NZ624069B2 - Vaccine for tumor immunotherapy - Google Patents
Vaccine for tumor immunotherapy Download PDFInfo
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- NZ624069B2 NZ624069B2 NZ624069A NZ62406912A NZ624069B2 NZ 624069 B2 NZ624069 B2 NZ 624069B2 NZ 624069 A NZ624069 A NZ 624069A NZ 62406912 A NZ62406912 A NZ 62406912A NZ 624069 B2 NZ624069 B2 NZ 624069B2
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
- A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
- A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
- A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55588—Adjuvants of undefined constitution
- A61K2039/55594—Adjuvants of undefined constitution from bacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/80—Vaccine for a specifically defined cancer
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/025—Enterobacteriales, e.g. Enterobacter
- A61K39/0258—Escherichia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
Disclosed is a vaccine comprising bacterial ghosts, tumour associated antigens and antigen presenting cells, for use in tumour immunotherapy. Where the antigen presenting cells are comprised of either dendritic cells or monocytes; the bacterial ghosts are comprised of gram negative bacteria; and the tumour associated antigens are either loaded, recombinantly expressed, or carried by the bacterial ghosts. tumour associated antigens are either loaded, recombinantly expressed, or carried by the bacterial ghosts.
Description
Vaccine for tumor immunotherapy
Description
The present invention relates lly to a vaccine comprising dendritic
cells and ial ghosts for tumor immunotherapy.
tic cell-based therapy uses tumor cells to stimulate anti-tumor
immunity. Thereby, dendritic cells are incubated with tumor cells, in
particular, with autologous tumor cells (i.e. the patient's own tumor cells) to
stimulate anti-tumor immunity. The dendritic cells incubated with tumor cells
t antigens directly from the tumor cells. The dendritic cells bearing the
tumor-associated antigens are then used as vaccine to stimulate the immune
system against the tumor. While dendritic cells are necessary to activate a
response against cancer, they are often ineffective without prior activation
because they fail to recognize growing cancers as dangerous. By activating
dendritic cells, using an external stimulus, dendritic cells are d which
present the relevant tumor-associated ns and, thus, induce an
effective anti-tumor response.
Such an approach is bed, for example, in U.S. 2008/0031900. Therein,
antigen-presenting cells such as dendritic cells are activated with GM-CSF
and interferon alpha in the presence of one or more cancer cells.
While considerable ss has been made with such compositions, further
improvement is still desired.
Accordingly, in one aspect the invention provides a composition comprising
(i) professional antigen-presenting cells,
(ii) tumor-associated antigens, and
(iii) bacterial ghosts.
In another aspect, the ion relates to the use of
(i) professional n-presenting cells,
(ii) tumor-associated antigens, and
(iii) bacterial ghosts in the preparation of a vaccine for tumor immunotherapy.
In another apsect, the invention provides a therapeutic vaccine comprising
the composition of the invention.
Certain statements that appear below are r than what appears in the
statements of the invention above. These statements are provided in the
interests of providing the reader with a better understanding of the invention
and its practice. The reader is directed to the accompanying claim set which
defines the scope of the invention.
Also described herein is a composition comprising
(i) antigen-presenting cells (APCs),
(ii) tumor-associated antigens (TAAs), and
(iii) bacterial ghosts (BGs).
According to the invention, it has been found that the effectivity of antigenpresenting
cells and, in particular, dendritic cells as tumor e can be
improved by providing a composition which additionally contains bacterial
ghosts.
Bacterial ghosts (BGs) are empty bacterial cell envelopes of bacteria, in
ular, of Gram-negative bacteria. Preferred ia are E. coli or
Shigella flexneri 2a or Mannheimia haemolytica and, in particular, E. coli
Nissle 1917.
BGs can be produced by controlled expression of heterologous gene causing
disruption of ial membrane integrities and leading to lysis of the
bacteria. An example of lytic gene is the bacteriophage 4 gene E
encoding a polypeptide triggering the fusion of the inner and outer
membranes of the ial cells and forming trans-membrane tunnel
structure ng the whole cell envelope, through which the entire
cytoplasmic t is expelled due to the change in osmotic pressures
between the cell interior and the culture medium, whilst the inner and outer
membrane structures are preserved and remain intact (cf. U.S. 7,968,323
B2). The size of the trans-membrane tunnel structure depends on the lysis
conditions and inner diameter is in the range of 20-400 nm. The empty body
of BGs is devoid of nucleic acids, ribosomes and other constituents, s
essential inner and outer membrane structures including the antigenic
molecules, e.g. outer membrane proteins, adhesins, lipopolysaccharide
(LPS) and peptidoglycans are non-denatured and remain intact. There is
absolutely no risk of reversal to pathogenic form after induction of controlled
lysis process.
Bacterial ghosts may be prepared by a method comprising the following
steps:
(a) providing egative bacterial cells comprising a gene encoding a
lytic protein capable of forming a tunnel structure in the bacterial cell
envelope
(b) optionally cultivating the bacterial cells under conditions n the lytic
gene is not expressed
(c) subjecting the bacterial cell to conditions wherein the lytic gene is
expressed and the cytoplasmic components of the bacterial cells are
liberated and
(d) ing the ing bacterial ghosts.
