U.S. patent application number 10/980384 was filed with the patent office on 2005-06-16 for microparticles supporting cells and active substances.
This patent application is currently assigned to INSERM. Invention is credited to Benoit, Jean-Pierre, Menei, Philippe, Montero-Menei, Claudia, Tatard, Valerie, Venier, Marie-Claire.
Application Number | 20050129776 10/980384 |
Document ID | / |
Family ID | 34655185 |
Filed Date | 2005-06-16 |
United States Patent
Application |
20050129776 |
Kind Code |
A1 |
Montero-Menei, Claudia ; et
al. |
June 16, 2005 |
Microparticles supporting cells and active substances
Abstract
Microparticles including a biodegradable, biocompatible material
having at least a portion of surface adapted to adhere to cells of
interest or fragments thereof; and at least one substance active on
the cells or their environment upon implantation of the
microparticles in a patient associated with the material wherein
the substances is released in a controlled and/or extended
manner.
Inventors: |
Montero-Menei, Claudia;
(Angers, FR) ; Menei, Philippe; (Angers, FR)
; Benoit, Jean-Pierre; (Avrille, FR) ; Tatard,
Valerie; (Angers, FR) ; Venier, Marie-Claire;
(Juigne-sur-Loire, FR) |
Correspondence
Address: |
IP GROUP OF DLA PIPER RUDNICK GRAY CARY US LLP
1650 MARKET ST
SUITE 4900
PHILADELPHIA
PA
19103
US
|
Assignee: |
INSERM
Paris Cedex
FR
|
Family ID: |
34655185 |
Appl. No.: |
10/980384 |
Filed: |
November 3, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10980384 |
Nov 3, 2004 |
|
|
|
PCT/FR03/01377 |
May 2, 2003 |
|
|
|
Current U.S.
Class: |
424/489 ;
424/85.1; 514/1.2; 514/19.3; 514/44R; 514/8.4 |
Current CPC
Class: |
A61K 38/185 20130101;
A61K 9/167 20130101; A61K 38/193 20130101 |
Class at
Publication: |
424/489 ;
514/012; 514/044; 424/085.1 |
International
Class: |
A61K 038/19; A61K
038/22; A61K 038/18; A61K 009/14; A61K 048/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 3, 2002 |
FR |
02/05574 |
Claims
1. Microparticles comprising: a biodegradable, biocompatible
material having at least a portion of a surface adapted to adhere
to cells of interest or fragments thereof; and at least one
substance active on the cells or their environment upon
implantation of the microparticles in a patient associated with the
material wherein the substances is released in a controlled and/or
extended manner.
2. The microparticles according to claim 1, wherein the at least
one active substance is located on the surface of and/or
incorporated in the microparticles.
3. The microparticles according to claim 1, wherein the
biodegradable, biocompatible material is a polymer or a
copolymer.
4. The microparticles according to claim 3, wherein the polymer or
copolymer is selected from the group consisting of
poly((.alpha.-hydroxyacids), polyesters, poly(phosphozenes), PLGA
and mixtures thereof.
5. The microparticles according to claim 4, wherein the
poly(.alpha.-hydroxyacid) is a polyactide or a polylactide
co-glycolide.
6. The microparticles according to claim 4, wherein the polyester
is a poly .epsilon.-coprolactone or poly(orthoester).
7. The microparticles according to claim 1, having a diameter
between about 1 and about 500 .mu.m.
8. The microparticles according to claim 1, having a diameter
between about 10 and about 500 .mu.m.
9. The microparticles according to claim 1, further comprising an
adhesive coating on the material facilitating adhesion of the
cells, said compound being active on said cells or fragments
thereof.
10. The microparticles according to claim 1, wherein the cells of
interest are selected from the group consisting of adult cells,
embryonic cells, transformed cell lines, stem cells, genetically
modified cells, cells producing defective recombinant viruses for
their replication, hepatocytes, islets of Langerhans, nerve cells,
muscle cells, hemopoietic cells and bone cells.
11. The microparticles according to claim 1, wherein the at least
one active substance is selected from the group consisting of
immunomodulators, factors promoting survival of cells, agents toxic
to cells, factors acting on tissue environment by diminishing
immune reactions and rejection or by promoting integration by
augmenting angiogenesis, and factors controlling expression of a
gene present in a genetically modified cell.
12. The microparticles according to claim 1, wherein the at least
one active substance is selected from the group consisting of
growth factors, hormones and cytokines.
13. The microparticles according to claim 1, wherein the cells of
interest are autologous tumor cells obtained from primoculture of
the tumor of the patient as a source of antigen.
14. The microparticles according to claim 1, wherein the fragments
are apoptotic bodies or membrane vesicles obtained from
primoculture of a tumor.
15. The microparticles according to claim 1, wherein the at least
one active substance is selected from the group consisting of
pro-inflammatory cytokines and adjuvants.
16. A pharmaceutical composition for tissue repair or gene transfer
comprising the microparticles according to claim 1.
17. An antitumor vaccine comprising the microparticles according to
claim 1.
18. A method of repairing tissue comprising administering a
therapeutically effective amount of the pharmaceutical composition
according to claim 16 to the patient.
