U.S. patent application number 10/516915 was filed with the patent office on 2005-07-21 for method and composition for producing a cellular allogeneic vaccine.
This patent application is currently assigned to Immunicum AB. Invention is credited to Andersson, Bengt, Karlsson-Parra, Alex, Wallgren, Anna Carin.
Application Number | 20050158328 10/516915 |
Document ID | / |
Family ID | 29738554 |
Filed Date | 2005-07-21 |
United States Patent
Application |
20050158328 |
Kind Code |
A1 |
Karlsson-Parra, Alex ; et
al. |
July 21, 2005 |
Method and composition for producing a cellular allogeneic
vaccine
Abstract
A method for the production of a cellular allogeneic vaccine,
which is based upon an allogeneic APC, includes the following
steps: a) isolation of an APC from a subject or providing an APC
already established and/or isolated from a myeloid leukemia cell
line, and b) modifying the APC with an antigen using any of the
following methods: pulsing, transfection, infection or fusion; c)
treatment using an agent capable of removing sialic acid on cell
surfaces; and optionally d) culturing the APC in a suitable medium,
an allogeneic vaccine obtainable by the above method, a composition
comprising the vaccine and a pharmaceutically acceptable carrier
and therapeutic use of the vaccine or the composition.
Inventors: |
Karlsson-Parra, Alex;
(Molndal, SE) ; Wallgren, Anna Carin; (Molndal,
SE) ; Andersson, Bengt; (Molndal, SE) |
Correspondence
Address: |
YOUNG & THOMPSON
745 SOUTH 23RD STREET
2ND FLOOR
ARLINGTON
VA
22202
US
|
Assignee: |
Immunicum AB
GOTEBORG
SE
SE-411 19
|
Family ID: |
29738554 |
Appl. No.: |
10/516915 |
Filed: |
December 6, 2004 |
PCT Filed: |
June 5, 2003 |
PCT NO: |
PCT/SE03/00936 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60385898 |
Jun 6, 2002 |
|
|
|
Current U.S.
Class: |
424/184.1 ;
424/277.1; 435/372 |
Current CPC
Class: |
A61K 2039/5154 20130101;
A61K 39/0011 20130101; C12N 2501/70 20130101; C12N 2501/599
20130101; C12N 5/0645 20130101 |
Class at
Publication: |
424/184.1 ;
424/277.1; 435/372 |
International
Class: |
A61K 039/00; C12N
005/08 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 6, 2002 |
SE |
021726-7 |
Claims
1. A method for the production of a cellular allogeneic vaccine,
based upon an allogeneic APC, comprising the following steps:
isolation of an APC from a subject, preferably from a normal blood
donor or from a patient suffering from myeloid malignancies, or
providing an APC already established and/or isolated from a myeloid
leukemia cell line; b) modifying the APC with an antigen using any
of the following methods: pulsing, transfection, infection or
fusion; and c) treatment of the APC with an agent capable of
removing sialic acid on the surface of said APC; and optionally d)
culturing the modified APC in a suitable medium.
2. A method according to claim 1 comprising a further step between
step a) and step b): cultivation of an APC, preferably a monocyte,
or differentiation of an APC, preferably a monocyte, in a suitable
medium into monocyte-derived DC, macrophage or macrophage-derived
DC.
3. A method according to claim 1 wherein the APC is an allogeneic
monocyte or an in-vitro differentiated cell which is directly or
indirectly derived from an allogeneic monocyte.
4. A method according to claim 1 wherein the antigen, as recited in
step b), is a cancer antigen, preferably in the form of a soluble
antigen, a tumor cell lysate or a viable tumor cell, all of
allogeneic origin.
5. A method according to claim 4 wherein step b) is performed
through incorporating the antigen within the APC using any one of
the following methods: pulsing with soluble antigen or tumor cell
lysate, transfection with genes coding for the antigen or fusion
with an allogeneic tumor cell.
6. A method according to claim 1 wherein the agent capable of
removing sialic acid on the cell surface of the APC, as recited in
step c), is neuraminidase (NAS), one or more genes coding for
neuraminidase or neuraminidase-producing viruses or bacteria; or an
antibody against CD43.
7. A method according to claim 6 wherein the treatment of step c)
is performed by any of the following methods: treating the APC with
NAS, transfecting the APC with genes coding for NAS or infecting
the APC with NAS-producing viruses or bacteria.
8. A method according to claim 1 comprising an additional step in
which the APC is exposed to hyperthermia during step b) or between
steps b) and c).
9. A method according to claim 8 wherein the hyperthermia is
performed at a temperature of from 39 to 42.degree. C. and during
from 2 to 6 hours.
10. A cellular allogeneic vaccine obtainable by a method according
to claim 1.
11. A composition comprising a vaccine according to claim 10 and a
pharmaceutically acceptable carrier.
12. A frozen container comprising a composition according to claim
11.
13. Therapeutic use of an effective amount of a vaccine according
to claim 10.
14. A vaccine according to claim 10 for medical use.
15. Use of a vaccine according to claim 10 for the manufacture of a
medicament for use against cancer.
Description
[0001] This invention concerns the technical field of molecular
immunology and medicine, in particular a new method for the
obtaining of a cellular allogeneic vaccine based upon allogeneic
antigen-presenting cells (APCs), preferably monocytes, compositions
comprising said APCs and use of said APCs. The vaccine is
preferably used for treatment of cancer.
BACKGROUND
[0002] During the first half of the 1990's many research groups
worked departing from the hypothesis that autologous tumor cells
(taken from the same human being) could be reformed to be potent
antigen-presenting cells (APCs) through genetic modification. In
vivo studies in mice were reported in which tumor cells,
transfected ex vivo with genes coding for the co-stimulatory
molecule B7, used as cancer vaccine by injecting these cells into
the recipient had some success. The hypothesis was that the tumor
cell not only would express signal 1 (MHC class I+tumor peptide)
for CD8+ tumor specific T cells but also signal 2, B7, which
theoretically could lead to an efficient activation of these tumor
reactive CD 8+ CTL (cytotoxic T lymphocyte).
[0003] During a critical survey of the underlying immunological
mechanisms responsible for the often very positive effects of such
vaccination protocol, it was clearly shown that professional APCs
of the host, rather than the vaccinating tumor cells themselves,
were responsible for CTL priming. Most likely tumor cells
expressing B7 were efficiently killed by natural killer (NK)-cells.
