U.S. patent application number 15/504554 was filed with the patent office on 2017-08-24 for genetically modified mesenchymal stem cells expressing an immune response-stimulating cytokine to attract and/or activate immune cells.
The applicant listed for this patent is apceth GmbH & Co. KG. Invention is credited to Christine Gunther, Felix Hermann, Ralf Huss, Stefanos Theoharis.
Application Number | 20170239297 15/504554 |
Document ID | / |
Family ID | 51352460 |
Filed Date | 2017-08-24 |
United States Patent
Application |
20170239297 |
Kind Code |
A1 |
Gunther; Christine ; et
al. |
August 24, 2017 |
GENETICALLY MODIFIED MESENCHYMAL STEM CELLS EXPRESSING AN IMMUNE
RESPONSE-STIMULATING CYTOKINE TO ATTRACT AND/OR ACTIVATE IMMUNE
CELLS
Abstract
A genetically modified mesenchymal stem cell (MSC) and medical
use thereof in the treatment of tumors, the MSC including one or
more exogenous nucleic acid molecule(s), wherein the exogenous
nucleic acid molecule(s) include a region encoding one or more
immune response-stimulating or immune response-modulating
cytokine(s) operably linked to a promoter or promoter/enhancer
combination. The invention encompasses the use of the cells in
modulating the tumor microenvironment in order to attract immune
effector cells and facilitate their activation and/or adoption of a
memory phenotype. One aspect of the invention relates to the use of
the cells in anti-tumor treatment including combined administration
of the mesenchymal stem cells with anti-tumor immunotherapies, such
as checkpoint inhibitors, immune cells, for example T cells, such
as T cells with artificial T cell receptors, for example a chimeric
antigen receptor (CAR-Ts) or exogenous T-Cell Receptor (TCR)
transduced cells, NK cells or macrophages/monocytes, or a cancer
vaccine.
Inventors: |
Gunther; Christine;
(Munchen, DE) ; Theoharis; Stefanos; (Munchen,
DE) ; Hermann; Felix; (Munchen, DE) ; Huss;
Ralf; (Waakirchen, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
apceth GmbH & Co. KG |
Munchen |
|
DE |
|
|
Family ID: |
51352460 |
Appl. No.: |
15/504554 |
Filed: |
August 18, 2015 |
PCT Filed: |
August 18, 2015 |
PCT NO: |
PCT/EP2015/068942 |
371 Date: |
February 16, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/54 20130101;
A61K 35/28 20130101; A61P 43/00 20180101; A61P 35/00 20180101; C07K
14/52 20130101; A61K 2035/124 20130101; A61K 35/17 20130101; C07K
14/555 20130101; C12N 5/0663 20130101; A61K 38/00 20130101; C07K
14/70521 20130101; C12N 2510/02 20130101; C07K 16/30 20130101 |
International
Class: |
A61K 35/28 20060101
A61K035/28; C07K 16/30 20060101 C07K016/30; C07K 14/705 20060101
C07K014/705; A61K 35/17 20060101 A61K035/17; C07K 14/54 20060101
C07K014/54; C07K 14/555 20060101 C07K014/555 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 18, 2014 |
EP |
14181283.4 |
Claims
1. A method of treating a tumor in a subject comprising
administering a genetically modified mesenchymal stem cell (MSC)
and an anti-tumor immunotherapy to the subject, wherein said MSC
comprises one or more exogenous nucleic acid molecule(s), wherein
said exogenous nucleic acid molecule(s) comprise a region encoding
one or more immune response-stimulating cytokine(s) operably linked
to a promoter or promoter/enhancer combination.
2. The method according to claim 1, wherein the promoter or
promoter/enhancer combination yields constitutive expression of the
exogenous nucleic acid.
3. The method according to claim 2, wherein the promoter yielding
constitutive expression is an EFlalpha promoter, a PGK promoter, a
CMV promoter, an SV40 promoters, a GAG promoter or a UBC
promoter.
4. The method according to claim 1, wherein the promoter or
promoter/enhancer combination is induced when the genetically
modified mesenchymal stem cell comes into proximity with a tumor
tissue or a tumor stromal tissue, or wherein the promoter or
promoter/enhancer combination is induced upon differentiation of
said cell, post-administration.
5. The method according to claim 1, wherein the promoter is the
RANTES promoter, the HSP70 promoter or the Tie2 promoter.
6. (canceled)
7. (canceled)
8. (canceled)
9. The method according to claim 1, wherein the immune
response-stimulating cytokine maintains or enhances the activity,
survival and/or number of immune cells within and/or in proximity
to a tumor tissue.
10. The method according to claim 1, wherein the one or more immune
response-stimulating cytokine(s) comprise IL-2, IL-7, IL-12, IL-15
and/or IL-21.
11. (canceled)
12. (canceled)
13. (canceled)
14. (canceled)
15. The method according to claim 1, wherein the one or more immune
response-stimulating cytokine(s) comprise IFN gamma and/or IFN
beta.
16. (canceled)
17. The method according to claim 1, wherein the exogenous nucleic
acid molecule comprises a region encoding an immune stimulatory
molecule that induces T-cell proliferation and/or differentiation
operably linked to a promoter or promoter/enhancer combination.
18. The method according to claim 17, wherein the immune
stimulatory molecule that induces T-cell proliferation and/or
differentiation is CD28.
19. The method according to claim 1, wherein the one or more immune
response-stimulating cytokine is/are a chemokine.
20. The method according to claim 19, wherein the chemokine has
chemotactic properties for attracting T cells.
21. The method according to claim 1, wherein at least one of the
one or more immune response-stimulating cytokine(s) is selected
from the group consisting of chemokine (C-C motif) ligand 1 (CCL1,
CCL2, CCL4, CCL17, CCL19, CCL22, CCL23, and stromal cell-derived
factor 1 (SDF-1).
22. The method according to claim 1, wherein the anti-tumor
immunotherapy comprises the administration of an immune cell.
23. The method according to claim 22, wherein the mesenchymal stem
cell and the immune cell are autologous to the subject of medical
treatment.
24. The method according to claim 22, wherein the immune cell is a
T cell.
25. The method according to claim 22, wherein the immune cell is a
T cell comprising an artificial T cell receptor, wherein said T
cell receptor binds specifically to a tumor antigen.
26. The method according to claim 22, wherein the immune cell is a
macrophage.
27. The method according to claim 1, wherein the anti-tumor
immunotherapy comprises the administration of one or more
checkpoint inhibitors.
28. The method according to claim 27, wherein said checkpoint
inhibitor is a PD-L1 inhibitor, PD-1 inhibitor and/or CTLA-4
inhibitor.
29. The method according to claim 1, wherein the anti-tumour
immunotherapy comprises the administration of tumor antigens or
patient-derived tumor material.
30. (canceled)
31. The method according to claim 1, wherein the anti-tumor
immunotherapy comprises the administration of an antibody or
antibody fragment targeted to a tumor specific antigen.
32. A genetically modified mesenchymal stem cell (MSC), wherein
said MSC comprises one or more exogenous nucleic acid molecule(s),
wherein said exogenous nucleic acid molecule(s) comprises one or
more regions encoding two or more immune response-stimulating
cytokines operably linked to one or more promoters or
promoter/enhancer combinations.
33. The genetically modified mesenchymal stem cell according to
claim 32, wherein at least one of said two or more immune
response-stimulating cytokines is selected from the group
consisting of IL-2, IL-7, IL-12, IL-15, IL21, IFN gamma and IFN
beta.
34. The genetically modified mesenchymal stem cell according to
claim 32, wherein the two or more immune response-stimulating
cytokines comprise at least IL-12, and one or more of IL-2, IL-7,
IL-15, and/or IL-21.
35. The genetically modified mesenchymal stem cell according to
claim 32, wherein the two or more immune response-stimulating
cytokines comprise at least IL-7 and IL-21.
36. (canceled)
37. (canceled)
38. (canceled)
39. The method according to claim 25, wherein the artificial T cell
receptor is a chimeric antigen receptor (CAR).
40. The method according to claim 1, wherein said exogenous nucleic
acid molecule(s) comprise a region encoding two or more immune
response-stimulating cytokine(s), selected from the group
consisting of IL-2, IL-7, IL-12, IL-15 and IL-21.
41. The method according to claim 40, wherein the two or more
immune response-stimulating cytokines comprise at least IL-7 and
IL-21.
Description
[0001] The invention relates to a genetically modified mesenchymal
stem cell (MSC), and their use as a medicament in the treatment of
a tumour, said MSCs comprising one or more exogenous nucleic acid
molecule(s), wherein said exogenous nucleic acid molecule(s)
comprise a region encoding one or more immune response-stimulating
or immune response-modulating cytokine(s) operably linked to a
promoter or promoter/enhancer combination. The invention further
relates to a genetically modified mesenchymal stem cell comprising
at least one exogenous nucleic acid molecule that comprises a
region encoding an immune stimulatory molecule, for example that
that induces T-cell proliferation and/or differentiation.
[0002] The invention encompasses the use of MSCs as a medicament in
the treatment of a tumour and/or tumour disease, for example by
modulating the tumour microenvironment in order to attract immune
effector cells and facilitate their activation and/or adoption of a
memory phenotype. One aspect of the invention relates to the use of
said MSCs in anti-tumour treatment comprising the combined
administration of said mesenchymal stem cells with an
immunotherapy, for example checkpoint inhibitors, including for
example antibodies against CTLA-4, PD-1, PD-L1 and other immune
co-stimulatory molecules, immune cells, for example T cells, such
as T cells with artificial T cell receptors, for example a chimeric
antigen receptor (CAR-Ts) or exogenous T-Cell Receptor (TCR)
transduced cells, NK cells or macrophages/monocytes, or active
immunotherapeutic drugs, for example, tumour antigens,
patient-derived tumour material and other therapeutic drugs aiming
to activate and/or direct the immune response against a tumour, or
features of a tumour.
BACKGROUND OF THE INVENTION
[0003] Mesenchymal stem cells (MSCs) are cells of
non-haematopoietic origin that reside in the bone marrow and other
tissues. MSCs are commonly considered to be multipotent adult
progenitor cells that have the ability to differentiate into a
limited number of cell lineages, such as osteoblasts, chondrocytes,
and adipocytes. Studies have been conducted on the use of MSCs as a
therapeutic entity based on a capacity to differentiate directly
into these end-stage phenotypes, including the use of MSCs to
promote or augment bone repair and for the repair of cartilage
defects (Vilquin and Rosset, Regenerative Medicine 2006: 1, 4, p
589, and Veronesi et al, Stem Cells and Development 2013; 22, p
181). The isolation and cultivation of MSCs fora number of
therapeutic indications has been described and represents a
promising approach towards treating inflammation-associated
disorders (for example WO 2010/119039).
[0004] MSCs are known to exhibit immune evasive properties after
administration to a patient. MSCs have been shown to exhibit a
beneficial immune modulatory effect in cases of transplantation of
allogeneic donor material (Le Blanc et al, Lancet 2004: 363, p
1439), thereby reducing a potentially pathogenic alloreactivity and
rejection. MSCs treatment can also play a therapeutic role in wound
healing. The therapeutic delivery of MSCs can be performed via
systemic injection, followed by MSC homing to and engraftment
within sites of injury (Kidd et al, Stem Cells 2009: 27, p 2614).
MSCs are also known to exhibit migratory properties with respect to
homing towards tumours in vivo.
[0005] MSC-based cellular therapy using genetically modified MSCs
enables the delivery of therapeutic gene products to a specific
region of interest in the body of a patient. For example, MSCs have
been shown to migrate to areas of inflammation, such as tumours,
and thereby locally exert therapeutic influence. MSCs typically
have immune-modulatory effects that lead to immune suppression in
the area of interest, thereby mediating or reducing inflammation to
enhance recovery. However, the present invention makes use of MSCs
as a cellular vehicle for the delivery of immunomodulatory
effectors for simulating an immune response, thereby utilising the
unique homing abilities of MSC to target regions of inflammation,
in particular tumours, and thereby exert local therapeutic effects
based on activation of an appropriate immune response.
[0006] Tumour growth can be countered by appropriate activation or
strengthening of an immune response directed against tumour tissue.
However, means for local enhancement of such an immune response,
without initiating unwanted systemic side effects, are needed in
the field of cancer therapy. There are many drugs for the treatment
of cancer that cannot be administered systemically without causing
significant unwanted side effects (for example fever, elevated
levels of liver enzymes or systemic inflammation, such as a
cytokine storm, potentially leading to death). One option for
avoiding such side effects is the administration of a significantly
reduced dose of such drugs, which although effective in reducing
side effects, often yields insufficient drug levels at the target
site and insufficient therapeutic benefit.
SUMMARY OF THE INVENTION
[0007] In light of the prior art the technical problem underlying
the present invention is to provide a novel strategy for
immunotherapy of cancers. In particular, a problem underlying the
invention is the provision of suitable means for local stimulation
of an immune response directed against tumour tissue in a subject.
A further problem underlying the invention is the provision of
means for administration of an effective dose of therapeutic
agents, such as immune modulatory or immune stimulatory agents,
that provides an effective local effect at the target tissue with
reduced levels of unwanted side effects.
[0008] This problem is solved by the features of the independent
claims. Preferred embodiments of the present invention are provided
by the dependent claims.
[0009] The invention therefore relates to a genetically modified
mesenchymal stem cell for use as a medicament in the treatment of a
tumour, wherein said MSCs comprise one or more exogenous nucleic
acid molecule(s), wherein said exogenous nucleic acid molecule(s)
comprise a region encoding one or more immune response-stimulating
cytokine(s) operably linked to a promoter or promoter/enhancer
combination, wherein said treatment comprises the combined
administration of said mesenchymal stem cells with an anti-tumour
immunotherapy.
[0010] Combined administration may relate to concurrent and/or
sequential administration of said mesenchymal stem cells prior to,
during and/or subsequent to said immunotherapy. Combined shall also
include a combination treatment regime comprising multiple
administrations of either therapeutic component of the treatment.
Further embodiments of combined administration are provided
herein.
[0011] The invention therefore encompasses the medical use of the
MSCs described herein, in particular for treating cancer, in
addition to methods for the treatment of a subject with a tumour,
or methods of treatment of a tumour and/or tumour disease.
