expression studies of tumors, for
example.
[0045] In particular cases the CAR is specific for CSPG4, and in
certain embodiments, the present invention provides chimeric T
cells specific for CSPG4 by joining an extracellular
antigen-binding domain derived from a CSPG4-specific antibody to
cytoplasmic signaling domains derived from the T-cell receptor
.zeta.-chain, optionally with the endodomains of the exemplary
costimulatory molecules CD28 and OX40, for examples. This CAR is
expressed in human cells, such as human immune cells, including
human T cells, and the targeting of one or more CSPG4-positive
cancers is encompassed herein.
[0046] In specific embodiments, there is a hinge region between the
V.sub.H and V.sub.L domains, and one can alter the length of the
hinge in a variety of CSPG4-CARs. One can utilize short hinges or
long hinges, in particular embodiments. In specific embodiments,
the hinge is between about 10 to 25 amino acids. In specific
embodiments, a full hinge is employed, such as, for example, the
hinge from IgG1. In certain embodiments, part or all of the hinge
of IgG1 is utilized. In certain embodiments, the hinge is rich in
glycine, to impact flexibility, and/or is rich in serine and/or
threonine, to impact solubility. In particular embodiments, the
hinge can either connect the N-terminus of the V.sub.H with the
C-terminus of the V.sub.L, or vice versa. Optimization of different
CSPG4-CARs may occur in vitro and/or in vivo, for example in mouse
models, such as in NSG tumor-bearing mice.
[0047] In some embodiments, the CAR utilizes a transmembrane
domain. In specific embodiments, the transmembrane is derived from
that of CD28 or 4-1BB or CD8 alpha, for example. In other
embodiments, the number and kind of co-stimulatory endodomains may
differ between CSPG4 CARs. For example, the CSPG4 CAR may utilize
CD28 co-stimulatory endodomain, 4-1BB co-stimulatory endodomain, or
both.
[0048] In specific embodiments, the CSPG4 CAR comprises the entire
IgG1 hinge and IgG1 C.sub.H2 C.sub.H3 domain with the CD28
transmembrane domain and CD28 endodomain or 4-1BB endodomain. In
specific embodiments, the CSPG4 CAR comprises the entire IgG1 hinge
but lacks the IgG1C.sub.H2 C.sub.H3 domain but comprises the CD28
transmembrane domain and CD28 endodomain or 4-1BB endodomain. In
certain embodiments, the CSPG4 CAR comprises the entire IgG1 hinge
but lacks the IgG1C.sub.H2 C.sub.H3 domain but comprises the CD8a
alpha transmembrane domain and comprises the CD28 endodomain or
4-1BB endodomain. In specific embodiments, the CSPG4 CAR comprises
the full CD8a alpha stalk, including hinge and transmembrane
domain, and comprises CD28 endodomain or 4-1BB endodomain. In some
embodiments, the CSPG4 CAR is a third generation CAR (comprises
multiple signaling domains) and further comprises CD28 and 4-1BB
endodomains.
IV. Cells
[0049] Cells of the disclosure include mammalian cells, such as
human cells, including immune cells that express a CSPG4-targeting
CAR. In specific embodiments, the cells are engineered to express a
CAR and, therefore, are not found in nature.
[0050] 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 a recombinantly introduced nucleic acid. In embodiments
of the invention, a host cell is 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 and NKT
cells are also encompassed in the invention.
[0051] 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 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 with the single vector.
[0052] 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.
[0053] The cells can be autologous cells, syngeneic cells,
allogenic cells and even in some cases, xenogeneic cells.
[0054] In many situations one may wish to be able to kill the
modified CTLs, 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, and/or another 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 (such as caspase
9).
[0055] In certain embodiments, the cells that express a
CSPG4-targeting CAR comprise recombinant expression of heparanase,
such as when there is no expression of endogenous heparanase in the
cell or wherein existing expression of heparanase is overexpressed
upon recombinant expression of heparanase. In specific embodiments,
the cells lack endogenous heparanase expression and the modifying
step restores heparanase expression or the cells have endogenous
heparanase expression and the heparanase is overexpressed. Such
cells may be capable of penetrating the extracellular matrix (ECM),
and also exhibit improved migration through the ECM. In certain
aspects, the modified cells are able to (or are able to more
effectively) degrade heparin sulphate proteoglycans (main
components of ECM and cell surface). In specific embodiments, the
cells comprise a vector that comprises an expression construct that
encodes heparanase and the vector may be viral (such as retroviral,
adenoviral, or adeno-associated viral) or non-viral, such as a
plasmid. The vector or expression construct that encodes heparanase
may also encode the CSPG4 CAR expression construct, although they
may be comprised on different expression constructs or vectors.
[0056] In specific embodiments, there are cells that harbor a
polynucleotide that encodes a CSPG4 CAR and also harbor a
polynucleotide that encodes one or more cytokines, such as IL-15,
IL-2, IL-7, IL-4, IL-12, and/or IL-21. In some embodiments, the
polynucleotide that encodes the CSPG4 CAR also encodes the one or
more cytokines, although in other embodiments the CSPG4 CAR and the
one or more cytokines are present on different polynucleotides.
V. Illustrative Exemplifications
[0057] By way of illustration, individuals with cancer or at risk
for cancer (such as having one or more risk factors) or suspected
of having cancer may be treated as follows. CTLs 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. In some embodiments, the
genetically modified cells are encapsulated to inhibit immune
recognition and placed at the site of the tumor.
[0058] In particular cases, an individual is provided with
therapeutic CTLs modified to comprise a CAR specific for CSPG4 in
addition to other types of therapeutic cells. The cells may be
delivered at the same time or at different times. The cells may be
delivered in the same or separate formulations. The cells may be
provided to the individual in separate delivery routes. The cells
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. Cells may be provided locally or
systemically.
VI. Introduction of Constructs into CTLs
[0059] Expression vectors that encode the CSPG4 CARs can be
introduced as a DNA molecule or construct, 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
constructs 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.
[0060] 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.
[0061] 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.
VII. Administration of Cells
[0062] The exemplary T 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.
[0063] 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.
[0064] 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.
[0065] The cells may be administered as desired. Depending upon the
response desired, the manner of administration, the life of the
cells, the number of cells present, various protocols may be
employed. The number of administrations will depend upon the
factors described above at least in part.
[0066] 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 patient, 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 which 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.
VIII. Nucleic Acid-Based Expression Systems
[0067] A polynucleotide encoding the CSPG4 CAR and optionally a
suicide gene may comprise an expression vector. In specific
embodiments, cells that harbor a polynucleotide that encodes a
CSPG4 CAR also harbor a polynucleotide that encodes one or more
cytokines, such as IL-15, IL-2, IL-7, IL-4, IL-12, and/or IL-21. In
some embodiments, the polynucleotide that encodes the CSPG4 CAR
also encodes the one or more cytokines, although in other
embodiments the CSPG4 CAR and the one or more cytokines are present
on different polynucleotides.