19. A method of transferring genes comprising administering a
therapeutically effective amount of the pharmaceutical composition
according to claim 16 to the patient.
20. A method of vaccinating against tumors comprising administering
an effective amount of the vaccine according to claim 17 to the
patient.
Description
RELATED APPLICATION
[0001] This is a continuation of International Application No.
PCT/FR03/01377, with an international filing date of May 2, 2003
(WO 03/092657, published Nov. 13, 2003), which is based on French
Patent Application No. 02/05574, filed May 3, 2002.
FIELD OF THE INVENTION
[0002] This invention pertains to the field of the preparation and
transplantation of cells useful in the framework of cell therapy
for tissue repair or gene transfer, or for vaccination. More
specifically, the invention relates to microparticles based on a
biocompatible, biodegradable material carrying the cells of
interest or fragments thereof and growth factors or cytokines.
BACKGROUND
[0003] Cell therapy by graft of autologous or nonautologous cells
constitutes a major therapeutic tool which is at present
essentially developed in hemobiology, but should be applicable to
other specialties based on the knowledge acquired regarding stem
cells and their identification in most tissues, ranging from muscle
to the central nervous system. The increasing identification and
characterization of cytokines and growth factors allow us to
envisage the possibility of in vitro and/or in vivo control of the
proliferation and differentiation of these cells and the modulation
of their tissue environment (immunologic rejection phenomena,
angiogenesis). Despite these advances in cell biology, the clinical
development of cell grafts remains limited at present, notably
because of the low survival rate of the implanted cells which can
be linked to a nonspecific mortality (cell death by necrosis or
apoptosis) due to the procedures employed for collection, storage,
transformation and administration or to an immunologic rejection
(in allografts and xenografts), i.e., the absence of integration in
the host tissue.
[0004] It has been proposed to use nonbiodegradable microbeads on
which the cells adhere thereby functioning as transporters or
microcarriers to reduce this cellular mortality. For example, the
survival and functioning of hepatocytes were improved when such
cells were grafted/adhered to glass or dextran (Cytodex.RTM.)
microbeads (Demetriou et al., 1986; Te Velde et al., 1992). This
strategy has made it possible to obtain more promising results than
with microencapsulated hepatocytes.
[0005] More recently, these same microbeads have been used for
cultivating and grafting human keratinocytes to reconstitute a
cutaneous cover in the nude mouse (Voigt et al., 1999). This
approach has also been used for grafting neurochromaffins or
dopaminergic embryonic neurons in a murine model of Parkinson's
disease. In this model, the survival of the transplanted cells in
the striatum is greatly increased when they are first adhered to
glass or dextran microparticles, thereby enabling behavioral
improvement of the animals (Cherskey et al., 1996; Saporta et al.,
1997; Borlongan et al., 1998).
[0006] It has also been observed (Saporta et al., 1997) that human
fetal cells adhered on dextran microbeads survive for at least
three months without immunosuppressant treatment, whereas such
cells without microparticles are rapidly rejected.
[0007] Another more recent approach enabling augmentation of the
survival of grafted cells is the administration of growth factors
in association with the graft. These proteins, which can act on
proliferation, differentiation, activation and survival of the
cells, constitute a major contribution to the field of cell grafts.
Although it is now possible to have available human recombinant
growth factors, their administration represents a challenge because
these products have a short half-life and do not cross certain
biological barriers. They moreover have a pleiotropic action which
can be the cause of undesirable side effects. The presently
developed modes of administration are not completely satisfactory
and/or applicable in clinical practice.
[0008] One of the first modes of administration proposed grafting
cells in a suspension containing growth factor. Although this
approach is simple, it does not enable long-term action on the
cells. A second mode of administration consists of co-transplanting
a tissue identified as producing the selected growth factor, e.g.,
peripheral nerve-chromaffin cell co-grafts (Date et al., 1996) or
hepatocyte-islets of Langerhans co-grafts (Kneser et al., 1999).
The sometimes limited survival of such co-grafts and the inability
to control the doses of growth factors considerably limits this
strategy. Progress made in molecular biology now allows for the
production of genetically modified cells producing a growth factor
which can be used in co-grafts or in grafts as usually defined
(Menei et al., 1998; Wood and Prior, 2001). Nevertheless, this
approach remains limited by ethical problems, biological risk and
control of the released doses. Grafts of nerve cells have been
reported, such as PC12 neuroendocrine cells (Menei et al., 1989;
Dehaut et al., 1993), and normal Schwann cells or Schwann cells
genetically modified to produce a neurotrophic factor
(Montero-Menei et al., 1992; Menei et al., 1998).
[0009] There have also been reports of biodegradable microparticles
releasing neuroactive molecules in a controlled and prolonged
manner (Menei et al., 1997; Benoit et al., 1999). These
microspheres are constituted of a biopolymer of the poly(lactic
acid-glycolic co-acid) (PLGA) type. They are biocompatible with
nerve tissue and totally degraded in several months (Menei et al.,
1993; 1994b; Vziers et al., 2000). Their size of several tens of
microns allows stereotactic implantation in the brain at the level
of their pharmacological target using the same microsyringes as for
cell implantations (Menei et al., 1994a). They were used
successfully in a phase I clinical study for the interstitial
chemotherapy of brain tumors (Menei et al., 1999).