[0012] The present invention also encompasses the genetically
modified mesenchymal stem cells as described herein, as such,
independent of their particular medical use.
[0013] The present invention enables the stimulation of cells
involved in an anti-tumour immune response and thereby the local
activation, support and/or strengthening of an anti-tumour immune
response. The MSCs as described herein migrate to tumour tissue due
to either their natural or an engineered capacity for homing to
areas of inflammation in vivo. The homing to and/or engraftment
into tumour tissue (tumour stroma) leads to local expression of
immune stimulating cytokines, thereby creating increased amounts
and/or activities of immune cells in the local tumour environment,
thereby enabling the immune system of the patient to attack tumour
cells and also providing support for a combined immunotherapy.
[0014] The present invention enables an effective and
therapeutically relevant dose of one or more immune stimulatory
cytokines to be administered via expression from transplanted MSCs
while avoiding the significant side effects that are inherent in
systemic administration of cytokines without an appropriate
targeting agent. The invention therefore relates to the utilization
of MSC as a targeting agent and/or vehicle for the local delivery
of immune modulatory, preferably immune stimulating signals in
regions of inflammation, preferably in and in proximity to tumour
tissue.
[0015] A crucial limitation in the successful development and
clinical use of immunotherapies is the ability of tumours to evade
and suppress the natural immune response against the tumour cells,
by establishing an immunosuppressive tumour microenvironment, This
phenomenon is known as tumour-mediated immunosuppression and is
mediated to a large extent by the secretion of anti-inflammatory
cytokines by immune cells present in the tumour that display a
regulatory phenotype (for example, Regulatory T-cells; TRegs and
Monocyte-Derived Suppressor Cells; MDSCs). The invention therefore
provides means to modify the tumour microenvironment, making it
pro-inflammatory promoting the activation of immune cells present
in the tumour and recruitment and activation of external immune
cells and thereby facilitating the broad activation of the immune
system against the tumour and/or enhance the efficacy of
anti-tumour immunotherapeutic treatments.
[0016] In one embodiment the MSCs as described herein can be
administered prior to an immunotherapeutic treatment in order to
modify and render the tumour microenvironment favourable and
conducive to immunotherapies.
[0017] The present invention makes use of MSCs as a cellular
vehicle for the delivery of immunomodulatory effectors for
simulating an immune response, thereby utilising the unique homing
abilities of MSC to target regions of inflammation, in particular
tumours, and thereby exert local therapeutic effects based on
activation of an appropriate immune response, wherein the immune
response relates preferably to the natural immune response of a
host (subject), and thereby enhance the efficacy and therapeutic
effect of immunotherapeutics, such as bi-specific antibodies,
adoptive immunotherapies, anti-tumour vaccines and/or checkpoint
inhibitors.
[0018] MSC are able to express various anti-inflammatory factors in
response to pro-inflammatory signals (for example, Transforming
growth factor beta (TGF-b), indoleamine 2,3-dioxygenase). For TGF-b
it has been shown that it can enhance survival of activated T cells
as it protects the cells from so called "activation induced cell
death". Surprisingly, the expression of pro-inflammatory cytokines
in MSCs allows the targeted and continuous activation of bystander
T cells, without inducing activation induced cell death (AICD) in
the T cells. This property of the cells described herein is due to
the presence of anti-inflammatory factors (e.g. TGF-b) which are
also expressed by MSC and prevent AICD. The combination of MSCs
expressing anti-inflammatory factors with the pro-inflammatory
transgene cytokines leads to an unexpected technical effect that is
advantageous in the stimulation of the immune response against
tumours.
[0019] Surprisingly, the MSCs modified with one or more immune
response stimulating cytokine(s) as described herein show
unexpectedly good expression and secretion of said cytokines both
in vitro and in vivo. A skilled person would not expect that these
particular cytokines could be expressed in sufficient quantities
and exported from the cells in sufficient quantities to induce or
enhance the desired local immune response, based on either the
innate response or and immunotherapy.
[0020] The invention also encompasses the expression of a
combination of immune activating cytokine and/or chemokines in
tumours via the MSC-based approach described herein, with the aim
to attract immune effector and helper cells, induce immune
activation, promote the maturation of memory immune cells and/or
suppress the emergence and persistence of suppressive and/or
regulatory immune cells.
[0021] In one embodiment, a combination of cytokines is used, in
order to promote the activation of different arms of the immune
response, including the innate and adaptive immune response,
effector, helper and/or antigen presenting cells.
[0022] It is envisioned that cytokines such as TNF-alpha will
activate multiple aspects of the immune system and that this effect
may however lack the necessary specificity for an anti-tumoral
response.
[0023] On the other hand, IL-2, IL-7, IL-15 and IL-21 specifically
activate cytotoxic lymphocytes such as T-cells and NK cells that
mount a specific response against tumour cells. Likewise, IL-12
will activate cytotoxic lymphocytes, but also monocytes and helper
cells.
[0024] The combination of IL-12, for example, with IL-2, IL-7,
IL-15, and/or IL-21, will have the effect of activating (i)
tumour-directed cytotoxic cells, (ii) helper cells that enhance the
activation of cytotoxic cells and/or (iii) monocytes that can
develop an additive immunological response against the tumour. A
combination of cytokines therefore yields synergistic effects, as
is seen in the natural immune response, and in the present
invention greatly increases the therapeutic efficacy. It was a
surprising result that the natural immune response could, in
effect, be mirrored, or analogously applied, in an enhanced manner
using a MSC-based transgenic approach.
[0025] The invention is therefore based on the surprising finding
that by providing a combination of transgenes encoding immune
response-stimulating cytokine(s) in one or more MSCs a more
effective local and safe anti-tumour response can be obtained. The
combination of multiple cytokines, in the MSC vehicle, leads to
unique local expression and secretion of the immune-stimulating
factors that leads to a local anti-tumour response, comprising
multiple arms of the immune response, without inducing systemic
toxicity as is often observed when systemically applying cytokines
in tumour patients.
[0026] Furthermore, the unique properties of MSCs, which home to
and engraft into tumour tissue, leads to maintained expression of
the therapeutic cytokine factors in order to maintain the immune
response for therapeutic effect. In one embodiment the use of an
inducible promoter, preferably expressed in proximity to or within
tumour tissue, or in inflamed tissue, enables local and
tumour-specific cytokine expression which is subsequently reduced
after the tumour (and therefore preferably the inflammation) in
that region) is subsequently reduced.
[0027] The present invention also relates to a genetically modified
mesenchymal stem cell (MSC), wherein said MSCs comprise one or more
exogenous nucleic acid molecule(s), wherein said exogenous nucleic
acid molecule(s) comprise a region encoding two or more immune
response-stimulating cytokines operably linked to one or more
promoters or promoter/enhancer combinations.
[0028] In a preferred embodiment the genetically modified
mesenchymal stem cell as described herein is characterised in that
the exogenous nucleic acid comprises a region encoding two or more
immune response-stimulating cytokines operably linked to one or
more promoters or promoter/enhancer combinations, wherein the
cytokines are selected from the group consisting of IL-2, IL-7,
IL-12, IL-15, IL21, IFN gamma and IFN beta.
[0029] In a preferred embodiment the genetically modified
mesenchymal stem cell as described herein is characterised in that
the two or more immune response-stimulating cytokines comprise at
least IL-12, and one or more of IL-2, IL-7, IL-15, and/or
IL-21.
[0030] In a preferred embodiment the genetically modified
mesenchymal stem cell as described herein is characterised in that
the two or more immune response-stimulating cytokines comprise
least IL-7 and IL-21.
[0031] In a preferred embodiment the genetically modified
mesenchymal stem cell as described herein is characterised in that
the two or more immune response-stimulating cytokines comprise at
least one chemokine and at least one immune response-stimulating
cytokine is selected from the group consisting of IL-2, IL-7,
IL-12, IL-15, IL21, IFN gamma and IFN beta.
[0032] The present invention encompasses in some embodiments the
combination of cytokine transgenes in the cells as described
herein, in particular any given specific combination of individual
cytokines or chemokines disclosed herein, preferably any given
specific combination of two or more of -2, IL-7, IL-12, IL-15,
IL21, IFN gamma, IFN beta, CD28, chemokine (C-C motif) ligand 1, 2,
4, 17, 19, 22, 23 (CCL1, CCL2, CCL4, CCL17, CCL19, CCL22, CCL23),
stromal cell-derived factor 1 (SDF-1).
[0033] The MSCs defined by two or more immune response-stimulating
cytokines and the method of tumour treatment comprising
administration of MSCs defined by one or more immune
response-stimulating cytokines in combination with other
anti-tumour immunotherapies are bound by the surprising and
beneficial concept of local immune system stimulation in an
anti-tumour immune response, either by the innate immune system or
by combined immunotherapies. It was unexpected that MSCs encoding
transgenic immune-stimulating cytokines may be used as an effective
anti-tumour adjuvant in stimulating an anti-tumour response. The
related prior art teaches only the use of MSC vehicles for the
local administration of (transgenic) cytotoxic agents, with a
direct effect. However, the use of MSCs encoding (potentially
multiple) transgenic immune-stimulatory cytokines, or the
combination of such MSCs with anti-tumour immunotherapies, to boost
the local anti-tumour immune response, represent special technical
features of the invention.
[0034] Surprisingly, the MSCs modified with multiple cytokines as
described herein show unexpectedly good expression and secretion of
said cytokines both in vitro and in vivo. A skilled person would
not expect that these particular cytokines could be expressed in
sufficient quantities and exported from the cells in sufficient
quantities to induce or enhance the desired local immune response,
based on either the innate response or and immunotherapy.
[0035] The invention therefore relates to a genetically modified
mesenchymal stem cell described herein comprising one or more
exogenous nucleic acid molecule(s), wherein said exogenous nucleic
acid molecule(s) comprise a region encoding two or more immune
response-stimulating cytokines for use as a medicament in the
treatment of a tumour. Any of the features disclosed herein with
respect to the method of treatment or medical administration or
application of the MSCs, or their combined administration with an
anti-tumour immunotherapy described herein, also apply to the MSCs
comprising transgenic material for at least two or more immune
response-stimulating cytokines.
[0036] The invention provides suitable means for local stimulation
of an immune response directed against tumour tissue in a subject.
This includes the natural immune response of the patient and
immunotherapeutic treatments aiming to direct the immune response
against the tumour (e.g. checkpoint inhibitors, CARTs against
tumour antigens and other tumour immunotherapies). Such support or
induction of the immune response may in various clinical settings
be beneficial in order to initiate and maintain the immune response
and evade the tumour-mediated immunosuppression that often blocks
this activation.
[0037] The cells of the present invention may be used to enhance
the activities and/or increase amounts of endogenous immune cells
that are already present in the subject. Alternatively or
additionally, additional immune cells (either autologous or
allogeneic) may be administered in combination with the MSCs (for
example concurrently or sequentially) of the invention in order to
enhance the desired therapeutic immune response.
[0038] The construction of the genetically modified MSCs described
herein may be carried out using techniques known to a person
skilled in the art.
[0039] In one embodiment, the genetically modified mesenchymal stem
cell as described herein is characterised in that the exogenous
nucleic acid comprises viral vector sequences, for example in the
form of a viral expression construct.
[0040] In one embodiment, the genetically modified mesenchymal stem
cell as described herein is characterised in that the exogenous
nucleic acid is a non-viral expression construct.
[0041] As used herein, "nucleic acid" shall mean any nucleic acid
molecule, including, without limitation, DNA, RNA and hybrids or
modified variants thereof. An "exogenous nucleic acid" or
"exogenous genetic element" relates to any nucleic acid introduced
into the cell, which is not a component of the cells "original" or
"natural" genome. Exogenous nucleic acids may be integrated or
non-integrated in the genetic material of the target mesenchymal
stem cell, or relate to stably transduced nucleic acids.
[0042] Any given gene delivery method is encompassed by the
invention and preferably relates to viral or non-viral vectors, as
well as biological or chemical methods of transfection, or
combinations thereof. The methods can yield either stable or
transient gene expression in the system used.
[0043] Genetically modified viruses have been widely applied for
the delivery of genes into stem cells. Adenoviruses may be applied,
or RNA viruses such as Lentiviruses, or other retroviruses.
Adenoviruses have been used to generate a series of vectors for
gene transfer in the field of gene therapy and cellular
engineering. The initial generation of adenovirus vectors were
produced by deleting the El gene (required for viral replication)
generating a vector with a 4 kb cloning capacity. An additional
deletion of E3 (responsible for host immune response) allowed an 8
kb cloning capacity. Further generations have been produced
encompassing E2 and/or E4 deletions. The use of any given
adenovirus vector, for example those according to those described
above, is encompassed by the present invention.
[0044] Lentiviruses are members of Retroviridae family of viruses
(M. Scherr et al., Gene transfer into hematopoietic stem cells
using lentiviral vectors. Curr Gene Ther. 2002 February;
2(1):45-55). Lentivirus vectors are generated by deletion of the
entire viral sequence with the exception of the LTRs and cis acting
packaging signals. The resultant vectors have a cloning capacity of
about 8 kb. One distinguishing feature of these vectors from
retroviral vectors is their ability to transduce dividing and
non-dividing cells as well as terminally differentiated cells.
[0045] Non-viral methods may also be employed, such as alternative
strategies that include conventional plasmid transfer and the
application of targeted gene integration through the use of
nuclease-based gene editing, integrase or transposase technologies.
These represent approaches for vector transformation that have the
advantage of being both efficient, and often site-specific in their
integration. Physical methods to introduce vectors into cells are
known to a skilled person. One example relates to electroporation,
which relies on the use of brief, high voltage electric pulses
which create transient pores in the membrane by overcoming its
capacitance. One advantage of this method is that it can be
utilized for both stable and transient gene expression in most cell
types. Alternative methods relate to the use of liposomes or
protein transduction domains. Appropriate methods are known to a
skilled person and are not intended as limiting embodiments of the
present invention.
[0046] The invention encompasses the use of more than one virus, or
a virus and other gene editing event or genetic modification,
including the use of or mRNA, siRNA, miRNA, or other genetic
modification in order to manipulate gene expression any given
relevant factor. The immune response-stimulating cytokine may, in
some embodiments of the present invention, relate to multiple
cytokines and/or chemokines or combinations thereof.