A preferred example of a gene encoding the lytic protein is the
bacteriophage phiX174 gene E.
Particularly preferred, the bacterial cells used for the above described
method of bacterial ghost preparation onally encode an enzyme
capable of hydrolyzing cytoplasmic components in the bacterial cell as
described in WO 630. The corresponding method of bacterial ghost
preparation comprises the following additional steps:
(a) optionally ating the bacterial cells under conditions wherein the
enzyme gene is not expressed
(b) subjecting the bacterial cell to conditions wherein the enzyme gene is
expressed and the cytoplasmic components of the bacterial cells are
degraded.
The gene encoding the hydrolytic enzyme is preferably a nuclease gene, in
particular a Staphylococcus aureus nuclease gene (WO 03/006630).
BGs show no cytotoxic and genotoxic s on the viability and metabolic
activity of a wide range of tested cells ing hages, dendritic cells,
tumor cells, endothelial cells and epithelial cells. BGs with their intact surface
structures are ently recognized and phagocytosed by professional
APCs, e.g. dendritic cells and macrophages through various e
receptors, e.g. complement receptors and Toll-like receptors. er,
further studies using dentritic cells (DCs) as model of the most professional
antigen-presenting cells (professional APCs) revealed that their phagocytic
activity and uptake of BGs depend on the bacterial strain used for the
production of BGs.
Although the lysis process is very effective, there still might be a potential
contamination with approximately one intact bacterial cell per 10,000 BGs.
To avoid the presence of any living cell in a BG preparation, in particular,
already before lyophilization of BG samples, an alkylating agent such as
beta-propiolactone reacting and causing alterations in nucleic acids is
preferably added to the tation system prior to final harvesting of BGs.
A production process using beta-propiolactone for final vation meeting
the criteria for application in human medicine and veterinary is sed in
Patent Application No. .
The use of bacterial ghosts as vaccine or adjuvant and the preparation of
recombinant bacterial ghosts carrying heterologous proteins in their cell
envelope ures are disclosed in Patent Application No.
PCT/EP98/04723.
The use of ial ghosts as carrier or targeting vehicle of active
nds is disclosed in Patent Application No. PCT/EP00/01906.
The composition bed herein comprises as component (i) antigenpresenting
cells (APCs), in particular, professional antigen- ting cells.
In a preferred embodiment, the composition comprises monocytes and, most
preferred, dendritic cells (DCs). In particular, the composition after incubation
and ready for administration comprises mature tumor-associated antigen
loaded DCs, preferably the tumor-associated antigen loaded DCs are tumorassociated
antigen- presenting dendritic cells.
DCs are the most potent professional APCs as well as potent initiators and
modulators of T cell ses in vivo including sensitization of MHC-
restricted T cells, development of T cell-dependent antibody production, and
induction of logical tolerance. DCs have high ytic activity in
both peripheral tissue and secondary lymphoid tissues, and capture antigens
(Ag) via several mechanisms including macropinocytosis and or
mediated endocytosis. The major role of DCs is related to the recognition of
potential danger signals provided by foreign antigens, their internalization,
processing and presentation within the complex of MHC class I and II
molecules. In most cases during normal physiologic conditions, DCs are
present in their immature state characterized by high phagocytic capacity,
low expression of co-stimulatory and Ag-presentation molecules, and low
cytokine production. Phagocytosis of soluble ns by mannose receptors
(uptake of ylated Ag) and Fc-receptors (uptake of immunoglobulins)
strongly enhances efficiency of n presentation. Furthermore, DCs can
present Ag in more than ld lower concentrations after Ag complex with
dy is internalized through Fc-receptors compared to soluble Ag
internalized via macropinocytosis. Effective T cell stimulation is strictly
connected to DC tion which affects cytokine production, expression of
co-stimulatory molecules and presentation of peptide-MHC complexes.
Endocytosis of extracellular antigens and their processing via endosomallysosomal
pathway usually results in the presention of n fragments
within MHC class II molecules. However, endocytosis of extracellular
ns mediated through Fc-receptors allows MHC class I and class II
restricted antigen presentation and induces DCs maturation. Presentation of
extracellular antigens in the context of MHC class I molecules is known as
presentation or cross-priming. Efficient tation of antigens by
MHC molecules along with expression of co-stimulatory molecules and
cytokine secretion leads to stimulation of s types of T cells, e.g Th1,
Th2, Treg or Th17. For activation of Th1 lymphocytes and their proliferation
ient production of IL-12 by mature DCs is ant. Polarization toward
Th1 type T cell immune response is considered as one of the most important
factors necessary for induction of effective anti-tumor immune responses
leading to recognition and elimination of tumor cells.