[0010] Microspheres releasing proteins, in particular growth
factors and cytokines, have also been developed. Nerve growth
factor (NGF) is a substance of interest because it was among the
earliest characterized. There have been descriptions of
microspheres that can release NGF over at least two months (Pan et
al., 1998; Pan et al., 1999). Their therapeutic value was
demonstrated on two animal models of neurodegenerative diseases:
the murine model of Alzheimer's disease (Pan et al., 2000) and the
murine model of Huntington's chorea (Menei et al., 2000).
[0011] In the field of tumors, PLGA microspheres have been
formulated which are capable of releasing immunostimulant cytokines
after intratumoral implantation (Mullerad et al. 2000; Pettit et
al., 1997). The use of biodegradable microspheres for the release
of cytokines in the framework of antitumor vaccine was therefore
proposed (Golumbek et al., 1993). However, in that study the
microspheres were simply mixed with the cells immediately prior to
injection. In fact, the preparation of vaccine constituted of
microspheres coated by bacterial antigens or membrane vesicles had
already been proposed, but without the microspheres having the
ability to release immunostimulant molecules (Mescher and Rogers,
1996; Mesher and Savelieva, 1997).
SUMMARY OF THE INVENTION
[0012] This invention relates to microparticles including a
biodegradable, biocompatible material having at least a portion of
its surface adapted to adhere to cells of interest or fragments
thereof, and at least one substance active on the cells or their
environment upon implantation of the microparticles in a patient
associated with the material, wherein the substance is released in
a controlled and/or extended manner.
[0013] This invention also relates to a pharmaceutical composition
for tissue repair or gene transfer including the
microparticles.
[0014] This invention further relates to an antitumor vaccine
including the microparticles.
[0015] This invention still further relates to a method of
repairing tissue including administering a therapeutically
effective amount of the pharmaceutical composition to the
patient.
[0016] This invention also further relates to a method of
vaccinating against tumors including administering an effective
amount of the vaccine to the patient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Other advantages and characteristics of the invention will
become apparent from the examples below pertaining to the
preparation and use of MPAs in the field of neurotransplantation in
which reference will be made to the attached drawings in which:
[0018] FIG. 1 is a schematic representation of MPAs in accordance
with aspects of the invention;
[0019] FIG. 2 shows PC12 cells adhered to the MPAs observed by
optical microscopy (A) or by scanning electronic microscopy
(B);
[0020] FIG. 3 is a histogram representing the amphetamine-induced
rotatory behavior of different groups of rats before and after
implantation of: PC12 cells with MPA (thus releasing NGF), PC12
cells with blank microparticles (not releasing NGF), PC12 cells
alone or after injection solely of culture medium; and
[0021] FIG. 4 is a schematic representation of MPA for an antitumor
vaccine according to aspects of the invention.
DETAILED DESCRIPTION
[0022] This invention provides a combination of cells (or cell
fractions) and substances active on these cells, such as growth
factors or cytokines, at the level of the same microparticles for
the grafting of cells in cell therapy or vaccination.
[0023] We developed pharmacologically active microcarriers,
designated below as "MPAs", which release growth factors in a
prolonged and controlled manner. These microparticles of several
tens of microns in diameter comprise biodegradable, biocompatible
polymers enabling the adherence of cells or cell fragments due to
the intrinsic properties of the polymer or of a coating which can
be biologically active. MPAs do not present a biological risk and
are remarkable because, among other things:
[0024] Their function as a support for the culture of cells (or
their fragments). The preferential adhesion of the cells to be
grafted on the microparticles allows their in vitro preparation as
well as transformation without the necessity of using for their
collection proteolytic enzymes of animal origin which is not
recommended because of obvious health safety reasons.
[0025] They function as a support for grafted cells (or cell
fragments) and degrade without toxicity after implantation, not
interfering with integration of the grafted cells (or their
fragments).
[0026] They release one or more growth factors or cytokines during
a programmed or specified duration of time and at a determined
dosage.
[0027] They promote the survival and differentiation of the grafted
cells, modify their microenvironment, as well as their integration
in the host tissue.
[0028] The invention thus provides microparticles based on a
biocompatible, biodegradable material that carries on their surface
the cells of interest or fragments thereof and they comprise
molecules of at least one substance active on the cells or their
environment upon implantation of the microparticles, the molecules
being released by the microparticles in a controlled and/or
prolonged manner.
[0029] The invention can be implemented in many ways in which the
molecules of at least one active substance are on the surface of
and/or incorporated in the microparticles. Incorporation of the
active molecule can be implemented during the encapsulation process
and/or after formation of the particles. The matrix can be porous
to varying degrees with an essentially spherical form according to
operating conditions.
[0030] The microparticles comprise a biodegradable, biocompatible
polymer or copolymer. Such a polymer or copolymer may be, e.g., at
least one selected from among the group comprising
poly(.alpha.-hydroxyacids) such as the polylactides, co-glycolide
polylactides, polyesters such as poly .epsilon.-coprolactones,
poly(orthoesters), poly(phosphozenes), PLGA and mixtures thereof.
