Field of the Invention: The present invention provides a composition comprising population of activated dendritic
cells that are adapted to produce immunogens useful in treatment of cancer and similar
conditions. The invention also provides a method for producing population of activated
dendritic cells.
Background of the Invention:
It is known that dendritic cells play an important role in generating immune response by
presenting antigen to the T lymphocytes. Dendritic cells can be found in tissues such as skin,
inner lining of the nose, lungs, stomach and intestines. These are immature dendritic cells that
may have originated in the bone marrow and migrate throughout the body. Such cells are
immature and lay dorinant waiting to interact with invading pathogens or other foreign
bodies.
Dendritic cells assume great importance as they are adapted to process antigenic material and
present it on the cell surface for recognition by other cells of the immune system. These cells
have a unique morphology and cell surface characteristics which enables them to initiate
appropriate immune responses, especially T cell specific responses. The branches/processes
of the dendritic cells are thin and long and these processes drape around the cell bodies of
lymphocytes which are adapted to bind to the dendritic cells, The processes of the dendritic
cells embrace other cells of the immune system and with their constant movement deliver
antigens and other signals that are key to initiate immune responses.
In the case of a wound accompanied by inflammation, dendritic cells are attracted to the area
of inflammation and stimulated to capture and internally process antigens. Hence, the primary
function of an immature DC is to find and capture foreign bodies and antigens. Once
captured, the antigen is processed either by an exogenous or endosomal pathway, or by
endogenous or proteosomal pathway. Dendritic cells grab antigens from viruses, bacteria, or
other organisms and process the antigen and present the antigenic portions to T cells to
recruit an initial T cell immune response.
In particular, it appears that dendritic cells present antigens that activate both CD4+ and
CD8+ T cells. The proteins expressed by dendritic cells include co-stimulatory molecules
such as CD80 and CD86 that bind CD28 on T cells or MHC class I or class 11. Normally, this
I
is achieved by presenting a class of chemicals such as MHC class I which stimulates CD8+
cytotoxic T cells, and the antigen or protein is taken up by phagocytosis or receptor mediated
endocytosis into the cytosol of the dendritic cell. The antigens are further degraded in the
cytosol via proteosome and enter the endoplasmic reticulum where peptides bind to newly
synthesized MHC class I molecules for presentation on the cell surface. In case of MHC class
I1 presentation, CD4+ T helper cells are stimulated, and antigen is taken up by phagocytosis
or receptor-mediated endocytosis to endosomes where some proteolysis occurs. The peptides
enter a vesicle containing MHC class I1 where they bind and are transported to the cell
surface.
All of these mechanisms work well in normal cells and normal pathogen invasions.
However, cancer cells are often capable of evading the selflnon-self detection system in the
body; for example by generating IL-10, TGF-P or similar compounds that deregulate the
immune system. Another problem is that dendritic cells are found in very low levels in the
peripheral blood as compared to white blood cells or other leukocytes, and hence insufficient
to elicit appropriate immune response against cancerous tissue.
Hence, there is a great deal of interest in trying to harvest the potential of dendritic cells as
immune educators and developing new therapies for treatment of diseases such as cancer.
Dendritic cells are being studied as adjuvants for vaccines or as a direct therapy to induce
immunity against cancer. That dendritic cells may prove useful in cancer has been most often
studied in animal models. By modifying dendritic cells, researchers are able to trigger a
special kind of autoimmune response that includes a T cell attack of the cancer cells. Because
a cancer antigen alone is not enough to rally the immune troops, scientists first fuse a
cytokine to a tumor antigen with the hope that this will send a strong antigenic signal. Next,
they grow a patient's dendritic cells in the incubator and let them take up this fused cytokinetumor
antigen. This enables the dendritic cells to mature and eventually display the same
tumor antigens as appear on the patient's cancer cells. When these special mature dendritic
cells are given back to the patient, they waive their newly acquired tumor antigens at the
patient's immune system, and those T cells that can respond mount an attack on the patient's
cancer cells.
As a result of these studies, dendritic cells loaded with tumor lysates, tumor antigen-derived
peptides, MHC class I restricted peptides, or whole protein have all been shown to generate
t'
I
O R I ~ ~ ~ ! ~ ~
'7 .d 7 7 DEI- 1 31 ' I
1 3 JUN 2011
anti-cancer immune responses and activity, including in some cases the ability to induce
regression of existing tumor as for instance in US 20120244620.