This NK-cell mediated immune response has further been shown to
induce a local recruitment of host antigen-presenting cells (APCs),
including dendritic cells (DCs), whereby these cells take up whole
cellular proteins released into the tumor's microenvironment and
present them indirectly to CTL, so called cross-priming. This
indirect pathway of antigen presentation also explain why
vaccination with tumor cells transfected with the gene coding for
granulocyte-macrophage colony stimulating factor (GM-CSF) induce a
reasonably good anti-tumor response in rodent models since local
GM-CSF production have been shown to induce a local production of
macrophage inflammatory protein (MIP)-1 alpha which is a strong
chemoattractant for DC-precursors such as monocytes and immature
DC:s. It has moreover been shown that a local injection of a
plasmide vaccine expressing GM-CSF induces a local accumulation of
immature DCs at the vaccination site that is followed by the
appearance of mature DCs in regional lymph nodes, consistent with
egression of maturating DC from the injection site and migration to
the draining lymph nodes. The indirect pathway of tumor antigen
presentation also solves the problem with the demand for CD4+ T
helper type-1 (Th1) cells in order to achieve an efficient and
long-lasting tumor-specific CTL immune response since also MHC
class II restricted CD4+ cells can be activated via the indirect
pathway.
[0004] With this knowledge at hand it was now open for refining
this principle, ex vivo and in vivo, which is based upon an
efficient indirect presentation of tumor antigen by host
(autologous) APC. An obvious approach has been to propagate potent
APCs ex vivo, in particular dendritic cells (DCs), and thereupon
load these cells with tumor-derived proteins either by pulsing or
transfection. Also so called dendritoma vaccines, where autologous
tumor cells have been fused with autologous monocyte-derived DCs,
have been developed. A problem with ex vivo propagation of
autologous DCs is however the minor migration of these cells to
draining lymph nodes (a prerequisite for the injected DCs to meet
naive T cells). Studies in humans have shown that 1% maximum of
subcutanously injected DCs migrates to regional lymph nodes.
[0005] An alternative approach has therefore been developed, above
all from the observations during vaccination with GM-CSF producing
tumor cells in rodent models, with the goal to induce an efficient
recruitment in vivo of host DCs to the vaccination site. Phase I
trials in humans with prostate and renal carcinoma and melanoma
using autologous GM-CSF transfected tumor cells vaccines have been
evaluated and found to be safe but without any obvious clinical
effect. Additionally, a vaccine based upon autologous tumor cells
have been shown to create several technical problems. Firstly, the
vaccine depends on the availability of adequate numbers of tumor
cells, which are rarely available because of the reactive process
that are found infiltrating tumor cells of many common cancers.
Secondly, the vaccine requires de novo gene transfer for treatment
of each patient, which is labor intensive and may cause variable
cytokine expression levels between different patient vaccines.
Thirdly, there is significant expense and time required to certify
each patient's lot of vaccine so that they meet accepted
administration guidelines. One way to circumvent these technical
obstacles is to use a vaccine strategy that is based on a panel of
cytokine-expressing allogeneic tumor cell lines that can be
formulated and stored before the initiation of clinical studies.
This is a particularly attractive approach for the majority of
common cancers for which specific tumor antigens have not yet been
identified. Two findings provide the immunologic rationale for an
allogeneic tumor-cell vaccine approach. Firstly, that DCs of the
host, rather than the vaccinating tumor themselves, are responsible
for priming of CD4+ cells and CD8+ CTL, both of which are required
for generating systemic antitumor immunity (see above). Secondly,
many tumor antigens are commonly expressed among different
patient's tumors. A GM-CSF vaccine based on this concept has been
developed in rodent models (Chang E. Y. et al, (International
Journal of Cancer, 2000, Vol 86. No. 5, pp 725-730) and was
recently studied in a Phase I trial and found to be safe but
without any obvious clinical effect. Most likely, this clinical
insufficiency was due to the production of one single cytokine
(GM-CSF) since theoretically several factors ought to be produced
locally in order to induce not only an efficient recruitment of
immature DCs but also an efficient maturation of these cells.
Necessary factors most probably include chemotactic cytokines such
as MIP-1 alpha and/or RANTES, maturation factors such as
interleukin (IL)-1 beta, IL-6 and/or tumor necrosis factor (TNF)
alpha and finally Th1-polarizating factors such as interferon (IFN)
gamma.
[0006] Within the transplantation and transfusion areas there is
daily struggling with the problem of allo-immunization, which is a
T-cell-mediated immunization against indirectly presented
donor-specific HLA-antigens. Such immunization is frequently
developed with a strong power after transfusions with allogeneic
blood products and after transplantation of solid organs. Not even
a powerful continuous immunosuppressive treatment after a primary
successful transplantation of a HLA-incompatible organ appears to
prevent a slowly progressing process referred to as chronic
rejection. This process is mediated by CD4+ cells of Th1-type,
which are activated by allogeneic HLA-peptides indirectly presented
by autologous APCs. These CD4+ T cells in turn activate
donor-specific antibody (IgG)-producing B-cells and cytotoxic T
cells and tissue macrophages, which constitute the different
effector mechanisms during chronic rejection. A very central actor
for the starting-up of an allo-immunization appears to be viable,
metabolically intact, donor-derived (allogeneic) APCs. If these are
depleted or inactivated by UVB-irradiation before a transfusion of
e.g. platelet concentrates then the immunization is usually
avoided. This also pertain passenger APCs in transplanted tissue;
if these allogeneic APCs are depleted before transplantation the
risk for a subsequent immunization with chronic rejections is
essentially decreased. For non-viable allogeneic tissue the same
immunization rules pertains as for other foreign protein-derived
antigens i.e. in order to achieve an essential immunization it is
necessary to administer the antigen in a relatively large amount
together with an adjuvant as e.g. Freuds complete adjuvant (FCA).
The same is valid for in vitro primary stimulation with non-viable
MHC-expressing allogeneic APCs or pure MHC-derived allo-peptides
which do not induce any substantial priming of naive T cells
indirectly recognizing allo-derived peptides. A substantial priming
of these T cells is however obtained by using viable allogeneic
MHC-expressing APC during the primary stimulation.
[0007] Something that differ (discriminates) primary stimulation
with viable allogeneic APCs from primary stimulation with
non-viable (lysed or apoptotic) allogeneic APCs in vitro is the
very powerful T cell proliferation in the responder (host) cell
population, only seen during stimulation with viable APCs. This
reaction which is called allogeneic mixed leukocyte reaction (MLR)
also leads to the production of certain chemokines and cytokines,
including MIP-1 alpha, RANTES, IL-1 beta, IL-6, TNF-alpha and
IFN-gamma. The MLR is induced in both naive and memory responder
T-cells that are cross-reacting with MHC molecules on allogeneic
APCs through experiencing these molecules as their own MHC+foreign
peptide sequences which the T-cells that were predestined to react
against. It has been shown that as many as 1 out of 20 of our
circulating T cells may participate in this preformed
allo-reactivity. It is further known that treatment of
stimulator-APC with agents that reduce or remove sialic acids from
glycoproteins on the cell membrane, such as neuraminidase and
anti-CD43 antibodies, increase the potential of APC to induce a
proliferative response in allogeneic T cells.