The polymer may preferably be selected from among polylactides.
[0031] The microparticles have a diameter between about 1 and about
500 .mu.m, advantageously between about 10 and about 500 .mu.m. The
MPAs are able to adapt the size of the microparticles as a function
of the adhered cells.
[0032] Adhesion of the cells on the microparticles is enabled by
inherent properties of the polymer and/or by a coating with a
compound or mixture of compounds enabling adhesion of the cells and
which can be biologically active. Thus, the invention may use
synthetic polymer enabling cellular adhesion by their
physicochemical properties, or synthetic copolymers on the
molecules from which are grafted the RGD sequences or lysine
(Varani et al., 1993). As examples of compounds for coating
microparticles enabling adhesion of cells, we can cite at least
those selected from the group comprising poly-D-lysine,
poly-L-lysine, polyornithine, polyethylene amine or other synthetic
or non-synthetic molecules belonging to the extracellular matrix
such as fibronectin-like agent, or mixtures thereof.
[0033] The microparticles comprise molecules of at least one
substance active on the cells or the environment of these cells
upon grafting. There are various methods to encapsulate these
molecules. We can cite a method of double emulsion or other
physicochemical, mechanical or chemical process. It is also
possible to employ a simple inhibition of the microparticles with
the molecules to affix them on the surface of the microparticles.
The microparticles release the molecules of this substance in a
controlled and/or prolonged manner.
[0034] The MPAs are useful for preparing pharmaceutical
compositions useful in cell therapy for tissue repair or gene
transfer. The cells of interest affixed to the surface of the
microparticles can vary according to the type of graft desired. For
example, they can be adult autologous cells, embryonic cells,
optionally transformed cell lines of stem cells or the like. Such
cells can be used in different pathologies requiring cell therapy.
This is, for example, the case of grafts of hepatocytes and islets
of Langerhans. In the case of neurotransplantation, this can
involve a PC12 cell line capable of secreting dopamine and of
differentiating itself into sympathetic-like neurons under the
effect of NGF.
[0035] As examples, we can also cite grafting of cells for repair
of the liver, the myocardium and the central nervous system,
grafting of islets of Langerhans for treatment of diabetes and
grafting of bone marrow cells in hemopathies, adult cells,
embryonic cells, optionally transformed cell lines, stem cells,
genetically modified cells, cells producing defective recombinant
viruses for their replication, hepatocytes, islets of Langerhans,
nerve cells, muscle cells, hemopoietic cells and bone cells. This
can therefore involve cells enabling in vivo gene transfer such as
cells containing a transgene and cells producing defective
recombinant viruses for their replication which will infect the
neighboring cells of the host. These cells are often obtained from
animal lines and the xenogenic immune reactions prevent their
survival and thus their long-term function. The use of MPAs
releasing an immunomodulator and/or a factor promoting the survival
of these cells makes it possible to prolong their function over
time. To the contrary, the MPA can release at a selected moment a
molecule that is toxic to the transported cell, thereby
programming/causing its death and its elimination.
[0036] The molecules of the substances incorporated within or on
the surface of the MPAs are thus advantageously growth factors,
cytokines, hormones, molecules of adherence or of the extracellular
matrix known for their action on the transported cells or their
tissue environment. As examples, we can cite growth factors,
cytokines, immunomodulators or factors acting on cell
differentiation, especially those selected from the group
comprising neutrophins such as NGF, BNDF, NT-3 and the like,
TGF.beta.s, the GDNF family, FGFs, EGF, PDGF, interleukins such as
IL-1, IL-2, the chemokines, retinoic acid, erythropoietin, and the
like, or mixtures thereof.
[0037] The molecules released by the microparticles alone or in
combination with the coating compound for the adhesion of the cells
promotes the survival of the cells, their function or orient the
differentiation of stem cells to a determined phenotype. They can
also modify the tissue environment by diminishing the immune
reactions and rejection or by promoting integration by augmenting
angiogenesis. These molecules can also serve to control the
expression of a gene present in a genetically modified cell and
which is under the control of a promoter responding to these
molecules.
[0038] The invention thus also may use microparticles based on a
biodegradable, biocompatible material to prepare a pharmaceutical
composition intended for tissue repair or gene transfer, in which
the microparticles comprise on their surface the cells of interest
and comprise molecules of at least one substance active on the
cells or their environment upon implantation of the microparticles,
the molecules being released by the microparticles in a controlled
and/or prolonged manner.
[0039] According to one aspect of the invention, the MPAs may also
be used to prepare vaccines particularly for antitumor vaccination.
This new approach in cancer treatment uses autologous tumor cells
obtained from primoculture of the patient's tumor as the source of
antigen. These cells can be genetically modified to release a
pro-inflammatory cytokine. The MPAs can play this role without
passing through the phase of transfection and selection which
lengthens the procedure.