[0068] A. Vectors
[0069] The term "vector" is used to refer to a carrier nucleic acid
molecule into which a nucleic acid sequence can be inserted for
introduction into a cell where it can be replicated. A nucleic acid
sequence can be "exogenous," which means that it is foreign to the
cell into which the vector is being introduced or that the sequence
is homologous to a sequence in the cell but in a position within
the host cell nucleic acid in which the sequence is ordinarily not
found. Vectors include plasmids, cosmids, viruses (bacteriophage,
animal viruses, and plant viruses), and artificial chromosomes
(e.g., YACs). One of skill in the art would be well equipped to
construct a vector through standard recombinant techniques (see,
for example, Maniatis et al., 1988 and Ausubel et al., 1994, both
incorporated herein by reference).
[0070] The term "expression vector" refers to any type of genetic
construct comprising a nucleic acid coding for a RNA capable of
being transcribed. In some cases, RNA molecules are then translated
into a protein, polypeptide, or peptide. In other cases, these
sequences are not translated, for example, in the production of
antisense molecules or ribozymes. Expression vectors can contain a
variety of "control sequences," which refer to nucleic acid
sequences necessary for the transcription and possibly translation
of an operably linked coding sequence in a particular host cell. In
addition to control sequences that govern transcription and
translation, vectors and expression vectors may contain nucleic
acid sequences that serve other functions as well and are described
infra.
[0071] B. Promoters and Enhancers
[0072] A "promoter" is a control sequence that is a region of a
nucleic acid sequence at which initiation and rate of transcription
are controlled. It may contain genetic elements at which regulatory
proteins and molecules may bind, such as RNA polymerase and other
transcription factors, to initiate the specific transcription a
nucleic acid sequence. The phrases "operatively positioned,"
"operatively linked," "under control," and "under transcriptional
control" mean that a promoter is in a correct functional location
and/or orientation in relation to a nucleic acid sequence to
control transcriptional initiation and/or expression of that
sequence.
[0073] A promoter generally comprises a sequence that functions to
position the start site for RNA synthesis. The best known example
of this is the TATA box, but in some promoters lacking a TATA box,
such as, for example, the promoter for the mammalian terminal
deoxynucleotidyl transferase gene and the promoter for the SV40
late genes, a discrete element overlying the start site itself
helps to fix the place of initiation. Additional promoter elements
regulate the frequency of transcriptional initiation. Typically,
these are located in the region 30 110 bp upstream of the start
site, although a number of promoters have been shown to contain
functional elements downstream of the start site as well. To bring
a coding sequence "under the control of" a promoter, one positions
the 5' end of the transcription initiation site of the
transcriptional reading frame "downstream" of (i.e., 3' of) the
chosen promoter. The "upstream" promoter stimulates transcription
of the DNA and promotes expression of the encoded RNA.
[0074] The spacing between promoter elements frequently is
flexible, so that promoter function is preserved when elements are
inverted or moved relative to one another. In the tk promoter, the
spacing between promoter elements can be increased to 50 bp apart
before activity begins to decline. Depending on the promoter, it
appears that individual elements can function either cooperatively
or independently to activate transcription. A promoter may or may
not be used in conjunction with an "enhancer," which refers to a
cis-acting regulatory sequence involved in the transcriptional
activation of a nucleic acid sequence.
[0075] A promoter may be one naturally associated with a nucleic
acid sequence, as may be obtained by isolating the 5' non-coding
sequences located upstream of the coding segment and/or exon. Such
a promoter can be referred to as "endogenous." Similarly, an
enhancer may be one naturally associated with a nucleic acid
sequence, located either downstream or upstream of that sequence.
Alternatively, certain advantages will be gained by positioning the
coding nucleic acid segment under the control of a recombinant or
heterologous promoter, which refers to a promoter that is not
normally associated with a nucleic acid sequence in its natural
environment. A recombinant or heterologous enhancer refers also to
an enhancer not normally associated with a nucleic acid sequence in
its natural environment. Such promoters or enhancers may include
promoters or enhancers of other genes, and promoters or enhancers
isolated from any other virus, or prokaryotic or eukaryotic cell,
and promoters or enhancers not "naturally occurring," i.e.,
containing different elements of different transcriptional
regulatory regions, and/or mutations that alter expression. For
example, promoters that are most commonly used in recombinant DNA
construction include the beta-lactamase (penicillinase), lactose
and tryptophan (trp) promoter systems. In addition to producing
nucleic acid sequences of promoters and enhancers synthetically,
sequences may be produced using recombinant cloning and/or nucleic
acid amplification technology, including PCR.TM., in connection
with the compositions disclosed herein (see U.S. Pat. Nos.
4,683,202 and 5,928,906, each incorporated herein by reference).
Furthermore, it is contemplated the control sequences that direct
transcription and/or expression of sequences within non-nuclear
organelles such as mitochondria, chloroplasts, and the like, can be
employed as well.
[0076] Naturally, it will be important to employ a promoter and/or
enhancer that effectively directs the expression of the DNA segment
in the organelle, cell type, tissue, organ, or organism chosen for
expression. Those of skill in the art of molecular biology
generally know the use of promoters, enhancers, and cell type
combinations for protein expression, (see, for example Sambrook et
al. 1989, incorporated herein by reference). The promoters employed
may be constitutive, tissue-specific, inducible, and/or useful
under the appropriate conditions to direct high level expression of
the introduced DNA segment, such as is advantageous in the
large-scale production of recombinant proteins and/or peptides. The
promoter may be heterologous or endogenous.
[0077] Additionally any promoter/enhancer combination could also be
used to drive expression. Use of a T3, T7 or SP6 cytoplasmic
expression system is another possible embodiment. Eukaryotic cells
can support cytoplasmic transcription from certain bacterial
promoters if the appropriate bacterial polymerase is provided,
either as part of the delivery complex or as an additional genetic
expression construct.
[0078] The identity of tissue-specific promoters or elements, as
well as assays to characterize their activity, is well known to
those of skill in the art.
[0079] A specific initiation signal also may be required for
efficient translation of coding sequences. These signals include
the ATG initiation codon or adjacent sequences. Exogenous
translational control signals, including the ATG initiation codon,
may need to be provided. One of ordinary skill in the art would
readily be capable of determining this and providing the necessary
signals.
[0080] In certain embodiments of the invention, the use of internal
ribosome entry sites (IRES) elements are used to create multigene,
or polycistronic, messages, and these may be used in the
invention.
[0081] Vectors can include a multiple cloning site (MCS), which is
a nucleic acid region that contains multiple restriction enzyme
sites, any of which can be used in conjunction with standard
recombinant technology to digest the vector. "Restriction enzyme
digestion" refers to catalytic cleavage of a nucleic acid molecule
with an enzyme that functions only at specific locations in a
nucleic acid molecule. Many of these restriction enzymes are
commercially available. Use of such enzymes is widely understood by
those of skill in the art. Frequently, a vector is linearized or
fragmented using a restriction enzyme that cuts within the MCS to
enable exogenous sequences to be ligated to the vector. "Ligation"
refers to the process of forming phosphodiester bonds between two
nucleic acid fragments, which may or may not be contiguous with
each other. Techniques involving restriction enzymes and ligation
reactions are well known to those of skill in the art of
recombinant technology.
[0082] Splicing sites, termination signals, origins of replication,
and selectable markers may also be employed.