BGs show an excellent capacity to be recognized and internalized by
professional APCs ing DCs. DNA loaded BGs stimulate more efficiently
both humoral and cellular Ag-specific immune responses than naked DNA in
mice. An increase of IFN-gamma producing Ag-specific CD8+ T cells was
observed in animals vaccinated with DNA loaded BGs in response to
restimulation by APCs pulsed with peptide containing the immunodominant
MHC class I epitope. Furthermore, BGs enhanced sion of MHC class I
molecules and co-stimulatory molecules on DCs. Cross-presentation of
tumor-associated Ag delivered to DCs by BGs could activate both CD4+ and
CD8+ T cells and stimulates the immune system to enhance an immune
response against tumor-associated Ag expressed by tumors.
Bacterial lipopolysaccharide (LPS) enhances maturation of DCs, affects
endosomal ication of DCs and also es presentation of Ag.
Inner and outer membrane structures of BGs including LPS remain intact
after protein E-mediated lysis of egative bacteria, therefore, besides
high loading capacity, BGs also “carry” on the e a LPS-highly effective
molecule for stimulation of cross-presentation by DCs.
Thus, interaction between APCs and BGs in the inventive composition
results in stimulation, activation and, thus, maturation of the APCs.
The composition according to the invention further comprises at least one
tumor-associated antigen (component (iii)). Tumor-associated antigens
(TAAs) can be provided e.g. by a tumor cell lysate. Preferably, an autologous
tumor cell lysate is provided, i.e. a lysate from a tumor derived from the
patient to be treated. However, it is also possible to use tumor-associated
antigens from a tumor cell line.
Preferably, a tumor cell line of the same tumor type as the tumor to be
treated is used. Preferably, at least two distinct tumor cell lysates are
ed in the composition according to the ion.
As a further alternative, the TAA may be carried by BGs. In one embodiment
the BGs are loaded with TAA. In another embodiment the BGs carrying TAA
are BGs carrying recombinant TAA (protein). Such BGs ng
inant TAA are derived from bacteria recombinantly expressing TAA.
Thus, the invention also relates to a composition wherein components (ii)
and (iii) are coupled, e.g. in the form of BGs carrying recombinant TAA
(protein).
The tumor or cancer cells are preferably from cancers selected from the
group consisting of fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, Kaposi's
sarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, elioma, Ewing's tumor,
leiomyosarcoma, rhabdomyosarcoma, rhabdosarcoma, colorectal
carcinoma, colorectal adenocarcinoma, pancreatic cancer, breast cancer,
n cancer, prostate cancer, us cell carcinoma, basal cell
carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland
carcinoma, papillary carcinoma, papillary adenocarcinomas,
cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal
cell carcinoma, hepatoma, bile duct oma, choriocarcinoma, seminoma,
embryonal oma, Wilms' tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, glioblastoma, astrocytoma, medulloblastoma,
craniopharyngioma, moma, pinealoma, hemangioblastoma, acoustic
neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma,
a, lymphoma, melanoma, and leukemia.
In an embodiment, the composition comprises a T98G tumor cell lysate.
More preferably, the composition comprises an autologous tumor cell lysate.
The composition of the ion may comprise tumor cells in a heat
shocked, chemically treated or/and killed form.
The composition of the invention further optionally contains a cytokine, in
particular, GM-CSF. Further, it ally contains interferon alpha.
Also described is an anti-tumor vaccine made of te-derived tic
cells (DCs), at least one, preferably at least two distinct tumor cell lysates
from the same tumor type, bacterial ghosts (BGs) and, optionally,
recombinant human granulocyte-macrophage colony-stimulating factor (GM-
CSF), and interferon alpha (IFN-α).
The antigen-presenting cells in the composition according to the invention
are activated and/or d by incubation with associated antigens
(TAAs) and ial ghosts (BGs). In particular, one or more antigenpresenting
cells are activated that present one or more cancer antigens and
induce T cell activation through incubation of the one or more antigenpresenting
cells in the presence of one or more cancer cells, preferably in
the presence of one or more tumor or cancer cell lysates, and in the
presence of bacterial ghosts.
An advantage of the t application is that DCs can be incubated in the
ce of tumor lysate obtained from autologous patient tumor cells and/or
tumor cell lines prepared from identical histological tumor type. Thus, the
DCs can be trained for the respective cancer type to be treated. Use of
autologous tumor cells minimized the risk of infections or transfer of other
diseases.
Another advantage is caused by incubation of DCs in the presence of tumor
lysates made of two or more distinct cell lines obtained from identical tumor
type. Thereby, the range of antigens presented by the DCs can be
increased.
Intact surface immunostimulatory structures of BGs, optionally together with
IFN-α and/or GM-CSF stimulate full maturation of DCs and elicit production
of IL-12. GM-CSF is the cytokine typically used for generation of mature
DCs.
Monocytes can be obtained from peripheral blood of patients by blood draw
or leukapheresis, which guarantees a sufficient amount of cells.
Incubation with one or more tumor s provides for the presence of
several tumoral tumor-associated antigens (TAA) and leads to the
stimulation of broader TAA-specific immune ses.
Tumor lysates can be ed either from autologous sample of tumor
tissue obtained during patient’s surgery or from tumor cell lines generated
from the identical tumor type. Tissue donors are screened before surgery for
sexually-transmitted diseases (STD), e.g. HIV, HCV, HBV, syphilis, and only
negative patients are considered as tissue .