[0040] However, one value of the MPAs is that, due to their surface
properties, they can also transport not only cells, but also cell
fragments preferably covered by cytoplasmic membrane or protein
extract or mRNA or DNA. Representative examples include but are not
limited to fragments of apoptotic bodies, exosomes or membrane
vesicles obtained from tumor primoculture. Apoptotic bodies are
readily obtained by the action of Na butyrate, by thermal treatment
of cells followed by UV B irradiation. Membrane vesicles are
obtained by cellular fragmentation followed by separation by
centrifugation in a biphasic system. The value of apoptotic bodies
or membrane vesicles is that they can be produced in large
quantities from a tumor sample, whereas autologous cells require a
phase of culture and expansion which considerably delays
treatment.
[0041] In this application for vaccination, the MPAs incorporate or
comprise on their surface molecules one or more pro-inflammatory
cytokines or adjuvant stimulating the action of dendritic cells and
the antitumor T response. We can cite as non-limiting examples of
such substances those selected from the group comprising Freund's
adjuvant, GM-CSF, IL12, IL4 and IL18.
[0042] The value of MPAs for vaccines is remarkable in the sense
that the immunogenic character results not only in the association
of the membrane antigen and the prolonged release of cytokine, but
also in the presentation of the antigen on a microparticle system.
This is an advantageous mode of presentation for the recognition of
the antigen by dendritic cells. This nonspecific immune stimulation
or the adjuvant role of the microparticles has been demonstrated
over a considerable period of time (Nakaoka et al., 1995; Scheicher
et al., 1995a, 1995b; Venkataprasaed et al., 1999).
[0043] The invention thus also comprises microparticles based on a
biodegradable, biocompatible material for preparing a
pharmaceutical vaccine composition, more specifically an antitumor
composition, in which the microparticles contain on their surface
the cells of interest or fragments thereof and comprise molecules
of at least one substance active on the cells or their environment
upon implantation of the microparticles, the molecules being
released by the microparticles in a controlled, prolonged
manner.
EXAMPLE 1
General Presentation of MPAs for Cell Grafts
[0044] The non-limiting examples below pertain to the field of
neurotransplantation (grafts of nerve cells in the central nervous
system). Biocompatible, biodegradable MPAs of a diameter of 60
.mu.m were prepared. They were constituted of PLGA and coated with
a fine layer of synthetic adherence molecules (poly-D-lysine and
fibronectin-like agent) and release NGF, a neurotrophic factor, on
a continuous basis (for at least 15 days). Cells responding to NGF,
such as the PC12 cell line capable of secreting dopamine and of
differentiating itself into sympathetic-like neurons under the
effect of this factor were used. The efficacy of these MPAs was
evaluated in vivo successfully using an animal model of Parkinson's
disease.
[0045] Neurotransplantations began clinically in the 1980s
essentially in the context of Parkinson's disease (Menei et al.,
1991a, 1991b). They continue at present in the form of clinical
research and remain essentially limited by the availability of
cells to be grafted (embryonic cells) and the low survival rate of
the cells after implantation (5 to 10%). Recent studies have shown
that most of the neurons die in the first week after
transplantation, probably due to a lack of trophic support, of
neuron connections and a limited vascularization (Emgard et al.,
1999; Mahalik et al., 1994; Zawada et al., 1998). It was
demonstrated that neurotrophic factors administered in parallel
with the grafted cells improved their survival. They also acted on
their differentiation and the development of synapses, thereby
aiding their better integration (Mahoney et al., 1999; Sautter et
al., 1998; Yurek et al., 1996). These factors can also act in a
beneficial manner on the environment of the grafted cells by
modifying the inflammatory and cellular reaction (Wei et al.,
1999).
[0046] 1) Selection of the Growth Factor
[0047] NGF was initially selected because it is a trophic factor
that has multiple advantages in the context of
neurotransplantation. In addition to its neurotrophic action, it
can protect in a nonspecific manner against the excitotoxicity
responsible for an early mortality of the grafted cells (Strijbos
and Rothwell, 1995; Carlson et al., 1999). NGF moreover has a
potential value in the context of graft rejections. It diminishes
the expression on microglial cells of the immunologic molecules
essential for the activation of T cells in the central nervous
system (Neumann et al., 1998; Wei and Jonakait, 1999; Aloisi et
al., 2000) thereby enabling the prevention of rejection and the
establishment of a local immunotolerance.
[0048] 2) Selection of the Cells
[0049] The chromaffin cells of the medullar-suprarenal have been
conventionally used for cell grafts in the context of Parkinson's
disease. Cells of the PC12 line were therefore used because they
are relatively easy to culture. These cells synthesize and release
dopamine. Furthermore, under the action of NGF, they stop
proliferation and differentiate themselves into sympathetic-like
neurons. PC12 cells also constitute a good model for determining
the production conditions of microcarriers because they do not have
natural adherence properties and only adhere to surfaces coated by
molecules promoting adherence.
[0050] The clinical development of embryonic cell grafts remains
limited by ethical problems. It is therefore necessary at present
and in the future to envisage other sources such as nerve stem
cells or mesenchymal stem cells of the bone marrow (MSC). These
latter cells that can be readily collected in humans, are capable
of differentiating themselves in vitro, in accordance with culture
conditions, into osteoblasts, chondrocytes, myocytes, adipocytes or
nerve stem cells. Thus, MSCs differentiated into nerve stem cells
by basic FGF express the high affinity NGF receptor (trka) and can
be used in the framework of this invention.