[0008] A method for inducing an antigen-specific immune response by
co-administration of allogeneic DCs with autologous DC, in which
the antigen was incorporated, to a subject is disclosed in WO
99/47687. The autologous DC were expected to present the antigen to
CTLs while the allogeneic DC were expected to induce a strong
reaction from alloreactive T cells resulting in the local release
of stimulatory molecules that would amplify the ability of
autologous DC to activate CTLs. No data supporting their hypothesis
was presented nor any immune response elicited.
[0009] Vaccination with hybrid cells consisting of autologous tumor
cells fused with allogeneic mature DC is described in "Regression
of human metastatic cell carcinoma after vaccination with tumor
cell-dendritic cell hybrids", A. Kugler et al. Nature Medicine, vol
6, No 3, March 2000, pp. 332-336. Theoretically, this method is
based upon the expectance that co-expression of allogeneic MHC
molecule on the semi-allogeneic tumor cell (expressing autologous
MHC class I molecules+tumor peptides) would activate alloreactive T
cells. This activation would result in a local release of
stimulatory cytokines that would help to trigger the activation of
CTLs recognizing the tumor peptide on autologous, tumor
cell-derived, MHC class I molecules. Using this vaccine approach a
limited number of patients with renal tumor exhibited a clinical
anti-cancer response.
[0010] In WO 9421798 it is mentioned that transfection of DNA
encoding neuraminidase protein into autologous APCs (but not
allogeneic APCs) could be used to boost their ability to present
tumor antigens directly to autologous, MHC-restricted, T cells (see
page 3 line 20-23 and line 28, page 4 line 2 and page 7 line
22-26). This concept is based upon the central dogma within
immunology put forward by Zinkernagl and Doherty in the middle of
the 70's: A T-ell only recognize foreign peptides (e.g.
tumor-derived peptides from an autologous or allogeneic tumor) if
they are presented by APCs which express own MHC molecules (i.e
MHC-compatible APC), so called "self MHC-restriction".
[0011] Methods utilizing the immunogenicity of allogneic APCs as
adjuvant when vaccinating with antigen-loaded autologdus APCs have
earlier been disclosed as said above. In WO 99/47687, autologous
antigen-loaded APC were expected to present the antigen to
MHC-restricted autologous CTLs while the co-administered allogeneic
APC were expected to induce an inflammatory allogeneic response
that would amplify the ability of aotologous DC to activate
CTLs.
[0012] A vaccine against other tumors, using dendritic cells fused
with cancer cells, is also suggested in "Smallpox, polio and now a
cancer vaccine?", D. W. Kufe, Nature Medicine, vol 6, No 3, March
2000, pp. 252-253. The methods of Kugler et al and Kufe above are
however limited by a number of factors. Firstly, an autologous
vaccine depends on the availability of adequate numbers of tumor
cells, which are rarely available because of the reactive process
that is found infiltrating tumor cells of many common cancers.
Secondly, an autologous vaccine requires de novo gene transfer for
treatment of each patient, which is labor intensive and may cause
variable cytokine expression levels between different patient
vaccines. Thirdly, there is significant expense and time required
to certify each patient's lot of vaccine so that they meet accepted
administration guidelines.
[0013] In U.S. 20020039583 A1 there is further mentioned allogeneic
and also xenogeneic APCs loaded with immune complex containing
stress proteins as a thinkable cellular vaccine.
[0014] The methods disclosed in WO 99/47687 and in Nature Medicine
by Kugler at al are however limited by a number of factors. First
both methods are dependent on the availability of adequate numbers
of autologous cells (APCs and/or tumor cells). Secondly, both
methods require labour-intensive manipulations of autologous cells
for treatment of each patient. Thirdly there is significant expense
and time required to certify each patent's lot of vaccine so that
they meet accepted administration guidelines.
[0015] Accordingly there is a need for a vaccine that creates a
better immune response and is better suited for storing, producing
in a large scale and is independent of supply limitations.
SUMMARY OF THE INVENTION
[0016] The present invention solves the above problems by
providing, according to a first aspect a method for the production
of a cellular allogeneic vaccine which is based upon an allogeneic
APC, and comprising the following steps:
[0017] a) Isolation of an APC from a subject, preferably from a
normal blood donor, or from a patient suffering from myeloid
malignancies, or providing an APC already established and/or
isolated from a myeloid leukemia cell line;
[0018] b) modifying the APC with an antigen using any of the
following methods: pulsing, transfection, infection or fusion;
and
[0019] c) treatment of the APC with an agent capable of removing
sialic acid from the surface of said APC; and optionally
[0020] d) culturing the modified APC in a suitable medium.
[0021] According to a second aspect, a cellular allogeneic vaccine
obtainable by a method according to the first aspect is provided.
According to a third aspect, a composition comprising a vaccine
according to the second aspect and a pharmaceutically acceptable
carrier is provided. According to a fourth aspect, therapeutic use
of a vaccine according to the second aspect or a composition
according to the third aspect is provided.
DETAILED DESCRIPTION OF THE INVENTION
[0022] It is intended throughout the present description that the
expression "cellular allogeneic vaccine" embraces any reagent, cell
or compound capable of eliciting an antigen-specific immune
response in a subject, wherein said reagent, cell or compound is of
allogeneic origin i.e. originates from a donor, preferably a human
donor, other than the recipient, preferably a human recipient, of
the cellular allogeneic vaccine.
[0023] As used in the present specification and claims, the
singular form "a", "an" and "the" include plural references unless
the context clearly dictates otherwise. For example the term "a
cell" includes a plurality of cells, including mixtures
thereof.
[0024] The terms "antigen-presenting cell(s)", "APC" or "APCs"
include both intact, whole cells as well as other molecules (all of
allogeneic origin) which are capable of inducing the presentation
of one or more antigens, preferably in association with class I MHC
molecules, and all types of mononuclear cells which are capable of
inducing an allogeneic immune response. Preferably whole viable
cells are used as APCs. Examples of suitable APCs are discussed in
detail below and include, but are not limited to, whole cells such
as monocytes, macrophages, dendritic cells, monocyte-derived
dendritic cells, macrophage-derived dendritic cells, B cells and
myeloid leukaemia cells e.g. cell lines THP-1, U937, HL-60 or
CEM-CM3. Myeloid leukaemia cells are said to provide so called
pre-monocytes.
[0025] The terms "cancer," "neoplasm" and "tumor" used
interchangeably and in either the singular or plural form, as
appearing in the present specification and claims, refer to cells
that have undergone a malignant transformation that makes them
pathological to the host organism. Primary cancer cells (that is,
cells obtained from near the site of malignant transformation) can
be readily distinguished from non-cancerous cells by
well-established techniques, particularly histological examination.
