U.S. patent application number 11/900670 was filed with the patent office on 2008-05-08 for vaccination with immuno-isolated cells producing an immunomodulator.
Invention is credited to Nicolas Mach.
Application Number | 20080107686 11/900670 |
Document ID | / |
Family ID | 29716769 |
Filed Date | 2008-05-08 |
United States Patent
Application |
20080107686 |
Kind Code |
A1 |
Mach; Nicolas |
May 8, 2008 |
Vaccination with immuno-isolated cells producing an
immunomodulator
Abstract
The present invention relates to immuno-protected encapsulated
cells producing an immunomodulator, for example GM-CSF
(granulocyte-macrophage colony stimulating factor). The cells of
the invention are particularly well adapted for providing an active
adjuvant or immunomodulator, for example in the context of
immunisation in humans and animals. These cells can be used for
vaccination where they provide the immunomodulator in an active
form, in a continuous, non-immunogenic manner in the immediate
vicinity of the vaccine antigen(s). The invention also relates to a
vaccine composition comprising immuno-protected encapsulated cells
producing an immunomodulator and an antigenic component. The
invention also relates to a kit comprising a cell as described and
an antigenic component. The strategy of the invention is perfectly
suited for both cancer immunotherapy and vaccination against
infectious agents.
Inventors: |
Mach; Nicolas;
(Collonge-Bellerive, CH) |
Correspondence
Address: |
MINTZ, LEVIN, COHN, FERRIS, GLOVSKY;AND POPEO, P.C.
ONE FINANCIAL CENTER
BOSTON
MA
02111
US
|
Family ID: |
29716769 |
Appl. No.: |
11/900670 |
Filed: |
September 12, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11058883 |
Feb 16, 2005 |
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11900670 |
Sep 12, 2007 |
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10464565 |
Jun 17, 2003 |
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11058883 |
Feb 16, 2005 |
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Current U.S.
Class: |
424/208.1 ;
424/184.1; 424/204.1; 424/228.1; 424/93.21; 435/325 |
Current CPC
Class: |
A61K 2039/55522
20130101; A61K 9/48 20130101; A61K 2039/5152 20130101; C12N 11/04
20130101; A61K 38/18 20130101; C12N 2740/16011 20130101; A61K
39/0011 20130101; C12N 2770/24211 20130101; C12N 7/00 20130101;
A61P 31/00 20180101; C12N 15/85 20130101; A61K 38/193 20130101;
A61P 43/00 20180101; A61K 39/21 20130101; Y02A 50/30 20180101; A61P
35/00 20180101; A61K 39/39 20130101; A61K 39/29 20130101 |
Class at
Publication: |
424/208.1 ;
435/325; 424/093.21; 424/184.1; 424/204.1; 424/228.1 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C12N 5/06 20060101 C12N005/06; A61P 43/00 20060101
A61P043/00; A61K 35/00 20060101 A61K035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 17, 2002 |
EP |
02013249.4 |
Claims
1. A cell producing at least one immunomodulatory agent, said cell
being immuno-isolated.
2. The cell according to claim 1, wherein said immunomodulatory
agent is an immunostimulatory agent.
3. The cell according to claim 1, wherein said immunomodulatory
agent is a cytokine.
4. The cell according to claim 3, wherein said cytokine is GM-CSF
or FL.
5. The cell according to claim 1, further producing another
immunomodulatory agent or an antigenic agent.
6. The cell according to claim 1, wherein said produced
immunomodulatory agent is secreted.
7. The cell according to claim 1, wherein said immuno-isolation is
achieved by a barrier device, microencapsulation or
macroencapsulation.
8. The cell according to claim 7, wherein said barrier device is
selectively permeable.
9. The cell according to claim 8, wherein said barrier device is
selectively permeable to molecules with molecular weight smaller
than 280 kDa.
10. The cell according to claim 1, wherein said cell is genetically
modified to express the immunomodulatory agent.
11. The cell according to claim 10, wherein the genetic
modification is achieved by transfection by a plasmid or infection
by a virus.
12. The cell according to claim 1, wherein said cell is a human
established cell line, for instance a fibroblast or an epithelial
cell line.
13. The cell according to claim 1, wherein said cell exhibits one
or more of the following characteristics: (a) is immortal or
immortalised; (b) is non tumoral; (c) is of mammal origin; (d) is
human; (e) is irradiated; (f) secretes between 80 and
960.times.10.sup.-15 g/24 hr of immunomodulatory agent; or (g)
secretes more than 10.times.10.sup.-15 g/24 hr, preferably more
than 100.times.10.sup.-15 g/24 hr of immunomodulatory agent.
14. A pharmaceutical composition comprising a cell according to
claim 1 and a physiologically acceptable carrier.
15. A vaccine composition comprising a cell according to claim 1
and an antigenic component.
16. The vaccine composition according to claim 15, wherein said
antigenic component is a tumour cell.
17. The vaccine composition according to claim 16, wherein said
tumour cell is irradiated.
18. The vaccine composition according to claim 15, wherein said
antigenic component is from an infectious agent.
19. The vaccine composition according to claim 18, wherein said
infectious agent is a virus.
20. The vaccine composition according to claim 19, wherein said
virus is Hepatitis C or HIV.
21. A kit comprising a cell according to claim 1 and an antigenic
component.
22. The kit according to claim 21, wherein the antigenic component
comprises a cell producing or releasing one or several
antigens.
23. The kit according to claim 22, wherein said cell producing or
releasing one or several antigens is a tumour cell.
24. The kit according to claim 23, wherein said tumour cell is
irradiated.
25. The kit according to claim 21, wherein the antigenic component
is from an infectious agent.
26. The kit according to claim 25, wherein said infectious agent is
a virus.
27. The kit according to claim 21 which is biocompatible.
28. A capsule containing at least one cell producing an
immunomodulator.
29. The capsule according to claim 28, wherein said immunomodulator
is GM-CSF.
30. The capsule according to claim 28 also containing a polymer
matrix.
31. The capsule according to claim 28 which is designed to be
removable after insertion.
32. The capsule according to claim 28, wherein said capsule is
irradiated.
33. The cell according to claim 1 for use in therapy or
vaccination.
34. A use of a cell according to claim 1 for the manufacture of an
adjuvant for enhancing immune response.
35. A use of a cell according to claim 1 for the manufacture of a
medicament for the treatment or prevention of cancer.
36. A use of a kit according to claim 21 for the manufacture of a
vaccine.
37. The use according to claim 36, wherein said vaccine is to be
implanted and subsequently removed.
38. The use according to claim 37, wherein removal is accomplished
between 2 to 7 days after implantation, preferably between 5 to 7
days.
39. The use according to claim 35, wherein said cells are
irradiated.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
11/058,883, filed Feb. 16, 2005, which is a continuation of U.S.
Ser. No. 10/464,565, filed Jun. 17, 2003, which claims priority to
European Patent Application No. 02013249.4, filed Jun. 17, 2002,
each of which is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to a new approach for
providing an active adjuvant or immunomodulator, for example in the
context of immunisation in humans and animals. According to this
approach, an immunomodulator, for example GM-CSF
(granulocyte-macrophage colony stimulating factor), is released
from immuno-protected encapsulated cells producing this protein.
This system is particularly well adapted to vaccination in that it
provides the immunomodulator in an active form, in continuous,
non-immunogenic manner in the immediate vicinity of the vaccine
antigen(s). The strategy of the invention is perfectly suited for
both cancer immunotherapy and vaccination against infectious
agents.
BACKGROUND OF THE INVENTION
[0003] In the field of vaccination, first generation vaccines
comprised only the antigen against which an immune response was
desired. However, because the presence of an antigen alone is in
most cases only weakly efficient, a second generation of vaccines
was developed, wherein the vaccinating composition includes one or
more adjuvants as immunomodulators to enhance this immune
response.
[0004] Several different techniques have been reported for
providing the adjuvant at the vaccination site, depending on the
context of the immunisation.
[0005] In the context of antigen-based vaccines (as opposed to
cell-based vaccines), a widely applicable technique used for
providing the necessary adjuvant is simply to combine the antigen
with the adjuvant in the vaccinating composition. The resulting
composition is administered directly to the subject, thereby
supplying the antigen and adjuvant in a simultaneous and
co-localised manner.
[0006] This simple approach cannot however be used in all
vaccination settings. For example, in most cancers, useful antigens
are often not known. This is the case for most of the common
cancers in human such as lung, colon, stomach, lymphoma, brain.
Therefore, new immunisation strategies are needed such as a
cell-based approach. Immunisation strategy involving cell-based
vaccines, where the antigen(s) to which an immune response is
required is (are) produced by whole cells implanted in a subject,
necessitates the use of more elaborate techniques to ensure the
efficient delivery of adjuvant.
[0007] One solution in this type of context is to directly inject
the immunomodulator at the vaccination site. The immunomodulator
may either be "naked", or alternatively may be administered in a
slow release formulation using pegylated, liposomal microspheres
(International patent application WO 98/33520, filed by Bystryn).
This strategy is however limited by technical and biochemical
difficulties, as well as some degree of systemic release inducing
potential toxicities.