[0047] In one embodiment the genetically modified mesenchymal stem
cell as described herein is characterised in that the promoter or
promoter/enhancer combination yields constitutive expression of the
exogenous nucleic acid. Due to the beneficial homing properties of
MSCs to tumours within the body of a subject post systemic
administration or after local administration, the use of a
constitutive promoter for expression of the one or more immune
response-stimulating cytokine(s) is preferred.
[0048] In one embodiment the genetically modified mesenchymal stem
cell as described herein is characterised in that the promoter is
an EF1alpha promoter, for example the EF1alphaS promoter.
[0049] In one embodiment the genetically modified mesenchymal stem
cell as described herein is characterised in that the promoter is
the PGK promoter.
[0050] In one embodiment the genetically modified mesenchymal stem
cell as described herein is characterised in that the promoter is
the CMV or SV40 viral promoters.
[0051] In one embodiment the genetically modified mesenchymal stem
cell as described herein is characterised in that the promoter is
the GAG promoter.
[0052] In one embodiment the genetically modified mesenchymal stem
cell as described herein is characterised in that the promoter is
the UBC promoter.
[0053] In one embodiment, the genetically modified mesenchymal stem
cell as described herein is characterised in that the immune
response-stimulating cytokine is expressed when the genetically
modified mesenchymal stem cell comes into proximity with tumour
tissue or tumour stromal tissue.
[0054] Given that mesenchymal stem cells can show a selective
migration to different tissue microenvironments in normal as well
as diseased settings, the use of tissue-specific promoters, or
other promoters linked to a particular disease microenvironment, or
promoters induced by a differentiation pathway initiated in the
recruited stem cell, is encompassed in the present invention and
can be used to drive the selective expression of therapeutic genes,
such as the cytokines described herein, specifically within a
defined biological context.
[0055] In a preferred embodiment, the genetically modified
mesenchymal stem cell as described herein is characterized in that
the promoter or promoter/enhancer combination is induced upon
differentiation of said cell post-administration. One example of
differentiation post-administration is endothelial differentiation,
wherein the MSC can engraft and subsequently differentiate into an
endothelial or endothelial-like cell in or in proximity to the
tumour tissue, thereby enabling expression of the stimulatory
cytokine in a local manner.
[0056] Stem cells that are recruited to other tissue niches, but do
not experience the disease region (the tumour environment), should
not express the therapeutic gene. This approach allows a
significant degree of potential control for the selective
expression of the therapeutic gene within a defined
microenvironment, thereby reducing the probability of the
occurrence of known toxicities associated with systemic
administration of pro-inflammatory cytokines, and has been
successfully applied to regulate therapeutic gene expression during
neovascularization. Potential approaches to such gene modifications
are disclosed in WO 2008/150368 and WO 2010/119039, which are
hereby incorporated in their entirety.
[0057] In one embodiment, the genetically modified mesenchymal stem
cell as described herein is characterised in that the promoter is
the Tie2 promoter.
[0058] Promoters can be introduced that are selectively regulated
in the context of inflammation or neovascularization. In this
regard the Tie2-promoter, Flkl promoter and intronic enhancer,
endothelin-1 promoter and the pre-proendothelin-1 promoter have
been studied for endothelial specific expression (Huss, R, von
Luttichau, I, Lechner, S, Notohamiprodjo, M, Seliger, C, Nelson, P
(2004) [Chemokine directed homing of transplanted adult stem cells
in wound healing and tissue regeneration]. Verh Dtsch Ges Pathol
88: 170-173).
[0059] Another embodiment of the invention provides mesenchymal
stem cells that comprise a promoter or promoter/enhancer
combination, which is inducible by inflammatory mediators and which
controls the transcription of the stimulatory cytokine (immune
response-stimulating cytokine). These inflammatory mediators can be
released by the tumour's stromal tissue so that the expression of
the cytotoxic protein in the mesenchymal stem cells is induced when
the stem cells come into proximity with the tumour's stromal
tissue. The inflammatory mediators can for example be cytokines,
such as TNF alpha or IFN gamma. In particular the promoter can be
the RANTES promoter, which can inter alia be induced by TNFOC or
IFN gamma (Nelson P J, Kim H T, Manning W C, et al. Genomic
organization and transcriptional regulation of the RANTES chemokine
gene. J Immunol 1993; 151 (5):2601-12; von Luettichau I, Nelson P.
J., Pattison J. M., et al. RANTES chemokine expression in diseased
and normal human tissues. Cytokine 1996; 8(I):89-98; Nelson P. J.,
Pattison J. M., Krensky A. M. Gene expression of RANTES. Methods
Enzymol 1997; 287:148-62; Duell E. J., Casella D. P., Burk R. D.,
et al. Inflammation, genetic polymorphisms in proinflammatory genes
TNF-A, RANTES, and CCRS, and risk of pancreatic adenocarcinoma.
Cancer Epidemiol Biomarkers Prey 2006; 15 (4):726-31;
Marfaing-Koka, A., et al., Regulation of the production of the
RANTES chemokine by endothelial cells. Synergistic induction by
IFN-gamma plus TNF- alpha and inhibition by IL-4 and IL-13. Journal
of Immunology, 1995. 154(4): p. 1870-8).
[0060] In one embodiment, the genetically modified mesenchymal stem
cell as described herein is characterised in that the promoter is
the RANTES promoter. The "RANTES" promoter is also known in the art
as the "CCLS" promoter.
[0061] Further examples of promoters, which are inducible by
pro-inflammation mediators are the NF-kB-responsive element and in
general promoters, which can be induced by TNF.
[0062] Additionally, promoters activated by anti-inflammatory
mediators (e.g. TGF-beta) can be used to achieve a targeted
expression the cytotoxic protein in the mesenchymal stem cells.
Examples are promoters which contain Smad-binding elements. Using
promoters, which are inducible by inflammation mediators, enables a
selective treatment of tumours, which have not yet undergone
angiogenesis.
[0063] Additionally, promoters activated in cancerous tissue, or
activated by signals released by cancerous cells, can be used in
the present invention to achieve selective expression of the
encoded cytokine in the relevant location within the patient in
order to avoid unwanted systemic effects. One example of a promoter
that is up-regulated in cancers is the HSP70 promoter. The HSP70
protein, which is the major stress-inducible heat shock protein, is
a chaperone protein abundantly and preferentially expressed in
human tumours and tumour cell lines. Owing to the ability of Hsp70
to protect cells from a wide range of apoptotic and necrotic
stimuli, it has been assumed that Hsp70 may confer survival
advantage to tumour cells (Rohde et al., Genes Dev. Mar. 1, 2005;
19(5): 570-582, Nylandsted et al., Ann N Y Acad Sci. 2000;
926:122-5, Ramp et al., Histol Histopathol. 2007 October;
22(10):1099-107, Ricaniadis et al., Eur J Surg Oncol. 2001
February; 27(1):88-93). The HSP70 promoter is therefore one option
for selective expression of the therapeutic cytokine encompassed by
the present invention. Further information on the HSP70 promoter
can be obtained from Wu et al., Proc. Natl. Acad. Sci. USA, Vol.
83, pp. 629-633, 1986.
[0064] The use of a "tumour-specific" promoter, or promoter
preferentially expressed or induced under inflammatory or
"cancer-like" conditions, may show a synergistic effect in
combination with the MSC homing properties with respect to
reduction of unwanted systemic effect. The MSCs of the present
invention migrate towards inflammatory, in particular tumour,
tissue, thereby providing effective means for avoiding systemic
expression of the encoded cytokine in the body of a patient. The
use of a promoter for the expression of the cytokine that is
preferentially expressed under conditions of inflammation or of
being present in tumour tissue further enhances the reduction in
systemic expression in a synergistic manner, thereby providing
surprising benefits in the MSC-based mode of administration of the
cytokines described herein.
[0065] In one embodiment the genetically modified mesenchymal stem
cell as described herein is characterised in that the immune
response-stimulating cytokine maintains or enhances the activity,
survival and/or number of immune cells within or in proximity to
tumour tissue.
[0066] As known in the art, an immune response-stimulating cytokine
is to be understood as any cytokine that leads to or produces
either directly or indirectly the induction, activation and/or
enhancement of an immune response, preferably directed against an
antigen, for example a tumour antigen. In particular, the immune
response-stimulating cytokines of the invention are preferably
considered as cytokines that leads to the induction, activation
and/or enhancement of an immune response beneficial for the
treatment of a tumour disease.
[0067] As used herein, the terms immune response-modulating
cytokine or immune response-associated cytokine may be used
interchangeably and relate to molecules that participate, modulate,
control and/or regulate the immune response and/or inflammatory
reactions including anti-tumour activity due to the
differentiation, maturation and activation of immune cells.
[0068] Cytokines are a diverse group of non-antibody proteins that
act as mediators between cells. Cytokines are currently being
clinically used as biological response modifiers for the treatment
of various disorders. The term cytokine is a general term used to
describe a large group of proteins. Particular kinds of cytokines
may include Monokines, namely cytokines produced by mononuclear
phagocytic cells, Lymphokines, namely cytokines produced by
activated lymphocytes, especially Th cells, Interleukins, namely
cytokines that act as mediators between leukocytes and Chemokines,
namely small cytokines primarily responsible for leucocyte
migration. Cytokine signaling is flexible and can induce both
protective and damaging responses. They can produce cascades, or
enhance or suppress production of other cytokines. Despite the
various roles of cytokines, a skilled person is aware of which
cytokines may be considered as immune response-stimulating and
therefore applied in the treatment of a tumour disease as described
herein.
[0069] Cytokines have the ability to modulate immune responses and
are often utilised by a tumour to allow it to grow and manipulate
the immune response. These immune-modulating effects allow them to
be used as drugs to provoke an immune response against the
tumour.
[0070] The following cytokines may be referred to as
immune-response stimulatory or immune response-modulatory
cytokines.
[0071] Two commonly used groups of cytokines in anti-tumour therapy
are the interferons and interleukins.
[0072] Interferons are cytokines produced by the immune system
usually involved in an anti-viral response, but also show
effectiveness in the treatment of cancer. There are three groups of
interferons (IFNs): type I (IFN alpha and IFN beta), type 2 (IFN
gamma) and the relatively newly discovered type III (IFN lambda).
IFN alpha has been applied in the treatment of hairy-cell
leukaemia, AIDS-related Kaposi's sarcoma, follicular lymphoma,
chronic myeloid leukaemia and melanoma. Type I and II IFNs have
been researched extensively and although both types promote the
anti-tumour effects of the immune system, only type I IFNs have
been shown to be clinically effective in cancer treatment so far.
IFN lambda has been tested for its anti-tumour effects in animal
models, and shows promise.
[0073] In a preferred embodiment the genetically modified
mesenchymal stem cell as described herein is characterised in that
the immune response-stimulating cytokine is IFN alpha.
[0074] In a preferred embodiment the genetically modified
mesenchymal stem cell as described herein is characterised in that
the immune response-stimulating cytokine is IFN gamma.
[0075] In a preferred embodiment the genetically modified
mesenchymal stem cell as described herein is characterised in that
the immune response-stimulating cytokine is IFN beta.
[0076] According to some embodiments of the present invention, the
immune-response stimulatory or immune response-modulatory cytokines
are preferably those involved in T cell regulation or with effector
function for T cells (T cell regulatory cytokines). These cytokines
exhibit desired properties with respect to inducing a
pro-inflammatory microenvironment and thereby facilitating the
activation of the immune system against the tumour and/or enhance
the efficacy of anti-tumour immunotherapeutic treatments. Such
cytokines may be able to attract immune effector cells, such as T
cells, and promote the maturation of memory immune cells. Examples
of these cytokines are IFN gamma, IL-2, IL-12, IL-23, IL-15 and
IL-21 (refer Kelley's Textbook of Rheumatology; Firestein et al,
8th ed. (ISBN 978-1-4160-3285-4), p 367 "Cytokines").
[0077] The specific molecules mentioned herein relate preferably to
mammalian molecules, preferably human molecules, for reasons of
suitability for administration in human subjects. For example, the
cytokines and/or chemokines mentioned herein relate preferably to
the human sequences, as can be obtained by a skilled person without
undue effort, for example from a sequence database such as those
maintained by the National Center for Biotechnology Information
(NCBI).
[0078] Protein sequences or protein-coding nucleic acid sequences
may be modified in comparison to commonly known sequences, for
example by making e. g. conservative amino acid substitutions in a
protein sequence, or by using the degeneracy of the genetic code in
order to change the coding sequence without changing the encoded
protein sequence. As a skilled person is aware, the sequences of
biological molecules can be changed (mutated) via standard
techniques, their properties thereby being improved or maintained
in comparison to the known original sequence. Any modified cytokine
sequence that maintains the basic properties of, or is functionally
analogous to, the known sequence is therefore encompassed in the
scope of the present invention.
[0079] In a preferred embodiment the genetically modified
mesenchymal stem cell as described herein is characterised in that
the immune response-stimulating cytokine is IL-2.
[0080] Interleukin-2 is an example of a cytokine that can enhance
the anti-tumour activity of the immune system and thus can be used
as a treatment in cancer. Interleukin-2 has been used for the
treatment of malignant melanoma (trade name, Proleukin.RTM.) and
renal cell carcinoma. In normal physiology it promotes both
effector T cells (cells that produce the immune response) and
T-regulatory cells (cells that repress the immune response), but
its exact mechanism in the treatment of cancer is unknown. Recent
work indicates a beneficial effect of IL-2 expression in cancer
treatment. IL-2 has been used in conjunction with adoptive
immunotherapies, such as CART therapies, in order to promote T-cell
activation. However, systemic administration of IL-2 carries the
risk of broad immune activation which contributes to the toxicities
associated with CART therapies.
[0081] In a preferred embodiment the genetically modified
mesenchymal stem cell as described herein is characterised in that
the immune response-stimulating cytokine is IL-7.
[0082] Interleukin 7 (IL-7) is a hematopoietic growth factor
secreted by stromal cells in the bone marrow and thymus. IL-7
stimulates the differentiation of multipotent (pluripotent)
hematopoietic stem cells into lymphoid progenitor cells. IL-7 as an
immunotherapy agent has been examined in pre-clinical animal
studies and more recently in human clinical trials for various
malignancies and during HIV infection (Fry T J, Mackall C L (June
2002). "Interleukin-7: from bench to clinic". Blood 99 (11):
3892-904).