The definition of a cancer cell, as used herein, includes not only
a primary cancer cell, but also any cell derived from a cancer cell
ancestor. This includes metastasized cancer cells, and in vitro
cultures and cell lines derived from cancer cells. When referring
to a type of cancer that normally manifests as a solid tumor, a
"clinically detectable" tumor is one that is detectable on the
basis of tumor mass; e.g., by such procedures as CAT scan, magnetic
resonance imaging (MRI), X-ray, ultrasound or palpation.
Biochemical or immunologic findings alone may be insufficient to
meet this definition.
[0026] The term "genetically modified" means, as appearing in the
present specification, containing and/or expressing a foreign gene
or nucleic acid sequence which in turn, modifies the genotype or
phenotype of the cell or its progeny. In other words, it refers to
any addition, deletion or disruption to a cell's endogenous
nucleotides. APCs modified using transfection, infection or fusion
are examples of genetically modified APCs. APCs that are only
pulsed are not genetically modified.
[0027] An "effective amount", as appearing in the present
specification and claims, is an amount sufficient to effect
beneficial or desired results. An effective amount can be
administered in one or more administrations, applications or
dosages. The vaccines of the present invention may be administered
or applied transdermally, orally, subcutaneously, intramuscularly,
intravenously or parenterally. For purposes of this invention, an
effective amount of the vaccines is that amount which provokes an
antigen-specific immune response in the subject. A suitable amount
may be approximately 10.times.10.sup.6 cells for administration to
one subject. An ampoule (which may be frozen and thawed before
using) may contain 20.times.10.sup.6 cells. Cells form a frozen
ampoule (or vial) may further be cultured again before
administrated to a subject. Obviously the amount will be determined
by the medical practitioner who will judge the nature and severity
of the condition of the subject in need of treatment. The condition
of the subject may also call for re-vaccination that may be
performed at any time point after the first vaccination, e.g. each
third to sixth week. Before administering the vaccine or the
composition according to the present invention said vaccine or
composition is preferably gamma-irradiated.
[0028] The term "an agent capable of removing sialic acid on cell
surfaces", as appearing in the present specification and claims,
refers to an agent that is capable of removing sialic acid from
cell surfaces. Examples of such cells are the APCs as set out
above. Agents which are capable of removing sialic acid from cell
surfaces can be exemplified by neuraminidase (NAS) and antibodies
raised against CD 43 (anti-CD43). Preferably said agent is NAS. NAS
is thought to cleave the sialic acid from glyco-conjugates and
anti-CD43 is thought to bring the sialic acid from the surface and
into the cell. Other examples of said agents are genes coding for
NAS or NAS-producing viruses or bacteria. The NAS may be obtained
from bacteria and viruses e.g. from Vibrio cholerae, Newcastle
virus, Influenza virus or Clostridium perfringens. A further
preferred agent is an antibody against CD43, i.e. the CD43 membrane
glycoprotein.
[0029] The term "culturing", as appearing in the present
specification and claims, refers to the in vitro propagation of
cells or organisms in media of various kinds. It is understood that
the descendants of a cell grown in culture may not be completely
identical (either morphologically, genetically, or phenotypically)
to the parent cell. A suitable culturing medium can be selected by
the person skilled in the art and examples of such media are RPMI
medium or Eagles Minimal Essential Medium (EMEM).
[0030] A "subject", as appearing in the present specification and
claims, is a vertebrate, preferably a mammal, more preferably a
human. Mammals include, but are not limited to, murines, simians,
humans, farm animals, sport animals, and pets. The subject may also
be a patient.
[0031] An "antigen", as appearing in the present specification and
claims, refers to any species, specimen or compound capable of
eliciting an immune response e.g. tumor specimen, a tumor cell, a
virus, a bacterium, a fungus, a parasite, a protein or a peptide.
The tumor specimen may further be obtained from any tumor.
According to a preferred embodiment of the present invention, the
tumor specimen is selected to represent as many other tumor types
as possible. If e.g. a vaccine against pancreatic cancer is
desirable, then a representative specimen of pancreatic cancer
(antigen) is used for the manufacture of a pancreatic cancer
vaccine. If instead e.g. a vaccine against breast cancer is
desirable, then a representative specimen of breast cancer
(antigen) is used for the manufacture of a breast cancer vaccine.
Said tumor specimen may further preferably be of allogeneic origin,
but also autologous tumor specimens are thinkable for use in the
present invention.
[0032] A "composition", as appearing in the present specification
and claims, is intended to mean a combination of active agent, i.e.
the vaccine, and another compound or composition, inert (for
example, a detectable agent or label) or active, such as an
adjuvant. In one particular aspect, a composition of this invention
comprises the vaccine and a pharmaceutically acceptable carrier
suitable for administration to the subject. A "pharmaceutical
composition", as appearing in the present specification and claims,
is intended to include the combination of an active agent with a
carrier, inert or active, making the composition suitable for
diagnostic or therapeutic use in vitro, in vivo or ex vivo.
[0033] As used herein, the term "pharmaceutically acceptable
carrier", as appearing in the present specification and claims,
encompasses any of the standard pharmaceutical carriers, such as a
phosphate buffered saline solution, water, and emulsions, such as
an oil/water or water/oil emulsion, and various types of wetting
agents. The compositions also can include stabilizers and
preservatives. For examples of carriers, stabilizers and adjuvants,
see Martin, REMINGTON'S PHARM. SCI., 15th Ed. (Mack Publ. Co.,
Easton (1975)).
[0034] The terms "major histocompatibility complex" or "MHC" refers
to a complex of genes encoding cell-surface molecules that are
required for antigen presentation to T cells and for rapid graft
rejection. In humans, the MHC complex is also known as the HLA
complex. The proteins encoded by the MHC complex are known as "MHC
molecules" and are classified into class I and class II MHC
molecules. Class I MHC molecules include membrane heterodimeric
proteins made up of a chain encoded in the MHC associated
non-covalently with .beta.2-microglobulin. Class I MHC molecules
are expressed by nearly all nucleated cells and have been shown to
function in antigen presentation to CD8+ T cells. Class I molecules
include HLA-A, -B, and -C in humans. Class I molecules generally
bind peptides 8-10 amino acids in length. Class II MHC molecules
also include membrane heterodimeric proteins. Class II MHC are
known to participate in antigen presentation to CD4+ T cells and,
in humans, include HLA-DP, -DQ, and DR. Class II molecules
generally bind peptides 12-20 amino acid residues in length. The
term "MHC restriction" refers to a characteristic of T cells that
permits them to recognize antigen only after it is processed and
the resulting antigenic peptides are displayed in association with
either a self class I or self class II MHC molecule.
[0035] The isolation of an APC of step a) in the first aspect of
the present invention may be performed in-vivo and/or in-vitro.
