NEW ANTIGEN PRESENTING CELLS, A PROCESS FOR PREPARING THE SAME AND THEIR USE AS CELLULAR VACCINES

NOUVELLES CELLULES PRESENTANT L'ANTIGENE, PROCEDE DE PREPARATION ASSOCIE, ET LEUR UTILISATION COMME VACCINS CELLULAIRES

English Abstract

The invention relates to monocytes derived antigen presenting cells (MD-APCs)
characterized in that they have the following
properties: they present on their surface: antigen CD14 and CD64 with a mean
intensity of about 5 to about 200; antigen CD80 and
CD86 with a mean intensity of about 20 to about 200; antigen CD40 and mannose
receptor with a mean intensity of 50 to 500; they are
substantially devoid of the surface antigens CD1a and CD1c; they present a
phagocytosis property; they have the property of stimulating
the proliferation of allogenic lymphocytes.

French Abstract

L'invention concerne des cellules présentant l'antigène dérivées des monocytes (MD-APC), caractérisées par les propriétés suivantes: elles présentent à leur surface les antigènes CD14 et CD64 avec une intensité moyenne comprise entre 5 et 200 environ, les antigènes CD80 et CD86 avec une intensité moyenne comprise entre 20 et 200 environ, l'antigène CD40 et le récepteur du mannose avec une intensité moyenne comprise entre 50 et 500; elles sont sensiblement dépourvues des antigènes de surface CD1a et CD1c; elles présentent une propriété de phagocytose; elles présentent la propriété de stimuler la prolifération de lymphocytes allogènes.

Claims

21
CLAIMS:
1. A monocyte derived antigen presenting cell (MD-
APC) having the following properties:
presents on its surface:
antigen CD80 and CD86, wherein said antigen CD80
and CD86 are present in an amount which corresponds to a
measured mean fluorescence intensity of about 20 to about
200 measured using an antibody of a given isotype relative
to a reference value, wherein said reference value is
derived from a parallel measurement using a non-specific
antibody of the same isotype,
antigen CD40 and mannose receptor, wherein said antigen CD40
and mannose receptor are present in an amount which
corresponds to a measured mean fluorescence intensity of 50
to 500 measured using an~antibody of a given isotype
relative to a reference value, wherein said reference value
is derived from a parallel measurement using a non-specific
antibody of the same isotype,
- is substantially devoid of the surface antigens CD1a
and CD1c
- is capable of phagocytosis, and
is capable of stimulating proliferation of allogenic
lymphocytes.
2. MD-APC according to claim 1, wherein said presence
and mean fluorescence intensities are determined by
immunofluorescence staining and flow cytometry analysis.
3. MD-APC according to claim 1 or 2, wherein said MD-
APC is capable of phagocytosis as determined by an
evaluation of uptake of formalin fixed yeast.

22
4. MD-APC according to claim 3, wherein said
evaluation of uptake of formalin fixed yeast comprises:
- culturing MD-APCs for 2 hours;
- adding yeast in a ratio of 1/10 MD-APC/yeast cell
ratio;
- incubating at 37°C, 5% CO2 atmosphere for 2-3 hours
- fixing by May-Grunwald Giemsa (MGG) staining and
- quantifying the percentage of phagocytic MD-APCs.
5. MD-APC according to claim 4, wherein quantifying
the percentage of phagocytic MD-APCs is done by microscopic
analysis.
6. MD-APC according to claim 5, wherein the
percentage of phagocytic.MD-APCs is of about 60%.
7. MD-APC according to any one of claims 1 to 6,
wherein said MD-APC is capable of stimulating proliferation
of allogenic lymphocytes as determined by a test comprising:
- an allogenic primary mixed lymphocytes reaction
(MLR) comprising addition of different numbers (2×10 3 to
2×10 5 in 100 µl medium/well) of MD-APCs to wells of 96 well
plates containing 2×10 5 in 100 µl/well of allogenic T cells
purified from buffy coats;
- incubation for 5 days at 37°C; and
- assessment of cell proliferation by a colorimetric
method.
8. MD-APC according to claim 7, wherein said
colorimetric method comprises hydrolysis of tetrazolium salt
WST-1 (slightly red) to Formozan (dark red).