[0008] An alternative approach which has been proposed to
circumvent the problems arising from the direct injection technique
is the use of "by-stander cells" to locally produce the
immunomodulators. According to this approach, cells producing the
adjuvant are implanted in proximity to the source of the antigen,
thereby providing an efficient local release of adjuvant at the
vaccine site. The efficacy of this approach has been demonstrated
in mice where the vaccine is a mix of GM-CSF secreting fibroblasts
and irradiated tumour cells (Aruga et al, 1997, Cancer Research,
57, 3230-3237).
[0009] However, even this approach is not totally without
drawbacks. Indeed, for human immunization, it is known that
multiple immunizations are required. Since syngeneic by-stander
cells are not easily available, allogenic cells are used in the
vast majority of cases. Consequently, after the first injection,
the "by-stander cells" are recognised by the immune system of the
host (allorecognition) and are rejected, thereby preventing further
production of immunomodulator. In addition, the allorecognition of
the "by-stander cells" jeopardizes the desired immune response
against the antigenic substance of the vaccine.
[0010] In order to avoid this allorecognition, Borrello et al
(Human Gene Therapy, 1999) has described a strategy in which the
GM-CSF supplying cell is a cell line, K-562, which fails to express
HLA class I or II antigens, potentially decreasing the magnitude of
the alloresponses generated on repeated immunisations. The K-562
cells, engineered to secrete GM-CSF do not express MHC molecules on
their surface. They are HLA negative. These cells are however human
cancer cells and are highly sensitive to potent rejection
mechanisms occurring without the involvement of HLA class I or II
proteins. These defence strategies are less specific but very rapid
and potent for cellular destruction. At least two subtypes of
lymphocytes known as .gamma..delta. T cells or natural killer (NK)
lymphocytes are known to attack and destroy foreign cells by
mechanism independent of HLA class I or II. Regarding K-562 they
are known to be very sensitive to NK cells and also to
.gamma..delta. T cells (J. Immunotherapy 2000 23:536-548 Schilbach
K. et al) and are therefore used as a positive control for NK cell
activity.
[0011] It is therefore likely that K-562 by-stander cells injected
at the vaccine site will be destroyed efficiently and quickly by
non-MHC dependent cytotoxic mechanisms. This may very well
significantly decrease the release of the immunomodulator.
[0012] Beside being very sensitive to rapid destruction by NK
cells, K-562 cells can express MHC class I upon Interferon .gamma.
exposure. It is possible that such cytokine could be present or
released at the vaccination site during the first or after repeated
immunizations. Such MHC class I upregulation will also lead to
rapid cell destruction via classical cellular immunity.
[0013] For these reasons, use of cells such as K-562 in vaccination
is associated with numerous drawbacks.
[0014] Another solution, widely used in the context of cell-based
vaccines, for example in cancer therapy, is to couple the
production of antigen and the release of immunomodulator, by
engineering the cell which is the source of antigen, to also supply
the immunomodulator. For example, in cancer vaccines, the source of
antigen is usually a whole tumour cell. This cell can be
engineered, for example by transfection, to simultaneously produce
the necessary adjuvant. Potent, specific and long lasting
anti-tumour immune responses have been reported in the mouse model
using this technique, relying on retroviral vectors as the gene
transfer method for engineering tumour cells delivering GM-CSF.
[0015] In view of the favourable results obtained in the mouse
model, the initial human trials used the same strategy (Simons J W.
et al. 1997 Cancer Research, 43(11); Soiffer R. et al. 1998, Proc.
Natl. Acad. Sci. USA, 95, 13141; Simons et al. 1999, Cancer
Research, 59(20)). However, the technique proved to be very labour
intensive and time consuming, because the patient's cells,
harvested surgically need to be expanded in vitro, for retroviral
infection, preventing a wide use of the method.
[0016] The use of other viral vector for infecting the tumour cells
has therefore been proposed to circumvent these difficulties.
Engineered viruses like adenovirus can infect tumour cells very
efficiently and with much simpler procedures. Because adenovirus
can infect non-dividing cells, the harvested tumour cells can be
infected right away, preventing the long and tedious primary
culture step required when using retroviral vectors.
[0017] The major problem of the new viruses tested is that in most
cases, some viral proteins will be expressed from the tumour cells
after infection. These viral proteins are strongly recognized by
the immune system as foreign, infectious agents. Therefore the
initial goal of mounting an immune response against weak tumour
antigens is skewed or diverted towards a viral protein. This
results in masking the anti-tumour immune response. It also primes
the recipient against subsequent immunization that will further
increase the destruction of the injected cells. These two
mechanisms will very likely decrease the efficacy of the
anti-tumour immunization scheme.
[0018] Thus, whilst the use of autologous engineered tumour cells
as combined source of antigen and adjuvant a priori minimizes the
risk of undesirable immune response, the step of viral infection
itself gives rise to significant problems.
[0019] In order to limit the problems arising from viral infection
of autologous cells, new strategies have been developed which do
not require patients' cells. According to these techniques, the
antigenic source is provided by cell-lines derived from other
patients with similar type of cancer. The patient is then immunized
with repeated injections of irradiated, GM-CSF secreting,
allogeneic (from another human being) tumour cells.
[0020] This strategy is based on the assumption that the cell-line
used for the immunization shares some relevant antigens with the
patient's own tumour and that these common antigens will allow the
development of an immune response that will recognize the patient
tumour cells and destroy them. A phase I clinical trial using such
strategy has been published (Jaffee E. et al. 2001 Journal of
Clinical Oncology, 19(1)).
[0021] However, the percentage of patients showing an immune
response is much lower than in previous report using autologous
cells secreting the same immunomodulator (GM-CSF) (Soiffer et
al.).
[0022] To date, no technique has been reported which provides both
a constant source of immunomodulator and an efficient immune
response, whilst being substantially free of undesirable
interactions with the natural or adaptive immune system.
SUMMARY OF THE INVENTION
[0023] The present invention provides a new approach overcoming the
drawbacks associated with the previous strategies. The invention is
based on the immuno-isolation of adjuvant producing cells by a
physical barrier such as a capsule.
[0024] The present invention provides a general protocol for
vaccination, named "Maxi-Vax" characterized by the delivery, in
close proximity, of antigens and of live cells engineered to
secrete immuno-activating cytokines (such as GM-CSF), these cells
being immuno-protected by macro- or micro-encapsulation in order to
sustain prolonged in situ delivery of the immune activator.
[0025] A particular case of the invention utilises
macro-encapsulation of cells in hollow fibers PES capsules, but
other forms of encapsulation, within devices or within gel
matrices, are included in the invention.
[0026] A particular case of the invention concerns the use of
cancer cells as the antigen, for the purpose of immunising against
cancer-specific antigens and establishing a systemic anti-tumour
cell protective immune response. The later case is referred to as
"Onco-Maxi-Vax".
[0027] Another particular case of the invention applies to
vaccination against infectious agents, such as viruses (including
HIV and Hepatitis C).
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1: Clinical immunization protocol for Onco-Maxi-Vax
[0029] The vaccination treatment with Onco-Maxi-Vax comprises
repeated immunizations. The immunization is repeated at two week
intervals at different sites, always sub-cutaneous. The total
number of vaccination is a minimum of five.
[0030] .gradient.: surgery and cell harvesting and processing of
patients own cells
[0031] .dwnarw.: Immunization with Onco-Maxi-Vax: encapsulated,
irradiated GM-CSF producing cells and irradiated autologous tumour
cells
[0032] FIG. 2: Protective immunity against wild-type tumor
challenged by various irradiated, cytokine secreting tumour
cells.
[0033] This figure compares the efficacy of various
immunomodulatory molecules in vaccination mouse model. Each block
represents the percentage of mice surviving a tumour challenge
after vaccination with irradiated tumour cells producing the
described molecule.
[0034] All of the mice vaccinated with irradiated tumour cells
secreting GM-CSF are protected. 25% of the mice vaccinated with
irradiated tumour cells secreting FLT3-L are protected.
[0035] FIG. 3: GM-CSF release from encapsulated, non irradiated
Renca-GM-CSF secreting cells.
[0036] This table shows an analysis of GM-CSF secretion outside the
capsule at various time points by encapsulated, GM-CSF secreting
Renca cells. GM-CSF release is expressed in ng/capsule/24 h.
[0037] Measurement of in-vitro release of murine GM-CSF from the
capsule containing GM-CSF secreting cells is carried out at Days 4,
7, 11, 14 and 21 post loading the cells into the capsule. This is
performed with standard monoclonal antibodies against murine GM-CSF
in Enzyme-Linked immunoabsorbent Assays (ELISA). (R&D systems).
The amounts of protein released as well as the reproducibility from
one capsule to the others are assessed.
[0038] FIG. 4: Survival at 50 days after B16 WT challenge.
[0039] This graph shows the survival at 50 days of mice immunized
at day-7 with [0040] either irradiated, encapsulated GM-CSF
secreting Renca cells only (group 1), [0041] either irradiated,
encapsulated GM-CSF secreting Renca cells plus irradiated B16 wt
melanoma cells (group 2); [0042] or irradiated, non-encapsulated
B16 melanoma cells engineered to secrete GM-CSF (group 3);
[0043] The graph represents the mean value over 5 mice per
group.