[0083] C. Plasmid Vectors
[0084] In certain embodiments, a plasmid vector is contemplated for
use to transform a host cell. In general, plasmid vectors
containing replicon and control sequences which are derived from
species compatible with the host cell are used in connection with
these hosts. The vector ordinarily carries a replication site, as
well as marking sequences which are capable of providing phenotypic
selection in transformed cells. In a non-limiting example, E. coli
is often transformed using derivatives of pBR322, a plasmid derived
from an E. coli species. pBR322 contains genes for ampicillin and
tetracycline resistance and thus provides easy means for
identifying transformed cells. The pBR plasmid, or other microbial
plasmid or phage must also contain, or be modified to contain, for
example, promoters which can be used by the microbial organism for
expression of its own proteins.
[0085] In addition, phage vectors containing replicon and control
sequences that are compatible with the host microorganism can be
used as transforming vectors in connection with these hosts. For
example, the phage lambda GEM.TM. 11 may be utilized in making a
recombinant phage vector which can be used to transform host cells,
such as, for example, E. coli LE392.
[0086] Further useful plasmid vectors include pIN vectors (Inouye
et al., 1985); and pGEX vectors, for use in generating glutathione
S transferase (GST) soluble fusion proteins for later purification
and separation or cleavage. Other suitable fusion proteins are
those with galactosidase, ubiquitin, and the like.
[0087] Bacterial host cells, for example, E. coli, comprising the
expression vector, are grown in any of a number of suitable media,
for example, LB. The expression of the recombinant protein in
certain vectors may be induced, as would be understood by those of
skill in the art, by contacting a host cell with an agent specific
for certain promoters, e.g., by adding IPTG to the media or by
switching incubation to a higher temperature. After culturing the
bacteria for a further period, generally of between 2 and 24 h, the
cells are collected by centrifugation and washed to remove residual
media.
[0088] D. Viral Vectors
[0089] The ability of certain viruses to infect cells or enter
cells via receptor mediated endocytosis, and to integrate into host
cell genome and express viral genes stably and efficiently have
made them attractive candidates for the transfer of foreign nucleic
acids into cells (e.g., mammalian cells). Components of the present
invention may be a viral vector that encodes one or more CARs of
the invention. Non-limiting examples of virus vectors that may be
used to deliver a nucleic acid of the present invention are
described below.
[0090] 1. Adenoviral Vectors
[0091] A particular method for delivery of the nucleic acid
involves the use of an adenovirus expression vector. Although
adenovirus vectors are known to have a low capacity for integration
into genomic DNA, this feature is counterbalanced by the high
efficiency of gene transfer afforded by these vectors. "Adenovirus
expression vector" is meant to include those constructs containing
adenovirus sequences sufficient to (a) support packaging of the
construct and (b) to ultimately express a tissue or cell specific
construct that has been cloned therein. Knowledge of the genetic
organization or adenovirus, a 36 kb, linear, double stranded DNA
virus, allows substitution of large pieces of adenoviral DNA with
foreign sequences up to 7 kb (Grunhaus and Horwitz, 1992).
[0092] 2. AAV Vectors
[0093] The nucleic acid may be introduced into the cell using
adenovirus assisted transfection. Increased transfection
efficiencies have been reported in cell systems using adenovirus
coupled systems (Kelleher and Vos, 1994; Cotten et al., 1992;
Curiel, 1994). Adeno associated virus (AAV) is an attractive vector
system for use in the cells of the present invention as it has a
high frequency of integration and it can infect nondividing cells,
thus making it useful for delivery of genes into mammalian cells,
for example, in tissue culture (Muzyczka, 1992) or in vivo. AAV has
a broad host range for infectivity (Tratschin et al., 1984;
Laughlin et al., 1986; Lebkowski et al., 1988; McLaughlin et al.,
1988). Details concerning the generation and use of rAAV vectors
are described in U.S. Pat. Nos. 5,139,941 and 4,797,368, each
incorporated herein by reference.
[0094] 3. Retroviral Vectors
[0095] Retroviruses are useful as delivery vectors because of their
ability to integrate their genes into the host genome, transferring
a large amount of foreign genetic material, infecting a broad
spectrum of species and cell types and of being packaged in special
cell lines (Miller, 1992).
[0096] In order to construct a retroviral vector, a nucleic acid
(e.g., one encoding the desired sequence) is inserted into the
viral genome in the place of certain viral sequences to produce a
virus that is replication defective. In order to produce virions, a
packaging cell line containing the gag, pol, and env genes but
without the LTR and packaging components is constructed (Mann et
al., 1983). When a recombinant plasmid containing a cDNA, together
with the retroviral LTR and packaging sequences is introduced into
a special cell line (e.g., by calcium phosphate precipitation for
example), the packaging sequence allows the RNA transcript of the
recombinant plasmid to be packaged into viral particles, which are
then secreted into the culture media (Nicolas and Rubenstein, 1988;
Temin, 1986; Mann et al., 1983). The media containing the
recombinant retroviruses is then collected, optionally
concentrated, and used for gene transfer. Retroviral vectors are
able to infect a broad variety of cell types. However, integration
and stable expression require the division of host cells (Paskind
et al., 1975).
[0097] Lentiviruses are complex retroviruses, which, in addition to
the common retroviral genes gag, pol, and env, contain other genes
with regulatory or structural function. Lentiviral vectors are well
known in the art (see, for example, Naldini et al., 1996; Zufferey
et al., 1997; Blomer et al., 1997; U.S. Pat. Nos. 6,013,516 and
5,994,136). Some examples of lentivirus include the Human
Immunodeficiency Viruses: HIV-1, HIV-2 and the Simian
Immunodeficiency Virus: SIV. Lentiviral vectors have been generated
by multiply attenuating the HIV virulence genes, for example, the
genes env, vif, vpr, vpu and nef are deleted making the vector
biologically safe.
[0098] Recombinant lentiviral vectors are capable of infecting
non-dividing cells and can be used for both in vivo and ex vivo
gene transfer and expression of nucleic acid sequences. For
example, recombinant lentivirus capable of infecting a non-dividing
cell wherein a suitable host cell is transfected with two or more
vectors carrying the packaging functions, namely gag, pol and env,
as well as rev and tat is described in U.S. Pat. No. 5,994,136,
incorporated herein by reference. One may target the recombinant
virus by linkage of the envelope protein with an antibody or a
particular ligand for targeting to a receptor of a particular
cell-type. By inserting a sequence (including a regulatory region)
of interest into the viral vector, along with another gene which
encodes the ligand for a receptor on a specific target cell, for
example, the vector is now target-specific.
[0099] 4. Other Viral Vectors
[0100] Other viral vectors may be employed as vaccine constructs in
the present invention. Vectors derived from viruses such as
vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar
et al., 1988), sindbis virus, cytomegalovirus and herpes simplex
virus may be employed. They offer several attractive features for
various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal
and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).
[0101] E. Delivery Using Modified Viruses
[0102] A nucleic acid to be delivered may be housed within an
infective virus that has been engineered to express a specific
binding ligand. The virus particle will thus bind specifically to
the cognate receptors of the target cell and deliver the contents
to the cell. A novel approach designed to allow specific targeting
of retrovirus vectors was developed based on the chemical
modification of a retrovirus by the chemical addition of lactose
residues to the viral envelope. This modification can permit the
specific infection of hepatocytes via sialoglycoprotein
receptors.
[0103] Another approach to targeting of recombinant retroviruses
was designed in which biotinylated antibodies against a retroviral
envelope protein and against a specific cell receptor were used.