[0083] In a preferred embodiment the genetically modified
mesenchymal stem cell as described herein is characterised in that
the immune response-stimulating cytokine is IL-15.
[0084] Interleukin 15 (IL-15) is a cytokine with structural
similarity to IL-2. Like IL-2, IL-15 binds to and signals through a
complex composed of IL-2/IL-15 receptor beta chain (CD122) and the
common gamma chain (gamma-C, CD132). IL-15 induces cell
proliferation of natural killer cells; cells of the innate immune
system whose principal role is to kill virally infected or
cancerous cells. IL-15 has been shown to enhance the anti-tumour
immunity of CD8+ T cells in pre-clinical models (Klebanoff Calif.,
et al. Proc. Natl. Acad. Sci. U.S.A. 101 (7): 1969-74).
[0085] In a preferred embodiment the genetically modified
mesenchymal stem cell as described herein is characterised in that
the immune response-stimulating cytokine is IL-21.
[0086] Interleukin-21 (IL-21) is a cytokine that has potent
regulatory effects on cells of the immune system, including natural
killer (NK) cells and cytotoxic T cells that can destroy virally
infected or cancerous cells. IL-21 has been approved for Phase 1
and 2 clinical trials in metastatic melanoma (MM) and renal cell
carcinoma (RCC) patients (Sondergaard H, Skak K, Tissue Antigens 74
(6): 467-79). It has been shown to be safe for administration with
flu-like symptoms as side effects. Dose-limiting toxicities
included low lymphocyte, neutrophil, and thrombocyte count as well
as hepatotoxicity.
[0087] Interleukin 12 (IL-12) is an interleukin that is naturally
produced by dendritic cells, macrophages and human B-lymphoblastoid
cells (NC-37) in response to antigenic stimulation. IL-12 is
involved in the differentiation of naive T cells into Th1 cells. It
is known as a T cell-stimulating factor, which can stimulate the
growth and function of T cells, thereby falling under the concept
of the present invention. IL-12 is known to stimulate the
production of interferon-gamma (IFN-.gamma.) and tumour necrosis
factor-alpha (TNF-.alpha.) from T cells and natural killer (NK)
cells, and reduces IL-4 mediated suppression of IFN-.gamma.. IL-12
also has a known anti-angiogenic activity. IL-12 functions by
increasing production of IFN gamma, which in turn increases the
production of the chemokine CXCL10.
[0088] Interleukin-12 (IL-12) plays a role in the interaction
between the innate and adaptive arms of immunity by regulating
inflammatory responses, innate resistance to infection, and
adaptive immunity, in particular with respect to immune responses
against cancer cells (Colombo et al., Cytokine Growth Factor Rev.
2002 April; 13(2):155-68). IL-12 is required for resistance to many
pathogens and to transplantable or chemically induced tumours. It
is known that recombinant IL-12 treatment shows anti-tumour effect
on transplantable tumours, on chemically induced tumours, and in
tumours arising spontaneously in genetically modified mice. Because
of this ability, IL-12 has a potent adjuvant activity in
cancer.
[0089] However, until the present time, excessive clinical toxicity
and modest clinical response has been observed in clinical trials,
thereby necessitating novel approaches and administration regimes
that minimize toxicity without affecting the anti-tumour effect of
IL-12. IL-12 has not been shown to have substantial activity in the
tumours tested to this date via systemic administration in doses
that are non-toxic to the subject.
[0090] The present invention therefore relates to the MSCs as
described herein and medical use thereof, wherein the exogenous
nucleic acid encodes IL-12. The MSC-based approach towards
site-specific expression of IL-12 represents a novel and
advantageous approach towards avoiding the toxicity inherent in
IL-12 systemic administration.
[0091] The present invention enables a surprising and advantageous
anti-tumour effect via the expression of an immune stimulatory
cytokine in the MSCs as described herein. The expression of a
stimulatory cytokine as described herein by the MSCs supports an
anti-tumour immune response and leads to reduction in tumour size
and/or growth, and shows a distinct reduction in and/or avoidance
of the side effects produced by systemic administration of such
cytokines known in the art. Side effects such as nausea and
vomiting, sores in the mouth or on the lips, diarrhoea, drowsiness,
allergic reactions, fever or chills, hives, itching, headache,
coughing, shortness of breath, or swelling of the face, tongue, or
throat, may be avoided by the MSC-based therapy described
herein.
[0092] The present invention therefore provides means for reducing
the side effects of cytokine therapy, and the concomitant use of
cytokines with immunotherapies, by enabling local (or locally
confined) tumour-specific effects, achieved preferably by systemic
administration of the cells, but exerted in a tissue specific
manner via cell therapy using MSCs that comprise and express said
cytokines under the appropriate tissue-specific conditions.
[0093] The present invention therefore relates to a genetically
modified mesenchymal stem cell for use as a medicament as described
herein, wherein the exogenous nucleic acid comprises a region
encoding two or more immune response-stimulating cytokines operably
linked to one or more promoters or promoter/enhancer combinations,
wherein the cytokines are selected from the group consisting of
IL-2, IL-7, IL-12, IL-15, IL21, IFN gamma and IFN beta.
[0094] The present invention therefore relates to a genetically
modified mesenchymal stem cell for use as a medicament as described
herein, wherein the exogenous nucleic acid comprises a region
encoding two or more immune response-stimulating cytokines operably
linked to one or more promoters or promoter/enhancer combinations,
wherein said cytokines comprise at least IL-7 and IL-21, IL-12 and
IL-2, IL-15 and IL-12, IL-7 and IL-12, or IL-21 and IL-12.
[0095] The invention also relates to a genetically modified
mesenchymal stem cell comprising an exogenous nucleic acid
molecule, wherein said exogenous nucleic acid molecule comprises a
region encoding an immune stimulatory molecule(s) that induce
T-cell proliferation and/or differentiation (and/or maturation to a
memory cell and avoidance of tumour-mediated immunosuppression)
operably linked to a promoter or promoter/enhancer combination.
[0096] The immune stimulatory molecule that induces T-cell
proliferation and/or differentiation may be a cytokine as described
herein or another immune stimulatory molecule, such as a chemokine
or combination of cytokines and chemokines. A combination may be
preferred to ensure that immune cells are attracted appropriately
by chemokines, activated appropriately (by the appropriate cytokine
leading to activation) and are directed toward a memory phenotype
(by an appropriate cytokine promoting the maturation of memory
effector immune cells).
[0097] In a preferred embodiment the genetically modified
mesenchymal stem cell is characterised in that the immune
stimulatory molecule that induces T-cell proliferation and/or
differentiation is CD28. CD28 (Cluster of Differentiation 28) is
one of the proteins expressed on T cells that provide
co-stimulatory signals, which are required for their activation.
CD28 has also been found to stimulate eosinophil granulocytes,
where its ligation with anti-CD28 leads to the release of IL-2,
IL4, IL-13 and IFN gamma.
[0098] In a preferred embodiment the genetically modified
mesenchymal stem cell is characterised in that the immune
response-stimulating cytokine is a chemokine.
[0099] The invention also relates to genetically modified
mesenchymal stem cells for use as a medicament as described herein,
wherein the exogenous nucleic acid comprises a region encoding two
or more immune response-stimulating cytokines operably linked to
one or more promoters or promoter/enhancer combinations, wherein at
least one immune response-stimulating cytokine is a chemokine.
[0100] The invention also relates to genetically modified
mesenchymal stem cells for use as a medicament as described herein,
wherein the exogenous nucleic acid comprises a region encoding two
or more immune response-stimulating cytokines operably linked to
one or more promoters or promoter/enhancer combinations, wherein at
least one immune response-stimulating cytokine is a chemokine and
at least one immune response-stimulating cytokine is selected from
the group consisting of IL-2, IL-7, IL-12, IL-15, IL21, IFN gamma
and IFN beta.
[0101] In one embodiment the genetically modified mesenchymal stem
cell is characterised in that the immune response-stimulating
cytokine is an inflammatory chemokine.
[0102] In one embodiment the genetically modified mesenchymal stem
cell is characterised in that the immune response-stimulating
cytokine is a chemokine with chemotactic properties for attracting
T cells, for example, CCL1, CCL2 and/or CCL17.
[0103] Chemokines refer to a sub-group of cytokines (signalling
proteins) secreted by cells. Cytokines have the ability to induce
directed chemotaxis in nearby responsive cells; they are
chemotactic cytokines. Proteins are classified as chemokines
according to shared structural characteristics such as small size
(typically approximately 8-10 kilodaltons in size), and the
presence of four cysteine residues in conserved locations that are
key to forming their 3-dimensional shape. Cytokines may be known
under alternative definitions, such as the SIS family of cytokines,
SIG family of cytokines, SCY family of cytokines, Platelet factor-4
superfamily or intercrines. Chemokines have been classified into
four main subfamilies: CXC, CC, CX3C and XC. All of these proteins
exert their biological effects by interacting with G protein-linked
transmembrane receptors called chemokine receptors, which are
selectively found on the surfaces of their target cells.
[0104] The major role of chemokines is to act as chemoattractants
to induce or direct migration of immune cells. Cells that are
attracted by chemokines follow a signal of increasing chemokine
concentration towards the source of the chemokine. Some chemokines
control cells of the immune system during processes of immune
surveillance, such as directing lymphocytes to the lymph nodes so
they can screen for invasion of pathogens by interacting with
antigen-presenting cells residing in these tissues. Some chemokines
have roles in development; they promote angiogenesis (the growth of
new blood vessels), or guide cells to tissues that provide specific
signals critical for cellular maturation. Other chemokines are
inflammatory and are released from a wide variety of cells in
response to bacterial infection, viruses and agents that cause
physical damage such as silica or the urate crystals that occur in
gout. Their release is often stimulated by pro-inflammatory
cytokines such as interleukin 1. Inflammatory chemokines function
mainly as chemoattractants for leukocytes, recruiting monocytes,
neutrophils and other effector cells from the blood to sites of
infection or tissue damage. Certain inflammatory chemokines
activate cells to initiate an immune response or promote wound
healing.
[0105] Chemokines are generally considered pro-inflammatory or
homeostatic. Pro-inflammatory cytokines can be induced during an
immune response to recruit cells of the immune system to a site of
infection, or other immune target, such as a tumour, whereas the
homeostatic cytokines are involved in controlling the migration of
cells during processes of tissue maintenance or development.
Inflammatory cytokines and chemokines are typically formed under
pathological conditions (on pro-inflammatory stimuli, such as IL-1,
TNF-alpha, LPS, or viruses) and actively participate in the
inflammatory response attracting immune cells to the site of
inflammation.
[0106] The invention therefore relates to the mesenchymal stem
cells described herein, wherein the exogenous nucleic acid encodes
an inflammatory chemokine. Such chemokines are known to a skilled
person. Examples of inflammatory chemokines relate to CXCL-8, CCL2,
CCL3, CCL4, CCL5, CCL11 and CXCL10, CXCL1, CXCL2.
[0107] In one embodiment of the invention, the chemokine is capable
of inducing migration of T cells (T-lymphocytes) to the site of the
chemokine expressing MSC. Examples of chemokines are involved in
the recruitment of T lymphocytes to the site of inflammation are
CCL2, CCL1, CCL22 and CCL17. These chemokines are known attractants
of T-cells and can therefore be used to attract endogenous patient
T-cells, or to enhance the homing of adoptive immunotherapeutics
(e.g. CARTs) to the tumour. Homing of adaptive immunotherapeutics
in tumours is a known deficit of many such treatments and this
invention therefore carries the potential to significantly enhance
the homing and subsequent therapeutic efficacy of adoptive
immunotherapies, such as CARTs.
[0108] In some embodiments the chemokine is a CXC chemokine,
preferably CXCL13.
[0109] CXC chemokines relate to chemokines in which the two
N-terminal cysteines of CXC chemokines (or a-chemokines) are
separated by one amino acid, represented in this name with an "X".
There are typically two categories of CXC chemokines, those with a
glutamic acid-leucine-arginine motif (ELR motif) immediately before
the first cysteine of the CXC motif (ELR-positive), and those
without an ELR motif (ELR-negative). An example of an ELR-positive
CXC chemokine is interleukin-8 (IL-8), which induces neutrophils to
leave the bloodstream and enter into the surrounding tissue. An
example of an ELR-negative chemokine is CXCL13, which shows
chemoattractant properties for lymphocytes.
[0110] In one embodiment of the invention the chemokine is CX3CL1,
which belongs to the group of chemokines termed CX3C chemokines.
The CX3C chemokines relate to chemokines that have three amino
acids between the two cysteines. CX3CL1 is both secreted and
tethered to the surface of the cell that expresses it, thereby
serving as both a chemoattractant and as an adhesion molecule.
[0111] In some embodiments of the invention, the chemokine is
selected from the group consisting of stromal cell-derived factor 1
(SDF-1; CXCL12), Chemokine (C-C motif) ligand 23 (CCL23; Macrophage
inflammatory protein 3 (MIP-3)), Chemokine (C-C motif) ligand 19
(CCL19; EBI1; ELC or macrophage inflammatory protein-3-beta
(MIP-3-beta)) and Chemokine (C-C motif) ligand 4 (CCL4; Macrophage
inflammatory protein-1.beta. (MIP-1.beta.)
[0112] The stromal cell-derived factor 1 (SDF-1) is also known as
C-X-C motif chemokine 12 (CXCL12). Stromal cell-derived factors
1-alpha and 1-beta are small cytokines that belong to the chemokine
family, members of which activate leukocytes and are often induced
by pro-inflammatory stimuli such as lipopolysaccharide, TNF, or
IL1. The chemokines are characterized by the presence of 4
conserved cysteines that form 2 disulfide bonds. They can be
classified into 2 subfamilies. In the CC subfamily, the cysteine
residues are adjacent to each other. In the CXC subfamily, they are
separated by an intervening amino acid. The SDF1 proteins belong to
the latter group. SDF-1 is produced in two forms,
SDF-1.alpha./CXCL12a and SDF-1.beta./CXCL12b, by alternate splicing
of the same gene. The invention therefore relates to the MSCs as
described herein, wherein the exogenous nucleic molecule encodes an
SDF-1 chemokine, such as SDF-1.alpha./CXCL12a and/or
SDF-1.beta./CXCL12b.