[0044] FIG. 5: Survival of mice after B16 WT challenge.
[0045] The experimental conditions are identical to the conditions
specified in FIG. 4 and exposed in example 4.
DETAILED DESCRIPTION
Definitions
[0046] In the context of the present application, the following
terms are defined in the following manner:
[0047] An immunomodulator or an immunomodulatory agent is a
compound or a composition which can enhance, amplify or decrease an
immune response to an antigen or an immunogen.
[0048] An immunostimulatory agent or an immuno-activator is an
immunomodulator which specifically enhances or amplifies the immune
response to an antigen or an immunogen. In the context of the
present invention, the term "immunostimulatory agent" or
"immuno-activator" is used synonymously with the term
"adjuvant".
[0049] Cells are considered to be immuno-isolated if, when
introduced into a host, they are physically protected against the
immune response of the host, i.e. there is no significant acquired
or natural immune response against any cell components, including
cell-surface antigens, secreted proteins etc., provided there is no
physical contact between the cells and the effectors of the immune
system. Consequently, no significant antibody or cell-mediated
immune response to the cell is seen in the host organism.
[0050] Consequently, immuno-isolated cells are not attacked or
destroyed by the immune response of the host, because they are
undetectable by the immune system, which prevents any immune
response against them and because they are physically protected
against any immune response.
[0051] Encapsulation is a particular means of immuno-isolating
cells in a device comprising a capsule of material, for example
plastic, which is non-immunogenic for the host organism. Preferred
polymers for the capsule are thermoplastic polyethersulfone (PES)
hollow fibers (OD:720 .mu.m; ID:524 .mu.m, molecular weight
cut-offs: 32 and 80 kDa; Akzo Nobel Faster AG, Wupperthal, Germany)
and AN-69 polymer (acrylonitrile and sodium metallysulphonate
anionic copolymer, Hospal R&D Int, Meyzieu, France). The
capsules may have various shapes and sizes.
[0052] The present invention relates to encapsulated cells
producing and secreting an immunomodulator (preferably GM-CSF), for
use in therapy and in vaccination (preferably with cancer cells).
Preferably, the encapsulated cells are engineered to produce the
immunomodulator, although the invention also encompasses the use of
cells and cell-lines which naturally produce the immunomodulator.
It also relates to pharmaceutical compositions, vaccines and kits
which can be used in this context. It finally relates to processes
for vaccinating or treating patients.
[0053] In a first aspect, the present invention relates to cells
which produce an immunomodulatory agent and which are physically
immuno-isolated. The immunomodulatory agent produced is preferably
a protein synthesized by the cells, but it can also be for example
a cell-component such as a lipid, or an exogenous molecule further
transformed by the cell, for example antigens processed by
antigen-presenting cells or metabolites.
[0054] In a preferred embodiment, the immunomodulatory agent is
immunostimulatory. Antigens are frequently too weak to trigger a
significant immune response and some molecules involved in this
response are known to enhance or amplify it.
[0055] An immunostimulatory agent may act in attracting
Antigen-presenting cells, for example dendritic cells. It may also
act in stimulating the activities of CD4 or CD8 T-cells.
[0056] Particularly potent immunostimulatory agents, which are
preferred in the context of the present application, belong to the
cytokine family. Preferred cytokines are IL-3, IL-4, IL-9, IL-1,
IL-2, IL-7 (interleukine), transmembrane receptors of IFN.gamma.,
SCF (Stem Cell Factor) soluble or membranous, FL (Flt3 Ligand),
G-CSF and GM-CSF (Granulocyte and Granulocyte-Macrophage
Stimulating Factor), and combinations thereof. Particularly
preferred immunostimulatory agents are FL and GM-CSF. Preferred
cytokines are human cytokines, for example human GM-CSF.
[0057] In the context of cancer therapy, GM-CSF is particularly
recommended as immunostimulatory agent because it has been
identified as the most potent cytokine for activating systemic
antitumor immunity (Dranoff et al, 1993).
[0058] In order to induce an adequate immune response, it can be
very advantageous to combine several immunomodulatory agents in
order to stimulate different pathways. A preferred combination is
the association of GM-CSF and FL. Other combinations of 2 or more
immunomodulatory agents are also envisaged by the present
application.
[0059] Although the main role of an immuno-isolated cell of the
invention is to produce an immunomodulatory agent, it can fulfil
other additional functions. It can for example play a role in the
detection of the magnitude or the localisation of the expected
immune response. Another major additional function is to provide
the antigenic agent. According to this variant, the same cell can
produce both the antigenic agent triggering the response and the
immunostimulatory agent enhancing the response. This is
particularly advantageous in the case of antigen-based vaccination,
or cell-based vaccination where the antigens are secreted or
released from the cell membrane. This approach ensures that
antigenic and immunostimulatory agents are co-localised.
[0060] In the case of cancer immunisation, the immuno-isolated
cells of the invention are possibly chosen among cancerous cells
expressing TAA (tumour associated-antigens) which preferably are
lineage specific and tumour-specific.
[0061] Because the cells of the invention are immuno-isolated, the
produced immunomodulatory agent is preferably soluble in order to
be secreted and released. The same applies for the antigenic agents
like TAA.
[0062] According to a particularly preferred variant, the source of
immunomodulator, i.e. the immuno-isolated cells of the invention
are dissociated from the source of antigenic agent. For example,
according to this variant, a first cell or group of cells
constitutes the antigen source and a second, immuno-isolated cell
or group of cells constitutes the adjuvant source.
[0063] A preferred way to immuno-isolate cells is to provide a
physical barrier "hiding" them from the general immune system. This
goal can be achieved by a barrier device. Different barrier devices
are suitable, in particular microcapsules and macrocapsules. The
actions of encasing a cell or population of cells in such a barrier
are referred to herein as microencapsulation and
macroencapsulation, respectively.
[0064] Immuno-isolation overcomes the significant disadvantages
associated with the implantation of free cells. In fact, the use of
free cells generally requires immuno-suppressing drugs in order to
protect them against the immune system of the host. By mechanically
blocking immune attacks, barrier devices around grafted cells
obviate the need for immunosuppressive therapy. Moreover, the cells
can be retrieved readily after a while, if need be. This last
property allows a switchable release of the immunomodulatory agent.
By retrieving the device, the release of the agent is stopped,
which prevents unwanted presence of a molecule after the end of the
immunization process. Capsules of the invention can be specifically
engineered to facilitate their withdrawal, for example by
incorporation of a string or other means to ease their
retrieval.
[0065] Immuno-isolation also overcomes the significant
disadvantages associated with the use of HLA-negative cells such as
K-562 cell line which fails to express HLA class I or II antigens.
Since the encapsulated cells are entirely protected against the
immune system, they are not destroyed by innate or cellular
immunity, whereas K-562 cells are involved in innate immunity
rejection. The capacity of encapsulated cells to survive, to
secrete protein for a prolonged period of time and to allow
multiple immunizations is directly linked to the physical barrier
of the capsule. Moreover, the amount of GM-CSF release into the
patient after capsule implantation is not likely to differ from one
individual to another depending on his or her innate immunity or
immunosuppression. In contrast, the stability of GM-CSF release is
likely to vary significantly not only in any given patient at the
first and subsequent immunizations but also from one patient to
another, with the injection of GM-CSF secreting K-562 cells.
[0066] The main property of the barrier device is to separate
living cells from the immune system of the host by a synthetic,
selectively permeable, non-immunogenic membrane.
[0067] The cells of the invention are living and preferably provide
the chosen immunomodulatory agent on a long-term basis. For
example, experimental results showed that encapsulated cells,
engineered to secrete GM-CSF, do release GM-CSF outside the capsule
for at least fifteen days. For this purpose, they must be supplied
with all the factors necessary for their survival, their growth and
the production of the immunomodulator of interest. In order to
allow free exchange of nutriments, proteins, oxygen and
biotherapeutic substances between exterior and interior, the device
preferably is selectively permeable. Small molecules can transit
via pores, especially molecules necessary for the survival of the
cells, whereas high-molecular-weight substances such as immunocytes
or antibodies are excluded. It moreover excludes inflammatory cells
and thereby protects the encapsulated cells from tissue
rejection.
[0068] Conversely, immunomodulatory agents produced by the cells of
the invention can be delivered through the pores into the external
medium. The diameter of the pores is preferably chosen in a range
such that small molecules or proteins and immunomodulators are
allowed to cross the barrier and that bigger ones like
immunoglobulins are not, in order for the device to retain its
immuno-protective property.
[0069] Clinical trials have already been performed showing that
such immuno-isolation, using capsules, is very efficient, allowing
the survival of allogeneic but also xenogeic (from other species)
cells for many months without immune rejection. The efficacy of the
physical immuno-isolation by a capsule has been validated by early
clinical trial.
[0070] For the manufacture of microcapsules, a mixture of cells and
sodium alginate are extruded, and solidified into beads generating
a matrix which allows free exchange of proteins, nutriments and
oxygen between encapsulated cells and the host. Advantages of this
device include great surface to volume ratio and ease of
implantation.