Anti-tumor vaccine based on DCs, tumor lysate and BGs can be applied as
treatment for patients with tumors, in particular, with glioblastoma, renal
carcinoma, ovarian carcinoma, prostate carcinoma, bladder carcinoma,
ctal carcinoma, colorectal adenocarcinoma, and melanoma.
Preferably, tumor lysates are prepared from the respective tumor type and/or
tumor cell lines.
The composition according to the invention preferably comprises empty
bacterial ghosts as component (iii). However, it is also possible to use
bacterial ghosts which are loaded, in particular, with an activator or silencer
of the immune system. The bacterial ghosts can be loaded, in ular, with
a pharmaceutical agent and/or DNA.
The composition may optionally contain bacterial lipopolysaccharide (LPS).
It was found that the APCs, in ular, dendritic cells ned in the
inventive composition and being incubated with TAAs and BGs produce
specific cytokines, in particular, IL-12, IL-23 and/or IL-1β.
When administering the matured APCs, in particular, DCs to a patient, an
immune response is induced. It was found, in particular, that the ive
composition induces a Th17 response which was not observed when
administering compositions of antigen-presenting cells which have not been
ted with bacterial ghosts.
The composition according to the invention preferably contains at least one
bacterial ghost, more preferably at least five bacterial ghosts and more
preferably at least 10 or at least 100 bacterial ghosts per antigen-presenting
cell, in ular, per dendritic cell. The composition may n up to
,000 bacterial ghosts, preferably up to 1,000 ial ghosts and more
preferably up to 500 bacterial ghosts per dendritic cell.
Also described is a vaccine comprising the composition described herein. By
administration of such a vaccine, an immune response against cancer cells
is evoked. Thus, the invention also relates to a vaccine for tumor
immunotherapy. When administered, the ition according to the
invention and, in particular, the antigen-presenting cells comprised therein
induce an increased tumor antigen recognition and, thus, tumor-specific
g by autologous T cells.
For administration, a vaccine preferably comprises from about 1 to 10 million
cells, in particular, from 4 to 6 million cells per dose. Preferably, 5 to 15, in
particular, 6 to 10 doses are administered. Administration can take place
over several weeks. ably, the first two to three doses are administered
weekly, followed by a monthly administration of the remaining doses.
For storage, the ition may be frozen or lyophilized. Preferably, the
composition is frozen.
Administration can be performed in any suitable way, e.g. systemically,
subcutaneously, odally, ermally or intratumorally. Preferably the
composition is administered subcutaneously, intranodally, intradermally or
umorally.
For stration, bacterial ghosts may contain an active agent for
activating or silencing the immune system. For maturating the APC, the
bacterial ghost may contain additional ingredients such as tumor lysates,
tumor es, tumor antigens or cytokines. Incubation of antigen-
presenting cells with bacterial ghosts and tumor-associated antigen
preferably takes place for a time range of from 10 min to 12 h, preferably
from 15 min to 6 h, and more preferably from 3 h to 5 h.
In a particularly preferred embodiment, dendritic cells are cultured in
cially available CellGro® DC Medium GMP Serum-free Medium
optimized for the generation of DCs, clinical ex vivo use, standardized,
ctured, tested and released in compliance with the relevant GMP-
guidelines.
Preparation of anti-tumor vaccine according to the above-mentioned
combination of stimulatory agents and DCs is preferably performed by
incubation of monocytes, in particular, DCs obtained from peripheral blood of
patients in a 5% CO2 humidified incubator at +37°C in o® culture
medium supplemented with DNAse for 2h, followed by 3 days incubation in
CellGro ® culture medium supplemented with recombinant human GM-CSF
and IFN-α in order to obtain a population of immature DCs. At least one
tumor lysate, and preferably at least two tumor lysates, prepared from at
least two distinct cell lines of identical tumor type are mixed together with
BGs, ed and incubated for 1 h at room temperature (RT) with gentle
shaking. Subsequently, recombinant human GM-CSF and IFN-α are mixed
with the blend of BGs and tumor lysates, and added to immature DCs and
incubated in a 5% CO2 fied incubator at +37°C.
After 10 min to 12h of incubation, more preferably 4 h of incubation, non-
internalized tumor lysates and BGs are carefully removed by gentle
collection of media from DCs to a sterile 50 ml tube and spun down at 700
RPM/5 minutes/RT. Meanwhile, fresh o® culture medium
supplemented with inant human GM-CSF and IFN-α is added to the
remaining cells. After centrifugation, the supernatant is quickly and carefully
removed, the pellet is resuspended in CellGro® culture medium
supplemented with recombinant human GM-CSF and IFN-α and the cells are
returned back to the culture flask. DCs are incubated for additional 6 h in a
% CO2 humidified tor at +37°C before deep freezing of vaccine.