However, most of the methods in the prior art use chemical agents to culture the dendritic
cells or use activators in order to stimulate/activate the dendritic cells. In addition the
methods in the prior art are antigen specific, in that the dendritic cells are primed with one or
more specific antigens. As a result the dendritic cells primed are adapted to present one or
more specific antigens to the body and accordingly appropriate immune response which is
limited to one or more specific antigen only, which sometimes inefficient in treating cancer.
It is therefore desirable to obtain populations of dendritic cells which are enriched and
adapted to express substantially all the antigens as may be found in the tumoral tissues. In
addition, it is desirable to provide methods for treatment of cancer which are not time
consuming, labor intensive, costly, having high specificity. The present invention seeks to
eliminate these disadvantages of the prior art and provide an efficient method for treatment of
cancer.
Detailed Description of the Figures:
Figure 1 is a graphical representation of the percentage of cells adhered in presence or
absence of ~ aK++ A TPase inhibitor.
Figure 2 is a graphical representation of the percentage of inhibition of monocytes in
presence of ~ aK++ A TPase inhibitor.
Figure 3 is a graphical representation of the percentage of cells adhered in presence or
absence of PPAR gamma agonists.
Figure 4 is a graphical representation of the percentage of inhibition of monocytes in
presence of PPAR gamma agonists.
Figure 5 is a graphical representation of the amplification of the immunogenicity of the
dendritic cells incubated with Oxidised tumor lysate.
Summary of the Invention:
It is an object of the invention to provide a composition comprising population of activated
dendritic cells that are adapted to produce immunogens useful in treatment of cancer and
similar conditions. It is also an object of the invention to provide a method for producing
population of activated dendritic cells and to provide a cost-effective and efficient method for
treatment of cancer.
4
A. ORIGINAL ,
Detailed Description of the Invention:
I 1 3 JUN 2011
Accordingly, in one aspect the invention provides a composition comprising a population of
mature activated dendritic cells adapted to express substantially all antigens as present in a
tumor lysate derived from a malignant tumour tissue, and adapted to evoke adaptive
immunity in a host.
The invention also provides a method for producing a population of mature activated
dendritic cells, comprising the steps of:
a. preparing an antigen enriched tumor lysate from a tumorous tissue;
b. isolating plasma blood mononuclear cells (PBMC) from blood sample and
isolating monocytes;
c. culturing the monocytes in a suitable growth media for about 2-4 hours at 3 7 ' ~
incubating with suitable nutrient medium containing only GM-CSF to obtain
immature dendritic cells (iDC);
d. contacting and pulsing the population of Immature Dendritic cells (iDc) with the
antigen enriched tumor lysate;
e. further culturing the iDC population for 2-days to obtain a population of mature
activated dendritic cells adapted to express substantially all antigens as present in
the tumor lysate derived from the malignant tissue.
According to the invention, the antigen enriched tumor lysate is prepared from tumoral cells
obtained from the malignant tissue of a patient. The tumoral tissue may be selected from any
of prostrate, lung, brain, pancreatic, liver, ovary, breast, colon, gastric tumor or any other
solid tumour. Preferably, the tissue may be obtained from the tissue extracted from the patient
during surgery; it may also be obtained through biopsy or any other mode. In the alternative,
a cell line from a tumor tissue may be employed as a source of antigens. In an embodiment
the tumor cell line may be grown in a SCID mouse using xenograft approach and extract
antigens after developing population of desired tumor.
Initially, after extraction of the tumor tissue, a homogenate is prepared which is subjected to
repeated freeze-thaw cycles to obtain a lysate, which is centrifuged. The supernatant which
comprising multiple proteinslantigens and is stored for further use and treated with oxidizing
agents such as NaOCl and HC104 and/or adjuvants. Such supernatant which comprises a
plurality of antigens as present in the humanlpatient is preferred over prior art techniques that
-, "r 4- (JkA (7j ,r. ~d'fr '\1'7 t ~ ~1 3D JUN 2E011 L
focus on singular antigen or expression of singular antigen. The said supernatant is referred to
"antigen enriched tumor lysate" hereafter.
According to one aspect of the invention, the peripheral blood of the patient is obtained by
apheresis or leukopheresis. In the alternative the mononuclear cells may also be derived from
bone narrow, umbilical' cord and similar sources from a patient. The peripheral blood
collected may comprise mononuclear cells and other cells. Optionally, an anti-coagulant
along with culture media and heparin may be added to the blood so obtained so as maintain a
healthy population of Mononuclear cells. The population of cells so obtained may comprise
substantial number of lymphocytes. An important advantage of the method of the invention is
that the patient is not required to undergo any treatment prior to peripheral blood collection.