[0051] 3) Selection of the MPAs
[0052] A preferred size in this application is on the order of
about 60 .mu.m. A smaller size has an insufficient surface for
allowing adherence of an acceptable number of cells. However, the
microspheres should not have too large a diameter so as to be
resorbed without difficulty and to be readily administered via a
needle.
[0053] The PLGA microparticles were coated with fibronectin-like
agent and poly-D-lysine for cell adhesion. The use of synthetic
molecules not of animal origin is indispensable for future clinical
application. These coated microspheres enable in vitro a continuous
release of NGF during at least fifteen days. In order to study in
vivo the effect of the pharmacologically active microcarriers, they
were implanted in the rat striatum after total dopaminergic
denervation by a neurotoxic agent (parkinsonian rat). After
implantation, the PC12 cells remained adhered to the blank
microparticles (without active product) or those releasing NGF. On
the animals treated with the microcarriers releasing NGF, the PC12
cells were differentiated and had prolongations probably in
response to the release of NGF. This differentiation was
accompanied by behavioral tests (rotation test).
[0054] PC12 cells were used in this study to improve the MPAs.
However, other cells can be used. Grafts of cells of embryonic
origin were studied in animal models of neurodegenerative diseases.
These studies led to the implementation of clinical trials in
Parkinson's disease and Huntington's chorea. However, the modest
effects of these grafts in relation to a high mortality of the
grafted cells led to a rethinking of the methodology (Lindvall,
1997). In the framework of Parkinson's disease and grafts of
dopaminergic embryonic neurons, studies showed that the exposure of
these cells to GDNF (Tornqvist et al., 2000) or the use of
microcarriers (Saporta et al., 1997) increased their survival.
Microparticles releasing GDNF can also be prepared to promote
grafting of embryonic dopaminergic neurons and study their efficacy
in the rat.
EXAMPLE 2
Preparation of PC12/NGF MPAs
[0055] 1) Preparation of the Microparticles
[0056] The microparticles were produced by double emulsion (H/L/H)
evaporation/extraction of solvent.
[0057] The aqueous phase was constituted of 60 .mu.l of citrate
buffer (16 mM, pH 6), 2.5 mg of HSA (or other molecule having a
surface-active power), 90 .mu.l of PEG 400 and 100 .mu.g of NGF. 50
mg-100 mg of PLGA 37.5/25 (poly D,L-lactide-co-glycolide, Mw 21,000
Da, I=1.7) or another biocompatible, biodegradable polymer were
dissolved in 2 ml of an organic solution constituted of 75% of
dichloromethane and 25% acetone. The primary emulsion was made from
these two phases by sonication at 0-4.degree. C. (15 s, 5-6 W).
This primary emulsion was added under mechanical agitation (200
rpm) to 30-150 ml of a polyvinyl alcohol solution (0.8-4.5%,
4-8.degree. C.) containing 10% (W/V) of NaCl containing 0 to 2% of
dichloromethane. Agitation was maintained for 1 to 7 minutes. The
secondary emulsion was then poured into 400 ml of a 10% aqueous
solution of NaCl under magnetic stirring (25-50 minutes). The
aqueous extraction phase can be added in parts to the secondary
emulsion. The microparticles were then filtered (0.45 .mu.m, HVLP,
Millipore), washed 5 times with 100 ml of distilled water then
lyophilized and stored at +4.degree. C.
[0058] The encapsulation yield was on the order of 85% but,
depending on the manufacturing conditions, it can reach 97% (Pan et
al., 1998).
[0059] 2) Coating the Microparticles
[0060] Coating of the microparticles with a combination of
fibronectin-like agent (16 .mu.g/ml) with poly-D-lysine (12
.mu.g/ml) constitutes the condition enabling adherence and an
optimal differentiation of the PC12 cells.
[0061] The conditions for coating the microparticles (agitation
velocity and time) were perfected by agitation of the microsphere
suspension in the presence of fibronectin-like agent and
poly-D-lysine. After multiple tests, we selected an agitation rate
of 15 rpm and a duration of coating of 2 hours at 37.degree. C.
[0062] 3) Adhesion of the Cells on the Microparticles
[0063] Different agitation rates and times of the cells in the
presence of the microparticles were tested to perfect the
conditions of adherences of the cells on the microcarriers. It was
thus found that adherence of the PC12 cells was maximized for
agitation at 3 rpm for 4 hours at 37.degree. C. in an incubator at
5% CO.sub.2.
[0064] The tests of separation of the cells alone from the cells
adhered to the microparticles led to centrifuging the
microparticle/cell suspension at 135 g for 10 seconds. This
processing, as well as the passage of the residue in a 10-.mu.l
Hamilton syringe used for the grafts, did not produce a decrease in
the number of microparticles having cells on their surface. Under
these conditions and after counting under the microscope of the
microparticle/cell suspension, it was possible to obtain about 90%
of microparticles with cells adhered to their surface. We found, on
average, 8 to 10 cells per microcarrier. In contrast, when the
microspheres were not coated, only 5% of them had cells adhered to
their surface. The coating of the microparticles by a combination
of fibronectin-like agent and poly-D-lysine was thus essential to
make the cells adhere to them. This adherence was confirmed by
scanning electronic microscope images (FIG. 2).