[0071] The macroencapsulation makes use of preformed macrocapsules,
initially empty units that are loaded with a matrix and all the
cells needed for treatment. The matrix is preferably a polymer, for
example polyvinyl alcohol or a biopolymer like alginate. The matrix
ensures a good ordering of the cells inside the capsule,
specifically a homogenous distribution, and prevents agglutination
at the walls. Macrocapsules are more durable and rugged than
microcapsules, they contain internal reinforcements, can be tested
for seal integrity before implantation and can be designed to be
refillable in the body. They can also be retrieved simply.
[0072] Preferred polymers for the capsule are thermoplastic
polyethersulfone (PES) hollow fibers (OD:720 .mu.m; ID:524 .mu.m,
molecular weight cut-offs: 32 and 80 kDa; Akzo Nobel Faster AG,
Wupperthal, Germany) and AN-69 polymer (acrylonitrile and sodium
metallysulphonate anionic copolymer, Hospal R&D Int, Meyzieu,
France).
[0073] The macrocapsules can have various sizes ranging from few
micrometers to three to four centimetres. Depending on the size of
the capsule and the size of the cells, as many as 200 000 cells can
be loaded into a 1 cm device.
[0074] Particularly preferred devices of the invention are
microcapsules and macrocapsules. According to the present
application, cells of the invention produce a immunomodulatory
agent. Either the cells naturally produce the agent, or this goal
is achieved by modifying the cells. In a preferred case, cells are
genetically modified to express the immunomodulatory agent. This is
particularly convenient for many reasons.
[0075] By modifying a cell which normally may not produce the
immunomodulatory agent, the use of cells of the invention is not
limited to those naturally producing it. It is particularly
advantageous that the agents are not limited to those naturally
occurring. As it is known that mutated proteins sometimes exert
improved activities, it is very advantageous to use this modified
version of the protein instead of the wild-type one. It is also
sometimes convenient to clone the soluble version of a membranous
protein, in order to achieve its secretion.
[0076] Moreover, by genetically modifying cells of the invention,
it is also possible to control the expression level of the
immunomodulatory agent. A particularly attractive situation is the
overexpression of the agent by cloning its sequence under the
control of a promoter known to be very strong in the used cell. The
modified cells become engineered factories producing high levels of
immunomodulatory agent. The promoter can be chosen according to its
activity such as to have a controlled level of immunomodulator
expression. According to another embodiment, cells may be used
which naturally contain the gene for the immunomodulatory agent,
said gene being transcriptionally silent in that particular cell.
Transcription can be activated by insertion of appropriate
regulatory sequences, for example by homologus recombination.
Inducible regulatory sequences which respond to specific stimuli
such as substances, light, etc. . . . may also be used.
[0077] According to the invention, immunomodulatory agent-secreting
cells secrete more than 10 ng/10.sup.6 cells/24 hr of
immunomodulatory agent. Preferred cells of the invention secrete a
quantity of immunomodulatory agent equal or superior to 100
ng/10.sup.6 cells/24 hr, preferably more than 500 ng/10.sup.6
cells/24 hr. If needed, several capsules may be implanted
simultaneously.
[0078] Consequently, a cell of the invention secretes more than
10.times.10.sup.-15 g of immunomodulatory agent per 24 hr,
preferably more than 100.times.10.sup.-15 g/24 hr of
immunomodulatory agent. A cell of the invention secretes for
example between 80 and 960.times.10.sup.-15 g/24 hr, preferably
more than 500.times.10.sup.-15 g/24 hr of immunomodulatory
agent.
[0079] Concerning the best way to genetically modify the cells of
the invention, methods and protocols are well known by one skilled
in the art. Particularly preferred methods make use of engineered
plasmids introduced by transfection and viruses introduced by
infection. Retroviruses are well suited in the context of the
present invention because they can be engineered to introduce a
gene coding for the immunomodulatory agent into the genome of host
cell.
[0080] As mentioned above, cells of the invention are not limited
to cells naturally producing an immunomodulatory agent of interest.
All sorts of cells can be used, particularly preferred are cells
which are easy to transduce or transfect, and to culture and
propagate. It is not necessary to use tumour cells as the
by-stander immunomodulator producer. Based on the medical
literature, different cell types can been used to produce for
example cytokines. These include immortalized non-tumoral
fibroblasts, myoblasts or tumour cells. Cells of the invention are
advantageously endothelial cells or fibroblasts.
[0081] Particularly preferred cells are in general immortal cell
lines. It is thus possible to genetically modify a cell line just
once and to use cells from it for all applications of the present
invention. Because the cells are immuno-isolated, they are not
endangered by the immune system of the host, but they also do not
endanger the other cells in their vicinity.
[0082] Another particularly interesting advantage is that cells are
not necessary autologous with respect to the host. It is easier to
employ allologeneic or heterologous cells, it can also be
advantageous to use non-human animal cells, without prejudice
thanks to the barrier device. Cells of the invention are preferably
human cells.
[0083] The immuno-isolation of the cells is thus very advantageous
over some prior systems because the sources of immunomodulatory
agent can be considered as "universal" and not limited to a unique
individual.
[0084] Cells of the invention are living. This ensures that the
immunomodulator is continuously produced, for at least several days
and even more if needed. In previous experiments using
encapsulation, encapsulated cells engineered to secrete a protein
have been implanted in patients for weeks or months. This
long-lasting release overcomes the common problem of short
half-life of immunomodulators.
[0085] According to a preferred immunization program, cells of the
invention are implanted for a few days, preferably less than 12
days, for example between 4 and 10 days, for example between 5 to 7
days. Implantation of encapsulated cells for such a short period
will not lead to marked fibrosis induced by the release of the
immunomodulatory agent. Inflammation, at the vaccination site,
around the capsule, will not induce a decrease of cell viability
within the capsule and therefore it will not prevent the production
and release of the immunomodulatory agent in that time frame.
[0086] Preferably, the cell-containing capsule is irradiated, for
example by X-Rays, before implantation. Firstly, this irradiation
ensures that, even if disruption of the capsule occurs, enclosed
cells are not capable of propagation. Secondly, this irradiation
ensures that the secretion of the immunomodulatory agent will stop
after around 10 days, due to irradiation-induced cell death. This
may be advantageous, should the secreted immunomodulatory agent
generate a violent inflammatory response. Specifically,
subcutaneous implantation of GM-CSF-secreting cells, encapsulated
or not, could induce cutaneous necrosis if implanted during a
period exceeding 15 days to 1 month. Irradiation of the capsule or
cells before implantation does not prevent subsequent retrieval of
the capsule.
[0087] In a preferred embodiment, cells of the invention are used
in the context of cancer therapy. This product has been designated
"Onco-Maxi Vax". Onco-Maxi-Vax is a therapeutic product
(therapeutic vaccine) made of two components that are physically in
close proximity during the immunization.
[0088] The first component of the Onco-Maxi-Vax system is the
source of tumour antigens, this antigenic load is made of
irradiated cells harvested from the patient to be treated, this
component is specific for each patient. In order to ensure maximal
antigen exposure the source of antigen is made of each patient's
own tumour cells. These are harvested surgically or endoscopically
from the patient, digested in order to obtain a cell-suspension and
then irradiated at 10000 Rad before storage in aliquots in liquid
nitrogen. Irradiation is a safety measure in order to prevent any
growth of tumour cells that will be re-injected to the patients.
This procedure has already been performed safely. This component of
the Onco-Maxi-Vax system is unique to each patient, as it will be
harvested from each individual and used in combination with the
second component of the vaccine.
[0089] The second component of the Onco-Maxi-Vax vaccine is common
(or "universal") to all patients, it is the immunomodulator
provider. It is composed either of a large capsule
(macro-encapsulation) or small capsules (micro-encapsulation) that
contain living cells. The capsule(s) is (are) required to
immuno-isolate allogeneic human cells. The capsule(s) is (are)
semi-permeable. It allows the survival of the cells inside by
nutrient migrations and it prevents cells exposure to an
environment which would normally destroy them.
[0090] In the Onco-Maxi-Vax system, the cells to be encapsulated
are genetically engineered to secrete immunostimulatory molecules.
It should be seen as an immuno-isolated bio-reactor that produces
and releases, at the site of vaccination, strong immuno-stimulatory
signals. So far Granulocyte-Macrophage Colony-Stimulating Factor
(GM-CSF) is probably the most potent immunostimulatory molecule for
anti-tumor immune response. Because of biochemical properties
GM-CSF needs to be produced locally at the vaccine site in order to
obtain a sustained release of the protein for at least five to
seven days. Initially the encapsulated cells are engineered to
secrete GM-CSF only. Depending on synergistic studies, other
immunomodulatory molecules can be added without difficulty (other
cytokines such as IL-12, IL-15, IL-4, Interferon gamma, chemokines
or dendritic growth factors).
[0091] To maximise safety, the capsule used in macro-encapsulation
is retrievable and the cells that it contains are irradiated prior
to implantation. Previous experiments have shown that irradiation
of GM-CSF producer cells does not prevent the production and the
release of the protein.
[0092] The two components of the Onco-Maxi-Vax vaccine are placed
under the patient's skin in close proximity or in contact. They are
implanted at sites distant from the primary tumour or metastasis in
order to perform the vaccination in an immunologically un-disturbed
location.