The antibodies were coupled via the biotin components by using
streptavidin (Roux et al., 1989). Using antibodies against major
histocompatibility complex class I and class II antigens, they
demonstrated the infection of a variety of human cells that bore
those surface antigens with an ecotropic virus in vitro (Roux et
al., 1989).
[0104] F. Vector Delivery and Cell Transformation
[0105] Suitable methods for nucleic acid delivery for transfection
or transformation of cells are known to one of ordinary skill in
the art. Such methods include, but are not limited to, direct
delivery of DNA such as by ex vivo transfection, by injection, and
so forth. Through the application of techniques known in the art,
cells may be stably or transiently transformed.
[0106] G. Ex Vivo Transformation
[0107] Methods for transfecting eukaryotic cells and tissues
removed from an organism in an ex vivo setting are known to those
of skill in the art. Thus, it is contemplated that cells or tissues
may be removed and transfected ex vivo using nucleic acids of the
present invention. In particular aspects, the transplanted cells or
tissues may be placed into an organism. In certain facets, a
nucleic acid is expressed in the transplanted cells.
IX. Kits of the Invention
[0108] Any of the CSPG4 CAR compositions described herein may be
comprised in a kit, including nucleic acids, proteins, peptides,
and/or cells. In a non-limiting example, one or more 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. Polynucletoides that encodes the CSPG4
CAR or portions thereof may be included in the kit. The kit
components are provided in suitable container means.
[0109] 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 invention also will typically include a means
for containing the components in close confinement for commercial
sale. Such containers may include injection or blow molded plastic
containers into which the desired vials are retained.
[0110] 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 useful. 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.
[0111] 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.
[0112] In particular embodiments of the invention, 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 CAR as described herein
and/or regulatory elements therefor.
[0113] In particular embodiments, there are one or more apparatuses
in the kit suitable for extracting one or more samples from an
individual. The apparatus may be a syringe, scalpel, and so forth.
One or more reagents and/or apparatuses for diagnosis of cancer may
be included in the kit, including for blood tests, sample
extraction, blood extraction, tumor marker tests (such as
particular antibodies), and so forth.
[0114] In some cases of the invention, the kit, in addition to cell
therapy embodiments, also includes a second cancer therapy, such as
chemotherapy, hormone therapy, and/or 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.
X. Combination Therapy
[0115] 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).
[0116] 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.
[0117] 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.
[0118] Various combinations may be employed, present invention is
"A" and the secondary agent, such as radio- or chemotherapy, is
"B":
TABLE-US-00003 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
[0119] 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.
[0120] A. Chemotherapy
[0121] 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 and also combinations thereof.
[0122] In specific embodiments, chemotherapy for the individual is
employed in conjunction with the invention, for example before,
during and/or after administration of the invention.
[0123] B. Radiotherapy
[0124] 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.
[0125] 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.
[0126] C. Immunotherapy
[0127] 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.
[0128] 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 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.
[0129] D. Genes
[0130] 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.
[0131] E. Surgery
[0132] 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.
[0133] 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.
[0134] 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.
[0135] F. Other Agents
[0136] 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 abilities 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-hyperproliferative
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
[0137] The following examples are included to demonstrate certain
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, 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
invention.
Example 1
MCSP-Chimeric Antigen Receptor (CAR)-Redirected T Cells Target
Multiple Solid Tumors
[0138] Melanoma-associated chondroitin sulphate proteoglycan (MCSP,
also known as CSPG4) participates in tumor migration, angiogenesis
and metastasis and is frequently over-expressed in tumors that
develop and require these functions to survive. Taking into
consideration its broad expression in cancer cells, its limited
expression in normal tissues (basal cells of epidermis) and its
pivotal role in tumor survival, targeting this antigen with
CAR-redirected T cells offers a useful therapeutic approach for
several solid tumors. As described in the Examples to follow,
second generation CAR (encoding the CD28 costimulatory endodomain,
as an example) targeting MCSP (CAR.MCSP) was therefore constructed
in a retroviral vector. After transduction, T lymphocytes (n=4)
stably and efficiently expressed the CAR (65%-88%) and lysed the
melanoma cells SEMNA significantly better (59%.+-.6%) than control
T cells (11%.+-.8%) even at the 5:1 effector-to-target ratio in
.sup.51Cr release assays. Furthermore, in long-term co-culture
assays, CAR.MCSP.sup.+ T cells efficiently and consistently
eliminated several MCSP.sup.+ targets including melanoma (SEMNA and
CLB, residual tumors: 0.1%.+-.0.06% and 0.1%.+-.0.1, respectively),
mesothelioma (PH1 and MILL: 3.8%.+-.3.1%; 3.2%.+-.5%), head and
neck carcinoma (PCI-30, 0.5%.+-.0.5%), and basal breast carcinoma
(UACC-812 and MDA-MB-231: 5.7%.+-.6.1%; 3.1%.+-.2.5%, respectively)
while having no effect on a MCSP.sup.- targets (38%.+-.10%). As
expected all tumor cells expanded in co-culture with control T
cells. The antitumor activity of CAR.MCSP.sup.+ T cells was
paralleled by release of Th1 cytokines, such as IL2 (from 6.+-.10
pg/.mu.L to 190.+-.98 pg/.mu.L) and IFNg (from 105.+-.48 pg/.mu.L
to 3710.+-.975 pg/.mu.L) upon coculture with different MCSP.sup.+
tumors. Both CAR.MCSP transgenic CD4.sup.+ and CD8.sup.+ cells
proliferated in response to SEMNA tumor cells as compared to
control T cells, as assessed by CFSE dilution assays. A third
generation CAR encoding CD28 and 4-1BB endodomains (as examples)
was also produced. However, since this construct did not show
superior function in at least certain cases in vitro as compared to
the CD28 endodomain, the latter was selected for the following in
vivo experiments. Using NSG mice (n=10/group) either melanoma
(SEMNA) or head and neck carcinoma (PCI-30) or basal breast
carcinoma (UACC-812) cells were engrafted s.c. Across all tumor
models, mice treated with CAR.MCSP.sup.+ T lymphocytes consistently
showed tumor control (753 mm.sup.3.+-.350 mm.sup.3; 18.5
mm.sup.3.+-.10 mm.sup.3; 28 mm.sup.3.+-.13 mm.sup.3) as compared to
mice receiving control T lymphocytes (7126 mm.sup.3.+-.2500
mm.sup.3; 190 mm.sup.3.+-.75 mm.sup.3; 166 mm.sup.3.+-.64 mm.sup.3)
by days 40-50 post tumor engraftment. In summary,
CAR.MCSP-redirected T cells can be used for the treatment of a
variety of solid tumors.
Example 2
Exemplary Materials and Methods
[0139] Cell Lines.
[0140] The previously described SENMA, CLB and P1143 tumor cell
lines were generated in the laboratory from melanoma biopsies.
MDA-MB-231 was originally obtained from American Type Culture
Collection (ATCC) and authenticated by the analysis of short tandem
repeat sequences performed at MD Anderson Cancer Center, Texas,
USA. UACC-812, PCI-30 and PHI cell lines were obtained and these
cells, when maintained in culture for several passages, retained
the same phenotypic expression of CSPG4 as the early cell passages.
Previously described melanoma cell lines PLAODE, NE-18732,
NE-18588, NE-8959, NE-4405 and NE-371952 were only used to confirm
the expression of CSPG4 in a broad array of melanoma cell lines.