The population of Peripheral Blood Mononuclear cells collected as a result of apheresis and
Leukopheresis undergoes Density gradient separation to obtain a pure Peripheral Blood
Mononuclear cells free of RBC contamination. As a result of Density gradient method, pure
peripheral blood mononuclear cells are separated substantially free of RBCs or Plasma. An
advantage of the invention is that it does not involve the use of any cytokines such as
interleukins in order to convert the monocytes into immature dendritic cells//activated
monocytes.
In one aspect the Peripheral Blood Mononuclear cells are cultured on a substrate coated with
an activator such as polylysine and cultured in suitable growth media for 2-4 hours at 37 OC
in C02 incubator. The culture is washed with the culture media as a result the other cells like
granulocytes, lymphocytes are washed away. The adhesion of monocytes to the substrate is
prevented by addition of PPAR gamma agonist and ~ aK++ A TPase Inhibitors. The Cells are
added with RPMI media along with GM-CSF and Growth factor and autologous plasma
(0.8%) and incubated at 3 7 ' ~in C02 incubator and the culture is replenished every alternate
day till the 5' Day. On the sixth day the culture is pulsed with enriched tissue lysate together
with an adjuvant such as po1yI:C. The addition of such adjuvant increases the immune
density of the antigen. The cells are grown so as to permit sufficient uptake of antigen and its
expression on cell surface. After the dendritic cells are "pulsed" with tissue lysate it may take
two days for the dendritic cells to process the antigen for presentation to nake and memory
T-cells.
Once the DC has engulfed and processed the antigen, mature antigen-primed dendritic cell
population is obtained.
The population of dendritic cells so obtained after approximately 8 days as mature activated
Dendritic cell population is then ready for administration patients.
This inventive method of treating cancer achieves a therapeutic effect that is greater than the
therapeutic effect achieved by administration of conventional dendritic cell compositions. In
this respect, the therapeutic effect is typically measured by a mean delay in the time of
progression of the cancer in a subject. Alternatively, the therapeutic effect or therapeutic
index can be determined according to the effect on tumor volume, which can be inferred from
tumor area, or staging of cancer in the patient's body. For example, stabilization of the tumor
after administration of few doses would represent a significant therapeutic effect as it would
be indicative of the fact that the administration of the population of dendritic cells prepared
according to the invention has arrested further growth or spread of the tumor and same may
be eventually controlled or reduced. Alternatively, the therapeutic effect can be measured by
observing the change in the one year survival rate, the five-year survival rate, or ten-year
survival rate. An increase in therapeutic effect may also mean decreasing the side-effects and
improving quality of life of the patient. In a further aspect, the dose of population of dendritic
cells administered to a patient is far less than what is administered through conventional
therapies. Hence decreasing the quantity of dendritic cell population administered to a patient
to achieve a therapeutic effect is a significant improvement because dendritic cell-mediators
are sometimes proven be toxic to the subject to which they are administered.
The present inventive method may be carried out by administering a predetermined
quantityldose of dendritic cell composition of the invention to a patientlsubject in need
thereof. Thus, the therapeutic method allows an increase in the therapeutic index by reducing
the number andlor severity of side-effects while still enjoying the anticancer benefit of
administering a dendritic cell-based composition/population.
Preferably the patient may be given 6 dosages with a gap of two weeks between two dosages.
Each dosage may comprise at least one million mature primed dendritic cells. This is far less
than conventional dendritic cell therapies that require administration of aleast 2 million cells
to achieve a therapeutic effect. The entire process of preparing the population of mature
I
active dendritic cells is completed within 8 days, as compared to conventional process which
take more than 10-1 5 days.
An advantage of the method of the invention is that the dendritic cells are primed with
substantially all the antigens as may present in the tumor tissue of the patient. In other words
it comprises a plurality of antigens and represents substantially all the antigens present in a
cancerous tissue. Such dendritic cells are found to provide better therapeutic effect as
compared to conventional compositions which employ a specific antigen and the dendritic
cells are primed accordingly.