[0093] Five to seven days after the implantation, the capsule is
removed, for example using an especially designed string attached
to it. The autologous irradiated tumour cells injected as a
component of the Onco-Maxi-Vax are progressively processed and
removed by the patient's immune system, via naturally occurring
mechanisms.
[0094] The vaccination treatment with Onco-Maxi-Vax comprises
repeated immunizations at two to three week intervals at different
sites, usually sub-cutaneous. The total number of vaccinations,
although adjustable is preferably a minimum of five.
[0095] The dose of autologous cells is adjusted to the amount of
cells harvested from the patients. It is recommended to have around
10.sup.7 to 10.sup.8 cells per immunization. When this dosage
cannot be repeated 5 times, the dose of autologous tumour cells is
reduced accordingly.
[0096] The ideal number of cells to be encapsulated is dependent on
the amount of immunomodulatory agent such as GM-CSF that is
required at the vaccination site. In previous studies, GM-CSF
producing autologous cells were releasing between 80 to 960
ng/10.sup.6 cells/24 hr. Release between 500 to 1000 ng/24 hr is
considered a reasonable goal.
[0097] According to the invention, immunomodulatory agent such as
GM-CSF-secreting cells secrete more than 10 ng/10.sup.6 cells/24 hr
of immunomodulatory agent. Preferred cells of the invention secrete
a quantity of GM-CSF equal or superior to 100 ng/10.sup.6 cells/24
hr, preferably more than 500 ng/10.sup.6 cells/24 hr. If needed,
several capsules may be implanted simultaneously. The total
quantity of delivered immunomodulatory agent, especially GM-CSF,
must be at least 1 microgram per 24 hours at the vaccination
site.
[0098] Consequently, a cell of the invention secretes more than
10.times.10.sup.-15 g of immunomodulatory agent per 24 hr,
preferably more than 100.times.10.sup.5 g/24 hr of immunomodulatory
agent. A cell of the invention secretes for example between 80 and
960.times.10.sup.-15 g/24 hr, preferably more than
500.times.10.sup.-15 g/24 hr of immunomodulatory agent.
[0099] Capsule according to the invention may contain between
2.times.10.sup.5 cells and 2, 3, 4 or 5.times.10.sup.7 cells,
preferably more than 10.sup.7 cells per capsule, for example
2.times.10.sup.7 cells per capsule.
[0100] This anti-tumour immunization is performed for a wide range
of tumour types. As discussed above, the encapsulated cells are
identical for every patient. Only in patients with tumour cells
that can be harvested from a solid primary tumour, a metastasis or
from fluid containing tumour cells (pleural, peritoneal, bone
marrow or blood) is it possible to generate the full vaccine
product.
[0101] In clinical oncology, most of the patients present a primary
tumour or metastatic lesion. Not all tumours or metastases are
equally easy to process. Therefore the type of tumour that will be
tested needs to fulfil various criteria in terms of feasibility.
Cancers that are likely to have a primary tumour that can be
harvested are: [0102] Central Nervous System tumour such as
Glioblastoma [0103] Lung tumour (non-small cell lung cancer) [0104]
Prostate tumour [0105] Gastric carcinoma [0106] Breast carcinoma
[0107] Lymphoma [0108] Pancreatic carcinoma [0109] Hepatocarcinoma
(Liver tumour) [0110] Colon carcinoma [0111] Renal cell carcinoma
[0112] Ovarian carcinoma [0113] Uterine carcinoma [0114] Sarcoma
(soft tissue) [0115] Leukemia (lymph node or blood) [0116] Multiple
myeloma (blood, bone marrow, lymph-node, soft tissue)
[0117] Cancers that are likely to have metastases that can be
harvested are dependant on the location of the metastasis. For
technical reasons it is more difficult to harvest bone metastases
than other localizations. These cancers may be for example: [0118]
Head and neck carcinoma (lymph-node metastasis) [0119] Lung cancer
(lung, liver, soft tissue, brain, adrenal metastasis) [0120]
Prostate (non-bone metastasis) [0121] Breast carcinoma (lung,
liver, soft tissue, pleural fluid) [0122] Gastric carcinoma (liver)
[0123] Pancreatic carcinoma (liver) [0124] Colon carcinoma (liver)
[0125] Melanoma (lung, lymph-node, soft tissue, liver, brain)
[0126] Renal Cell carcinoma (lung, liver) [0127] Ovarian carcinoma
(peritoneal or pleural fluid, liver) [0128] Germinal tumors (lung)
[0129] Bladder carcinoma (liver, lymph-node)
[0130] In addition to these requirements, patients to be enrolled
in a phase I study must have failed or refused standard treatment
for their cancer type and their tumour stage.
[0131] Regarding this aspect, the following tumours could be
preferably treated with Phase 1 Onco-Maxi-Vax after the specified
treatment:
CNS tumour (glioblastoma): Relapse after surgery
+/-radiochemotherapy;
Head and neck tumor: Metastatic disease after surgery
+/-radiotherapy;
Lung cancer: NSCLC/Mesothelioma: First line metastatic disease or
after progression post chemotherapy;
Small cell lung cancer: In metastatic patients after first line of
chemotherapy for either localized or extensive disease;
Breast/Ovarian cancer: After two lines of chemotherapy for
metastatic disease;
Esophagus/Gastric/Colon carcinoma/Bladder, Uterine cancer:
Metastatic, progressing after one line of chemotherapy;
Pancreatic/Hepato-cellular cancer: Post-surgery when incomplete or
with loco-regional or distant metastasis;
Renal cell carcinoma/Melanoma: Metastatic disease first line;
Lymphoma/Multiple myeloma/Leukemia: Progression despite multiple
chemotherapy regimen in advance disease, not treatable with
currently available recognized therapy (autologous bone marrow
re-infusion, antibody);
Sarcoma, germinal tumors: Progressive disease after multiple
chemotherapy regimens;
Prostate cancer: after failure of hormone therapy and when
metastasis can be harvested (liver, lymph-node, others.)
[0132] In order to evaluate potential response using classical
radiological criteria, patients must have measurable disease or
evaluable disease. Follow-up period is until progression of
disease. Follow-up includes radiological studies mainly with CT
scan to record any change in tumour volume.
[0133] Toxicity and feasibility are recorded during the
immunization period and also during the follow-up period by
bi-weekly visit at the Oncology center and evaluation by an
oncologist.
[0134] Serological tumour markers such as CA 153, CA 19-9, CEA,
AFP, NSE, CA 125, are monitored when elevated prior to
vaccination.
[0135] Whenever possible surgical resection of metastasis are
attempted in order to document any changes in tumour structure. It
is well described that classical tumour evaluation by
bi-dimensional measurement may not be the best evaluation method to
assess potential efficacy of immunization treatment. Destruction of
the tumour cells can be very efficient and replaced with fibrous or
inflammatory cells without detectable changes in size on
radiological examination. Metabolic activity as assess by PET scan
may be of relevance in this setting. The analysis of post
immunization tumour lesion is of great interest for immunological
analysis such as the characterization of the potential tumour
antigen targeted by the treatment.
[0136] In another preferred embodiment, cells of the invention are
used in the context of immunisation against various infectious
diseases. This product of the invention can thus be named "IA-Maxi
Vax" (infectious agent).
[0137] Similarly to the Onco-Maxi-vax approach "IA-Maxi-Vax" is a
therapeutic product (vaccine) made of two components that are
physically in close proximity during the immunization.
[0138] One component represents the source of antigen(s). For this
anti-infectious vaccination the antigen comprises one or more
components from the infectious agents. Therefore all patients with
a specific infection will be treated with the same product. Many
known antigen components have been described in infectious diseases
caused by viral, bacterial or parasitic pathogens and are used
currently for immunization strategies. These antigens can be used
as inactivated pathogens, infectious agent's lysates, protein
extracts, recombinant proteins, peptides, DNA or other forms. In
some conditions depending on the infectious agent or the host
medical condition immunization is weak or non-protective leading to
significant morbidity or mortality.
[0139] HIV is an example of failure to naturally eradicate a viral
pathogen.
[0140] Immunosuppression after bone marrow transplantation is an
example where the usually benign CMV infection can be
life-threatening.
[0141] The first component of the vaccine comprises a combination
of the antigen or a pool of antigens. Encapsulated, bystander cells
of the invention that release locally, at the vaccination site, a
very potent immunomodulatory signal constitute the second component
of the vaccine.
[0142] The second component of the vaccine is the same
("universal") for all patients. It is made of a semi-permeable
capsule (macro or micro) that contains live cells. The capsule is
required to immuno-isolate allogeneic human cells. It allows the
survival of the cells inside by nutrients migrations and it
prevents cells exposure to the environment that would normally
destroy them.
[0143] The cells to be encapsulated are genetically engineered to
secrete immunostimulatory molecules. So far Granulocyte-Macrophage
Colony-Stimulating Factor (GM-CSF) is probably one of the most
potent immunostimulatory molecule for boosting the immune response.