[0036] The present invention thus provides a method for provoking
antigen-specific immune responses, and in particular, immune
responses against tumor antigens.
[0037] According to a further embodiment of the first aspect of the
present invention there is provided a vaccine wherein said APC is
an allogeneic monocyte, i.e. a monocyte derived from a second part
(not derived from the same subject which is to be treated with the
vaccine according to the present invention).
[0038] In one aspect of the invention, the APCs, preferably
monocytes, may be isolated from the white blood cell fraction of a
mammal, such as a murine, simian or a human, preferably a human.
The white blood cell fraction can be from the peripheral blood of
the mammal, preferably a normal blood donor. This method includes
the following steps: (a) providing a white blood cell fraction
obtained from a mammalian source by methods known in the art such
as leukapheresis; (b) separating the white blood cell fraction of
step (a) into four or more sub-fractions by counter current
centrifugal elutriation. Monocytes can also be recovered from PBMC
by elutriation, Ficoll and Percoll gradients, through their ability
to adhere to plastic or through negative or positive selection
using antibodies linked to beads e.g. magnetic beads.
[0039] According to a further embodiment of the first aspect of the
present invention there is provided a method comprising a further
step between step a) and step b): cultivation of an APC, preferably
a monocyte, or differentiation of an APC, preferably a monocyte, in
a suitable medium into monocyte-derived DC, macrophage or
macrophage-derived DC. This may be accomplished by incubating
monocytes or myeloid leukemia cells in the presence of
differentiation factors (growth factors) such as GM-CSF and/or IL4
and/or TNF-alpha and/or macrophage colony forming factor (M-CSF)
and/or IFN-gamma in different combinations.
[0040] According to a further embodiment of the first aspect of the
present invention there is provided a method wherein the APC is an
allogeneic monocyte or an in-vitro differentiated cell which is
directly or indirectly derived from an allogeneic monocyte.
[0041] According to a further embodiment of the first aspect of the
present invention there is provided a method wherein the antigen,
as recited in step b), is a cancer antigen, preferably in the form
of a soluble antigen, a tumor cell lysate or a viable tumor cell,
all of allogeneic origin.
[0042] According to a further embodiment of the first aspect of the
present invention there is provided a method wherein step b) is
performed through incorporating the antigen within the APC using
any one of the following methods: pulsing with soluble antigen or
tumor cell lysate, transfection with genes coding for the antigen
or fusion with an allogeneic tumor cell. As a result there is
provided a vaccine adapted for use against cancer. Also a mixture
of different cancer antigens may be used.
[0043] According to a further embodiment of the first aspect of the
present invention there is provided a method wherein the agent
capable of removing sialic acid from the cell surface of the APC,
as recited in step c), is neuraminidase (NAS), one or more genes
coding for neuraminidase or neuraminidase-producing viruses or
bacteria; or an antibody against CD43.
[0044] According to a further embodiment of the first aspect of the
present invention there is provided a method wherein the treatment
of step c) is performed by any of the following methods: treating
the APC with NAS, transfecting the APC with genes coding for NAS or
infecting the APC with NAS-producing viruses or bacteria as recited
above. Preferably the APCs may be transfected with NAS after
modification, e.g. transfection, with the antigen as set out above
earlier.
[0045] According to a further embodiment of the first aspect of the
present invention there is provided a method comprising an
additional step in which the APC is exposed to hyperthermia (heat
stress) during step b) or between steps b) and c).
[0046] According to a further embodiment of the first aspect of the
present invention there is provided a method wherein the
hyperthermia recited above is performed at a temperature of from 39
to 42.degree. C. and during from 2 to 6 hours. This heat stress is
preferably performed at a temperature of from 39 to 42.degree. C.
during approximately 3 hours. Preferably pulsing, i.e. one way of
modifying the APCs, is performed at the same time i.e. in
parallel.
[0047] According to a further embodiment of the third aspect of the
present invention there is provided a frozen container (such as a
test tube, an ampoule or a vial) comprising a composition according
to the third aspect.
[0048] According to a further embodiment of the fourth aspect of
the present invention there is provided therapeutic use of an
effective amount of a vaccine according to the second aspect of the
invention or a composition according to the third aspect of the
invention.
[0049] According to a further embodiment of the fourth aspect of
the present invention there is provided a vaccine according to the
first aspect for medical use.
[0050] According to a further embodiment of the fourth aspect of
the present invention there is provided use of a vaccine according
to the first aspect for the manufacture of a medicament for use
against cancer.
[0051] An explanation to the unexpected effect, which the present
inventors are not bound to in any way, of the present invention
might be that the allogeneic modified APCs, preferably monocytes,
when injected into a subject generally induces a local MLR which
creates an environment which is favorable for recruitment and
maturation of DC-precursors which during their
differentiation/maturation also acquire e.g. tumor-derived peptides
from locally lysed allogeneic hybrid-cells i.e. modified APCs. Thus
the normally negative effect induced by a transplanted or
transfused allogeneic APC (allo-immunization) can be used in a
positive way e.g. immunization against other foreign antigens such
as cancer antigens.
[0052] For an immunologist/a person skilled in the art the concept
of the present invention is completely different from the technique
disclosed in WO 9421798, i.e. the supplying of tumor antigens
within a NAS-treated but MHC-incompatible allogeneic APC, is
completely different from the technique disclosed in WO 9421798
where MHC-compatibility between the NAS-transfected APC and T cells
is a prerequisite. For the person skilled in the art our concept
may even prima facie appear as lunacy (even if the APC would be
NAS-treated). This would be the case if the present strategy of the
present invention departed from the hypothesis that the NAS-treated
allogeneic APC would be able to present the tumor antigen for the
tumor specific T cell of the recipient. It is thus an immunological
matter of course that in WO 9421798 they have not included
allogeneic APC as thinkable APC. The present concept of the present
invention on the contrary is based upon using NAS-treated
allogeneic APCs as "carriers" of tumor antigen and at the same time
an adjuvant which within the recipient commences an inflammatory
reaction (of the same type but very likely stronger than the one
leading to priming of alloreactive T cells after transplantation of
allogeneic organs) which initially is not tumor specific.
[0053] The allogeneic APCs of the present invention are thus not
intended for presenting the tumor antigen for the recipients
T-cells self. On the contrary they operate as an adjuvant through
the induction of one, without regarding the tumor, unspecific
activation (alloactivation) of the recipients immune system. This
in turn leads to that they themselves become lysed (via activation
of alloreactive T cells/NK cells) so that their contents of tumor
antigen become accessible for APCs of the recipients which due to
the inflammatory process have been recruited to the vaccination
area.