23
9. MD-APC according to any one of claims 1 to 7,
which presents, on its surface, antigen major
histocompatibility complex II (MHC-II) with a mean
fluorescence intensity of about 100 to about 400, being
determined by immunofluorescence staining and flow cytometry
analysis.
10. MD-APC according to any one of claims 1 to 9,
which is substantially devoid of surface antigen CD83, the
absence.of said surface antigen being determined by
immunofluorescence staining and flow cytometry analysis.
11. MD-APC according to any one of claims 1 to 10,
which presents adherent properties.
12. MD-APC according to claim 11, wherein said
adherent properties are determined by a test comprising
culturing MD-APCs for 2 h in culture medium in plastic
flasks and quantifying the percentage (%) of adherent cells.
13. MD-APC according to claim 12, wherein said culture
medium is I.M.D.M. or R.P.M.I.
14. MD-APC according to claim 12, wherein said
percentage of adherent cells is quantified by microscopic
analysis.
15. A population of cells comprising monocyte derived
antigen presenting cells (MD-APCs) wherein:
- about 30% to about 100% of the MD-APCs present
antigens CD80 and CD86 on their surface;
- about 80% to about 100% of the MD-APCs present
antigen MHC-II on their surface;
- about 70% to about 100% of the MD-APCs present
adherent properties; and

24
- about 30% to about 100% of the MD-APCs present a
phagocytosis property:
wherein antigens CD80 and CD86 are expressed according to
the intensities specified in any one of claims 1 to 14 and
wherein antigen MHC-II is expressed according to the
intensities specified in any one of claims 9 to 14.
16. Population of cells according to claim 15, wherein said
cells are capable of phagocytosis as determined by an
evaluation of uptake of formalin fixed yeast, said
evaluation comprising:
- culturing MD-APCs for 2 hours:
- adding yeast in a ratio of 1/10 MD-APC/yeast cell
ratio;
- incubating at 37°C, 5% CO2 atmosphere for 2-3 hours:
- fixing by May-Grunwald Giemsa (MGG) staining: and
- quantifying the percentage of phagocytic MD-APCs.
17. Population of cells according to claim 15 or 16,
wherein said MD-APC is capable of stimulating proliferation
of allogenic lymphocytes as determined by a test comprising:
- an allogenic primary mixed lymphocytes reaction
(MLR) comprising addition of different numbers (2×10 3 to
2×10 5 in 100 µl medium/well) of MD-APCs to wells of 96 well
plates containing 2×10 5 in 100 µl/well of allogenic T cells
purified from buffy coats:
- incubation for 5 days at 37°C: and
- assessment of cell proliferation by a colorimetric
method.

25
18. Population of cells according to claim 17, wherein
said colorimetric method comprises hydrolysis of tetrazolium
salt WST-1 (slightly red) to Formozan (dark red).
19. Population of cells according to any one of claims
15 to 18, which presents, on its surface, antigen major
histocompatibility complex II (MHC-II) with a mean
fluorescence intensity of about 100 to about 400, being
determined by immunofluorescence staining and flow cytometry
analysis.
20. Population of cells according to any one of claims
15 to 19, which is substantially devoid of surface antigen
CD83, being determined by immunofluorescence staining and
flow cytometry analysis.
21. Population of cells according to any one of claims
15 to 20, which presents adherent properties.
22. Population of cells according to claim 21, wherein
said adherent properties are determined by a test comprising
culturing MD-APCs for 2 h in culture medium in plastic
flasks and quantifying the percentage (%) of adherent cells.
23. A process for preparing a composition comprising
monocyte-derived antigen presenting cells (MD-APCs), said
process comprising:
- starting from a composition containing leukocytes
derived from healthy donors or from patients and obtained
from peripheral blood by apheresis;
- removal of platelets and anticoagulant from the
apheresis product, such as by centrifugation of the
apheresis products;
- isolation of mononuclear cells (monocytes +
lymphocytes) from red cells and granulocytes in order to