All these cells, including SENMA, CLB, and P1143, when maintained
in culture for several passages, retained the same phenotypic
expression of CSPG4 as the early cell passages. SENMA, CLB,
UACC-812, MDA-MB-231, and PCI-30 cell lines were cultured in DMEM
(Invitrogen Grand Island, N.Y.) or RPMI 1640 (P1143, UACC-812, and
PHI) (Cambrex, East Rutherford, N.J.) medium supplemented with 10%
heat inactivated fetal calf serum (FCS) (HyClone, Thermo Fisher
Scientific Inc., Wyman, Mass.), 200 IU/mL penicillin, 200 mg/mL
streptomycin (Invitrogen), and 2 mmol/L GlutaMAX (Invitrogen) at
37.degree. C. in a 5% CO.sub.2 atmosphere. Tumor cell lines were
transduced with a gamma retroviral vector encoding eGFP to obtain
GFP+ tumor cells (>98% GFP+). Primary epithelial cells from
normal small airway, kidney and prostate were purchased from ATCC
and kept in culture according to ATCC recommendation.
[0141] Tissue Microarrays and Immunohistochemistry (IHC).
[0142] Antigen retrieval was performed by placing the samples in
1.times. Dako Citrate Buffer followed by incubation at 90.degree.
C. in a pressure cooker for 45 minutes. After blocking with normal
goat serum diluted in Tris-Buffered Saline, samples were incubated
with the CSPG4 mAb (Abcam, Anti-NG2 antibody [LHM 2], ref#
ab104535) (1:300 dilution) either overnight at 4.degree. C. or at
room temperature for 1 hour. Detection of CSPG4 was then assessed
using the VECTASTAIN.RTM. ABC kit (Vector Laboratories, Inc. ref#
PK-4001) following the manufacturer's protocol. Tissue arrays were
obtained from Cybrdi Inc (Rockville, Md.) for breast cancer
(CC08-10-001) and HNSCC (CC34-01-001), while melanoma (ME2082b),
neuroblastoma (MC602), mesothelioma (T392), and normal tissue (FDA
808b) arrays were obtained from US Biomax, Inc. (Rockville, Md.).
Each array contained a range of 4 to 192 cores of tumor or 5 to 18
cores of normal tissue samples in duplicate, triplicate or
quadruplicate in the case of mesothelioma. Expression of CSPG4 in
tumor cells was scored in blind fashion by the pathologist Dr
Michael Ittmann based on both intensity (0-3+) and extent of
staining (1-3+). A multiplicative staining score was calculated by
multiplying the intensity and extent scores to yield scores on a 10
point scale from 0-9. In microarrays with multiple cores per
patient, the individual scores were averaged to obtain a final
score. In some cores, tumor was not identified due to artifacts. In
the vast majority of cases, IHC showed uniform staining (3+) within
a given core and in most cases cores from different patients were
highly concordant. Areas of necrosis or acellular keratin were not
included in the scoring. Cases were divided based on staining
scores into three groups: negative/weak (0-3), moderate (4-6) or
strong (7-9).
[0143] Generation of the CSPG4-Specific CAR and Transduction of T
Lymphocytes.
[0144] The hybridoma 763.74 was generated from a BALB/c mouse
immunized with cultured human melanoma cells. The scFv 763.74 was
isolated from the hybridoma and then cloned in frame with the human
IgG1-CH2CH3 domains, the CD28 costimulatory endodomain and the
CD3.zeta. chain into the SFG retroviral backbone (CAR.CSPG4), as
previously described. The control CAR specific for the CD19 antigen
(CAR.CD19) has been previously described.
[0145] Transient retroviral supernatant was generated by
co-transfection of 293T cells with the RD114 envelope (RDF
plasmid), the MoMLV gag-pol (PegPam3-e plasmid) and the retroviral
vector, as previously described. For the generation of CAR-T cells,
peripheral blood mononuclear cells (PBMCs) were isolated from buffy
coat preparations (Gulf Coast Regional Blood Center, Houston, Tex.)
using Ficoll-Paque (Amersham Biosciences, Piscataway, N.J.). PBMCs
were activated with OKT3 and CD28 (BD Biosciences PharMingen, San
Diego, Calif.) mAbs, transduced with the retroviral supernatant by
day 3 of culture and then expanded in complete medium containing
45% RPMI 1640 and 45% Click's medium (Irvine Scientific, Santa Ana,
Calif., USA) supplemented with 10% FCS, 100 IU/mL penicillin, 100
mg/mL streptomycin, and 2 mmol/L GlutaMAX. Cells were fed with IL-2
(50 U/mL) (PeproTech; Rocky Hill, N.J.) twice a week for 2
weeks.
[0146] Flow Cytometry.
[0147] Conjugated CD3, CD4, CD8, CD45RO, CD62L and CCR7 mAbs (BD
Biosciences) were used to identify T lymphocytes, while the CSPG4
mAb (Miltenyi-Biotech Inc, Auburn, Calif.) was used to label tumor
cells. CAR expression in T lymphocytes was assessed using an
antibody recognizing the human IgG1-CH.sub.2CH.sub.3 fragment
(Jackson ImmunoResearch, West Grove, PN). Analyses were performed
on a FACsCaliber flow cytometer using the BDFACs CellQuestPro
software (BD Biosciences, San Jose, Calif.).
[0148] Cytotoxicity and Co-Culture Assays.
[0149] The cytotoxic activity of control and CAR.CSPG4+ T
lymphocytes was determined using a standard .sup.51Cr release assay
at different effector-to-target (E:T) (40:1, 20:1, 10:1 and 5:1)
ratios using a gamma counter (Perkin-Elmer, Waltham, Mass.)(Vera et
al., 2006). For the co-culture experiments, control and CAR.CSPG4+
T lymphocytes were plated at 1.times.10.sup.6 cells/well in 24-well
plates at different E:T ratios according to the kinetic growth of
each tumor cell line. Tumor cell lines with a slow kinetic growth
were plated at higher tumor ratio (T cells:tumor cells 3:1)
compared to tumor cell lines with a fast kinetic growth (T cells:
tumor cells 5:1). Supernatant was collected at 24 hours of culture
to measure IFN.gamma. and IL-2 release using specific ELISAs
(R&D system, Minneapolis, Minn.). Following 72 hours of culture
at 37.degree. C., adherent tumor cells and T cells were collected
and residual tumor cells and T cells assessed by FACs analysis
based on GFP and CD3 expression, respectively.
[0150] Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE)
Assay.
[0151] One week post transduction, control and CAR.CSPG4.sup.+ T
lymphocytes were labeled with 1.5 .mu.mol/L CFSE (Invitrogen) and
plated with irradiated tumor target (SENMA) at an E:T ratio of 5:1.
CFSE dilution was measured on CD4.sup.+ and CD8.sup.+ cells by flow
cytometry by day 4 of co-culture.
[0152] Xenogenic Mouse Models.