The term ‘comprising’ as used in this specification and claims means
‘consisting at least in part of’. When interpreting statements in this
specification and claims which includes the ‘comprising’, other features
besides the features prefaced by this term in each statement can also be
present. d terms such as ‘comprise’ and ised’ are to be
interpreted in similar manner.
In this specification where reference has been made to patent specifications,
other al documents, or other sources of information, this is generally
for the purpose of providing a t for discussing the features of the
invention. Unless specifically stated otherwise, reference to such external
documents is not to be construed as an admission that such documents, or
such sources of information, in any jurisdiction, are prior art, or form part of
the common general dge in the art.
The invention is further described by the enclosed Figures and the following
Examples.
FIGURE 1. The cell surface markers expression on DCs. Immature DCs
were ed by tion of monocytes obtained from peripheral blood of
normal healthy donors in CellGro® DC medium supplemented with IFN-α
(3000 IU/mL) and rhGM-CSF (1000 IU/mL) for 3 days. Maturation markers of
DCs were analyzed by multicolor flow cytometry 48h after short (4h)
stimulation of immature DCs with tumor lysate and BGs from E. coli Nissle
1917 (10 and 100 BGs/1DC) in the presence of IFN-α and rhGM-CSF.
Immature DCs ted with IFN-α and rhGM-CSF and tumor lysate
supplemented with Lipopolysaccharide (LPS) (200 ng/mL) or without extra
maturation stimuli served as controls. The y-axis represents the percentage
of cells expressing specific differentiation antigen. Data represent the mean
± SD of 4 independent experiments performed using cells obtained from
different .
CD14 is an indicator of maturation of dendritic cells. While monocytes show
CD14 expression, matured dendritic cells show no or little CD14 expression.
CCR7 is a marker for attractants of the cells to the lymph node. As can be
seen from Fig. 1, expression of marker CCR7 increases for dendritic cells
ted with bacterial ghosts showing their mobility.
CD83 is a major marker of mature dendritic cells.
CD80, CD86, CD1a, CD11c and HLA-DR are maturation markers for
dendritic cells.
Figure 2. Migratory capacity of DCs after incubation with tumor lysate
and LPS or short stimulation with BGs. Chemotaxes of distinct DC
tions matured in in the presence of IFN-α, rhGM-CSF and tumor
lysate supplemented with BGs from E. coli Nissle 1917 (10 and 100
BGs/1DC), LPS (200 ng/mL) or without extra maturation i in response
to CCL21 (chemokine eliciting its effects by binding to a cell surface
chemokine receptor CCR7) were determined as the number of cells which
migrated into the lower part of a transwell system containing different
concentrations of CCL21 and counted by flow ter. Each bar
represents the mean number of migrated cells ± SD of 4 independent
ments performed using cells obtained from different normal healthy
donors.
FIGURE 3. Cytokine profile of DCs matured in the presence of tumor
lysate and LPS or short time incubation with BGs. Immature DCs were
stimulated with IFN-α, rhGM-CSF and tumor lysate for short time (4h) in the
presence of pure LPS (200ng/mL) or BGs from E. coli Nissle 1917 (10 and
100 BGs/1DC) prior to measuring cytokine ed into supernatants after
24h and 48h incubations. Cells incubated t additional stimuli served as
negative control. The levels of cytokines released from DCs were measured
using FACSArray Bioanalyzer. Data represent the mean ± SD of 4
independent experiments performed using cells obtained from various
normal healthy donors. P values <0.05 were considered significant and are
indicated with asterisks (*, P<0.05; **, P<0.01; ***, P<0.001).
IL-12 and IFN-γ are major activation markers for differentiation of T
lymphocytes into Th1 type lymphocytes.
IL-23 (the growth and stabilization factor) and IL-6 (the entiation factor)
are cytokines involved in the development of Th17 lymphocytes. Fig. 3
shows that T helper cells are d by compositions according to the
invention.
IL-1ß and TNF-α are pro-inflammatory s.
IL-10 is an nflammatory cytokine.
IL-2 is a T cell growth factor.
FIGURE 4. Allogeneic (A) and autologous (B) stimulatory
capacities of analysed DCs. Short time incubation of immature DCs with
IFN-α, rhGM-CSF, tumor lysate and BGs (4h) significantly enhanced
capacity of DCs to stimulate proliferation of autologous T cells compared to
DCs matured with IFN-α, rhGM-CSF and tumor lysate mented with
pure LPS or t extra maturation stimuli. Autologous and allogeneic
immunostimulatory ties of analyzed DC populations were determined
after 6 days of incubation in the presence of scence-labeled (CFSE-
labeled) allogenic or autologous T cells at the ratio DCs:T cells - 1:10.
ated cells were stained after incubation with a panel of monoclonal
antibodies (anti-CD3, anti-CD4, and anti-CD8) and proliferation of both
autologous and allogeneic T cells was determined by multicolor flow
try. Values were calculated as percentage of T cells erated
spontaneously subtracted from the percentage of cells proliferated after
stimulation with distinct populations of DCs. T cells incubated with
phytohemaglutinin (PHA) (5 µg/ml) served as positive control. Data represent
the mean ± SD of 4 independent experiments performed using cells obtained
from different donors. P values <0.05 were considered significant and are
indicated with asterisks (*, P<0.05; **, P<0.01; ***, P<0.001).