Examples:
Example 1: Preparation of tissue lysate
Histopathologically confirmed tumour tissue obtained from biopsy is used as starting
material for tissue lysate. Tumour tissue is washed with Phosphate Buffer Saline (PBS) and
tissue lysate is obtained using tissue homogenizer. The lysate is subjected to 5 cycles of
freeze and thaw. The vial is kept for Induction on a hot plate at 60°C for 1-2 hr. Total protein
content of the lysate is estimated using Bradford protein assay.
Western blotting of the tissue lysate is also carried out for detecting marker protein for the
specific cancer (Atleast one protein from the list of proteins, specified in table 1). Tumour
lysate in which total protein content and presence of cocktail of tumour associated/specific
protein is confirmed, is further filtered through 0.22pm syringe filter and preserved. This
tissue lysate is treated with different concentrations (10- 100 micro molars) of oxidizing
agent (sodium hypochlorite- NaOCl and perchloric acid HC104).This tissue lysate is used
krther for priming immature Dendritic cells (iDC).
Type of Cancer
Breast Cancer
Prostate Cancer
Colorectal carcinoma
Ovarian Cancer
Uterine Cancer
Cervical Cancer
Tumour Marker Antigens
ER, PR, Hedlneu, MUC-1, BRCAI,
PSMA, ERK5,
CEA, CA242, TK
CA125, CA15.3
CA 125
CA125, CEA
Example 2: Culture of Dendritic cells from Blood
120 ml of Peripheral Blood Mononuclear cells (PBMCs) consisting of Lymphocytes,
Monocytes and Macrophages are separated from whole blood of patient by Leukapheresis. If
desired, a nutrient media (Nutriprep), 50ml consisting of PBS, RPMI in the ratio of 1: 1 and
20p1 Heparin is employed for Leukapheresis. Nutriprep may be added if required.
Liver Cancer
Pancreas
Lymphoma and Melanoma
Renal Cell Carcinoma , Thyroid
cancer, Head and Neck
Gastrointestinal Cancer
Lung Cancer
Smooth muscle1 Skeletal muscle
Sarcoma
Brain Tumour
Bladder Cancer
Multiple Myeloma
The PBMCs so obtained are subjected to differential centrifugation to separate the
monocytes from the patient blood from buffy coat (Middle layer) obtained after step 2 is
carefully removed and mixed with PBS (1 :9 ratio) and further centrifuged.
CA125, 19.9
CA125, CA 19.9
Lactic dehydrogenase
CEA
15.3
NSE (neuron-specific enolase), CYFRA
21 -1 , ProGRP
Desmin
Glial fibrillary acidic protein(GFAP)
Bladder Tumour Antigen (BTA).
Bence-Jone Proteins
After two cycles of centrifugation and washing with PBS, monocytes are obtained as pellet.
The monocytes (obtained as pellet) are suspended in RPMI 1640 media to obtain single cell
suspension. True count of cells (Monocytes) in the single cell suspension is done through
Fluorescence Activated Cell Sorting (FACS) technique; 15 million cells plated onto a Poly
Lysine coated culture plate. Plated cells are incubated for 2-4hrs at 37°C and 5% COz, Cells
are washed with PBS.
The adhesion of inactivated monocytes on solid surfaces was prevented by using two
different agents. The two agents used are PPAR-Gamma Agonists and Sodium Potassium
ATPase Inhibitor. The use of these agents in the culture media along with a plasma
concentration of 0.8% prevents the activation of monocytes. The inactivated monocytes are
thus cultured in media containing GM-CSF alone without any cytokine for a sufficient time
10
i
A
ranging between 2-8 days. This culturing of monocytes in culture media allows the
conversion of monocytes into immature dendritic cells.
This immature DC primed with Poly IC and lysate obtained from autologous tumour tissue
enriched by Oxidizing agents i.e. NaOCl and HC104. After 2-3 days of incubation, Dendritic
cells (DC) are harvested. DC characterized for cell surface marker CD 80, CD 83, CD 86,
CCR7, DC205 etc and sterility testing (LAL assay). Such autologous tumour lysate primed
mature DC can be used for evoking cancer specific adaptive immune response.
Example 3: Inhibition of monocyte activation by Sodium Potassium ATPase Inhibitor
The cells were cultured in 6 well crystal grade gamma sterlised polystyrene cell culture plates
with 1x10~ce lls per well in 3 ml media. Prior to addition of the cell, each well is thoroughly
rinsed with the media. Addition of cells is immediately followed by addition of three
different concentration of Na+ K+ ATPase inhibitor (Ouabain and Digoxin) lnm , 1
micromolar and 1 millimolar. A control is also kept where the Na+ K+ ATPase inhibitor is not
added into the cells. 0.8% of Plasma was added in all the cells before incubation into the COz
incubator at 5% C02 and 37' C. The cells were incubated in C02 incubator for 4 hours. After
4 hours of Incubation the supernatant from each cell is counted for number of cells.