[0153] In vivo experiments were performed in accordance with Baylor
College of Medicine's Animal Husbandry guidelines. Antitumor
activity of control and CAR.CSPG4.sup.+ T lymphocytes was evaluated
using NOG/SCID/.gamma.c.sup.-/- mice (Jackson Lab, Bar Harbor, Me.)
engrafted with tumor cells. Eight to 9 week old mice were
subcutaneously injected with 0.5.times.10.sup.6 SENMA,
3.times.10.sup.6 UACC-812 or 3.times.106 PCI-30 cells resuspended
in Matrigel (BD Biosciences, San Jose, Calif.). On days 4, 6, and 8
following tumor cell injection, 1.times.10.sup.7 control or
CAR.CSPG4+ T lymphocytes were injected i.v. by tail vain. In
summary, for the melanoma xenograft model 3 different preparations
of CAR.CSPG4 T cells were generated from 3 different donors. Three
doses, given two days apart, of 1.times.10.sup.7 were infused i.v.
into 5 mice per group. In total, 15 animals were treated for each
group. The endpoint of the experiment was to examine differences in
tumor volume up to day 30-post tumor injection. For the xenograft
models of breast cancer and HNSCC 2 different preparations of T
cells generated from 2 different donors were used. Two doses, given
two days apart, of 1.times.10.sup.7 were infused i.v. into 5 mice
per group. In total, 10 animals were treated per group. In all
tumor models, mice were sacrificed at 30 days or in accordance with
institution's guidelines for the handling of sick animals. Weekly
manual caliper measurements were performed post-treatment to
evaluate tumor growth. Tumor volume was calculated using the
modified ellipsoidal formula: tumor volume
(mm.sup.3)=(width).sup.2.times.length/2.
[0154] Statistical Analysis.
[0155] In vitro data are presented as mean.+-.standard deviation
(SD) and a paired student's t-test was used to determine
statistical significance. The in vivo data are presented as
mean.+-.standard error of the mean (SEM) and a paired student's
t-test was used to identify significant differences between
CAR-treated and control-treated groups. Public gene expression
profiling datasets of human tumors were queried for CSPG4,
including data from The Cancer Genome Atlas (TCGA Data Portal;
http://tcga-data.nci.nih.gov/tcga), Bittner multi-cancer dataset
(unpublished, from www.oncomine.org) and GeneAtlas U133A data set
(http://niogps.org).
Example 3
CSPG4 is Expressed on a Variety of Solid Tumors
[0156] As CSPG4 was originally identified as a melanoma associated
antigen, its expression was first independently validated using IHC
in a melanoma tissue array containing multiple primary cutaneous
and visceral melanomas and metastatic lesions. Examples of either
strong or negative/low staining are shown in FIG. 1A. Consistent
expression of the antigen was documented in all types of lesions,
regardless of their primary or metastatic origin, or their
cutaneous and visceral source. Melanomas were therefore analyzed as
a whole group. Overall, 59% of melanomas showed strong staining and
25% displayed moderate staining (FIG. 1B). The analysis was
extended to include multiple samples of additional solid tumors
including breast cancer, HNSCC, neuroblastoma, and mesothelioma.
For the breast cancer array, staining was seen in invasive ductal
and lobular carcinomas as well as in the small number of Paget's
disease and ductal in situ carcinoma cases present on the array.
Staining for representative invasive ductal carcinomas (either
strong or negative/low) are shown in FIG. 1A and summarized in FIG.
1B, while other lesions were not sufficient in number for a
comparative analysis. Staining for CSPG4 in invasive ductal
carcinoma was remarkable with 77% of cases showing moderate or
strong staining. HNSCC most predominantly (50%) expressed moderate
staining for CSPG4, with only 20% showing strong staining. Although
neuroblastoma exhibited the weakest overall staining, there was
still a fraction of cases with moderate to strong expression.
Finally, despite the limited number of mesotheliomas, these lesions
all consistently expressed CSPG4. In certain embodiments it was
considered that, at the protein level, all these malignancies
exhibited variable but in most cases significant expression of
CSPG4. To examine the expression of CSPG4 in a broader array of
tumors publically available databases were examined for mRNA
expression data. As shown in FIG. 1C, examination of TCGA datasets
showed over expression of CSPG4 transcripts in melanoma and in
glioma as anticipated based on the previously reported expression
of the protein. Concordant with the protein expression data, CSPG4
mRNA expression was increased in HNSCC. Increased mRNA expression
in clear cell renal carcinomas by in silico analysis, and overall,
despite some intratumor variability, significant increased mRNA
levels in all these tumor types relative to the corresponding
normal tissues (FIG. 1D). Examination of the large Bittner
multi-cancer dataset (www.oncomine.org) confirmed high CSPG4 mRNA
expression in melanoma, clear cell renal carcinoma, HNSCC, multiple
sarcoma types (chondrosarcoma, leiomyosarcoma, liposarcoma),
gastrointestinal stromal tumors, skin, and vulvar squamous cell
carcinomas. Of note several sarcoma cell lines have been previously
reported to express CSPG4 protein. A number of other common
malignancies such as colorectal, ovarian, and endometrial carcinoma
did not show increased CSPG4 transcripts, consistent with the mRNA
expression from the TCGA data sets. CSPG4 protein expression in an
array of normal tissues was negative (FIG. 6). In addition to the
normal tissues represented in FIG. 7, CSPG4 expression was
evaluated on a total of 33 different types of tissues, all of which
were negative. Using the public Novartis GeneAtlas
(http://biogps.org) and TCGA databases, CSPG4 mRNA expression was
observed in a number of normal tissues. However, the levels of
expression are remarkably lower than those of cancer tissues (FIG.
7).
[0157] CSPG4 expression was examined in a series of cell lines from
a variety of tumor types analyzed above. Expression of CSPG4 was
detected in 8 of the 9 melanoma cell lines screened (FIG. 1E).
Importantly, CSPG4 was detected on tumor cell lines representative
of the above identified solid tumors such as mesothelioma (MILL and
PHI), HNSCC (PCI-30) and breast cancer [MDA-MB-231 (adenocarcinoma)
and UACC-812 (ductal carcinoma)] (FIG. 1F) all consistent with
analysis of human tumor samples.
Example 4
T Lymphocytes Expressing the CSPG4-Specific Car are Cytotoxic
Against CSPG4+ Tumor Cell Lines but not Against Primary Normal
Tissues
[0158] To target CSPG4.sup.+ tumors, a CSPG4-specific CAR was
generated containing the CD28 costimulatory endodomain (CAR.CSPG4)
(FIG. 2A). T lymphocytes from 4 healthy donors were engineered to
express the CAR.CSPG4 using a gamma retroviral vector. Transduction
efficiency was 80%.+-.3%, and both CD4 and CD8 T cells stably
expressed the CAR (26%.+-.9% and 51%.+-.16%, respectively), as
assessed by phenotypic analysis by day 7 of culture (FIG. 2B). The
majority of CAR.CSPG4+ T cells were CD45RO+ (76%.+-.7%) and a
fraction retained CD62L expression (51%.+-.7%) and CCR7
(13%.+-.2%), indicating that they were mainly composed of
effector-memory T cells (FIG. 2C). The expression of CAR.CSPG4 by T
cells was comparable to that obtained with a previously described
CD19-specific CAR (CAR.CD19) (FIG. 9), which was used as an
irrelevant-CAR control population.
[0159] Cytotoxic activity of control and CAR.CSPG4.sup.+ T cells,
after 1-2 weeks of culture, was assessed against K562, to measure
natural killer cell-mediated activity, and against the melanoma
derived cells lines P1143 (as CSPG4.sup.- target) and SENMA (as
CSPG4+ target) (FIG. 1E) at various E:T ratios (FIG. 3A).