FIGURE 5. Recognition of tumor cells and cytotoxic effects of allogenic
(A) and autologous (B) T cells induced by DCs loaded with tumor lysate
after short time stimulation with BGs. Allogenic or autologous T cells were
incubated for 6 days in the presence of tumor cell lysate (T98G cells) loaded
DCs pre-stimulated with pure LPS (200ng/mL), short time incubation (4h)
with BGs from E. coli Nissle 1917 (10 and 100 BGs/1DC) or without extra
tion stimuli. Subsequently, stimulated T cells were added to fresh
fluorescence-labeled (CFSE-labeled) T98G tumor cells at the Effector:
Target ratio 10:1. Specific lysis of tumor cells was ined 24h after
mutual co-incubation by flow cytometry. Lysis of tumor cells after incubation
with 10% Et-OH served as positive control (100%). Tumor cells incubated
without the presence of pre-stimulated effector cells served as negative
control for spontaneous cell death. Data represent the mean ± SD of 4
independent experiments performed using cells obtained from different
donors. P values <0.05 were considered significant and are indicated with
asterisks (*, P<0.05; **, ; ***, P<0.001).
In particular, the s of ent of T98G tumor cells show a
considerable ement for DCs+BGs compared to DCs alone or
In all experiments shown in Figs. 1 to 5 human dendritic cells are included.
Examples
Example 1. Bacterial Ghosts.
Bacterial ghosts are prepared in accordance with Patent ation No.
Example 2. Tumor lysate preparation.
Tumor tissue obtained from a cancer patient or tissue culture plates (flasks)
are stored in tubes filled with NaCl (0,9% sodium chloride in water for
injection „ Fresenius“). The tube with tumor tissue or cells are stored at +4°C
and processed up to 3 days after the surgery. The tube is irradiated (120 Gy)
before processing of tissue. The donors (patients) have to be screened for
sexually-transmitted diseases (STD), e.g. HIV, HCV, HBV, syphilis, and
patients with positive detection of any of the mentioned diseases are
ed. Tumor tissue obtained from the patient is transferred to a sterile
Petri dish filled with 5-10 ml HBSS (Hanks´ Balanced Salt Solution; Lonza,
No.: 10-547F or F; manufactured in ance with cGMP
regulations). Both ic and connective tissues are removed by scalpel
and tweezers. Remaining tumor tissue is cut into small pieces of
approximately 5 mm and ground by sterile e plunger. The obtained cell
suspension is additionally homogenized by passing the suspension h
a 20G (0.9 mm) needle attached to a sterile syringe l times until a
homogenized suspension is obtained. The cell suspension is subsequently
filtered through a nylon strainer (100 µm) and collected in a sterile 50 ml
tube. The Petri dish is washed with remaining HBSS, filtered through a nylon
strainer and combined with filtered cells. The cell suspension is spun down
at 1600 RPM for 7 min at +4°C. The supernatant is quickly and carefully
decanted and a pellet is resuspended in 2ml of CellGro ® culture medium.
Single cell suspension is equally divided into microtubes. The microtubes are
placed into liquid N2 or a mixture of dry ice and methanol for approximately 3
minutes. Subsequently, frozen cells are thawed at room temperature (RT) for
-30 minutes until the cell pellet becomes completely melted (cells should
not be kept at RT too long in order to prevent degradation of tumor cell
proteins). The freezing-thawing procedure should be repeated 5 times. The
cell lysate is then sonicated in an ultrasonic bath for 5 minutes. Cell debris
er with cell lysate is collected in one tube, spun down at 10000-15000
RPM for 10 min, followed by quick and careful collection of the supernatant
(cell lysates) using a fine needle separating off the pellet. Cell lysates should
not be filtered and all steps have to be done under sterile conditions. Nonfiltered
cell lysates are aliquoted and stored at -80°C until further use. The
cell lysate is diluted with 10x DPBS (Dulbecco´s Phosphate Buffered Saline,
10x PBS, Lonza, No.:17-515F). A spectrophotometer is used for
determination of protein concentration.
Example 3. Culture of monocyte-derived dendritic cells for anti-tumor
vaccine preparation.
Monocytes obtained from peripheral blood of patients are isolated either by
elutriation (Elutra Cell Separation System, CaridianBCT Europe NV/SA) or
magnetic separation (CliniMACS, Miltenyi Biotec GmbH). GM-CSF is
dissolved in CellGro® culture medium to obtain a final concentration of 1x10 5
IU/ml and ted into ubes (700 µl per tube) and stored at -80°C.