26
have less than 10% granulocytes and less than 5% red cells;
and
- culture of mononuclear cells obtained at the
previous stage by placing them in hydrophobic bags in an
appropriate culture medium containing, as sole chemical
ligands having receptors on the membrane of mononuclear
cells;
- histamine or agonist of histamine receptor (H1 in
action) and a H2 antagonist
- or IL-13
in combination with "additional" GM-CSF, for a time
sufficient to obtain differentiated MD-APCs, preferably for
about 5 to 15 days, and possibly separating the MD-APCs from
the lymphocytes, and recovering the MD-APCs and lymphocytes.
24. A process according to claim 23, wherein removal of
platelets and anticoagulant from the apheresis product is
such as obtained by centrifugation of the apheresis products
for 10 mn at 280g.
25. A process according to claims 23 or 24, wherein
the culture medium contains
- histamine or agonist of histamine receptor (H1 in
action)
- a H2 antagonist
in combination with "additional" GM-CSF.
26. A process according to claims 23 or 24, wherein
the culture medium contains IL-13 in combination with
"additional" GM-CSF.
27. A process according to any one of claims 23 to 25,

27
wherein the culture medium contains histamine and a H2
antagonist, in combination with additional GM-CSF, histamine
being present at a concentration of about 10-2 M to about
10-6 M, and preferably of about 10-4 M, the H2 antagonist
being present at a concentration of about 10-4 M to about
109 M, and preferably of about 10-6 M, and additional GM-CSF
being present at a concentration of about 50 U/ml to about
1000 U/ml, preferably of about 500 U/ml.
28. A process according to any of claims 23 to 25
or 27, wherein:
- the agonist of histamine receptor (H1 in action) is 2
methyl-histamine, and/or
- the H2 antagonist is chosen among the group
consisting of: cimetidine, tiotidine, burinamide, metiamide
and ranitidine.
29. Process according to any one of claims 23, 24
or 26, wherein the culture medium contains IL-13 and
additional GM-CSF, said additional GM-CSF being present at a
concentration of about 50 U/ml to about 1000 U/ml, and
preferably of about 500 U/ml.
30. Process according to any one of claims 23 to 29,
wherein the culture medium of MD-APCs is added with at least
one of the following elements:
- crude antigens, for instance autologous tumor
membrane, killed tumoral cells, bacterial capsides, viral
homogenates cleared from nucleic acids,
- specific peptides against which an immune response
is desired,
- cDNA or genetic material linked to vectors (for
example gluconated polylysine) to allow transfection of the

28
MD-APCs with material coding for the relevant peptide or
protein to be presented on the MD-APC membrane and against
which an immune response is desired,
- bispecific antibodies targeting on the one side, a
surface antigen of the MD-APCs and, on the other side, a
relevant antigen against which an immune response is
desired.
31. Monocyte-derived antigen presenting cells (MD-
APCs) such as obtained according to a process of any one of
claims 23 to 30.
32. MD-APCs according to claim 31 which have the
following properties:
- they present on their surface
* antigen CD14 and CD64 with a mean intensity of about
20 to about 200,
* antigen CD80 and CD86 with a mean intensity of about
20 to about 200,
* antigen CD40 and mannose receptor with a mean
intensity of about 50 to about 500,
* antigens CD1a and CD1c with a mean intensity lower
than 20,
the presence and mean intensities respectively of CD14,
CD64, CD80, CD86,CD40, mannose receptor, CD1a and CD1c being
determined by immunofluorescence staining and flow cytometry
analysis,
- they present a phagocytosis property such as
determined by the following test:
said phagocytosis capacity being evaluated by an uptake