[0065] 4) Characterization In Vitro
[0066] The coating was characterized by atomic force microscopy and
showed that the coating was distributed in a homogeneous manner on
the particle and diminished the porosity of its surface. Under the
previously specified coating conditions, the thickness of the
coating was around 20 nm. The coating remained intact after
lyophilization and maintained its efficacy of adhesion and
differentiation in an in vitro adhesion test.
[0067] Under the previously specified adhesion conditions, when
1.5.times.10.sup.5 cells were brought into the presence of the
MPAs, an average of 5.times.10.sup.4 cells adhered on each MPA.
EXAMPLE 3
Results of the NGF/PC12 MPAs on Animals
[0068] The MPAs release NGF in a controlled, prolonged manner. In
fact, the initial results of the kinetic of release in vitro showed
that for 200 .mu.g of encapsulated NGF, 15% was released in a
continuous manner during the first two weeks. The implantation of
0.5 mg of MPAs thus leads to the release of 5-10 ng of NGF per day
which is in agreement with the quantities necessary for action of
NGF on the cells.
[0069] After implementation in the denervated striatum of
parkinsonian rats, the PC12 cells remained strongly adhered on the
microparticles. The transported cells continued to express tyrosine
hydroxylase and were thus capable of producing dopamine. The
microparticles still had not degraded and still functioned as cell
supports two weeks after implantation. In fact, the microparticles
were still spherical and had a rather smooth appearance without
vacuolization pores. Generally speaking, the cells adhered to the
microparticles with or without NGF had a differentiated appearance
compared to the cells grafted unaided in the striatum. Observing
these cells at high degrees of magnification, it was noted that on
the microparticles releasing NGF, the extensions were longer,
comprising more or less 2 to 3 times the size of the cell body
(FIG. 2B). At the behavioral level, the initial results of the
amphetamine-induced rotation tests showed that only the rats having
received the cells with the microparticles releasing NGF had
improved (FIG. 3). Among these results it would appear that the
PC12 cells adhered to the microparticles not releasing NGF stop
proliferating.
[0070] Results on Animals
[0071] Immunolabeling with an antibody recognizing the active site
of NGF two weeks after implantation of the microspheres in the
striatum of the parkinsonian rat showed that NGF was clearly
released in a biologically active form. At two weeks, NGF was still
detected in the MPAs and released all around them in a homogeneous
manner and a distance of at least 40 .mu.m. In certain sites around
MPAs releasing NGF, axon networks were also observed, clearly
demonstrating the release of the growth factor which thereby
stimulated the differentiation of the PC12 cells.
[0072] PC12 cells, like other cell lines or even stem cells, can
form tumors after implantation. Proliferation markers showed that
in grafts of PC12 cells adhered to MPAs, there was a decrease in
the number of cells that proliferate. This effect was more
pronounced with the MPAs releasing NGF.
[0073] Cell death by apoptosis was also diminished in the grafting
of PC12 cells adhered to MPAs releasing or not releasing NGF.
[0074] At the behavioral level, the amphetamine-induced rotation
test showed that only the rats having received the cells with MPAs
releasing NGF were improved, thereby confirming the efficacy of the
MPAs.
EXAMPLE 4
Preparation of MPAs Releasing GDNF Transporting Dopaminergic
(E-dopa) Embryonic Cells)
[0075] After incubation of MPAs coated with poly-D-lysine with
cells at a rotation rate of 6 rpm for a minimum of one hour, E-dopa
cells adhered to the surface of 70 to 90% of the MPAs. The number
of adhered cells was quite variable, in the range of 5-30 cells per
MPA.
EXAMPLE 5
General Presentation of the MPAs for Antitumor Vaccination
[0076] Our clinical experience with antitumor vaccination as well
as the results published in the literature confirm that the culture
of tumor cells is a limiting step in this type of approach. The
yield is low and the elapsed time for obtaining the requisite
number of cells is often incompatible with the rapidity of
evolution of the tumor. Autologous cells still remain the most
suitable source of specific tumor antigens (STAs) whether for
loading dendritic cells in vitro or subcutaneous vaccination. It is
therefore useful to develop a system that can be prepared rapidly
from a small number of cells capable of presenting the majority of
tumor antigens as well as an adjuvant of the dendritic cells.
[0077] We formulated for this bio-artificial particles based on the
concept of the pharmacologically active microcarriers (MPAs) ERIT-M
0104 developed at INSERM. These are microspheres with a diameter of
approximately 5 to 30 .mu.m, constituted of a biodegradable,
biocompatible copolymer (such as poly[lactic acid-co-glycolic acid]
or PLGA) that can release in a controlled, prolonged manner an
adjuvant such as a pro-inflammatory cytokine (such as GM-CSF, IL2,
IL12 or IL18). Due to their surface properties (film-coated by
synthetic cell adhesion molecules), these microparticles can carry
on their surface tumor antigens prepared from autologous tumor
cells (membrane vesicles, apoptotic bodies, exosomes, protein
extract, mRNA and DNA).