GM-CSF has been shown to increase immunity against various
parasitic or viral agents. Because of bio-chemical properties
GM-CSF needs to be produced locally at the vaccine site in order to
obtain a sustained release of the protein for at least five to
seven days. Initially the encapsulated cells are engineered to
secrete GM-CSF only. Depending on synergistic studies, other
immunomodulatory molecules could easily be added (other cytokine
such as IL-12, IL-15, IL-4, Interferon gamma, chemokines or
dendritic cells growth factors)
[0144] To maximize safety, the capsule is retrievable and the cells
that it contains are irradiated prior to implantation. Previous
experiment have shown that irradiation of GM-CSF producer cells
does not prevent the production and the release of the protein.
[0145] The two components of the infectious-agent vaccine need to
be placed under the patients skin in close proximity or contact. It
may be beneficial to have sequential administration of the two
components, the capsule being implanted first, followed by the
antigenic stimulus.
[0146] Five to seven days after the implantation the capsule is
removed, for example using an especially designed string attached
to it.
[0147] The vaccination treatment with "IA-Maxi-Vax" comprises
repeated immunizations at two to three week intervals at different
sites, always sub-cutaneous. The total number of vaccination is
dependant of the protocol and dosages and must be adjusted in each
particular case.
The "IA-Maxi-Vax" addresses non-exhaustively the following
conditions:
[0148] Viral infections: [0149] Target: HIV patients at various
stage of their disease (early stage may be better candidates, with
stronger immune system) [0150] Target: CMV infection in specific
condition (pre or post organ transplantation) [0151] Target:
recurrent herpes infection [0152] Hepatitis B or Epstein Bar virus
[0153] Hepatitis C
[0154] Bacterial infections such as:
[0155] Target: Mycobacterial infection is specific population such
as HIV patients [0156] Target Elicobacter pylori. E. Pylori is the
causative agent in the majority of the stomach ulcer or
gastritis.
[0157] Parasitic infections such as [0158] Malaria, Toxoplasma,
Pneumocystis.
[0159] According to a second aspect, the present invention relates
to pharmaceutical compositions, vaccines and kits comprising cells
which produce an immunomodulatory agent and which are physically
immuno-isolated.
[0160] All these products are particularly well suited for
industrial production because they are easy to produce in large
amount thanks to the `universal` characteristic of the device. As
the cells are immuno-isolated, the same encapsulated cells are
suitable for all the patients. This `universal` characteristic is
particularly interesting for cancer therapy where multiple
injections of the vaccine or the pharmaceutical composition are
required.
[0161] A pharmaceutical composition according to the present
invention comprises immuno-isolated cells producing an
immunomodulator combined with a physiologically acceptable carrier.
The density of cells of the invention to incorporate in the
composition must be defined for each different case. In particular,
the density of cells to incorporate is function of the
immunomodulator dose required. Examples of suitable physiologically
acceptable carriers would consist of a device that would fulfil the
following needs: non-toxic (local and systemic), efficient
immuno-isolation, permeable allowing sustained release of the
immuno-modulator. A macro or microcapsule made of PES or AH-69
fulfil these requirements as well as the products developed by
Theracytes Inc.
[0162] A vaccine composition of the present invention comprises
immuno-isolated cells producing an immunomodulator combined with an
antigenic component. This second component of the vaccine
represents the molecule against which an immunization reaction is
expected. The first one secretes the immunomodulator necessary for
enhancing the immunization process. The antigenic component can be
represented by a protein, for example a viral protein which is
known to be immunogenic, or by a whole cell expressing antigens at
its surface, or by an extract containing several antigenic
substances. In the context of cancer, the antigenic component is
preferably a tumour cell (therapeutic vaccine). Tumour cells are
known to express at their surface tumor-associated antigens against
which an immune response is desired.
[0163] When tumour cells are used, as in the cancer context, the
tumour cells are preferably irradiated before incorporation into a
composition. Irradiation is a safety measure in order to prevent
any growth of tumour cells that will be re-injected to the
patients. In addition to safety, irradiation may be a very good way
to allow the release of potentially useful antigenic
determinants.
[0164] In the context of vaccination against infectious agents, the
antigenic component is preferably from the agent against which an
immune response is desired, for example from a virus. Preferred
viruses are Hepatitis C and HIV.
[0165] The present invention also provides kits comprising
immuno-isolated cells producing an immunomodulator combined with an
antigenic component. The first component of the kit secretes the
immunomodulator necessary for enhancing the immunization process
against the second antigenic component.
[0166] As already mentioned for the vaccine composition, the
antigenic component of a kit according to the present invention
preferably comprises a cell producing, secreting or releasing an
antigen. Alternatively, the antigenic component can be a molecule,
for example a protein, or a cellular extract.
[0167] In the context of cancer, the antigenic component of the kit
is preferably a tumour cell. The same safety measures are
preferably taken by irradiating the tumour cell before use.
[0168] According to the present invention, the kit will generally
be used for implantation into human body. For this reason, many
constraints are imposed on the properties of the kit. In
particular, this kit must be designed to be as small as possible.
It must also be biocompatible. A kit according to the invention can
be placed for a time as long as several weeks or months. The kit
must be safe throughout the duration of this period. In particular,
in numerous uses of the kit, the encapsulation device needs to be
sealed.
[0169] According to a third aspect of the present application, the
invention encompasses uses of immuno-isolated cells producing an
immunomodulatory agent, and uses of pharmaceutical compositions,
vaccines and kits as described above in the therapeutic domain as
well as in the vaccination field.
[0170] The invention relates in particular to a process for
vaccinating a subject in need of such a treatment. This process
comprises the administration of a composition comprising cells of
the invention. The subject is preferably a human patient, but
animals are also possible. A subject can be in need of vaccination
for different reasons, because a preventive immunization is
preferable, like for benign disease, or is necessary for example in
the case of severe epidemics.
[0171] In a preferred case of vaccination, the immune response is
raised against a particular antigenic component. In this
predominant case, the process of the invention comprises also the
step of administrating the antigenic component to the subject.
[0172] A patient can be preventively vaccinated because he will be
in contact with the antigenic component sooner or later. He can
also be therapeutically immunized by the process of the invention
because he is already in contact with the antigenic component but
not able to generate an adequate immune response by himself.
[0173] When the process comprises the administration of cells of
the invention and of an antigenic component, different protocols
are envisaged. Both administrations can be made simultaneously, or
they can be made separately, or sequentially. It can be very
advantageous to temporally dissociate the administrations. In fact,
cells of the invention, thanks to their barrier device, have a
long-lasting effect. On the contrary, antigenic components injected
are likely to be processed and eliminated very rapidly by the host
immune system. In such a case, when administrations are
dissociated, the administration of antigenic component can be
repeated whereas there is a single administration of cells of the
invention.
[0174] The antigenic and the immunomodulatory agents should
preferably be co-localised in order to produced an optimised
effect.
[0175] Preferably, cells of the invention are irradiated before
administration. When the immuno-isolation consists in a capsule, it
is preferred to irradiate the capsule. This irradiation step is
preferably done shortly prior administration, but may occur a few
days before.
[0176] Preferably, for an optimised immunization process, the
administration is repeated several times, preferably the
administrations of cells of the invention as well as antigenic
component are repeated. In a preferred case, the administration is
repeated more than twice, preferably between 3 and 6 times,
preferably 5 times.
[0177] It is crucial that the repeated immunisations must not
generate an increased immune response against the immunomodulator
or its means of delivery, at the risk of annulling the benefit of
the repeated immunisations or generating a dangerous immune
response. The immuno-isolation device of the cells producing the
immunomodulator, as proposed in the present application, avoids
these problems. It represents a technical advantage over the
systems developed in the prior art.
[0178] As discussed above, when the vaccinating process of the
invention is used in the field of cancer, the antigenic component
is preferably a whole tumour cell, advantageously irradiated.
Because it is known that some antigens are present on many tumours
from the same lineage, the used tumour cells may be an allogeneic
ones. However, the tumour cells are preferably autologous with
respect to the patient.
[0179] Preferably, the tumour cells, which are the source of
antigenic component, and the immuno-isolated cells, which are the
source of immunomodulatory component, are distinct. This situation
is advantageous over the prior systems coupling both, because the
tumour cells need not be manipulated, except the harvest and
irradiation steps, contrary to prior solutions requiring genetic
manipulation of the tumour cells.
[0180] The administration mode of cells of the invention, according
to the present process, is chosen to be the most efficient. In
particular, the mode is adapted to the desired localisation for the
cells and the antigenic component. A preferred localisation is
sub-cutaneous because this region is rich in dendritic cells. The
administration can be made intradermally. Other localisations
likely to favour the expected immune response are also
preferred.
[0181] According to the invention, the immunomodulatory secreting
cells are loaded in a capsule, which is to be implanted. In a
preferred embodiment, the capsule is removed after 2 to 7 days,
preferably after 5 to 7 days.
[0182] The invention also relates to a process for treating a
patient suffering from cancer. Particularly well suited cancers and
states are described in detail above. This process comprises the
administration of a composition containing immuno-isolated cells
producing an immunomodulatory agent. According to the present
invention, the immuno-isolated cells may be autologous or
allogeneic, in particular, they may be autologous tumour cells.
[0183] The administered composition may be injected, ingested,
implanted, applied, or any other administration means. The
composition may comprises any pharmaceutical additive necessary for
the survival of the cells and for the success of the administration
or implantation. The composition may also contain an antigenic
component which is generally irradiated whole tumour cells. It is
preferably administered at a site distant from the tumour location,
where no previous immune response is supposed to have taken place.