[0113] Chemokine (C-C motif) ligand 23 (CCL23) is a cytokine
belonging to the CC chemokine family that is also known as
Macrophage inflammatory protein 3 (MIP-3) and Myeloid progenitor
inhibitory factor 1 (MPIF-1). CCL23 is predominantly expressed in
lung and liver tissue, but is also found in bone marrow and
placenta. CCL23 is chemotactic for resting T cells and
monocytes.
[0114] Chemokine (C-C motif) ligand 19 (CCL19) is a cytokine
belonging to the CC chemokine family that is also known as EBI1
ligand chemokine (ELC) and macrophage inflammatory protein-3-beta
(MIP-3-beta). CCL19 is expressed abundantly in thymus and lymph
nodes, with moderate levels in trachea and colon and low levels in
stomach, small intestine, lung, kidney and spleen. CCL19 is known
to play an important role in trafficking of T cells in thymus, and
in T cell and B cell migration to secondary lymphoid organs.
[0115] CCL4, also known as Macrophage inflammatory
protein-1.beta.(MIP-1.beta.) is a CC chemokine with specificity for
CCR5 receptors. It is a chemoattractant for natural killer cells,
monocytes and a variety of other immune cells.
[0116] All immunotherapies rely on the local and timely activation
of the immune response and this, in turn, relies largely on the
presence of effector cells to exert their function. In the case of
oncology, this function results in the killing of tumour cells. In
many cases, however, only insufficient amounts of immune effector
cells are attracted to the tumour and the effect of the
immunotherapy is curtailed.
[0117] The chemokines described herein are capable of exhibiting T
cell recruiting properties that are beneficial in an immune
response against a tumour disease. The use of such chemokine
molecules in the context of MSC-based expression and delivery of
the molecules enables surprising benefits with respect to
site-specific T cell recruitment and reduction in side effects
associated non-target effects caused by systemic administration of
such cytokines/chemokines.
[0118] In another aspect of the invention the use of the
genetically modified mesenchymal stem cells as a medicament as
described herein is characterised in that said treatment comprises
the combined administration of said mesenchymal stem cell with a
checkpoint inhibitor.
[0119] Normally, cells that are potentially cancerous are destroyed
by the immune system. All cancer cells undergo changes that
differentiate them from their neighbours, the most obvious change
being the ability to multiply without inhibition. Cancer cells
utilise mechanisms that avoid regular immune system control.
Checkpoint proteins have been shown to function by communicating to
the immune system that a potentially cancerous cell is not to be
destroyed. There may be other molecules signalling that the cell is
cancerous, but if there are enough checkpoint proteins on the cell
surface, the immune system may overlook cancerous signals.
[0120] A ligand-receptor interaction that has been investigated as
a target for cancer treatment is the interaction between the
transmembrane programmed cell death 1 protein (PD-1; also known as
CD279) and its ligand, PD-1 ligand 1 (PD-L1). In normal physiology
PD-L1 on the surface of a cell binds to PD1 on the surface of an
immune cell, which inhibits the activity of the immune cell. It
appears that up-regulation of PD-L1 on the cancer cell surface may
allow them to evade the host immune system by inhibiting T cells
that might otherwise attack the tumour cell. Antibodies that bind
to either PD-1 or PD-L1 and therefore block the interaction may
allow the T-cells to attack the tumour.
[0121] Checkpoint inhibitors (also known as immune checkpoint
modulators, or CPMs) are designed to lessen the effectiveness of
checkpoint proteins. They may have a variety of mechanisms of
action, but if effective, they enable the immune system to
recognize other molecules on the surface of the cancer cells.
[0122] In one embodiment the medical use of the genetically
modified mesenchymal stem cell as described herein is characterised
by the combined administration of a checkpoint inhibitor,
preferably a PD-L1 and/or PD-1 inhibitor, with said MSCs. Examples
include Nivolumab (BMS-936558, MDX-1106, ONO-4538), a fully human
Immunoglobulin G4 (IgG4) monoclonal PD-1 antibody, Lambrolizumab
(MK-3475), a humanized monoclonal IgG4 PD-1 antibody, and
BMS-936559, a fully human IgG4 PD-L1 antibody.
[0123] In one embodiment the medical use of the genetically
modified mesenchymal stem cell as described herein is characterised
by the combined administration of a checkpoint inhibitor,
preferably a CTLA-4 inhibitor, with said MSCs. Examples include
Tremelimumab (Pfizer, N.Y., USA) and ipilimumab, two fully human
monoclonal antibodies against CTLA-4.
[0124] The combined administration of the MSCs expressing an immune
response-stimulating cytokine and/or an immune stimulatory molecule
that induces T-cell proliferation and/or differentiation together
with a checkpoint inhibitor leads to a synergistic effect with
respect to the desired anti-cancer effect. The cytokine or other
immune stimulator provides local enhancement of the T cell response
against the cancer tissue, whilst the checkpoint inhibitor also
enables the T cells to more effectively attack and destroy
cancerous tissue. The effects of these two agents are combined in a
synergistic manner, resulting in a technical effect greater than
the sum of these two aspects when considered alone.
[0125] In a further embodiment, the invention relates to the
genetically modified mesenchymal stem cell for use as a medicament
as described herein, wherein the immunotherapy comprises the
administration of tumour antigens.
[0126] The present invention is used to enhance the therapeutic
efficacy of cancer vaccines. Cancer vaccines direct the immune
system to mount an attack against cancer cells in the body. Instead
of preventing disease, like vaccines used to prevent infection,
they activate the immune system to attack a disease that already
exists. Some cancer treatment vaccines are made up of cancer cells,
parts of cells, or pure antigens. Sometimes a patient's own immune
cells, such as T-cells, or antigen presenting cells, are removed
and exposed to these substances in the lab to create the vaccine.
Once the vaccine is ready, it's injected into the body to mediate
the immune response against cancer cells. Vaccines are often
combined with other substances or cells called adjuvants that help
boost the immune response even further. Cancer vaccines cause the
immune system to attack cells with one or more specific antigens.
Activation of the cellular adaptive immune response leads to
immunological memory, so, it's hoped that the vaccine might
continue to work long after it's given. In many cases, the efficacy
and immune activation of cancer vaccines has been hindered by the
tumour-mediated immunosuppression. The combined of the cancer
vaccine with said mesenchymal stem cells is expected to remove or
downgrade the immunosuppression, or promote immune activation in
spite of any persisting immunosuppression.
[0127] The MSCs as described herein may therefore be considered a
therapeutic agent themselves, or as an adjuvant for a
co-administered immunotherapeutic agent or agents.
[0128] A further aspect of the invention relates to a genetically
modified mesenchymal stem cell for use as a medicament as described
herein, wherein said treatment comprises the combined
administration of said mesenchymal stem cell with immune cells. It
is encompassed within the invention that administration of
genetically modified MSCs prior to immune cells (e.g. CARTs) will
enhance the chemoattraction of CARTs and other immune effector
cells due to the expression of appropriate chemokines. The
expression of stimulating cytokines will enhance the activation of
T-cells only locally, preferably within or in proximity to the
tumour, and subsequently lead to a memory effector cell phenotype,
thereby prolonging the therapeutic effect of the treatment.
[0129] The response to pathogens or cancerous tissue is
orchestrated by complex interactions and activities of a large
number of diverse cell types involved in an immune response. Immune
cells as used herein may relate to any of the following cell types,
as described in the context of the following processes: The innate
immune response is the first line of defence and occurs soon after
pathogen exposure or exposure to "foreign" or cancerous matter. It
is carried out by phagocytic cells such as neutrophils and
macrophages, cytotoxic natural killer (NK) cells, and granulocytes.
The subsequent adaptive immune response includes antigen-specific
defence mechanisms and may take days to develop. Cell types with
critical roles in adaptive immunity are antigen-presenting cells
including macrophages and dendritic cells. Antigen-dependent
stimulation of various cell types including T cell subsets, B
cells, and macrophages all play critical roles in host defense.
Immune cells as described herein relate to biological cells
involved in the immune response in a subject. Immune cells are
preferably selected from T Cells, B Cells, Dendritic Cells,
Granulocytes, Innate Lymphoid Cells (ILCs), Megakaryocytes,
Monocytes/Macrophages, Natural Killer (NK) Cells, Platelets, Red
Blood Cells(RBCs) and/or Thymocytes.
[0130] The subject from which the mesenchymal stem cells and/or
immune cells are obtained may be the same subject, to whom the
cells are intended to be administered after cultivation. Such cells
may therefore be considered autologous. The cells may however be
obtained from a subject distinct from the intended patient,
therefore being considered allogenic. As used herein, a cell is
"allogenic" with respect to a subject if it or any of its precursor
cells are from another subject of the same species. As used herein,
a cell is "autologous" with respect to a subject if it or its
precursor cells are from the same subject. In a preferred
embodiment, the immune cells are autologous to the subject of
medical treatment.
[0131] In a preferred embodiment, the immune cell is a T cell. In
other embodiments, the immune cell is a T cell comprising an
artificial T cell receptor, such as a chimeric antigen receptor
(CARTs), wherein said T cell receptor binds specifically to a
tumour antigen (Lee, DW et al., Clin Cancer Res; 2012;18(10);
2780-90). In further embodiments the immune cell is a macrophage,
preferably an M1 macrophage, and/or a monocyte.
[0132] Monocytes are a type of white blood cells (leukocytes). They
are the largest of all leukocytes. They are part of the innate
immune system of vertebrates including all mammals (humans
included). Monocytes are produced by the bone marrow from
precursors called monoblasts, bipotent cells that differentiated
from hematopoietic stem cells. Monocytes circulate in the
bloodstream for about one to three days and then typically move
into tissues throughout the body. They constitute between three to
eight percent of the leukocytes in the blood. In tissues monocytes
mature into different types of macrophages at different anatomical
locations.
[0133] Macrophages, sometimes called macrophagocytes, are cells
produced by the differentiation of monocytes in tissues. Monocytes
and macrophages are phagocytes. Macrophages function in both
non-specific defense (innate immunity) as well as help initiate
specific defense mechanisms (adaptive immunity) of vertebrate
animals. Macrophages predominantly expressing the killer phenotype
are called M1 macrophages, whereas those involved in tissue repair
are called M2 macrophages. The role of macrophages is to
phagocytose, or engulf and then digest, cellular debris and
pathogens, either as stationary or as mobile cells. They also
stimulate lymphocytes and other immune cells to respond to
pathogens. They are specialized phagocytic cells that attack
foreign substances, infectious microbes and cancer cells through
destruction and ingestion.
[0134] In one embodiment the invention therefore relates to cancer
immunotherapy and approaches involving adoptive cell transfer,
which encompasses T cell-based cytotoxic responses to attack cancer
cells.
[0135] Cancer immunotherapy attempts to stimulate the immune system
to reject and destroy tumours. Initially, immunotherapy treatments
involved administration of cytokines such as Interleukin.
Thereafter, due to the adverse effects of intravenously and
systemically administered cytokines, alternative approaches were
attempted using the extraction of lymphocytes from the blood and
expanding these cells in vitro against a tumour antigen before
injecting the cells with appropriate stimulatory cytokines. The
cells would then specifically target and destroy the tumour
expressing antigen against which they have been raised. Despite
these approaches, methods for appropriate stimulation of such cells
with cytokines were not sufficiently effective to produce
convincing tumour-reduction. Significant side effects also arose
from the large quantities of cytokine required.
[0136] One approach to these previous attempts at immunotherapy
involves engineering patients' own immune cells to recognize and
attack their tumours. And although this approach, called adoptive
cell transfer (ACT), has been restricted to clinical trials so far,
treatments using these engineered immune cells have generated
therapeutic responses in patients with advanced cancer.
[0137] The present invention therefore encompasses adoptive cell
transfer in combination with administration of the MSCs described
herein. It is encompassed within the invention that administration
of genetically modified MSCs prior to immune cells (e.g. CARTs)
will enhance the chemoattraction of CARTs and other immune effector
cells administered during adoptive cell transfer due to the
expression of appropriate chemokines. The expression of stimulating
cytokines will enhance the activation of T-cells only locally,
preferably within or in proximity to the tumour, and subsequently
lead to a memory effector cell phenotype, thereby prolonging the
therapeutic effect of the treatment.
[0138] Adoptive cell transfer uses T cell-based cytotoxic responses
to attack cancer cells. T cells that have a natural or genetically
engineered reactivity to a patient's cancer are generated in vitro
and then transferred back into the cancer patient. Autologous
tumour-infiltrating lymphocytes have been used as an effective
treatment for patients with metastatic melanoma. This can be
achieved by taking T cells that are found with the tumour of the
patient, which are trained to attack the cancerous cells. These T
cells may be referred to as tumour-infiltrating lymphocytes (TIL).
Such T cells may be stimulated to multiply in vitro using high
concentrations of IL-2, anti-CD3 and allo-reactive feeder cells.
Traditionally, these T cells are then transferred back into the
patient along with exogenous administration of IL-2 to further
boost their anti-cancer activity.
[0139] The present invention encompasses in one embodiment the
administration of IL-2 via the genetically modified MSC as
described herein. Through this approach the side effects of
systemic administration of IL-2 are avoided, whilst the beneficial
effects of the T cell transfer are maintained and/or enhanced.
Similar effects are obtained for IL-12, IL-7, IL-21 and/or IL-15,
alone or in combinations thereof, to achieve activation and
establishment of immunological memory.
[0140] The present invention therefore provides novel means for
local and tissue-specific cytokine production for the stimulation
of an anti-tumour immune response, mediated preferably by cytotoxic
T cells and/or NK cells.
[0141] Artificial T cell receptors (also known as chimeric T cell
receptors, chimeric immunoreceptors, chimeric antigen receptors
(CARs)) are engineered receptors, which graft an arbitrary
specificity onto an immune effector cell. Typically, these
receptors are used to graft the specificity of a monoclonal
antibody onto a T cell; with transfer of their coding sequence
facilitated by retroviral vectors.
[0142] Genetically engineered T cells may, in one embodiment, be
created by infecting patient's cells with a virus that contain a
copy of a T cell receptor (TCR) gene that is specialised to
recognise tumour antigens. The virus is not able to reproduce
within the cell however integrates into the human genome. This is
beneficial as new the TCR gene remains stable in the T-cell. A
patient's own T cells are exposed to these viruses and then
expanded non-specifically or stimulated using the genetically
engineered TCR. The cells are then transferred back into the
patient and ready to have an immune response against the tumour.