IFN-α , ROFERON-A 12x106 IU/ml) is aliquoted into microtubes (17.5
µl per tube) and stored between +2°C and +8°C. Cryopreservation freeze
media CryoStor CS2 or CryoStor CS5 are aliquoted under sterile conditions
into microtubes (1.5 ml per tube). Separated tes are resuspended in
culture medium and added into a culture flask, approximately 167x106
monocytes in 70 ml of culture medium. imately 150 µg of tumor lysate
is required for one culture flask or tumor lysate from cells corresponding to a
number of tumor cells 3 times that of immature DCs (monocytes). Monocyte
cell suspension from culture flasks is centrifugated at 1500 RM for 10 min at
RT. After decantation of supernatant the cell pellet is resuspended in a small
volume of CellGro® culture medium and completed with CellGro® culture
medium up to 35 ml. The cell suspension is transferred to a culture flask.
GM-CSF (700 µl/7x104 IU) and IFN-α (17.5 µl/2.1x105 IU) in 35 ml of
CellGro ® culture medium are added to the monocyte cell suspension. The
ts of the culture flask are carefully mixed by gently moving the flask
from side to side. The cells are incubated in a 5% CO 2 humidified incubator
at +37°C for 3 days.
The prepared cell suspension of re DCs is collected and distributed
into 3 sterile 50 ml tubes. Fresh CellGro® culture medium (5ml) is added
meanwhile into each culture flask to avoid death of cells attached to the
surface of culture flask. The cell suspension is spun down at 1500 RPM for
minutes at RT. Supernatant is quickly and carefully decanted and the cell
pellets are ended in 2 ml of CellGro® culture medium and the cells
from the culture flasks are transferred to 1 tube. Each of the tubes used for
spinning of cells is gently rinsed with 4 ml of CellGro ® culture medium and
the content is transferred to the tube with collected cells. Tumor lysate (5ml)
obtained from tumor tissue or tumor cell lines (ratio of tumor cells:DCs = 3:1;
501x10 6:167x10 6) is mixed with 167x10 8 ial ghosts prepared from E.
coli Nissle 1917 (ratio of BGs:DCs = 100:1), vortexed thoroughly and
incubated with gentle shaking at RT for 60 min. The mix of BGs and tumor
cell lysate supplemented with GM-CSF (25 µl/2.5x104 IU) and IFN-α (6.25
µl/7.5x10 4 IU) is added to the DC suspension. Subsequently, the cell
suspension with all reagents is transferred to a culture flask. The tube which
contained the cell suspension is rinsed with 5 ml of CellGro® culture medium
and the medium is transferred to the culture flask and carefully mixed with
gentle shaking from side to side. The cells are incubated in a 5% CO2
humidified incubator at +37°C for 4 hours to allow alization of BGs and
tumor lysate and to start the maturation process. After 4h of incubation, the
cells are transferred to a 50 ml tube and the culture flask is gently rinsed with
CellGro ® culture medium. The medium used for g of flasks is
transferred to the tube with cell sion. Fresh CellGro® culture medium
(5ml) is added meanwhile into the e flask to avoid death of cells
attached to the surface of the culture flask. The cell suspension is spun
down at 1500 RPM for 10 minutes at RT. The supernatant is quickly and
carefully ed and cell pellets are resuspended in 20 ml of fresh
CellGro ® culture medium mented with GM-CSF (25 µl/2.5x10 4 IU) and
IFN-α (6.25 x104 IU). The cell suspension is transferred into the original
culture flask which is carefully and gently shaken from side to side, and the
cells are incubated in a 5% CO2 humidified incubator at +37°C for additional
Example 4. Storage of anti-tumor vaccine preparations.
A culture flask with stimulated DCs obtained in Example 3 is ghly
shaken to release cells adhered to the flask walls. The cell suspension is
completely transferred to a labeled 50 ml tube. The culture flask is rinsed
with two additional volumes of the same HBSS (10 ml) used for tumor lysate
preparation, if possible, and the whole volume of HBSS is transferred to the
tube with cell suspension. The original culture flask is filled with 5 ml of
HBSS and placed back into an incubator. The volume of cell suspension is
filled up to 50 ml with HBSS and gently mixed by tube overturns. The cell
suspension is spun down at 1500 RPM for 10 minutes at +4°C. The
supernatant is quickly and carefully decanted and cell pellets are
resuspended first in 5 ml HBSS, ed by on of additional 45 ml of
HBSS and mixed well by tube overturns. 10 µl of cell sion is used to
determine the cell number, using a Bürkner counting r. If the
concentration of cell within the suspension is below 1.4x106/ml, accutase
should be used to release the remaining cells from culture flask, otherwise
cell aliquots each containing of 5x106 cells will be frozen for future
administration to the patient. It is mandatory to make at least 6 aliquots for
administration to the patient, one aliquot for quality control, one aliquot for
monitoring, one aliquot for testing of mycoplasma and three aliquots
for arbitrage. The cell suspension is spun down at 1500 RPM for 10 minutes
at +4°C. Cryotubes are transferred to a pre-cooled MiniCooler. The
supernatant is quickly and carefully decanted; cell pellets are resuspended in
cryopreservation freeze media CryoStor CS2 and transferred to cryotubes.
Immediately after aliquoting of anti-tumor vaccine ation, all bes
are placed in an isopropanol box at -80°C. After 24h in -80°C, all frozen
vaccine preparation is transferred to a Dewar container filled with liquid
nitrogen specifically set for anti-tumor vaccine preparation purposes use
only.