[0054] Patent application U.S. 20020039583 includes
antigen-"loaded" allogeneic APCs (and xenogeneic APC) as potential
cellular components of a cellular vaccine. However the inventors
appear to believe that T cells recognize peptide/protein/immune
complexes which are located on a cell surface without necessity of
self MHC-restriction. Apparently not even an APC is necessary as
the inventors believe that a non-cell-associated complex may also
stimulate T cells (see page 13, right column "T cells may be
stimulated with stress protein complexes, polynucleotide encoding a
stress protein complex and/or and antigen presenting cell (APC)
that express such a stress protein complex"). It is never mentioned
that an allogenic APCs would be capable of operating as an adjuvant
in order to commence an immunologic reaction that would lead to the
recruitment of the patients (the subjects) own APCs. For a person
skilled in the art it must appear unlikely that the use of
antigen-"loaded" allogeneic APCs, as set out in U.S. 2002` would
lead to a direct activation of MHC-incompatible T cells. Further,
it appears even more unlikely that a person skilled in the art
would combine this concept with the NAS-concept disclosed in WO
9421798.
[0055] When comparing the technique disclosed in Chang E. Y. et al,
(International Journal of Cancer, 2000, Vol 86. No. 5, pp 725-730),
one of the aims therein is the same as in the present invention,
namely the recruitment of the patients APC to the vaccination site.
In the concept of the present invention we additionally obtain
production of chemokines such as MIP-1 alpha and RANTES, recruiting
monocytes/immature DCs and maturating factors such as IL-1 beta,
IL-6, TNF-alpha and TH-1 controlling IFN-gamma (see the
accompanying figures of the present specification).
[0056] Preferred features of each aspect of the invention are as
for each of the other aspects mutatis mutandis. The prior art
documents mentioned herein are incorporated to the fullest extent
permitted by law. The invention is further described in the
following examples in conjunction with the appended drawings, which
do not limit the scope of the invention in any way. Embodiments of
the present invention are thus described in more detail with the
aid of examples of embodiments and figures, the only purpose of
which is to illustrate the invention and are in no way intended to
limit its extent.
DESCRIPTION OF THE FIGURES
[0057] FIG. 1 illustrates allogeneic monocytes as carriers of tumor
antigens.
[0058] FIG. 2 illustrates the vaccine concept of the present
invention.
[0059] FIG. 3 illustrates the results of the pretreatment using
neuraminidase (NAS).
[0060] FIG. 4 shows an optimum formula for in vivo propagation of
mature DCs carrying phagocytosed tumor antigens, i.e. one preferred
embodiment of the present invention.
[0061] FIG. 5 shows that monocytes cultured in media containing 50%
(v/v) MLR conditioned media (MLR-CM) develop into CD83+ cells with
low expression of CD14 after 6 days (lower graph). The upper graph
depicts a control experiment without addition of MLR-CM.
[0062] FIG. 6 shows that MLR-CM (50 v/v %) induce a phenotypical
maturation (CD14 low, CD83+, CD86+, HLA-DR+ and CCR7+) of
monocyte-dervied immature DCs as illustrated in the graphs to the
right. The graphs to the left depict the cellular phenotype of
untreated monocytes.
[0063] FIG. 7 shows the cytokine profile determined in an MLR-CM
using Luminex.
[0064] FIG. 8 shows the impact of NAS on allogeneic tumor cell and
allogeneic tumor-APC chemokine production during MLR.
[0065] FIG. 9 shows chemokine and cytokine production in autologous
vs allogeneic MLR, using NAS-treated stimulator APCs.
[0066] FIG. 10 shows the impact of NAS-treatment on APC chemokine
production.
[0067] FIG. 11 shows the impact of anti-CD43 on monocyte chemokine
production.
EXAMPLES
Example 1
[0068] Preparation of Monocytes from Leukocytes
[0069] Monocytes may be obtained using peripheral blood of a human.
This method includes the following steps: (a) providing a white
blood cell fraction obtained from a human source through
leukapheresis; (b) separating the white blood cell fraction of step
(a) into four or more sub fractions by counter current centrifugal
elutriation. Monocytes can also be recovered from PBMC by
elutriation, Ficoll and Percoll gradients or through their ability
to adhere to plastic. Negative selection of monocytes using
magnetic beads linked to certain antibodies available from Dynal or
Miltenyi is also possible.
[0070] Modification of Monocytes
[0071] The monocytes may then undergo pulsing treatment whereby the
monocytes are treated (incubated) in a solution with pure soluble
tumor protein (antigen) at a protein concentration of 1-10 mg/ml
during 3 hours at 37.degree. C. Pulsing may also be used when the
antigen is tumor cell lysates obtained e.g. from a) repeated
heat--thawing or b) sonication both with subsequent centrifugation.
Then the protein concentration in the supernatant is measured and
adjusted to 100-200 .mu.g/ml.
[0072] Monocytes may further be fused (whereby using PEG, i.e.
polyethylene glycol, as fusion chemical; the PEG solution is
preferably 50% and is diluted subsequently) with tumor cells thus
creating immortalized cells (hybrids) and thus vaccines. The fusion
is performed between allogeneic tumor cells and allogeneic
monocytes from a second part (see further below) Transfection with
genes coding for antigens may also be used to create modified
monocytes i.e. vaccines. (see FIGS. 1 and 2).
[0073] Neuraminidase (NAS) Treatment
[0074] The modified monocytes may be treated with 25 mU/ml of NAS.
The NAS treatment has been shown to increase most of the
chemokines/cytokines (see FIG. 3). The monocytes may also be
pre-transfected with a suitable gene coding for a NAS e.g. from V.
cholerae.
[0075] Effect of Neuraminidase (NAS)-Treatment on Direct
Allorecognition and on Chemokine/Cytokine Production
[0076] It has been known for over a decade that treatment of
stimulator APC with neuraminidase, which leads to the removal of
sialic acid on the cell membrane, significantly enhance their
capacity to stimulate allogeneic T cells in an allogeneic MLR. It
has been suggested that increased intercellular adhesiveness
induced by decreased cell-surface charge is a likely cause of this
neuraminidase effect. It has also been suggested that neuraminidase
treated APC might provide some unidentified accessory factor
required by T cells for activation. It has now been observed that
allogeneic peripheral blood mononuclear cells (PBMC) treated with
NAS from Vibrio cholerae (25 mU/mL) induce a stronger proliferative
response among responder T cells in a one-way MLR than untreated
PBMC. Moreover, by measuring chemokine and cytokine production
during a one-way allogeneic MLR it was discovered that MIP-alpha,
RANTES, IL1-beta, TNF-alpha and IFN-gamma are produced in
several-fold higher amounts when the stimulator cells (irradiated
monocytes) have been treated with NAS (FIG. 3).