Second- and third-generation CARs typically consist of a piece of
monoclonal antibody, called a single-chain variable fragment
(scFv), that resides on the outside of the T-cell membrane and is
linked to stimulatory inside the T cell. The scFv portion guides
the cell to its target antigen. Once the T cell binds to its target
antigen, the stimulatory molecules provide the necessary signals
for the T cell to become fully active. In this fully active state,
the T cells can more effectively proliferate and can attack cancer
cells.
[0143] The combined administration of the MSCs expressing an immune
response-stimulating cytokine and/or an immune stimulatory molecule
that induces T-cell proliferation and/or differentiation together
with immune cells, preferably T cells, such as those described
herein, and optionally in further combination with a checkpoint
inhibitor, leads to a synergistic effect with respect to the
desired anti-cancer effect. Checkpoint inhibition can be combined
with the MSCs expressing stimulatory cytokines, in order to enhance
the anti-tumour effect of additionally administered immune cells,
thereby creating a combination of factors associated with
unexpected efficacy in anti-tumour treatment.
[0144] In a further embodiment, the invention relates to the
genetically modified mesenchymal stem cell for use as a medicament
as described herein, wherein the immunotherapy comprises the
administration of patient-derived tumour material.
[0145] In a further embodiment, the invention relates to the
genetically modified mesenchymal stem cell for use as a medicament
as described herein, wherein the immunotherapy comprises the
administration of an antibody or antibody fragment targeted to a
tumour-specific antigen.
[0146] For example, antibody-dependent cell-mediated cytotoxicity
(ADCC) is a mechanism of cell-mediated immune defense whereby an
effector cell of the immune system actively lyses a target cell,
such as a cancer cell, whose membrane-surface antigens have been
bound by specific antibodies. It is one of the mechanisms through
which antibodies, as part of the humoral immune response, can act
to limit and contain cancerous growth. Previous reports have
indicated that ADCC is an important mechanism of action of
therapeutic monoclonal antibodies, including trastuzumab and
rituximab, against tumours. The combined administration of the MSC
as described herein with an anti-tumour antibody treatment is
encompassed by the invention.
[0147] For example, bi-specific antibodies simultaneously targeted
to a tumour-specific antigen and the CD3 molecule on T-cells may be
administered as the anti-tumour immunotherapy.
DETAILED DESCRIPTION OF THE INVENTION
[0148] An important role of the immune system is to identify and
eliminate tumours. The transformed cancerous cells of tumours
express antigens that are not found on normal cells. To the immune
system, these antigens appear foreign, and their presence causes
immune cells to attack the transformed tumour cells. The antigens
expressed by tumours have several sources. Some are derived from
oncogenic viruses like human papillomavirus, which causes cervical
cancer, while others are the organism's own proteins that occur at
low levels in normal cells but reach high levels in tumour cells.
One example is an enzyme called tyrosinase that, when expressed at
high levels, transforms certain skin cells (e.g. melanocytes) into
tumours called melanomas. A third possible source of tumour
antigens are proteins normally important for regulating cell growth
and survival, that commonly mutate into cancer inducing molecules
called oncogenes.
[0149] The main response of the immune system to tumours is to
destroy the abnormal cells using killer T cells, sometimes with the
assistance of helper T cells. Tumour antigens are presented on MHC
class I molecules in a similar way to viral antigens. This allows
killer T cells to recognize the tumour cell as abnormal. NK cells
also kill tumorous cells in a similar way, especially if the tumour
cells have fewer MHC class I molecules on their surface than
normal; this is a common phenomenon with tumours. Some tumour cells
also release products that inhibit the immune response; for example
by secreting the cytokine TGF-.beta., which suppresses the activity
of macrophages and lymphocytes.
[0150] The present invention therefore provides means for
supporting an anti-tumour immune reaction by the expression of an
immune-stimulating cytokine from the genetically modified MSCs
described herein.
[0151] Immunotherapy is to be understood in the context of the
present invention to encompass any therapeutic agent that uses the
immune system to treat cancer. Immunotherapy exploits the fact that
cancer cells have subtly different molecules on their surface that
can be detected by the immune system. These molecules, known as
cancer antigens, are most commonly proteins, but also include
molecules such as carbohydrates. Immunotherapy provokes or enhances
the immune system in attacking the tumour cells by using these
antigens as targets.
[0152] Immunotherapy encompasses, without limitation, cellular and
antibody therapy.
[0153] Cellular therapies typically involve the administration of
immune cells isolated from the blood or from a tumour of the
patient. Immune cells directed towards the tumour to be treated are
activated, cultured and returned to the patient where the immune
cells attack the cancer. Cell types that can be used in this way
are, without limitation, natural killer cells, lymphokine-activated
killer cells, cytotoxic T cells and dendritic cells. Dendritic cell
therapy provokes anti-tumour responses by causing dendritic cells
to present tumour antigens. Dendritic cells present antigens to
lymphocytes, which activates them, priming them to kill other cells
that present the antigen.
[0154] Antibodies are proteins produced by the immune system that
bind to a target antigen on the cell surface. Those that bind to
cancer antigens may be used to treat cancer. Cell surface receptors
are common targets for antibody therapies and include for example
CD20, CD274, and CD279. Once bound to a cancer antigen, antibodies
can induce antibody-dependent cell-mediated cytotoxicity, activate
the complement system, or prevent a receptor from interacting with
its ligand, all of which can lead to cell death. Multiple
antibodies are approved to treat cancer, including Alemtuzumab,
Ipilimumab, Nivolumab, Ofatumumab, and Rituximab.
[0155] Antibody-dependent cell-mediated cytotoxicity (ADCC) is a
mechanism of attack by the immune system that requires antibodies
to bind to target cell surfaces. Antibodies are formed of a binding
region (Fab) and the Fc region that can be detected by immune cells
via their Fc surface receptors. Fc receptors are found on many
immune system cells, including natural killer cells. When natural
killer cells encounter antibody-coated cells, the latter's Fc
regions interact with their Fc receptors, leading to the release of
perforin and granzyme B. These two chemicals programmed cell death
(apoptosis) in the tumour cell. Effective antibodies include
Rituximab, Ofatumumab, and Alemtuzumab.
[0156] The complement system includes blood proteins that can cause
cell death after an antibody binds to the cell surface. Generally,
the system deals with foreign pathogens, but can be activated with
therapeutic antibodies in cancer. The system can be triggered if
the antibody is chimeric, humanized or human; as long as it
contains the IgG1 Fc region. Complement can lead to cell death by
activation of the membrane attack complex, known as
complement-dependent cytotoxicity; enhancement of
antibody-dependent cell-mediated cytotoxicity; and CR3-dependent
cellular cytotoxicity. Complement-dependent cytotoxicity occurs
when antibodies bind to the cancer cell surface, the C1 complex
binds to these antibodies and subsequently protein pores are formed
in the cancer cell membrane.
[0157] Tumour-associated antigens, or Tumour-specific antigens, may
be targeted by the preferably cellular or antibody-based
anti-tumour immunotherapy and include, without limitation, those
antigens known to a skilled person or identifiable by a skilled
person that are expressed solely or predominantly by tumour cells
and may be targeted by immune therapy. As non-limiting examples,
tumour associated or tumour specific antigens encompass proteins
produced in tumour cells that have an abnormal structure due to
mutation, such as proto-oncogenes, abnormal products of ras and p
53 genes, or other proteins associated with tumour cells, such as
tissue differentiation antigens, cluster of differentiation (often
abbreviated as CD) cell surface molecules, mutant protein antigens,
oncogenic viral antigens, cancer-testis antigens and vascular or
stromal specific antigens. Glycoproteins, glycolipids,
carbohydrates or growth factor receptors may also be considered
tumour associated or tumour specific antigens as targets of
anti-tumour immunotherapy.
[0158] The MSCs of the present invention are capable of supporting
and/or enhancing the immunotherapies described herein through their
unique properties derived from a combination of immune-response
stimulating transgene cytokines and the MSCs inherent
anti-inflammatory properties.
[0159] As used herein, "tumour" shall include, without limitation,
a prostate tumour, a pancreatic tumour, a squamous cell carcinoma,
a breast tumour, a melanoma, a basal cell carcinoma, a
hepatocellular carcinoma, a choloangiocellular carcinoma,
testicular cancer, a neuroblastoma, a glioma or a malignant
astrocytic tumour such as glioblastma multiforme, a colorectal
tumour, an endometrial carcinoma, a lung carcinoma, an ovarian
tumour, a cervical tumour, an osteosarcoma, a
rhabdo/leiomyosarcoma, a synovial sarcoma, an angiosarcoma, an
Ewing sarcoma/PNET and a malignant lymphoma. These include primary
tumours as well as metastatic tumours (both vascularized and
non-vascularized).
[0160] The "mesenchymal stem cells" disclosed herein can give rise
to connective tissue, bone, cartilage, and cells in the circulatory
and lymphatic systems. Mesenchymal stem cells are found in the
mesenchyme, the part of the embryonic mesoderm that consists of
loosely packed, fusiform or stellate unspecialized cells. As used
herein, mesenchymal stem cells include, without limitation,
CD34-negative stem cells.
[0161] In one embodiment of the invention, the mesenchymal stem
cells are plastic-adherent cells, defined in some embodiments as
multipotent mesenchymal stromal cells and also include
CD34-negative cells. For the avoidance of any doubt, the term
mesenchymal stem cell encompasses multipotent mesenchymal stromal
cells that also includes a subpopulation of mesenchymal cells,
[0162] MSCs and their precursors, which subpopulation is made up of
multipotent or pluripotent self-renewing cells capable of
differentiation into multiple cell types in vivo.
[0163] As used herein, CD34-ngeative cell shall mean a cell lacking
CD34, or expressing only negligible levels of CD34, on its surface.
CD34-negative cells, and methods for isolating such cells, are
described, for example, in Lange C. et al., "Accelerated and safe
expansion of human mesenchymal stromal cells in animal serum-free
medium for transplantation and regenerative medicine". J. Cell
Physiol. 2007, Apr. 25.
[0164] Mesenchymal stem cells can be differentiated from
hematopoietic stem cells (HSCs) by a number of indicators. For
example, HSCs are known to float in culture and to not adhere to
plastic surfaces. In contrast, mesenchymal stem cells adhere to
plastic surfaces. The CD34-negative mesenchymal stem cells of the
present invention are adherent in culture.
[0165] The genetically modified cell(s) described herein may
comprise different types of carriers depending on whether it is to
be administered in solid, liquid or aerosol form, and whether it
need to be sterile for such routes of administration as injection.
The present invention can be administered intravenously,
intradermally, intraarterially, intraperitoneally, intralesionally,
intracranially, intraarticularly, intraprostaticaly,
intrapleurally, intratracheally, intranasally, intravitreally,
intravaginally, intrarectally, topically, intratumorally,
intramuscularly, intraperitoneally, subcutaneously,
subconjunctival, intravesicularlly, mucosally, intrapericardially,
intraumbilically, intraocularally, orally, topically, locally,
inhalation (e.g., aerosol inhalation), injection, infusion,
continuous infusion, localized perfusion bathing target cells
directly, via a catheter, via a lavage, in cremes, in lipid
compositions (e.g., liposomes), or by other method or any
combination of the forgoing as would be known to one of ordinary
skill in the art (see, for example, Remington's Pharmaceutical
Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein
by reference).
[0166] The present invention encompasses treatment of a patient by
introducing a therapeutically effective number of cells into a
subject's bloodstream. As used herein, "introducing" cells "into
the subject's bloodstream" shall include, without limitation,
introducing such cells into one of the subject's veins or arteries
via injection. Such administering can also be performed, for
example, once, a plurality of times, and/or over one or more
extended periods. A single injection is preferred, but repeated
injections over time (e.g., quarterly, half-yearly or yearly) may
be necessary in some instances. Such administering is also
preferably performed using an admixture of CD34-negative cells and
a pharmaceutically acceptable carrier. Pharmaceutically acceptable
carriers are well known to those skilled in the art and include,
but are not limited to, 0.01-0.1 M and preferably 0.05 M phosphate
buffer or 0.8% saline, as well as commonly used proprietary
cryopreservation media.
[0167] Administration may also occur locally, for example by
injection into an area of the subject's body in proximity to a
tumour disease. MSCs have been shown to migrate towards cancerous
tissue. Regardless, the local administration of the cells as
described herein may lead to high levels of the cells at their site
of action.
[0168] Additionally, such pharmaceutically acceptable carriers can
be aqueous or non-aqueous solutions, suspensions, and emulsions,
most preferably aqueous solutions. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions and suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringers dextrose, dextrose and sodium chloride,
lactated Ringers and fixed oils. Intravenous vehicles include fluid
and nutrient replenishers, electrolyte replenishers such as Ringers
dextrose, those based on Ringers dextrose, and the like. Fluids
used commonly for i.v. administration are found, for example, in
Remington: The Science and Practice of Pharmacy, 20th Ed., p. 808,
Lippincott Williams S-Wilkins (2000). Preservatives and other
additives may also be present, such as, for example,
antimicrobials, antioxidants, chelating agents, inert gases, and
the like.
[0169] As used herein, a "therapeutically effective number of
cells" includes, without limitation, the following amounts and
ranges of amounts: (i) from about 1.times.10.sup.2 to about
1.times.10.sup.8 cells/kg body weight; (ii) from about
1.times.10.sup.3 to about 1.times.10.sup.7 cells/kg body weight;
(iii) from about 1.times.10.sup.4 to about 1.times.10.sup.6
cells/kg body weight; (iv) from about 1.times.10.sup.4 to about
1.times.10.sup.5 cells/kg body weight; (v) from about
1.times.10.sup.5 to about 1.times.10.sup.6 cells/kg body weight;
(vi) from about 5.times.10.sup.4 to about 0.5.times.10.sup.5
cells/kg body weight; (vii) about 1.times.10.sup.3 cells/kg body
weight; (viii) about 1.times.10.sup.4 cells/kg body weight; (ix)
about 5.times.10.sup.4 cells/kg body weight; (x) about
1.times.10.sup.5 cells/kg body weight; (xi) about 5.times.10.sup.5
cells/kg body weight; (xii) about 1.times.10.sup.6 cells/kg body
weight; and (xiii) about 1.times.10.sup.7 cells/kg body weight.