However, the composition may be administrated in the vicinity of
tumour cells of the patient to be treated.
[0184] In the context of the present invention, immuno-isolated
cells, producing or secreting an immunomodulatory agent, as
described above, are used in therapy, particularly in cancer
therapy. These cells are also possibly used in the field of
vaccination.
[0185] Another use which is perfectly adapted to the cells of the
invention, is in the manufacture of an adjuvant. In this particular
case, the function of the adjuvant is to enhance an immune response
which is considered too weak. If so, the immunomodulatory agent
produced by the immuno-isolated cells of the invention is an
immunostimulator.
[0186] Another use is in the manufacture of a medicament for the
prevention or the treatment of cancer. The characteristics of such
an application are already well documented above.
[0187] Kits comprising cells of the invention are described above.
The present application also envisages the use of at least one of
these kits for the manufacture of a vaccine.
EXAMPLES
Example 1
Testing the Secretion of Human GM-CSF Release by Various Cells
Lines after Transfection
[0188] This example shows the ability of different cell lines to
secrete human GM-CSF with and without being irradiated.
Transfection Methods:
[0189] Human GM-CSF cDNA was cloned into a retroviral vector (MFG)
as described in the literature (Danos Olivier and Mulligan Richard
1988 PNAS Vol. 85 p6460-64; Jaffe Elizabeth et al. 1993 Cancer
research Vol. 53 p2221-26).
[0190] The human GM-CSF is inserted in-frame into the retroviral
vector. The MFG-hGM-CSF construct was sequenced in order to ensure
correct in-frame cloning.
[0191] Using transient transfection technique with lipofectamine
MFG-hGM-CSF construct was transfected into 293-gpg cells as
described in the literature (Ory Daniel et al. 1996 PNAS Vol. 93 p
11400-06). With adequate selection, these cells produce pseudotyped
retroviral particle containing the hGM-CSF gene. These viral
particles are replication defective but can infect a wide range of
mammalian cells.
[0192] The supernatant of transfected 293-gpg cells is used to
infect dividing cells. This is performed with polybrene and no
selection is performed. Therefore the whole cell population is used
for subsequent analysis.
Tests for Secretion:
[0193] The infected cells are then tested for their ability to
secrete human GM-CSF.
[0194] A classical ELISA is performed to measure the amount of
GM-CSF in the supernatant of the various cell type tested.
[0195] Murine and human cell lines were tested for their ability to
secrete human GM-CSF at various time points and also after
irradiation 3500 rads. The different tested cell lines are the
following:
[0196] Renca: murine renal cancer cell line from Balb/c
background
[0197] B16 F10: melanoma cell line from C57BL/6 background
[0198] Cell line A: human tumorogenic fibroblasts
[0199] Cell line B: human immortalized fibroblasts
[0200] 10.sup.6 cells are plated on a culture dish. After 48 hours
the supernatant is harvested and 0.2 u filtered and frozen at
-20.degree. C. For Elisa the antibodies and the protein standards
are purchased at R&D system.
[0201] Non-transfected cells are also tested as negative
control.
Results:
[0202] The Results are expressed in ng of h-GM-CSF by 10.sup.6
cells at 48 hours TABLE-US-00001 no irradiation irradiation Renca
wt 0 0 B16-wt 0 0 Renca h-GM-CSF 23400 9300 B16-h-GM-CSF 37650 Cell
line A 0.9 Cell line A h-GM-CSF 18600 14900 Cell line B 1.2 Cell
line B h-GM-CSF 6300 2950 Media only 0 0
[0203] Murine cancer cell lines and human fibroblast cell lines are
able to secrete large amount of human GM-CSF for prolonged period
of time in-vitro. No decrease has been observed in the production
with time (at least three weeks after infection, for non-irradiated
cells).
[0204] Altogether these experimental data show that human
fibroblasts can secrete high level of human GM-CSF for several days
without obvious toxicities to the cells. They are therefore good
candidates for the clinical application as suggested in the
Maxi-Vax approach; that is encapsulated, immunoprotected,
allogeneic cells releasing immunomodulator at the vaccination
site.
Example 2
Experimental Protocol Assessing the Efficacy of Onco-Maxi-Vax.
Autologous Irradiated Tumour Cells+Encapsulated GM-Producers Cells.
Pre-Clinical Development in Mouse Model
[0205] This example concerns the reproduction, using Onco-Maxi Vax,
of the vaccination efficacy observed in the classical setting, when
GM-CSF is produced by the irradiated tumour cells, in both
wild-type mice and GM-CSF deficient mice. It also enables
documentation of any new toxicity related to the use of the
capsule, its manipulation or the cells it contains.
[0206] Furthermore, characterization of the response by standard
techniques (Histology of vaccine site, cytokines profile, dendritic
cell staining at the vaccination site) is carried out.
Experimental Design
A) Measurement of In-Vitro Release of Murine GM-CSF from the
Capsule Containing GM-CSF Secreting Cells.
[0207] This is performed with standard monoclonal antibodies
against murine GM-CSF in Enzyme-Linked immunoabsorbent Assays
(ELISA). (R&D systems). The amounts of protein released as well
as the reproducibility from one capsule to the others are assessed
and results are shown in FIG. 3.
B) Direct Comparison of the Two Immunization Procedures:
The negative control group: Vaccination with irradiated,
un-manipulated wild-type B16 melanoma cells (B16 WT).
The positive control group: The standard technique used in the
laboratory: Vaccination with irradiated, GM-CSF secreting B16
melanoma cells. (B16-GM)
The investigational group: Vaccination with irradiated,
un-manipulated B16 melanoma cells (B16 WT) in close contact with a
sub-cutaneoulsy implanted capsule containing cells releasing murine
GM-CSF.
[0208] The vaccine combines irradiated wild type B16 melanoma cells
injected in close contact to macrocapsule made of PES
(polyethersulfone). This capsule contains 200 000 Renca cells
retrovirally engineered to secrete GM-CSF. The encapsulated cells
are mixed with a collagen-based matrix.
[0209] The B16 GM and the Renca GM cells are generated using the
same transfection technique. Briefly, the murine GM-CSF cDNA was
inserted into the MFG retroviral vector. Retroviral particles
containing the mGM-CSF cDNA were obtained after lipofectamine
transfection of 293-CPG cells. This infectious, non-replicative
retroviral particles were harvested, centrifuged, concentrated and
used to infect B16 and the Renca cells respectively. The amount of
GM-CSF released by the B16-GM and the Renca-GM is measured by
standard Elisa technique.
[0210] Three additional groups to study the potential effect of the
capsule itself. [0211] a) The positive control group+capsule
containing non-secreting cells, to test if the capsule decrease the
effect. [0212] b) The negative control group+capsule containing
non-secreting cells, to test if the capsule have an effect by
itself, without GM-CSF. [0213] c) Vaccination with encapsulated GM
secreting cells only, to test if the GM-CSF+allogeneic cells have
any effect.
[0214] These three additional groups ensure that the effect
observed with the investigational arm is GM-CSF dependant and
tumour specific.
Protocol
Mice: C57Bl/6 strain at least 8 weeks of age
[0215] Cells: Culture in DMEM media with 10% inactivated calf
serum+penicillin and streptomycin. Harvest from cell culture plates
is performed one to two hours before injection. B16 WT and B16 GM
adherent cells are washed with PBS .times.1, detached with Trypsin
EDTA (Life tech) then washed .times.3 in HBSS. Cells are then
counted and resuspended in HBSS at the described concentration.
Renca GM and Renca Wt are the cells loaded into the capsules. These
cells are cultured in the same DMEM media as above.
Day 0: Vaccination:
[0216] B16-WT or B16 GM are harvested, resuspended at the
concentration of 2.times.10.sup.6/ml and irradiated at 3500 rad.
Capsules are implanted subcutaneously on the abdomen in the
appropriate groups after irradiation (3500 rad).
[0217] Vaccination is performed sub-cutaneously with
1.times.10.sup.6 cells into 500 ul on the abdomen. Groups with the
capsule and the B16 cells co-injection, the cells are injected in
close contact with the capsule. Superficial anesthesia is used to
ensure reproducibility of the procedure.
Day 7: Challenge:
[0218] B16 WT are harvested from culture dishes and resuspended at
a concentration of 1.times.10.sup.6/ml.
[0219] Injection of 5.times.10.sup.5 cells in 500 ul is performed
on the upper-back.
[0220] Superficial anesthesia is used to ensure reproducibility of
the procedure.
Follow-Up.
[0221] Animals are checked daily for tumour growth. Day of visible
tumour is recorded. Animal are sacrificed when tumour is larger
then 1 cm or ulcerates. Day of sacrifice is recorded. Tumour free
animals are observed until day 80 for potential late tumour
growth.
[0222] This model has been well described in the past and the mice
that do not develop tumour at the sub-cutaneous challenge site will
not develop distant metastasis.
[0223] Mice free of tumour at day 80 have long-lasting specific
anti-tumour immunity as long as the control groups gave the results
that allow validation of the experiments.