[0077] Hyperthermia
[0078] Hyperthermia (heat stress treatment) of modified cells may
be performed by subjecting the modified monocytes to heating at
40.degree. C. during 3 hours. The fever treatment has further been
shown to upregulate the amount of HSP-coupled tumor antigen (in the
lysosome and/or in the cytoplasm) within the monocytes. This
treatment has also been shown to increase the amount of
immunogeneic tumor peptides. The presentation is not negatively
affected.
[0079] The Effect of Heat Stress on Direct Allorecognition
[0080] Cytosolic as well as lysosomal proteins are known to be
associated to heat shock proteins (HSP) during heat stress and it
is well established that HSP associated peptides become more
immunogenic in vivo. This is likely due to a more efficient uptake
by DC (receptor-mediated endocytosis/phagocytosis). Moreover
certain HSPs have been shown to induce maturation of immature DC in
vitro. It has been found that a mild hyperthermic stress
(41.degree. C. for 2 hours), known to induce a substantial
up-regulation of cellular HSP-expression, do not negatively affect
the stimulatory capacity of irradiated PBMC to induce allbgeneic T
cells responses as determined by proliferation in a one-way MLR
(data not shown).
[0081] Phenotypical Studies by FACS and Cytokine/Chemokine
Measurements
[0082] Earlier studies by others have shown that monocyte
conditioned medium (supernatants taken from monocyte cultures
activated by their Fc-receptors) as well as T-cell conditioned
medium (supernatants taken from T cell cultures activated by
stimulation with anti-CD3 antibodies) efficiently induce DC
maturation. Our hypothesis was that the immune interaction between
allogeneic monocytes and responder T cells leads to the production
of a chemokine/cytokine "cocktail" that also could induce a final
maturation of immature DC in vitro. Through performing primary
phenotypic studies during a traditional MLR an interesting
phenomenon was discovered. During a so-called one-way MLR
(irradiated stimulator cells) but not during two-way MLR or during
stimulation with nectrotic or apoptotic allogeneic monocytes a
subpopulation of CD83 positive cells with low expression of CD14
but high expression of CD86 and HLA-DR was found. By adding MLR-CM
(50% v/v) taken at day 6 from one-way allogeneic MLRs using
irradiated plastic-adherent mononuclear cells as stimulators
(1.times.10.sup.5 cells/ml) and allogeneic PBMCs (1.times.10.sup.6
cells/ml) to adherent monocytes in culture medium it was further
observed that these "bystander" cells developed a similar phenotype
(CD83+/CD14 low) after 6 days as illustrated in FIG. 5. MLR-CM (50
v/v %) were then added to immature, monocyte-derived DCs
(differentiated in GM-CSF/IL4 for 6 days). Two days later (day 8),
the cells where phenotyped by FACS. It was found that the monocytes
had differentiated into phenotypically fully mature DCs as
illustrated in FIG. 5 where the graphs to the left depict the
cellular phenotype at day 0 and the graphs to the right depict the
phenotype at day 8. Of particular interest is that a high
expression of CCR7 could be shown on these mature DCs which is of
central importance for the migration to regional lymphatic glands.
By using a multi-analyte profiling technique (Luminex) the cytokine
profile was determined in an MLR-CM and several cytokines with
known effect on monocyte differentiation and maturation of DCs were
found including TNF-alpha, IFN-gamma, IL-1 beta. The results are
depicted in FIG. 7. Additionally several chemokines are produced
including MIP-1 alpha. Through pre-treatment of the stimulatory
cells with neuraminidase (NAS) from Vibrio cholerae an additional
increase of the chemokine/cytokine production in a MLR was achieved
(see FIG. 3).
[0083] To conclude, the above discovery could well explain why
viable passenger APCs are so important for the allo-immunization.
The preformed T-ell reactivity against directly presented
allo-antigen on these cells thus leads to a favourable environment
for recruitment and local maturation of DCs of the transplanted
individual. This accumulation and maturation of DCs is a favourable
prerequisite for a subsequent efficient priming of naive
alloreactive T-cells (which recognizes allopeptides via the
indirect route) in secondary lymphoid organs.
Establishment of Monocytoma Cell Lines by Fusion of Human Cancer
Cell Lines with Allogeneic Peripheral Blood Monocytes
[0084] Human tumor cell lines (TCLs) including the breast cancer
cell line MCF7 and the prostate cell line DU 145, purchased from
the American Tissue Culture Collection, may be used. The TCLs may
be cultured in RPMI 1640 medium supplemented with 10%
heat-inactivated human AB serum. 2 mM L-glutamine, 100 U/mL
penicillin, and 100 microgram/mL streptomycin until fusion.
Isolated allogeneic peripheral blood monocytes from healthy blood
donors may be incubated with the TCL for 5 minutes at a ratio of
10:1 in serum-free RPMI 1640 medium containing 50% polyethylene
glycol (PEG). RPMI 1640 is then added slowly to dilute the PEG.
After washing and elimination of unfused cells by density gradient
the monocytomas may be resuspended in RPMI 1640 medium supplemented
with human AB serum, L-glutamine, penicillin and streptomycin as
stated above.
[0085] Selection of Monocytoma Cell Lines from Non-Hybrid Cells
[0086] The generation of cell hybrids may be conducted in analogue
with the generation of hybridomas producing monoclonal antibodies.
This relies on that the HGPRT can be fused for both positive and
negative selection. At a first, 6-thioguanine may be used to select
cancer cell lines that have mutated the HGPRT gene. This chemical
is converted to a toxic intermediate by the HGPRT gene product. The
mutated cells are subsequently fused to monocytes/dendritic cells
using PEG or electrofusion, and when grown in HAT media only fused
cells will survive. This is due to that HAT media will kill the
cancer cells lacking the HGPRT gene and that monocytes/dendritic
cells are not long-lived in culture although they have an active
gene.
[0087] In Vitro Priming of Human Th1 Cells Against Tumor
Antigens
[0088] The ability of a primary allogeneic MLR to induce a
substantial priming of antigen-specific human CD4+ Th1 cells may be
assessed a follows:
[0089] Monocytes may be pulsed with either soluble tumor proteins
(1-10 mg/mL for 3 hours) including prostatic soluble antigen (PSA)
and cancer antigen (CA)-125 or repeteadly freeze-thawed tumor cell
lysates (100-200 microgram cancer cell protein/mL for 3 hours) from
tumor cell lines including breast cancer and prostate cancer cells.
After washing and gamma-irradiation the pulsed monocytes may be
used as stimulator cells in a primary MLR using allogeneic PBMC as
responders. After 6 days the cell culture may be washed and
replaced into fresh culture medium containing 10 U/mL interleukin
(IL)-2. Two days later the cells from the primary MLR may be
restimulated by adding irradiated PBMC, autologous to the responder
cells in the primary MLR, pulsed with the antigen that was used
during the primary MLR. The number of CD4+ Th1 cells that were
primed during the primary MLR can then be recorded 1-2 days later
by using an ELISPOT assay specific for IFN-gamma producing
cells.