Human body weights envisioned include, without limitation, about 5
kg, 10 kg, 15 kg, 30 kg, 50 kg, about 60 kg; about 70 kg; about 80
kg, about 90 kg; about 100 kg, about 120 kg and about 150 kg. These
numbers are based on pre-clinical animal experiments and human
trials and standard protocols from the transplantation of CD34+
hematopoietic stem cells. Mononuclear cells (including CD34+ cells)
usually contain between 1:23000 to 1:300000 CD34-negative
cells.
[0170] As used herein, "treating" a subject afflicted with a
disorder shall mean slowing, stopping or reversing the disorders
progression. In the preferred embodiment, treating a subject
afflicted with a disorder means reversing the disorders
progression, ideally to the point of eliminating the disorder
itself. As used herein, ameliorating a disorder and treating a
disorder are equivalent. The treatment of the present invention may
also, or alternatively, relate to a prophylactic administration of
said cells. Such a prophylactic administration may relate to the
prevention of any given medical disorder, or the prevention of
development of said disorder, whereby prevention or prophylaxis is
not to be construed narrowly under all conditions as absolute
prevention. Prevention or prophylaxis may also relate to a
reduction of the risk of a subject developing any given medical
condition, preferably in a subject at risk of said condition.
[0171] Combined administration encompasses simultaneous treatment,
co-treatment or joint treatment, and includes the administration of
separate formulations of MSCs with immunotherapies, such as
checkpoint inhibitors and/or immune cells, whereby treatment may
occur within minutes of each other, in the same hour, on the same
day, in the same week or in the same month as one another.
Sequential administration of any given combination of combined
agents (for example MSCs, immune cells and/or checkpoint
inhibitors) is also encompassed by the term "combined
administration". A combination medicament, comprising one or more
of said MSCs with another immunotherapeutic, such as checkpoint
inhibitors and/or immune cells, may also be used in order to
co-administer the various components in a single administration or
dosage.
[0172] A combined immunotherapy may precede or follow treatment
with genetically modified stem cells by intervals ranging from
minutes to weeks. In embodiments where the other immunotherapeutic
agent and genetically modified stem cells are administered
separately to the site of interest, one would generally ensure that
a significant period of time did not expire between the time of
each delivery, such that the agent and the genetically modified
stem cell would still be able to exert an advantageously combined
effect on a treatment site. In such instances, it is contemplated
that one would 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, with a delay time of only about 12 h being most
preferred. In some situations, it may be desirable to extend the
time period for treatment significantly, however, where several
days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or
8) lapse between the respective administrations.
[0173] The term "stroma" as used herein refers to the supportive
framework of a tissue or an organ (or gland, tissue or other
structure), usually composed of extracellular matrix (ECM) and
stromal cells. The stroma is distinct from the parenchyma, which
consists of the key functional elements of that organ. Stromal
cells (in the dermis layer) adjacent to the epidermis (the very top
layer of the skin) release growth factors that promote cell
division. Stroma is made up of the non-malignant host cells. Stroma
provides an extracellular matrix on which tumours can grow or
maintain existence or separate themselves from the immune
environment.
[0174] As used herein, the term "tumour microenvironment" relates
to the cellular environment in which any given tumour exists,
including the tumour stroma, surrounding blood vessels, immune
cells, fibroblasts, other cells, signalling molecules, and the
ECM.
[0175] As used herein "cell migration" or "homing" is intended to
mean movement of a cell towards a particular chemical or physical
signal. Cells often migrate in response to specific external
signals, including chemical signals and mechanical signals. The
MSCs as described herein are capable of homing to tumour tissue or
other inflammation signals.
[0176] Chemotaxis is one example of cell migration regarding
response to a chemical stimulus. In vitro chemotaxis assays such as
Boyden chamber assays may be used to determine whether cell
migration occurs in any given cell.
[0177] For example, the cells of interest may be purified and
analysed. Chemotaxis assays (for example according to Falk et al.,
1980 J. Immuno. Methods 33:239-247) can be performed using plates
where a particular chemical signal is positioned with respect to
the cells of interest and the transmigrated cells then collected
and analysed. For example, Boyden chamber assays entail the use of
chambers isolated by filters, used as tools for accurate
determination of chemotactic behaviour. The pioneer type of these
chambers was constructed by Boyden (Boyden (1962) "The chemotactic
effect of mixtures of antibody and antigen on polymorphonuclear
leucocytes". J Exp Med 115 (3): 453). The motile cells are placed
into the upper chamber, while fluid containing the test substance
is filled into the lower one. The size of the motile cells to be
investigated determines the pore size of the filter; it is
essential to choose a diameter which allows active transmigration.
For modelling in vivo conditions, several protocols prefer coverage
of filter with molecules of extracellular matrix (collagen, elastin
etc.) Efficiency of the measurements can be increased by
development of multiwell chambers (e.g. NeuroProbe), where 24, 96,
384 samples are evaluated in parallel. Advantage of this variant is
that several parallels are assayed in identical conditions.
[0178] As used herein "engraftment" relates to the process of
incorporation of grafted or transplanted tissue or cells into the
body of the host. Engraftment may also relate to the integration of
transplanted cells into host tissue and their survival and under
some conditions differentiation into non-stem cell states.
[0179] Techniques for assessing engraftment, and thereby to some
extent both migration and the bio-distribution of MSCs, can
encompass either in vivo or ex vivo methods. Examples of in vivo
methods include bioluminescence, whereby cells are transduced to
express luciferase and can then be imaged through their metabolism
of luciferin resulting in light emission; fluorescence, whereby
cells are either loaded with a fluorescent dye or transduced to
express a fluorescent reporter which can then be imaged;
radionuclide labelling, where cells are loaded with radionuclides
and localized with scintigraphy, positron emission tomography (PET)
or single photon emission computed tomography (SPECT); and magnetic
resonance imaging (MRI), wherein cells loaded with paramagnetic
compounds (e.g., iron oxide nanoparticles) are traced with an MRI
scanner. Ex vivo methods to assess biodistribution include
quantitative PCR, flow cytometry, and histological methods.
Histological methods include tracking fluorescently labelled cells;
in situ hybridization, for example, for Y-chromosomes and for
human-specific ALU sequences; and histochemical staining for
species-specific or genetically introduced proteins such as
bacterial .beta.-galactosidase. These immunohistochemical methods
are useful for discerning engraftment location but necessitate the
excision of tissue. For further review of these methods and their
application see Kean et al., MSCs: Delivery Routes and Engraftment,
Cell-Targeting Strategies, and Immune Modulation, Stem Cells
International, Volume 2013 (2013).
[0180] Progenitor or multipotent cells, such as the mesenchymal
stem cells of the present invention, may be described as gene
delivery vehicles, essentially enabling the localization and
expression of therapeutic gene products in particular tissues or
regions of the subject's body. Such therapeutic cells offer the
potential to provide cellular therapies for diseases that are
refractory to other treatments. For each type of therapeutic cell
the ultimate goal is the same: the cell should express a specific
repertoire of genes, preferably exogenous nucleic acids that code
for therapeutic gene products, thereby modifying cell identity to
express said gene product and provide a therapeutic effect, such as
an immune stimulatory effect. The cells of the invention, when
expanded in vitro, represent heterogeneous populations that include
multiple generations of mesenchymal (stromal) cell progeny, which
lack the expression of most differentiation markers like CD34.
These populations may have retained a limited proliferation
potential and responsiveness for terminal differentiation and
maturation along mesenchymal and non-mesenchymal lineages.
[0181] As used herein "inducible expression" or "conditional
expression" relates to a state, multiple states or system of gene
expression, wherein the gene of interest, such as the immune
stimulatory cytokine, is preferably not expressed, or in some
embodiments expressed at negligible or relatively low levels,
unless there is the presence of one or more molecules (an inducer)
or other set of conditions in the cell that allows for gene
expression. Inducible promoters may relate to either naturally
occurring promoters that are expressed at a relatively higher level
under particular biological conditions, or to other synthetic
promoters comprising any given inducible element. Inducible
promoters may refer to those induced by particular tissue- or
micro-environments or combinations of biological signals present in
particular tissue- or micro-environments, or to promoters induced
by external factors, for example by administration of a small drug
molecule or other externally applied signal.
[0182] As used herein, in "proximity with" a tissue includes, for
example, within 50 mm, 10 mm, 5 mm, within 1 mm of the tissue,
within 0.5 mm of the tissue and within 0.25 mm of the tissue.
[0183] The cytokines described herein may relate to any mammalian
cytokine corresponding to the cytokine named herein. Preferably,
the cytokines relate to the human cytokines, or mouse
cytokines.
[0184] Given that stem cells can show a selective migration to
different tissue microenvironments in normal as well as diseased
settings, the use of tissue-specific promoters linked to the
differentiation pathway initiated in the recruited stem cell is
encompassed in the present invention and could in theory be used to
drive the selective expression of therapeutic genes only within a
defined biologic context. Stem cells that are recruited to other
tissue niches, but do not undergo the same program of
differentiation, should not express the therapeutic gene. This
approach allows a significant degree of potential control for the
selective expression of the therapeutic gene within a defined
microenvironment and has been successfully applied to regulate
therapeutic gene expression during neovascularization. Potential
approaches to such gene modifications are disclosed in WO
2008/150368 and WO 2010/119039, which are hereby incorporated in
their entirety.
FIGURES
[0185] The following figures are presented in order to describe
particular embodiments of the invention, by demonstrating a
practical implementation of the invention, without being limiting
to the scope of the invention or the concepts described herein.
[0186] Short description of the figures:
[0187] FIG. 1: Genetically modified human MSC expressing cytokines
in vitro.
[0188] FIG. 2: Preferred viral expression constructs of the present
invention.
[0189] FIG. 3: Intracellular FACS sorting of cells transduced with
viral expression constructs.
[0190] FIG. 4: ELISA detection of transgenic cytokines
DETAILED DESCRIPTION OF THE FIGURES:
[0191] FIG. 1: Genetically modified human MSC expressing cytokines
in vitro: Primary human MSC were transduced (MOI of 0.25) with
retroviral vectors expressing the cytokines indicated in the
graphic and the pac puromycin resistance gene. After transduction,
the transduced MSC were selected using puromycin and the average
vector copy number per cell was determined by qPCR. The selected
cells were then seeded for ELISA-measurements (Human IL-2 Thermo
ScientificEH2IL2, Human IL-7 Thermo Scientific EHIL7, Human IL-21
Thermo Scientific EHIL21).1.times.10.sup.5 cells were seeded 72 h
after seeding, the supernatants were collected and tested
subsequently. The generated data were normalized on 10E5 cells and
one vector copy number.
[0192] FIG. 2: Preferred viral expression constructs of the present
invention. The constructs were cloned into a y-retroviral backbone
(pSERS11, EP2019134A1). All constructs are driven by the elongation
factor short (pEFS) promoter. There are two Interleukins which are
separated by a P2A. The .alpha.- & .beta.-chain of IL-12 have
to be separated by a T2A. Human Interleukin sequences are used
(IL-2: GenBank: DQ861285.1, IL-76: GenBank: EF064721.1, IL-12a:
GenBank: AF101062.1, IL-12.beta.: NCBI Reference Sequence:
NM_002187.2, IL-15: GenBank: AY720442.1, IL-21: GenBank:
BC066260.1). A pac-gene and an oPRE-sequence are located behind an
IRES-sequence. On the 5'- and 3'-end of the constructs are LTRs.
The backbone contains LTRs, which are located on the 5'- &
3'-end of the constructs. The LTR at the 5'-end of the constructs
contains a SV40 Enhancer, RSV promoter, R Region and U5 Region. The
LTR at the 3'-end of the constructs has a deletion in the U3
Region, R Region, U5 Region and PolyA signal. The backbone contains
a bacterial part: a lacZ promoter, an origin of replication,
bla-gene and LacZ gene (pUC19).
[0193] FIG. 3: Intracellular FACS (ic-FACS) sorting of cells
transduced with viral expression constructs. Viral particles
encoding the designated vectors were generated by transfection of
293T cells. Primary human MSCs were transduced with a MOI of 0.25.
Transduced cells were selected with puromycin (Sigma Aldrich,
P9620-10ML, 10 mg/mL, final conc.: 3 .mu.g/mL). The selected cells
were analysed by is-FACS. To enhance detection of the expressed
cytokines, cells were treated GolgiPlug (BD, 555029) for 16 h to
enrich the proteins. Cells were permeabilized using (BD, 554722)
according to instructions of manufacturer; afterwards an
HA-Tag-specific antibody (Miltenyi, 130-092-257) was used to stain
the cytokines (1.33 pL per staining). Cells were analysed by flow
cytometry (FC500, Beckman coulter).
[0194] 3A: Non-transduced cells
[0195] 3B: Construct comprising IL7 and IL21, replicate 1
[0196] 3C: Construct comprising IL7 and IL21, replicate 2
[0197] 3D: Construct comprising IL2 and IL12, replicate 1
[0198] 3E: Construct comprising IL2 and IL12, replicate 2
[0199] 3F: Construct comprising IL15 and IL12, replicate 1
[0200] 3G: Construct comprising IL15 and IL12, replicate 2
[0201] 3H: Construct comprising IL7 and IL12, replicate 1
[0202] 3I: Construct comprising IL7 and IL12, replicate 2
[0203] 3J: Construct comprising IL21 and IL12, replicate 1
[0204] 3K: Construct comprising IL21 and IL12, replicate 2
[0205] FIG. 4: ELISA detection of transgenic cytokines. Human MSC
were transduced with the indicated constructs. The cells were
Puromycin-selected cells and 1.times.10e5 cells seeded into 6-well
plates. The supernatants were collected after 72 h and analysed by
ELISA. The generated data were normalized to 10E5 cells and vector
copy number. The used ELISA Kits were used according to the
instructions of manufacturer: Human IL-2 (Thermo Scientific,
EH2IL2), Human IL-7 (Thermo Scientific, EHIL), Human IL-15
(BioLegend, 431707), Human IL-21 (Thermo Scientific, EHIL21).