Preliminary Results:
[0224] Toxicity evaluation of the prolonged GM-CSF release by the
encapsulated cells and of the capsule by itself under the skin.
[0225] This is assessed by the analysis of mice with implanted
capsule containing increasing numbers of irradiated GM secreting
cells. The effect of empty capsule is also analysed. This is
performed by observation of the animal for any local or systemic
toxicity. Serum level of GM-CSF are assessed by ELISA. Histological
analysis is performed on the vaccination site. This toxicity
evaluation is performed with 2 mice per group.
Example 3
Protocol for the Preparation of "Onco-Maxi-Vax" for Use in
Humans
[0226] This example concerns the preparation of Onco-Maxi Vax, for
the vaccination of a human patient. The protocol gives detailed
information regarding the preparation of the antigenic load, the
generation of the immuno-isolated cytokine provider and the
immunization with the two components from the Onco-Maxi-Vax.
[0227] Every step of the vaccine preparation for the two components
(irradiated autologous cells and encapsulated GM-CSF producing
cells) and the immunizations are performed according to clinical
GMP guidelines.
1) Harvest of Autologous Tumor Cells (Antigenic Load)
[0228] A tumour mass (primary lesion or metastasis) from the
patient to be treated is surgically harvested. An standard
pathological examination is performed on a portion of the mass in
order to confirm the malignant nature of the harvested material. It
is then processed in order to obtain a single cell suspension. This
is performed by both mechanical and enzymatic methods.
[0229] The tumour mass is first cut in smaller pieces using
dissecting microscope, then the tumor is put into a sterile bag
with a sterile solution containing various enzymes (collagenase).
The bag is inserted into a cell blender (Stomacher Lab System) that
will process the product into a cell suspension. The combination of
enzymatic and mechanical activities at 37.degree. C. for few hours
allows the efficient dissociation of the extra-cellular matrix of
the tumour and turn it into single cell suspension. This is
performed in serum free solution.
[0230] The cells are then washed three time with HBSS using a
refrigerated centrifuge (Sorvall) 4.degree. C., 5 minutes, 700 rpm,
and resuspended in HBSS. Cells are then counted using Trypan blue
(Fluka) solution and a Neubauer chamber.
[0231] The cells are resuspended at a chosen concentration,
irradiated at 10000 rads in an irradiator devoted for clinical use,
aliquoted and frozen in freezing media containing 10% DMSO.
2) Immuno-Isolated Cytokine Provider
[0232] a) Generation of GM-CSF Producing Cells.
[0233] The cells to be introduced into the capsules are allogeneic
(obtained from a human cell line). In order to prevent un-predicted
toxicity, we use cell lines that have already been approved in
clinical protocols such as immortalized fibroblasts or myoblasts.
These cells are first stably transfected with human GM-CSF
cDNA.
[0234] Two methods of transfection can be used: retroviral and
electroporation. For retroviral transfection, hGM-CSF cDNA is
inserted in-frame into the MFG retroviral vector and transcription
is driven by the LTR of the virus. The plasmid does not contain any
selection marker or antibiotic resistance gene.
[0235] For the transfection by electroporation, hGM-CSF cDNA is
under the CMV promoter and the plasmid contains a selective marker
(such as an antibiotic resistance gene).
[0236] The invention therefore includes the use of different types
of cells for transfection and of different GM-CSF plasmids. This
leaves more flexibility with respects to local health department
regulations.
[0237] The cytokine producing cells is cultured in serum free media
at 37.degree. C. with 5% CO2 using standard techniques. Harvesting
is performed as follow: The supernatant of confluent, adherent
cells in a 10 cm culture plate is removed and the cells are washed
once with 5 ml of autoclaved Phosphate buffered Saline (PBS) for 5
minutes at 37.degree. C. PBS is then removed and 2 ml of
Trypsin-EDTA 0.5% (Life Technologies N.degree.25300054) is added
and the cells are incubated for four minutes at 37.degree. C. The
trypsin/EDTA allows the detachment of the adherent tumor cells. The
cells are then harvested with a 2 ml pipet and diluted into 5 ml of
Hank's balanced salt solution (HBSS Life Technologies
N.degree.24020091). The cells are washed three time with HBSS using
a refrigerated centrifuge (Sorvall) 4.degree. C., 5 minutes, 700
rpm) and resuspended in HBSS. Cells are then counted using Trypan
blue (Fluka) solution and a neubauer chamber.
[0238] The quantity of hGM-CSF produced and secreted by the cells
is evaluated Elisa (R&D system and Pharmingen kits) on filtered
cell's supernatant. This analysis allows the selection of the best
cytokine producing cell-line.
[0239] b) Immuno-Isolation of Cytokine Producing Cells
[0240] In order to ensure sustained release of cytokine by
allogeneic cells and allow repeated immunization it is necessary to
immuno-isolate the cytokine producing cells from the recipient's
immune system. This is performed by either macro or
micro-encapsulation.
[0241] The cytokine producing cells are loaded into macrocapsules
or embedded in microcapsules. The capsules can be made of various
polymers with various sizes and pores, such as PES and TF10/10
capsules with and without PVA (polyvinyl alcohol) matrix. The
capsule is loaded with the cell suspension at a rate of 10.5
ul/min. Sealing of the capsule is obtained by polymer glue, but can
also be done by heating or surgical clips. Analysis from
supernatant of encapsulated cells containing GM-CSF secreting cells
showed that a stable, continuous release of GM-CSF is achieved for
at least fifteen days after loading, with cytokine levels that are
around 70 ng/10.sup.5 cells/24 hrs.
3) Immunization:
[0242] Immunization with Onco-Maxi-Vax requires the sub-cutaneous
injection in close contact of the two components of the
vaccine.
[0243] The capsule containing the cytokine producing cells is
placed in the sub-cutaneous tissue using a small skin incision
under local anaesthesia. The skin is closed with surgical tape.
[0244] The irradiated tumor cells from the patient (=antigenic
load) are thawed, washed two time with 0.9% NaCl, sterile solution
and then injected, sub-cutaneously, in very close vicinity to the
capsule, using a 24 gauge needle.
[0245] Vaccination is repeated every two weeks four times (and more
if enough autologous tumour cells are available). The site of
vaccination is different at each immunization (abdominal wall,
upper arms, thighs, thorax, etc).
Example 4
Onco-Maxi-Vax. Autologous Irradiated Tumour Cells+Encapsulated
GM-Producers Cells. In Vivo Results in Mouse Model Showing Survival
at 50 Days after B16 Wt Challenge
[0246] This example concerns in vivo data in mice, which are
protected from death induced by the melanoma tumor cell line
B16.
Protocol
Mice: C57Bl/6 strain at least 8 weeks of age
[0247] Cells: Culture in DMEM media with 10% inactivated calf
serum+penicillin and streptomycin. Harvest from cell culture plates
is performed one to two hours before injection. B16 WT and B16 GM
adherent cells are washed with PBS .times.1, detached with Trypsin
EDTA (Life tech) then washed .times.3 in HBSS. Cells are then
counted and resuspended in HBSS at 2.times.10.sup.6 cells/ml for
vaccination and 4.times.10.sup.5 cells/ml for tumor challenge.
[0248] Renca GM (GM-CSF secreting Renca cells) are the cells loaded
into the capsules. The capsules were PES capsules with PVA
(polyvinyl alcohol) matrix. These cells are cultured in the same
DMEM media as above.
[0249] Three groups of 5 mice C57BL/6 are immunized.
Group 1 (Negative control group): Mice are treated with irradiated,
encapsulated GM-CSF secreting Renca cells only (no B16 melanoma
cells).
Group 2 (Study group). Mice are immunized with the same irradiated
encapsulated Renca-GM-CSF cells plus irradiated B16 wt melanoma
cells.
Group 3 (Positive control group). Mice are immunized with
irradiated, non encapsulated B16 melanoma cells engineered to
secrete GM-CSF.
[0250] Mice were immunized with either irradiated capsule only
(group 1), capsule+irradiated B16 wt (group 2) or irradiated
B16-GM-CSF (group 3) on day-7. Each capsule containing 10.sup.5
GM-CSF secreting Renca cells was irradiated (3500 rad) prior to
implantation. Mice from groups 1 and 2 were implanted with 2
capsules each, put sub-cutaneously in a V-shape on the abdomen.
After 3 hours, the groups 2 and 3 were injected with irradiated B16
cells, 10.sup.6 B16 wt cells for group 2, 10.sup.6 B16-GM-CSF cells
for group 3, sub-cutaneously between the two capsules.
[0251] On day 0, all the mice were challenged with live B16 wt
2.times.10.sup.5 cells on the upper back. Mice with growing tumor
superior to 1 cm or showing tumor ulceration were sacrificed. All
these were tumor-related. Non-sacrificed mice remained tumor-free.
Survival represents percentage of tumor-free mice in each
group.
[0252] This animal experiment shows a very good efficacy of
encapsulated GM-CSF secreting cells on survival at 50 days (see
FIG. 4).
[0253] This experiment was repeated and the results are shown in
FIG. 5 with a different graphical representation. This second
experiment also illustrates the efficiency of vaccination with
encapsulated GM-CSF secreting cells on survival over more than two
months.
* * * * *