29
of formalin fixed yeast, by culturing macrophages for 2
hours, adding yeast in 1110 macrophages/yeast ratio and
incubating at 37°C, 5% CO2 atmosphere for 2-3 hours fixing by
the May-Grünwald-Giemsa (MGG) staining, and the percentage
of phagocytic MD-APCs being quantified for instance by
microscopic analysis,
- they have the property of stimulating the
proliferation of allogenic lymphocytes such as determined by
the following test:
allogenic primary mixed lymphocytes reaction (MLR) was
carried out in 96-well microtiter plates by adding different
numbers (2×10 3 to 2×10 5 in 100 µl medium/well) of MD-APCs to
2×10 5 in 100 µl medium/well of allogenic T cells purified
from buffy coats and after 5 days incubation at 37°C, cell
proliferation was assessed by a colorimetric method, such as
the hydrolysis of tetrazolium salt WST-1 (Boehringer
Mannheim; Germany), (slightly red) to Formozan (dark red).
33. MD-APCs according to claim 31 or 32, presenting a
capacity of phagocytosis of 80%.
34. MD-APCs according to any one of claims 31 to 33,
which present, on their surface, antigen MHC-II with a mean
intensity of about 100 to about 400, such as determined by
immunofluorescence staining and flow cytometry analysis.
35. MD-APCs according to any one of claims 31 to 34,
which are substantially devoid of surface antigen CD83, such
as determined by immunofluorescence staining and flow
cytometry analysis.
36. MD-APCs according to any one of claims 31 to 35,
which present adherent properties such as determined by the
following test:

30
the macrophages are cultured for 2h in culture medium
(I.M.D.M. or R.P.M.I.) on plastic flasks and the percentage
(%) of adherent cells is quantified for instance by
microscopic analysis.
37. Monocyte-derived antigen presenting cells
(MD-APCs) which have the following properties:
- they present on their surface:
* antigen CD14 and CD64 with a mean intensity of about
20 to about 200,
* antigen CD80 and CD86 with a mean intensity of about
20 to about 200,
* antigen CD40 and mannose receptor with a mean
intensity of 50 to 500,
* antigens CD1a and CD1c, with a mean intensity lower
than 20, the presence and mean intensities respectively of
CD14, CD64, CD80, CD86, CD40, mannose receptor, CD1a and
CD1c being determined by immunofluorescence staining and
flow cytometry analysis,
- they present a phagocytosis property such as
determined by the following test:
said phagocytosis capacity being evaluated by an uptake
of formalin fixed yeast, by culturing macrophages for 2
hours, adding yeast in 1/10 macrophages/yeast ratio and
incubating at 37°C, 5% CO2 atmosphere for 2-3 hours fixing by
the May-Grünwald-Giemsa (MGG) staining, and the percentage
of phagocytic MD-APCs being quantified for instance by
microscopic analysis;
- they have the property of stimulating the
proliferation of allogenic lymphocytes such as determined by

31
the following test:
allogenic primary mixed lymphocytes reaction (MLR) was
carried out in 96-well microtiter plates by adding different
numbers (2×10 3 to 2×10 5 in 100 µl medium/well) of MD-APCs to
2×10 5 in 100 µl medium/well of allogenic T cells purified
from buffy coats and after 5 days incubation at 37°C, cell
proliferation was assessed by a colorimetric method, such as
the hydrolysis of tetrazolium salt WST-1 (Boehringer
Mannheim, Germany), (slightly red) to Formozan (dark red).
38. MD-APCs according to claim 37, presenting a
capacity of phagocytosis of 80%.
39. MD-APCs according to claim 37 or 38, which
present, on their surface, antigen MHC-II with a mean
intensity of about 100 to about 400, such as determined by
immunofluorescence staining and flow cytometry analysis.
40. MD-APCs according to any one of claims 37 to 39,
which are substantially devoid of surface antigen CD83, such
as determined by immunofluorescence staining and flow
cytometry analysis.
41. MD-APCs according to any one of claims 37 to 40,
which present adherent properties such as determined by the
following test:
the macrophages are cultured for 2h in culture medium
(I.M.D.M. or R.P.M.I.) on plastic flasks and the
percentage (%) of adherent cells is quantified for instance
by microscopic analysis.
42. Pharmaceutical compositions containing as active
substance, MD-APCs according to any one of claims 31 to 41.
43. Cellular vaccine compositions containing as active
substance, MD-APCs according to any one of claims 31 to 41.