Since monocytes has a tendency to stick to culture plate it is assumed that the cells which did
not stick to the culture plate remains in supernatant. The cells are calculated using flow
cytometer using CD 14 antibody.
All the concentration of Na+ K+ ATPase inhibitor is added in duplicate and mean of each
concentration is taken.
The percentage inhibition of cell is calculated as : % of cell adhered to plate in control(C)- %
of cells adhered to plate in Drug treated (D)/ % of cell adhered to the plate in control (C)
~100%in hibition of activation of monocytes: C-D/D x100. The results are shown in figure 1
and in table 2 wherein the use of adhesion inhibitors (ovabain and digoxin) effectively
prevents adhesion of the cells to the substrate as compared to the control and figure 2 and
Table 3 shows the percentage inhibition of activation of monocytes.
Table-2: % of cells adhered in presence or absence of NaK ATPase inhibitors.
Table 3: % inhibition of activation of monocytes by PPAR gamma antagonist
Ovabain
Digoxin
Example 4: Inhibition of monocyte activation
The cells were cultured in 6 well crystal grade gamma sterlised polystyrene cell culture plates
with 1x10~ce lls per well in 3 ml media. Prior to addition of the cell each well is thoroughly
rinsed with the media. Addition of cells is immediately followed by addition of three
different concentration of PPAR gamma agonist (Pioglitazone and Rosiglitazone) lnrn, 1
micromolar and 1 millimolar. A control was also kept where the PPAR gamma agonist is not
added into the cells. 0.8% of Plasma was added in all the cells before incubation into the COz
incubator at 5% C02 and 37' C. The cells were incubated in COz incubator for 4 hours. After
4 hours of Incubation the supernatant from each cell is counted for number of cells.
% of Cells adhered to cell culture plate. @ different
concentrations
Ovabain
Digoxin
Since monocytes has a tendency to stick to culture plate it is assumed that the cells which did
not stick to the culture plate remains in supernatant. The cells are calculated using flow
cytometer using CD14 antibody. All the concentration of PPAR gamma agonist is added in
duplicate and mean of each concentration is taken.
The percentage of Cells adhered is calculated as below. Initial concentration of Cells added to
the culture plate- Concentration of cells in the supernatant after 4 hrs incubation x100. The
percentage inhibition of cell is calculated as : % of cell adhered to plate in control(C)- % of
cells adhered to plate in Drug treated (D)/ % of cell adhered to the plate in control (C) ~100%
1 Milli molar
15
18
Control
75
78
% inhibition of activation of monocytes.
lnm
20
16
lnm
60
65
1 Micromolar
45
40
1 Micromolar
40
48
1 Milli molar
80
76
inhibition of activation of monocytes: C-DID x100. The results are shown in Table 4 and 5
and also in Figure 3 & 4.
Table 4: % of cells adhered in presence or absence of PPAR gamma agonists.
Table 5: % inhibition of activation of monocvtes in presence of PPAR gamma agonist
Rosiglitazone
Pioglitazone
Example 5: Amplification of the Immunogenicity by oxidizing the Tumor Lysate by
Oxidizing agents.
% of Cells adhered to cell culture plate. @ different concentrations .
Rosiglitazone
Pioglitazone
The harvested mature dendritic cells were tested for Immunogenicity based on their ability to
proliferate T cell. (Mixed Lymphocyte Reaction Assay).
Three types of Dendritic cells were prepared:
a) Immature Dendritic Cells were loaded with tumor lysate (Control).
b) Immature Dendritic Cells were loaded with tumor lysate treated with 3 concentrations of
NaOCl(25pMo1, 50 pMol, and 100 pMol).
c) Immature Dendritic Cells were loaded with HC1O4 (25pMo1, 50 pMol, and 100 pMol).
The Dendritic Cells so prepared were matured Dendritic Cells and they were incubated with
T cell culture to check their ability to proliferate T cells. Tests were performed in 96 well flat
bottom plates, as same medium were used that served for T cell culture: RPMI 1640 medium
was supplemented with 2 mM L-glutamine, Primocin and 5% heat-inactivated human serum.