Table 1- Standards for acceptability of anti-tumor vaccine ations
before stration to a patient.
Test Criterion to pass quality control
Sterility of anti-tumor vaccine preparation STERILE
Detection of mycoplasma NEGATIVE
Cell count (x106) 1x106 - 5x106
Viability (%) 70-100%
DCs purity (%) 70-100%
Cell CD3 (%) Total amount (CD3+ + CD19+)
contamination CD19 (%) 0-30%
CD80 (%) 60-100%
CD86 (%) 60-100%
DCs phenotype* MHC class II (%) 60-100%
CD83 (%) 60-100%
CD14 (%) 0-40%
Production IL-12 (pg/ml) ≥ 100 pg/ml
MC s
≥ 30%
Ratio 1:5
Allogenic MLR -
DCs:PBMC s
ted ≥ 30%
Ratio 1:10
T-lymphocytes (%)**
DCs:PBMC s
≥ 15%
Ratio 1:20
* at least 3 of 5 phenotypic markers should meet the criteria to pass quality control
before administration of anti-tumor vaccine preparation to a patient
** at least 2 of 3 examined ratios DCs:PBMCs in allogenic MLR tests should meet
the criteria to pass quality control before administration of anti-tumor vaccine
preparation to a patient
Claims (23)
- We claim: 5 1. Composition comprising (i) professional antigen-presenting cells, (ii) tumor-associated antigens, and (iii) bacterial ghosts. 10
- 2. Use of (i) professional antigen-presenting cells, (ii) tumor-associated antigens, and (iii) bacterial ghosts in the preparation of a vaccine for tumor immunotherapy.
- 3. The composition according to claim 1 or the use ing to claim 2, wherein the antigen- presenting cells comprise monocytes.
- 4. The composition or the use according to any one of the preceding 20 claims, wherein the antigen-presenting cells comprise dendritic cells.
- 5. The composition or the use ing to any one of the ing claims, comprising one or more tumor lysates. 25
- 6. The ition or the use according to any one of the preceding claims, comprising bacterial ghosts obtained from bacterial cells sing a gene encoding a lytic protein.
- 7. The composition or the use of any one of the preceding claims, 30 comprising bacterial ghosts which have been treated with β- propiolactone.
- 8. The composition or the use according to any one of the ing claims, comprising bacterial ghosts from Gram-negative bacteria.
- 9. The composition or the use ing to claim 8, comprising bacterial ghosts from E. coli. 5
- 10. The composition or the use according to claim 8 or claim 9, comprising bacterial ghosts from E. coli Nissle 1917.
- 11. The composition or the use according to any one of the preceding claims, comprising bacterial ghosts loaded with an activator or silencer 10 of the immune system, a pharmaceutical agent and/or DNA.
- 12. The composition or the use according to any one of the preceeding claims comprising bacterial ghosts carrying recombinant tumorassociated antigens.
- 13. The composition or the use according to claim 12, wherein the bacterial ghosts are loaded with tumor-associated antigens.
- 14. The composition or the use according to claim 12, wherein the bacterial 20 ghosts are derived from bacteria inantly sing tumorassociated antigens.
- 15. The composition or the use according to any one of the ing claims, comprising from 1 to 10,000 bacterial ghosts per antigen- 25 presenting cell.
- 16. The composition or the use according to claim 15, comprising from 10 to 1,000 bacterial ghosts per antigen-presenting cell. 30
- 17. The composition or the use according to any one of the preceding claims, sing 1 to 10 n antigen-presenting cells per dose.
- 18. The use according to claim 2, sing antigen-presenting cells which induce tumor-antigen recognition and tumor-specific killing by T cells. 5
- 19. The use ing to claim 2, comprising autologous antigenpresenting cells.
- 20. Therapeutic vaccine comprising the composition of any one of claims 1, or 3 to 19.
- 21. A composition according to any one of claims 1 or 3 to 17, susbtantially as herein described with reference to any example thereof.
- 22. A use according to any one of claims 2 to 19, substantially as herein 15 described with reference to any example thereof.
- 23. A therapeutic vaccine according to claim 20, substantially as herein described with reference to any e thereof.
Applications Claiming Priority (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161557532P | 2011-11-09 | 2011-11-09 | |
EP11188494.6A EP2591798B1 (en) | 2011-11-09 | 2011-11-09 | Vaccine for use in tumor immunotherapy |
US61/557,532 | 2011-11-09 | ||
EP11188494.6 | 2011-11-09 | ||
US13/665,145 | 2012-10-31 | ||
US13/665,145 US9790260B2 (en) | 2011-11-09 | 2012-10-31 | Vaccine for tumor immunotherapy |
PCT/EP2012/072040 WO2013068406A1 (en) | 2011-11-09 | 2012-11-07 | Vaccine for tumor immunotherapy |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ624069A NZ624069A (en) | 2015-10-30 |
NZ624069B2 true NZ624069B2 (en) | 2016-02-02 |
Family
ID=
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