32
44. Medium containing elements necessary for the
growth and differentiation of monocytes into MD-APCs
according to a process of any one of claims 23 to 30,
containing as sole chemical ligands having receptors on the
membrane of mononuclear cells:
- histamine or agonist of histamine receptor (H1 in
action), and a H2 antagonist such as cimetidine,
- or IL-13,
in combination with additional GM-CSF.
45. Medium according to claim 43, containing:
- histamine or an agonist of histamine receptor (H1 in
action), and
- a H2 antagonist, and
additional GM-CSF.
46. Medium according to any one of claims 43 to 45,
containing histamine and an H2 antagonist, in combination
with additional GM-CSF, histamine being present at a
concentration of about 10-2 M to about 10-6 M, and preferably
of about 10-4 M, the H2 antagonist being present at a
concentration of about 10-4 M to about 10-9 M, and preferably
of about 10-6 M, and additional GM-CSF being present at a
concentration of about 50 U/ml to about 1000 U/ml,
preferably of about 500 U/ml.
47. Medium according to any one of claims 43 to 46,
wherein:
- the agonist of histamine receptor (H1 in action) is 2
methyl-histamine, and/or
- the H2 antagonist is chosen among the group

33
consisting of: cimetidine, tiotidine, burinamide, metiamide
and ranitidine.
48. Medium according to claim 47, wherein the H2
antagonist is cimetidine.
49. Medium according to claim 44, wherein IL-13 and
GM-CSF are added in the culture medium, additional GM-CSF
being present at a concentration of about 50 U/ml to about
1000 U/ml, and preferably of about 500 U/ml.
50. Cell processor or kit containing:
- means for the recovery of lymphocytes and monocytes
free of contaminants,
- appropriate buffer and wash solutions and possibly
appropriate means for the conservation of monocyte-derived
antigen presenting cells (MD-APCs),
- means for preparing a culture for the monocytes and
possibly the lymphocytes and containing as sole chemical
ligands having receptors on the membrane of mononuclear
cells,
- histamine or an agonist of histamine receptor (H1 in
action), and a H2 antagonist such as cimetidine,
- or IL-13,
in combination with GM-CSF,
- possibly means for transfection of cultured cells
and means for targeting antigens to MD-APCs.
51. Cell processor or kit according to claim 50,
containing:
- histamine or an agonist of histamine receptor (H1 in

34
action), and
- a H2 antagonist,
in combination with GM-CSF.
34
52. Cell processor or kit according to claim 50,
containing IL-13 and GM-CSF.
53. Cell processor or kit according to any one of
claims 48 to 50, containing means for preparing a culture
for the monocytes and possibly the lymphocytes, and
containing histamine and cimetidine.
54. Cell processor or kit according to claim 52,
containing means for preparing a culture for the monocytes
and possibly the lymphocytes, and containing IL-13 and
GM-CSF.
55. Cell processor.or kit according to any one of
claims 50 to 54, containing:
- means for recovering and cetrifuging blood to obtain
a leukocyte concentrate,
- means for separating lymphocytes and monocytes from
the other white cells and for eliminating the contaminating
red cells,
- culture medium for MD-APCs and possibly lymphocytes
with complements and particularly, as sole chemical ligands
having receptors on the membrane of mononuclear cells
histamine or an agonist of histamine receptor (H1 in action),
and a H2 antagonist such as cimetidine, or IL-13 in
combination with GM-CSF,
- appropriate means for the conservation of MD-APCs,
- appropriate buffer and wash solution.

35
56. Cell processor or kit according to claim 50,
containing culture medium for MD-APCs and possibly
lymphocytes, with complements being histamine and
cimetidine, in combination with GM-CSF.
57. Cell processor or kit according to claim 49,
containing culture medium for MD-APCs and possibly
lymphocytes, with complements being IL-13 and GM-CSF.
58. Products containing MD-APCs according to any one
of claims 31 to 39, and lymphocytes, as a combined
preparation for simultaneous, separate or sequential use in
cell therapy.
59. Products according to claim 58, characterized in
that they contain the MD-APCs and the lymphocytes in a ratio
of at least 20% to 50% of MD-APCs expressed in cell number.
60. Use of MD-APCs according to any one of claims 31
to 39, for the preparation of a drug, for the treatment of
cancer and of infections by pathogens.
61. Use of MD-APCs according to any of claims 31 to
39, for the preparation of a drug, for the treatment of a
disease involving phagocytosis, and the stimulation of the
proliferation of T-lymphocytes.
62. Use according to claim 60 or 61, chracterized in
that said drug contains from about 10 8 to about 5×10 9
MD-APCs.
63. Use according to any one of claims 60 to 62,
characterized in that said drug also contains lymphocytes.
64. Use of histamine or an agonist of histamine
receptor (H1 in action), and a H2 antagonist, in particular
cimetidine, or of IL-13, in combination with GM-CSF, for the
preparation of monocyte-derived antigen presenting cells