T cell enriched fractions were obtained as 1 h non adherent fraction of PBMC. T-cells were
brought to a concentration of 2x10~ cellslml, DC of all three types were used at 2x10'
cellslml. A ratio of 20: 1 of T-cells to DC, in triplicates were carried out. T cells with DC not
1 Milli molar
44.7
48
% inhibition of activation of monocytes.
1 Micromolar
5 8
64.5
Control
75
7 8
1 nm
10.7
15.0
1 nm
66.9
66.7
1 Micromolar
22.6
18.0
1 Milli molar
40.4
40.0
treated with NaOCl and HC104 served as control. Cells were pulsed for 4 days of incubation
at 37'~. The assay is performed by the addition of a premixed optimized Dye Solution.
Culture wells of a 96-well plate usually containing various concentration test substance.
During a 4-hour incubation, living cells convert the tetrazolium component of the Dye
Solution into a formazan product. The solubilization solution/stop mix is then added to the
culture wells to solubilize the formazan product, and the absorbance at 595nrn is recorded
using a 96-well plate reader. The 595nrn absorbance reading is directly proportional to the
number of cells normally used in proliferation assays. Although the absorbance maximum for
the formazan product is 595nrn and pure solutions appear blue, the color at the end of the
assay may not be blue and depends on the quantity of formazan present relative to other
components (including serum, acidified phenol red and unreduced MTT) in the culture
medium. The results are shown in Table 6 and Figure 5. Figure 5 indicates the proliferation of
T cells when dendritic cells primed different oxidizing agents(NaOC1 and HC104) at different
concentrations.
Storage
Final formulation (six doses) as obtained in example 2 are stored in liquid nitrogen (-196°C).
According to the population obtained after culturing mature Dendritic cell 2x10 6 cells are
suspended in 1 ml volume (50% plasma, 5 % DMSO, 45% RPMI 1640) and kept for gradient
cooling ( 2 hours at 4 0 C, 4 hour in -20 degree and 6 hours at -80 degree then it is
transferred to liquid nitrogen tank and before administering the cells are taken out from
liquid nitrogen tank then rapidly thawed at 37 0 C and then it is washed with PBS and
centrifuged at 7000 rpm for 10 minutes to remove the DMSO, the pellets obtained after
centrifugation are resuspended in 15 ml RPMI 1640 nutrient medium which is then ready for
use.
-
ABSORBANCE
at 595 nm
Control
1.04
1.09
1-07
1.07
NaOCl
2 5
pM
1.45
1.49
1.42
1.45
HC104
25pM
1.13
1.17
1.19
1.16
50
pM
1.68
1.71
1.67
1.69
100pM
1.72
1.74
1.79
1.75
50pM
1.39
1.41
1.43
1.41
100pM
1.63
1.67
1.64
1.65
14
\ +
/ ., .;
" '?' C.: :!'-? '' d4 4L \ We Claim : \ JUN ?D\I
1. A composition comprising mature activated dendritic cells and adapted to express
substantially all antigens present in a tumor lysate, prepared by a process comprising
the steps:
i. preparing an antigen enriched tumor lysate from a tumorous tissue by
contacting the tumorous tissue with an Oxidising agent and poly I:C as
adjuvant ;
ii. isolating plasma blood mononuclear cells (PBMC) from blood sample and
isolating monocytes;
iii. culturing the monocytes in a growth medium for about 5 days at 37'~, the
nutrient medium comprising only GM-CSF and cell adhesion inhibitors to
obtain immature dendritic cells (iDC);
iv. contacting and pulsing the population of Immature Dendritic cell (iDc) with
the antigen enriched tumor and culturing the Immature Dendritic cell
population for 2-days to obtain a population of mature activated dendritic cells
adapted to express substantially all antigens as present in the tumor lysate
derived from the malignant tissue.
2. A composition as claimed in claim 1 wherein the oxidizing agent is sodium
? a
hypochlorite and. HC104,,
3. A composition as claimed in claim 1 wherein tissue may be selected from any of
prostrate, lung, brain, pancreatic, liver, ovary, breast, colon, gastric tumor or any other
solid tumor.
4. A composition as claimed in claim 1 wherein the nutrient medium comprises GM
CSF and either of PPAR gamma agonist or NaK ATPase inhibitor in the range of 1
mM to 1 micromolar.
5. A composition and method substantially as herein described and illustrated.