[0090] Using the same assay principle the ability of allogeneic
monocytomas to prime CD4+ Th1 cells during a primary MLR can be
studied after restimulation with autologous PBMC pulsed with a cell
lysate from the monocytoma used in the primary MLR.
[0091] Comparison with Prior Art Techniques
[0092] A comparison was made with the prior art techniques
disclosed in WO 9421798 and Chang E. Y. et al, (International
Journal of Cancer, 2000, Vol 86. No. 5, pp 725-730.
[0093] In Chang et al it is described that through injecting tumor
cells which produces GM-CSF they try to induce an inflammatory
environment which leads to the recruitment of the patients own APC
to the site of the vaccination (further studies around the effect
of GM-CSF have shown that local GM-CSF production induces MIP-1
alpha production which could explain the observed recruitment of
immunological cells, including APC to the vaccination site). As
these GM-CSF-producing tumor cells are not intended to operate self
as APC for tumor specific T cells you may use autologous as well as
allogeneic tumor cells as antigen source. A precondition is however
that they contain the same tumor antigen which is expressed in the
tumor of the patient. The selection of allogeneic tumor cells in
Chang et al is essentially of pure practical nature even though
they mention that allogeneic vaccines (i.e. vaccines which
comprises allogeneic tumor cells, not APC) may give a better immune
response compared with autologous tumor cell vaccines (not APC).
The obtained vaccine effect in the study of Chang E. Y. et al are
essentially not to be ascribed, according to the authors, to that
the tumor cells were allogeneic (most of the tumors are not
comprised by APC-derived cells and thus they miss MHC class II
which is the most important component in order to induce an
inflammatory reaction against allogeneic cells) but to their
capability to produce GM-CSF. In their publication there was also a
formal comparison done between immunogenicity of allogeneic tumor
cells (B16E7) and GM-SCF producing B16E7 cells (B16GME7). The
result disclosed shows clearly that allogeneic tumor cells which do
not produce GM-SCF have a minimal vaccination effect in comparison
with GM-CSF producing cells. These findings corresponds with
results which we now have obtained in vitro through comparing the
inflammatory reaction (chemokine/cytokine production) which arises
when mixing immune cells (mononuclear cells from peripheral blood)
with allogeneic tumor cells (THP1 cells which comprises precursors
of monocytes) or allogeneic APC (THP1 cells which via culturing in
GM-CSF and IFN-alpha differentiated out to functional APC), see
FIG. 8. Allogeneic tumor cells (including precursors to APC as
THP-1 cells) thus appear to be weak immune stimulators if they are
compared with fully matured allogeneic APC. Which can be seen in
FIG. 8, NAS-treatment of allogeneic APC leads to a further strong
increase in chemokine/cytokine production.
[0094] We have in an in-vitro model observed that the inflammatory
reaction which arises when a potent APC (the vaccine component) is
brought into contact with allogeneic mononuclear cells (corresponds
to immune cells of the patient), then, besides MIP-1 alpha, several
other chemokines with well known ability of recruiting APC are
produced. Additionally, several pro-inflammatory cytokines are
produced, with well known ability to additionally make recruited
immature DCs to develop into fully mature DCs (neither GM-CSF nor
MIP-1 alpha have the ability for this). Through incubating immature
DCs with medium from the above reaction we have also been able to
show that this medium really induces a DC maturation. Finally,
through pre-treating allogeneic APCs with NAS we have observed that
chemokine- and cytokine-production multiplies in the above
model.
[0095] Further we studied if there were any difference between
NAS-treated autologous and allogeneic APCs regarding the induction
of inflammatory chemokines and och cytokines. In a comparative
study, the concept of WO 9421798 was used in comparison (with the
present invention), wherein the inventors in WO 9421798 got a
vaccine effect. In our comparative study (see FIG. 9) it was shown
that NAS-treated APCs induce a more powerful inflammatory response
of allogeneic responder cells in comparison with autologous
responder cells. Which is apparent from FIG. 9 (Auto A, Auto B
represent autologous MLR, Allo A and Allo B represent allogeneic
MLR) is that above all the IFN-gamma production is influenced (an
approximately 100 times more powerful production in the allogeneic
situation). In addition we have observed that NAS-treated APCs
normally induce 50 to 100-times stronger proliferation of
allogeneic responder cells than autologous. As IFN-gamma is one of
the most important cytokines in order to control the immune
response against a so called TH1-response, a response which you aim
for during cancer vaccination, the potential of our vaccine-concept
is even more apparent. As also can be seen is that allogeneic tumor
cells in themselves are poorly immunogenic (see FIG. 9). An
indication of an assessable activation in vitro of allogeneic
responder cells could not be found, not even if the "stimulating"
tumor cells were NAS-treated. If on the other side the tumor cells
are comprised by fully matured APCs then NAS-treatment leads to a
very powerful inflammatory response with the production of several
of the above mentioned cytokines and chemokines.
[0096] The Impact of NAS-Treatment on Different APCs as to CC
Chemokine Production
[0097] It has been reported that immature DCs produce higher levels
of CC chemokines than mature DCs upon stimulation with
lipopolysaccharide and that macrophage-derived DCs are more potent
than monocyte derived DCs in this aspect. We have compared freshly
isolated monocytes with macrophages (monocytes differentiated in
M-CSF media for 7 days) and immature DCs (monocytes differentiated
in GM-CSF+IL4 for 7 days) and found that monocytes were more potent
than macrophages or DCs to produce CC chemokines upon NAS-treatment
(1.times.10.sup.6 cells/ml stimulated with Vibrio cholerae
neuraminidase 25 mU/ml for 24 hours). These findings are
illustrated in FIG. 10.
[0098] The Effect of Anti-CD43 on Monocyte Chemokine Production
[0099] Treatment of monocyte-derived dendritic cells with
antibodies against the CD43 membrane glycoprotein have been shown
to down-regulate sialic-acid expression on the cell membrane and to
induce production of pro-inflammatory cytokines such as IL1-beta,
11-6 and TNF-alpha. We have studied the impact of anti-CD43 on
monocyte chemokine production and found that anti-Cb43 also induce
a substantial production of chemokines which is illustrated in FIG.
11.
[0100] Various embodiments of the present invention have been
described above but a person skilled in the art realizes further
minor alterations, which would fall into the scope of the present
invention. The breadth and scope of the present invention should
not be limited by any of the above-described exemplary embodiments,
but should be defined only in accordance with the following claims
and their equivalents. For example, any of the above-noted
compositions and/or methods can be combined with known therapies or
compositions. Other aspects, advantages and modifications within
the scope of the invention will be apparent to those skilled in the
art to which the invention pertains.
* * * * *