EXAMPLES
[0206] The following examples are presented in order to describe
practical and in some cases preferred embodiments of the invention,
by demonstrating a practical implementation of the invention,
without being limiting to the scope of the invention or the
concepts described herein.
[0207] Experimental Models:
[0208] Mesenchymal stem cells can be extracted according to either
Lange C. et al. ("Accelerated and safe expansion of human
mesenchymal stromal cells in animal serum-free medium for
transplantation and regenerative medicine", J. Cell Physiol. 2007,
Apr. 25) or Soleimani ("A protocol for isolation and culture of
mesenchymal stem cells from mouse bone marrow", Nat Protoc.
2009;4(1):102-6).
[0209] The cells grow adherently and continuously in cell culture.
MSCs may be transformed with retroviral or lentiviral vectors
comprising cytokine encoding gene sequences. Viral constructs can
be engineered according to standard protocols and produced that
express genes encoding IL-2, IL-7, IL-15, IL-21, IL-12, IFN gamma,
IFN beta, SDF-1, CCL23, CCL19, CCL1, CCL2, CCL17, CCL22 and/or CCL4
and combinations therefrom.
[0210] Transformed cells are selected and cultured further before
harvesting for administration. All vectors can for example comprise
of an antibiotic resistance gene, such as a puromycin resistance
gene. Puromycin may therefore be used to select for transfected
cells at a concentration of 0.1-10 .mu.g/ml, or preferably 3-5
.mu.g/ml. Prior to injection into the mice or other subjects, the
cells are detached from the culture flasks, washed twice with PBS,
and re-suspended in PBS, or other suitable buffer.
[0211] Suitable experiments may be performed in either an
endogenous mouse breast cancer model (as described in WO2008150368)
or an orthotopic pancreatic carcinoma model (as described in
WO2010119039). In parallel experiments, mice with growing tumours
are injected with the various engineered MSCs, either with or
without T cells isolated from syngeneic subjects, and/or checkpoint
inhibitors. After five days, the animals may be sacrificed and the
tumours examined. Preliminary results indicate a reduction in
tumour size/growth in subjects of the aforementioned treatment in
comparison to appropriate controls.
[0212] Preparation of Human Mesenchymal Stem Cells:
[0213] In the present example, human MSCs are isolated from bone
marrow by plastic adherence and are cultured in growth medium e.g.
FBS containing DMEM as described by Pittinger, M. F. (2008)
Mesenchymal stem cells from adult bone marrow, In D. J. Prockop, D.
G. Phinney, B. A. Bunnell, Methods in Molecular Biology 449,
Mesenchymal stem cells, Totowa: Humana Press).
[0214] Generation of Vectors for the Expression of Cytokines and
Chemokines
[0215] The transgene expression cassettes consisting a promoter and
a gene (e.g. cDNA) for an immunostimulatory factor or factor
supporting immune response are constructed using standard cloning
techniques as described in Julia Lodge, Peter Lund, Steve Minchin
(2007) Gene Cloning, New York: Tylor and Francis Group. The
promoters may be constitutive promoters like the CMV promoter or
the PGK promoter or inducible promoters like Tie2, RANTES or the
HSP70 promoter.
[0216] Examples for genes encoding immunostimulatory factors or
factors supporting immune responses are IL-2, IL-7, IL-15, IL-21,
IL-12, IFN gamma, IFN beta, SDF-1, CCL23, CCL19, CCL1, CCL2, CCL17,
CCL22 and/or CCL4 (Strengell et al., M, The Journal of
Immunology,2003, 170: 5464-5469; Borish et al., J Allergy Clin
Immunol. 2003February; 111(2 Suppl): S460-7). The gene may or may
not be fused with tag-sequences (e.g. marker proteins/peptides like
the hemagglutinin-tag or the HIS-tag) to allow easy detection of
expression later on (Hinrik Garoff, 1985, Annual Review of Cell
Biology, Vol. 1: 403-445).
[0217] The transgene is then inserted into a suitable vector system
(e.g. lentiviral or gamma-retroviral vector) by standard cloning
techniques. A suitable vector is for example described by Baum (EP
1757703 A2). The vector may or may not include a second transgene
cassette consisting of a promoter and a selectable marker gene
(cell surface marker or resistance gene, for example the pac gene
to confer puromycin resistance) to allow enrichment of genetically
modified cells later in the process (David P. Clark, Nanette J.
Pazdernik, 2009, Biotechnology: Applying the Genetic Revolution,
London: Elsevier).
[0218] Preferred constructs according to the present invention are
shown in FIG. 2.
[0219] Genetic Modification of Mesenchymal Stem Cell:
[0220] The transduction is performed with modifications as
described by Murray et al., 1999 Human Gene Therapy. 10(11):
1743-1752 and Davis et al., 2004 Biophysical Journal Volume 86
1234-1242. In detail:
[0221] 6-well cell culture plates (e.g. Corning) are coated with
Poly-L-Lysine (PLL) (e.g. Sigma-Aldrich, P4707-50ML): The PLL
solution (0.01%) is diluted to final concentration between 0.0001%
and 0.001% with PBS. 2 ml of the diluted PLL are used for each
well. The plate is incubated at least for 2 h at room temperature.
After incubation, the plates are washed carefully with PBS.
[0222] Viral vector supernatant in a final volume of 2 ml is added
to each PLL-coated well. The number of particles should between
2.times.10e3 and 1.times.10e6 per well, which will result in
multiplicity of infection of 0.25 and 10. The loaded plate is
centrifuged for 2000 .times.g, 30 min, 4.degree. C. Afterwards the
supernatant is discarded and 1.times.10e5 mesenchymal stem cells
are seeded per well. The plates are incubated at 37.degree. with 5%
CO2 for further use.
[0223] Analysis of Transgene Expression in MSC:
[0224] Flow Cytometry:
[0225] To show that the immunostimulatory factors are expressed in
the MSC intracellular flow cytometry assays are performed. 3 days
after transduction MSC medium is exchanged for medium containing 1
.mu.l BD Golgi Plug (Cat. No. 555029) per 1 ml Medium to enrich the
expressed factors in the Golgi apparatus of the transduced cells.
Cells are incubated for 16 h at 37.degree. C. and are then
immunostained for the expression of the factors. MSC are harvested
and permeabilized using the BD Cytofix/Cytoperm Cell
Permeabilization/Fixation Solution (Becton Dickinson, 554722)
according to the manufacturer's instructions to allow intracellular
staining of the target proteins. A hemagglutinin-tag specific
antibody labelled with Phycoerythrin (PE) (Milteny, 120-002-687) is
used for detection of the expressed factor. 2.times.10e5 MSC are
stained with 100 .mu.l of antibody (1:75 diluted with Perm/wash
solution, Becton Dickinson, 554723). Alternatively, antibodies
directly directed against the factor may be used according to the
instruction of the manufacturer (e.g. anti-IL2 antibody labelled
with PE, ebiosience 12-7029-41). The stained cells are washed and
resuspended in PBS. The cells are then analysed on an FC500 flow
cytometer (Beckman Coulter).
[0226] Expression of the cytokine transgenes are shown in FIG.
3.
[0227] ELISA:
[0228] Transduced MSC are seeded in 6 well plates (1.times.10e5 MSC
per well). Transduced MSC, which carry the pac puromycin resistance
gene, are enriched by puromycin selection. For this puromycin (3
.mu.g/ml medium) is added to the medium and cells are cultivated
over a period of 5 days at 37.degree. C. and 5% CO2 with medium
exchanges every 2 days to deplete non-transduced cells from the
culture. Afterwards puromycin-free medium is used for the culture.
MSC are reseeded at a defined cell number of 1.times.10e5 cells per
well in a 6 well-plate and are incubated for 48 h. Medium is
collected and used for immune factor specific ELISA for
quantification according to the manufacturer's instructions (e.g.
IL-7 ELISA: Thermo Scientific, EHIL7; IL-15 ELISA: Thermo
Scientific, EHIL15).
[0229] Expression of the cytokine transgenes are shown in FIG.
4.
[0230] Monitoring of T Cell and Macrophage Activation In Vitro by
ELISA:
[0231] Peripheral blood mononuclear cells (PBMC) are isolated from
human blood using ficoll gradient centrifugation as described by
Ivan J. Fuss, Marjorie E. Kanof, Phillip D. Smith, Heddy Zola, 2009
Curr. Protoc. Immunol. 85: 7.1.1-7.1.8. To assess the
immune-stimulatory effect of the factors expressed in the MSC in
vitro, co-culture assays are performed. 1-5.times.10e5 PBMC are
seeded into a well of a 12-well together with 0.2-1.times.10e5
transduced MSC, untransduced MSC (control) or without MSC.
Unspecific suboptimal stimulation of the T-cells in the culture
mimicking engagement of the T cell receptor is performed:
therefore, prior to cell seeding the wells of the plates may be
coated with the stimulatory anti-CD3 antibody (e.g. OKT3,
Janssen-Cilag). The antibody solution should have a concentration
0.5-0.1 .mu.g/mL. Alternatively, PHA may be added to the coculture
in concentration of 20 .mu.g/ml (Ngoumou et al., Cytokine 25 (2004)
172-178). The wells are incubated at 37.degree. C. and 5% CO2 for
2-5 days prior to analysis.
[0232] Compared to wells with untransduced MSC or wells without
MSC, MSC transduced with immunostimulatory factors leads to an
increased activation of the cultured T cells. Activation status of
the T cells is assessed by measuring INF gamma concentration in the
cultures as these cytokines are indicative for T cell activation
(Boehm et al., Annu Rev Immunol. 1997;15:749-95.). To assess
activation status of monocytes, medium is collected and the release
of tumour necrosis factor alpha (TNFa) is determined. Medium is
collected and used for IFN gamma or TNF alpha specific ELISA for
quantification according to the manufacturer's instructions (e.g.
ELISA: IFN gamma, Thermo Scientific, EHIFNG; TNF alpha ELISA:
Thermo Scientific, EH3TNFA).
[0233] Monitoring of T Cell and Macrophage Activation In Vitro by
Flow Cytometry:
[0234] Peripheral blood mononuclear cells (PBMC) are isolated from
human blood using ficoll gradient centrifugation as described by
Ivan J. Fuss, Marjorie E. Kanof, Phillip D. Smith, Heddy Zola, 2009
Curr. Protoc. Immunol. 85: 7.1.1-7.1.8. To assess the
immune-stimulatory effect of the factors expressed in the MSC in
vitro, co-culture assays are performed. 1-5.times.10e5 PBMC are
seeded into a well of a 12-well together with 0.2-1.times.10e5
transduced MSC, untransduced MSC (control) or without MSC.
Unspecific suboptimal stimulation of the T-cells in the culture
mimicking engagement of the T cell receptor is performed:
therefore, prior to cell seeding the wells of the plates may be
coated with the stimulatory anti-CD3 antibody (e.g. OKT3,
Janssen-Cilag). The antibody solution should have a concentration
0.5-0.1 .mu.g/mL. Alternatively, PHA may be added to the coculture
in concentration of 20 .mu.g/ml (Ngoumou et al., Cytokine 25 (2004)
172-178). The wells are incubated at 37.degree. C. and 5% CO2 for
2-5 days prior to analysis.
[0235] Compared to wells with untransduced MSC or wells without
MSC, MSC transduced with immunostimulatory factors lead to an
increased activation of the cultured T cells and macrophages.
Activation status of the T cells and macrophages is assessed by
intracellular flow cytometry. 24 h prior to harvest of the cells,
the cells are treated with medium containing 1 .mu.l BD Golgi Plug
(Cat. No. 555029) per 1 ml Medium. Afterwards cells are harvested
and stained with fluorophore-labelled antibodies specific for T
cells (e.g. anti-CD4, ebioscience 17-0048 or anti-CD8, ebioscience
9017-0087) or macrophages/monocytes (anti-CD14, ebioscience,
9017-0149) according to the manufacturer's instruction. Next cells
are permeabilized using the BD Cytofix/Cytoperm Cell
Permeabilization/Fixation Solution (Becton Dickinson, 554722)
according to the manufacturer's instructions to allow intracellular
staining of IFNg (ebioscience, 11-7319) or TNFa (ebioscience,
11-7349). Antibodies are used according to manufacturer's
instructions. Afterwards cells are analysed using an FC500 flow
cytometer (Beckman Coulter).
[0236] Monitoring of Anti-Tumoral Effect of MSC Administration in
Animal Model:
[0237] Tumours from human tumour cell lines are grown in immune
deficient mice (e.g. SCID mice) for 2 weeks and engineered MSCs are
administered intravenously, for example via the tail vein.
Following that, PBMCs are administered intravenously. The tumour
sizes are then compared with the tumour sizes of untreated animals,
or animals treated with MSCs only, or PBMCs only.
[0238] In another experiment, tumours from human tumour cell lines
combined with engineered MSCs are grown in immune deficient mice
(e.g. SCID mice) for 2 weeks and PBMCs are administered
intravenously. The tumour sizes are then compared with the tumour
sizes of untreated animals, or animals treated with MSCs only, or
PBMCs only.
[0239] The above experiments can be performed whereas instead of
PBMCs, purified cytotoxic T Lymphocytes (CTLs) are used, or CART
cells that carry CARs directed at tumour antigens present on the
tumours. Likewise, a checkpoint inhibitor (e.g. anti-PD-1, or
anti-PD-L1 antibody) can be used together with MSCs and PBMCs, or
MSCs and CTLs, or MSCs and CARTs.
[0240] The tumours from the above experiments are analysed
histologically to assess the amount of expression of the cytokines
and cytokine combinations by MSCs, by using antibodies reactive
against these cytokines. The extent of infiltration of the tumour
by PBMCs, CTLs and CARTs is assessed using a hematoxylin and eosin
dye (H&E). The extent of infiltration of the tumour by T cells
can be assessed by using immunohistochemistry with antibodies
against CD3. The extent of infiltration of the tumour by monocytes
can be assessed by using immunohistochemistry with antibodies
against CD19. The extent of activation of the infiltrating T cells
in the tumour can be assessed by using immunohistochemistry with
antibodies against CD69, as well as IFN-gamma.
[0241] To confirm the experiments above, these experiments are
repeated with different types of tumours, grown using different
human tumour cell lines and CARTs with specificity against
respective tumour-associated antigens.
* * * * *