CAR.CSPG4.sup.+ but not control T lymphocytes significantly lysed
the CSPG4.sup.+ target (59%.+-.5% vs. 11%.+-.8% at 20:1
ratio)(p<0.01), while both CAR.CSPG4.sup.+ and control T cells
showed minimal activity against K562 (12%.+-.9% vs. 13%.+-.11%) and
the CSPG4- target (<10% in both cases). The antitumor activity
of CAR.CSPG4.sup.+ T lymphocytes was also evaluated in a 72 hour
co-culture assay (FIGS. 3B and C). CAR.CSPG4.sup.+ and control T
lymphocytes were co-cultured with GFP-expressing tumor cell lines
at an E:T ratio ranging from 5:1 to 3:1 according to the kinetic
growth of each cell line. CAR.CSPG4.sup.+ T cells significantly
controlled the growth of all CSPG4.sup.+ cell lines tested: SENMA
(residual tumor cells=0.1%.+-.0.06%), CLB (0.1%.+-.0.1%), UACC-812
(6%.+-.6%), MILL (3%.+-.5%), MDA-MB-231 (3%.+-.3%), PHI (4%.+-.3%),
and PCI-30 (0.5%.+-.0.5%), but not of the CSPG4- target P1143
(residual tumor cells 38%.+-.10%). As expected, all tumor cell
lines tested rapidly grow in the presence of control T lymphocytes
(residual tumor cells for: SENMA=62%.+-.3%, CLB=70%.+-.6%,
UACC-812=47%.+-.15%, MILL=50%.+-.8%, MDA-MB-231=42%.+-.11%,
PHI=29%.+-.6%, PCI-30=17%.+-.3%, and P1143=45%.+-.10%). In all
cases, the effects of CAR.CSPG4.sup.+ T cells were significantly
greater than those of control T cells (from p<0.05 to
p<0.001). T cells expressing the control CAR.CD19 showed
cytotoxic activity neither against CSPG4.sup.+ nor CSPG4.sup.-
targets (FIG. 8). As illustrated in FIG. 3, commercially available
primary normal epithelial cell lines (small airway, kidney and
prostate) derived from tissues found to express low levels of CSPG4
mRNA (FIG. 7) did not express detectable levels of the protein by
flow cytometry (FIG. 3D), and were not lysed by CAR.CSPG4.sup.+ T
cells when tested in .sup.51Cr release assays (FIG. 3E).
Example 5
CAR.CSPG4+ T Lymphocytes Secrete Th1 Cytokines and Proliferate in
Response to CSPG4+ Tumors
[0160] Because CAR.CSPG4 contains the CD28 costimulatory
endodomain, CAR.CSPG4+ T lymphocyte proliferation was studied in
response to CSPG4.sup.+ tumor cells using a CFSE dilution assay.
When CFSE-labeled control and CAR.CSPG4.sup.+ T cells were cultured
with irradiated SENMA tumor cells for 96 hours, a significant CFSE
dilution occurred for CAR.CSPG4.sup.+ T cells, with both CD4 and
CD8 T cells proliferating at a higher percentage (66%.+-.12% and
68%.+-.8%, respectively) compared to control CD4 and CD8 T cells
(8%.+-.7% and 14%.+-.10%, respectively) (p<0.05 and p<0.01,
respectively) (FIGS. 4A and B). T cells transduced with the control
CAR.CD19 also containing the CD28 endodomain did not show
significant proliferation in response to CSPG4.sup.+ targets (FIG.
8). It was evaluated whether the inclusion of a "late"
co-stimulatory endodomain, such as 4-1BB, in addition to CD28
(third generation construct) provided these T cells with additional
proliferative and cytotoxic activity, but found no further benefits
(FIG. 9).
[0161] The IL-2 and IFN.gamma. cytokines released in response to
the antigen was quantified by co-culturing control and
CAR.CSPG4.sup.+ T lymphocytes with CSPG4.sup.+ or CSPG4.sup.- tumor
cells. As expected CAR.CSPG4.sup.+ T lymphocytes secreted
significantly more IL-2 than control T cells only tail vein
injection with either control or CAR.CSPG4.sup.+ T lymphocytes, and
tumor growth quantified by sequential tumor volume measurements. In
all three models, CAR.CSPG4.sup.+ T lymphocytes inhibited tumor
growth significantly better than control T lymphocytes (FIG. 5). By
day 30, melanoma tumors reached a volume of 879 mm.sup.3.+-.124
mm.sup.3 in mice receiving CAR.CSPG4.sup.+ T lymphocytes versus
8359 mm.sup.3.+-.958 mm.sup.3 in mice receiving control T cells
(p<0.001) (FIG. 5A), and this corresponded to improved overall
survival (FIG. 10). Although HNSCC and breast carcinoma tumors were
not as aggressive as melanoma in vivo, in both models
CAR.CSPG4.sup.+ T lymphocytes controlled tumor growth. By day 30
the size of HNSCC tumors was 19 mm.sup.3.+-.10 mm.sup.3 in treated
mice versus 190 mm.sup.3.+-.75 mm.sup.3 in control mice
(p<0.001) (FIG. 5B) and the size of breast carcinoma tumors was
28 mm.sup.3.+-.13 mm.sup.3 in treated mice versus 166
mm.sup.3.+-.64 mm.sup.3 in control mice (p<0.001) (FIG. 5C).
Example 6
Significance of Certain Embodiments
[0162] The involvement in several signaling pathways associated
with cell proliferation, survival, migration, and suggested high
expression in various types of cancers highlight the critical role
that CSPG4 has in promoting tumor growth and simultaneously make it
an attractive target for immunotherapy. By IHC, CSPG4 protein
expression was independently validated in several solid tumors with
poor prognosis, such as melanoma, breast cancer, mesothelioma and
HNSCC. In silico analysis of microarray expression data confirmed
overexpression of CSPG4 in tumors that were validated by IHC as
compared to normal tissues, and also disclosed CSPG4 overexpression
in other important malignancies including glioblastoma, clear cell
renal carcinoma and sarcomas indicating that targeting this antigen
has a major impact on a broad array of solid tumors, in at least
particular cases.
[0163] Because CSPG4-specific mAbs can control tumor growth of
CSPG4.sup.+ tumor cells in both melanoma and breast cancer tumor
models, it was considered to improve the therapeutic benefits of
this antibody-based approach by generating a CAR that targets the
CSPG4 molecule. In contrast to mAb-based therapy, CAR-T cells
should produce long-lasting effects, as engineered T cells can
expand at the tumor site upon antigen stimulation if an appropriate
co-stimulatory endodomain, derived from CD28, CD137 or CD134, is
incorporated within the CAR. In contrast to a previous report, the
CSPG4-specific CAR obtained from the same 763.74 single chain has
potent antitumor activity. These striking differences were traced
to two critical components introduced in the construct. First, the
scFv in the CAR is coupled with the CD3-.zeta. endodomain of the
TCR rather than the Fc.epsilon.RI-.gamma. chain, which is known to
promote a much weaker and less durable signaling. Second, the CD28
costimulatory endodomain was incorporated within the CAR to
accomplish sustained IL-2 production and proliferation in response
to CSPG4.sup.+ tumor cells, thus recapitulating previous
observations for other CAR molecules. Of note, the inclusion of a
second costimulatory endodomain derived from CD137 did not further
improve the function of the CAR in vitro.