36
(MD-APCs) having the following properties:
- they present on their surface:
antigen CD14 and CD64 with a mean intensity of
about 20 to about 200,
antigen CD80 and CD86 with a mean intensity of
about 20 to about 200,
antigen CD40 and mannose receptor with a mean
intensity of 50 to 500,
CD1a and CD1c with a mean intensity larger than
20, the presence and mean intensities respectively of CD14,
CD64, CD80, CD86, CD40, mannose receptor, CD1a and CD1c
being determined by immunofluorescence staining and flow
cytometry analysis,
- they present high phagocytosis property such as
determined by the following test:
said phagocytosis capacity being evaluated by an
uptake of formalin fixed yeast, by culturing MD-APCs for 2
hours to select adherent cells, adding yeast in 1/10 mMD-
APCs/yeast ratio and incubating at 37°C, 5% CO2 atmosphere
for 2-3 hours fixing by the May-Grünwald-Giemsa (MGG)
staining, and the percentage of phagocytic MD-APCs being
quantified for instance by microscopic analysis,
- they have the property of stimulating the
proliferation of allogenic lymphocytes such as determined by
the following test:
allogenic primary mixed lymphocytes reaction (MLR)
was carried out in 96-well microtiter plates by adding
different numbers (2×10 3 to 2×10 5 in 100 µl medium/well) of
MD-APCs to 2×10 5 in 100 µl medium/well of allogenic T cells

37
purified from buffy coats and after 5 days incubation at
37°C, cell proliferation was assessed by a colorimetric
method, such as the cleavage of tetrazolium salt WST-1
(slightly red) to Formozan (dark red) or such as Brdu
incorporation during DNA synthesis.

Description

CA 02252505 2003-08-O1
1.
NEW ANTIGEN PRESENTING CELLS, A PROCESS FOR PREPARING THE
SAME AND THEIR USE AS CELLULAR VACCINES.
The invention relates to new antigen presenting
cells, a process for preparing the same and their use as
cellular vaccines.
Macrophages are recognized since their
description by Metchnikoff ~almmunity in Infective
Diseases, Cambridge Press, 1905 as cells which very
effectively phagocytose (z.nterio:rise) and digest
exogenous particles (antigens, cell. debris, bacteria).
They also secrete a variety of .immuraoeffector monokines.
They have therefore a central role in initiating the non-
specific and the specific immune responses. The recovery
of large quantities of human macrophages differentiated
in culture from blood monocytes has already been
described (International application WO 99/26875,
published November 24, 1999). These macrophages
express typical antiger~s and functions and have been
fully characterized.
Macrophages activated i,n the presence of IFNY
(Macrophage .Activated Killers * MAKE elicited selective
cytostasis and cytotoxicity for a large number of human
tumors even at low effector/targe°t ratio. In murine
models, murine activated macrophages given locally in the
tumor or its vicinity ~.nfiltrated the tumor mass,
inhibited tumor growth and decreased metastatic
development. Human macrophages also inhibited the growth
of human tumors engrafted in nude or SCID mice; this
effect was achieved after local or systemic injection of
a low number of macrophages (less than 1 millioi°a MAK) for
mice with macroscopic tumors.