[0078] These MPAs carrying tumor antigens on their surface and
releasing in a controlled manner an adjuvant such as a cytokine,
may be:
[0079] brought into contact with dendritic cells in vitro (in the
framework of vaccination with dendritic cells obtained from blood
stem cells), or
[0080] administered to a patient directly via the subcutaneous,
intradermal or intramuscular route.
[0081] The immunogenic character of the MPA is due not only to the
combination with the tumor antigen and the prolonged release of
cytokine, but also to the presentation of the antigen on a
microparticle system. This is an ideal mode of presentation for the
recognition of the antigen by the dendritic cells. This nonspecific
immune stimulation or the adjuvant role of the microparticles has
been demonstrated for a long time (Mescher and Rogers, 1996;
Mescher and Savelieva, 1997; Nakaoka et al., 1995; Rogers and
Mescher, 1992; Scheicher et al., 1995a, 1995b; Venkataprasad et
al., 1999).
[0082] PLGA microspheres were already described as being able to
release immunostimulant and/or antitumor cytokines (Golumbek et
al., 1993; Mullerad et al., 2000; Pettit et al., 1997) and we have
already demonstrated our capacity in the laboratory to develop
microspheres releasing antitumor agents or recombinant proteins
(Menei et al., 1996, 1999, 2000; Pan et al., 2000).
EXAMPLE 6
MPA Carriers of Plasma Membranes in Antiglioma Vaccination
[0083] The PLGA microspheres used (with and without GM-CSF, an
activating cytokine) were prepared by the solvent evaporation
technique as previously described (Menei et al., 1993; Menei et
al., 1996; Pan et al., 2000). Filtration was used to select the
smallest microspheres (5 to 30 .mu.m in diameter).
[0084] Purification of the plasma membranes of the GS-9L cells was
performed by incubating 1.5.times.10.sup.8 cells for 15 minutes at
4.degree. C. in a hypotonic buffer (KCl 42 mM, Hpes 10 mM pH 7,
MgCl.sub.2 4.5 mM and 1% of protease inhibitors) by performing
about 50 passages in a 30-gauge syringe. The cells broken in this
manner were then centrifuged (250 g; 10 minutes; 4.degree. C.) to
separate the membranes in the supernatant, the non-lysed cells and
the nuclei collected in a residue. The membranes were then
recovered after an ultracentrifugation step (100,000 g; 90 minutes;
4.degree. C.). They were then placed in a biphasic polyethylene
glycol (PEG 8000)/Dextran T500 system equilibrated 48 hours in
advance and then centrifuged (3000 g; 15 minutes; 4.degree.
C.).
[0085] The plasma membranes were then arranged at the interphase of
the biphasic system by affinity for the two phases. After recovery,
2 washings in a sucrose 0.25 M, Tris HCl 1 M buffer were performed
(100,000 g; 30 minutes; 4.degree. C.). The membranes were stored in
this same buffer at -80.degree. C. until their use. Among the
adsorption and formulation protocols tested, that which seemed to
be the most effective brought together the membranes and the
microspheres without prior coating in the Tris pH 6.8 buffer. Other
adsorption protocols optimal for protein fractions, membrane
vesicles, apoptotic bodies and exosomes are under development.
These protocols were validated by radiotagging the protein fraction
by I.sup.125 by direct immunofluorescence of membrane markers in
confocal microscopy then by analysis of the enzymatic activity of
the adsorbed structures.
[0086] Example of adsorption of the plasma membranes of GS-9L cells
on PLGA microspheres: The purified plasma membrane preparation was
treated by sonication for 30 seconds to reduce the size of the
aggregates of membrane vesicles. The equivalent of 10 .mu.g of
membrane proteins was then brought into the presence of 20,000 PLGA
microspheres. This was then placed under rotation (60 rpm)
overnight at 4.degree. C.
[0087] Quantitative determination using Lowry's test was performed
to determine the amount of membrane proteins recovered. Using a
calibration range of BSA (comprised between 0.2 and 1 .mu.g/.mu.l),
the protein concentration of the samples was calculated. The
aliquot, called the initial aliquot (Ai), collected at the
beginning of purification (just after lysis) and containing all of
the proteins of the cell, had a protein concentration between 7000
and 10,000 .mu.g/ml. In contrast, at the end of enrichment, we
obtained a protein concentration comprised between 200 and 300
.mu.g/ml. We thus recovered 3% of the total proteins.
[0088] The profile of the isolated proteins was analyzed by
polyacrylamide gel migration showing an intermediary migration
profile of the profiles of the soluble and insoluble fractions of
the cell lysates. It was the insoluble fraction that contained the
majority of the membrane proteins. The determination of the
specific activity of 5'-nucleotidase and of Fas-ligand by Western
blot indicated an enrichment of these membrane proteins, proving
the membrane nature of the enriched material. Direct
immunofluorescence of membrane markers with confocal microscopy
(5'-nucleotidase, integrin, ICAM-1) confirmed fixation of the
membrane proteins on the microparticles.
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