[0164] A significant improvement in the field by this work is the
applicability of CAR.CSPG4.sup.+ T cells not only to target
melanoma, but more broadly to other solid tumors generally
characterized by poor prognosis with conventional treatments such
as breast carcinoma, HNSCC and mesothelioma. It was demonstrated
herein that CAR.CSPG4.sup.+ T cells produce IFN.gamma. and promote
tumor elimination not only when challenged with tumor cells with
high CSPG4 expression but also with tumor cell lines characterized
by moderate/low CSPG4 expression, such as the breast carcinoma
tumor cell lines UACC-812 and MBA-MB-231. This further supports the
advantages of antibody specificity coupled with the T-cell effector
function, as mastered by CAR-modified T cells, which can target
tumors neglected by naked corresponding antibodies due to the
suboptimal expression of the targeted antigen.
[0165] Antitumor effects mediated by CAR.CSPG4+ T cells
significantly limit tumor growth in xenograft mouse models of
melanoma, HNSCC and breast carcinoma, strongly validating the in
vitro findings. The lack of sustained and complete tumor
eradication in these models was not caused by selection of
CSPG4-negative tumor cells, as harvested tumors retained the
expression of the antigen (FIG. 10), but conversely are likely to
be attributed to an intrinsic limitation of the models, as T cells
do not persist long term in these immunodeficient mice (FIG.
10).
[0166] To fully translate this approach, the differential
expression of CSPG4 in tumor cells versus normal tissues is
addressed in order to limit potential toxicities. The expression of
CSPG4 was absent or negligible in normal tissue arrays as assessed
by IHC. The analysis of publically available data sets indicates
that there is some level of CSPG4 mRNA expression in several normal
tissues. However, when one compared normal tissues with cancer
tissues, the cancers show consistent and dramatically higher
expression of CSPG4 at mRNA levels, and the in vitro analyses
illustrate that primary epithelial cells derived from some of these
tissues do not express significant amount of the protein and are
not targeted by CAR.CSPG4+ T cells. Even though the tissue
screening, bioinformatics analysis and lack of toxicity by in vitro
experiments support the relevance of CSPG4 as a targetable antigen
in cancer patients, in some cases the low levels of mRNA in normal
tissues, as reported in public data sets, may promote sufficient
protein expression in specific physiological conditions to become a
target for CSPG4-specific CAR-T cells. Thus, in some embodiments,
the inclusion of a suicide gene within the vector cassette is
useful to allow the rapid elimination of CAR-modified T cells in
case of undesired toxicity.
[0167] In summary, ample data is provided to support the use of
CAR.CSPG4.sup.+ T cells to treat a broad range of solid CSPG4.sup.+
tumors for which the prognosis remains poor with conventional
treatments. The combination of this approach with other biological
agents further increases their activity and thus clinical benefits,
in at least specific embodiments.
[0168] Although the present invention and its advantages have been
described in detail, it should be understood that various changes,
substitutions and alterations can be made herein without departing
from the spirit and scope of the invention as defined by the
appended claims. Moreover, the scope of the present application is
not intended to be limited to the particular embodiments of the
process, machine, manufacture, composition of matter, means,
methods and steps described in the specification. As one of
ordinary skill in the art will readily appreciate from the
disclosure of the present invention, processes, machines,
manufacture, compositions of matter, means, methods, or steps,
presently existing or later to be developed that perform
substantially the same function or achieve substantially the same
result as the corresponding embodiments described herein may be
utilized according to the present invention. Accordingly, the
appended claims are intended to include within their scope such
processes, machines, manufacture, compositions of matter, means,
methods, or steps.
Sequence CWU 1
1
21777DNAArtificial SequenceSynthetic Primer 1atggagtttg ggctgagctg
gctttttctt gtggctattt taaaaggtgt ccagtgctct 60agaatggccc aggtcaaact
gaaggagtct ggacctgagc tgaagaagcc tggagagaca 120gtcaagatct
cctgcaaggc ttctggttat accttcacag actattcaat gcactgggtg
180aagaagactc caggaaaggg tttaaagtgg ctgggctgga taaacactgc
gactggtgag 240ccaacatatg cagatgactt caagggacgg tttgccatct
ctttggaaac ctctgccagg 300actgtctatt tgcagatcaa taatctcaga
aatgaggaca cggctacata tttctgtttt 360agttactacg actactgggg
ccaaggcacc acggtcaccg tctcctcagg tgggggcggt 420tcaggcggag
gtggctctgg cggtggcgga ttggacatca agctcactca gtctccatcc
480atcctgtctg tgactccagg tgaaacagtc agtctttcct gtagggccag
ccagactatt 540tacaagaacc tacactggta tcaacagaaa tcacatcggt
ctccaaggct tctcatcaag 600tatggttctg attccatctc tgggatcccc
tccaggttca ctggcagtgg atcagggaca 660gattacactc tcaatatcaa
cagtgtgaag cccgaagatg aaggaatata ttactgtctt 720caaggttaca
gtacaccttg gacgttcggt ggagggacca agctggaaat aaaacgg
7772259PRTArtificial SequenceSynthetic Peptide 2Met Glu Phe Gly Leu
Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys
Ser Arg Met Ala Gln Val Lys Leu Lys Glu Ser Gly Pro 20 25 30 Glu
Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser 35 40
45 Gly Tyr Thr Phe Thr Asp Tyr Ser Met His Trp Val Lys Lys Thr Pro
50 55 60 Gly Lys Gly Leu Lys Trp Leu Gly Trp Ile Asn Thr Ala Thr
Gly Glu 65 70 75 80 Pro Thr Tyr Ala Asp Asp Phe Lys Gly Arg Phe Ala
Ile Ser Leu Glu 85 90 95 Thr Ser Ala Arg Thr Val Tyr Leu Gln Ile
Asn Asn Leu Arg Asn Glu 100 105 110 Asp Thr Ala Thr Tyr Phe Cys Phe
Ser Tyr Tyr Asp Tyr Trp Gly Gln 115 120 125 Gly Thr Thr Val Thr Val
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 130 135 140 Gly Ser Gly Gly
Gly Gly Leu Asp Ile Lys Leu Thr Gln Ser Pro Ser 145 150 155 160 Ile
Leu Ser Val Thr Pro Gly Glu Thr Val Ser Leu Ser Cys Arg Ala 165 170
175 Ser Gln Thr Ile Tyr Lys Asn Leu His Trp Tyr Gln Gln Lys Ser His
180 185 190 Arg Ser Pro Arg Leu Leu Ile Lys Tyr Gly Ser Asp Ser Ile
Ser Gly 195 200 205 Ile Pro Ser Arg Phe Thr Gly Ser Gly Ser Gly Thr
Asp Tyr Thr Leu 210 215 220 Asn Ile Asn Ser Val Lys Pro Glu Asp Glu
Gly Ile Tyr Tyr Cys Leu 225 230 235 240 Gln Gly Tyr Ser Thr Pro Trp
Thr Phe Gly Gly Gly Thr Lys Leu Glu 245 250 255 Ile Lys Arg
* * * * *
References