CA 02252505 2003-08-O1
~a
Patients with metastat~..c: cancer were infused
systemically or intraper~.toneal.~.y with 10~ to 4x109
autologous MAK. Tumoricidal monocytes ~AKM) were also
infused intraperitoneally in pat~.ents with colorectal
carcinomas. The clinical tolerance of MAK was excellent
with minor side effects such as law grade fever and
chills and no autoimmune r~or acute phase reactivity. No
complete antitumoral response was reported; irnproved
prognosis and. prolonged dz.sease free intervals were
described after intraperitoneal ~.r~jecaion of AKM, while
tumor necrosis, stabilisation, reduction of ascitic: fluid
and change z.n chemioresist:ance were seen after MAK
therapy.
The recognized limitation of these macrophages
is that they are not very potent ira the priming of a
specific immune response against a specific exogenous
antigen or tumor by stimulat~.on of MHO class Z restricted
cytotoxic

CA 02252505 2003-08-O1
~°t
l.....
T lymphocytes (CD$ +). They are r~~ore efficient in presenting antigens in the
context of MHC class Il molecules to T helper Iympht~cytes (CD4 +).
However these macrophages have: the potential to process and present
soluble antigens by the exogenous pathway of phagocytes but high
concentrations of proteins or antigens are required .
Cells expressing these functions of phagacytosis, digestion, processing,
and presentation at a high level would be required for the development of
cellular vaccines.
Dendritic cells are characterized by their morphology (dendrites) and a
to few membrane antigens, and are considered as professional antigen -
presenting
cells resident in tissues. They are the most potent cells for the stimulation
of
primary T - lymphocyte immune responses {L7entz~tic cells in fundamental and
clinical immunology. 1995, Plenum Press l~r.Y., f3anclmreau and Schmitt
Editors).
1 s Dendritic cell precursors arise from bane marrow and can be found in
blood and lymph. Dendritic cells derived from these origins can be obtained by
culture in the presence of GM - CAF + 'lid + Thfp. They will then exhibit
differences related to their maturation state and microenviranment.
The dendritic cells which can be derived from blood are most potent to
2o induce allogenic mixed lymphocyte reactions and to stimulate naive T
lymphocytes. However, these classical dendritic cells are technically
relatively
difficult to obtain and are poorly phagocytozing.
In contrast to macrophages, they do not express of CD14 and CD64 (high
affinity FY receptor).
25 Ta_ circumvent the problem of poor phagocytoses and processing of
particular antigens by dendritic cells, these cells have been pulsed with
small
peptides fixed on MHC-I molecules to induce primary immune reaction and
vaccination against new antigenic peptides .
Obtaining dendritic cells by in vitro differentiation requires the presence
30 of GM-~CSF and of a second cytokine that can be IL ~1 o r I L 13 ~ 1 a s
TNF.
One of the aims of the invention is to provide cells with high
phagocytosis, and efficient antigen presentation.
Another aim of the invention is to provide very potent antigen presenting
35 cells derived from human blood monacytes.
Another aim of the invention is to provide cells presenting membrane
receptors, allowing targeting and increased antigen presentation with the use
of
bispecific antibodies.

CA 02252505 2003-08-O1
Another aim of flue invention is to provide cells which can be transfected
with cDl~lA to be used in gene therapy .
Another aim of the invention is to provide a method allowing the recovery
from human blood of cells of the macrophage lineage with high phagocytosis,
digestive activity, processing MIIC class 1 and class Ii antigen presentation
.
Another aim of the invention is to provide a reproducible method allowing
to obtain the above defined cells, said process not requiring combination of
exogenous cytokines, defined media and recipients constituting a cell
processor.
to Another aim of the invention is to provide cellular vaccines demonstrating
the high phagocytosis, processing, M'.HC-II peptide presentation of the
macrophages and also the potent MHC-l T lymphocyte stimulation with high
level of accessory molecules for presentation of the dendritic cells.
The invention relates to macrophages which have the following properties
t s - they present on their surface
* antigen CD 14 with a mean intensity of about ~0 to about 200,
* antigen CDfi4 with a mean intensity of about 20 to about 200,
- they are substantially devoid of the surface antigens CDIa and CDIc,
the presence and mean intensities respectively of CD14, CD64 and the
2o absence of CDla and CDlc being for instance determined by
immunafluorescence staining and flow cytametry analysis,
- they present a phagocytosis property such as determined by the following
test:
said phagocytosis capacity being evaluated by an uptake of formalin fixed
yeast,
far example by culturing macrophages for ? hours , adding yeast in 1 f 10
2s macrophage/yeast ratio and incubating at 3'~°C,, 5~ C'02 atmosphere
for
2-3 hours fixing by the May-~runwald-Giemsa (MGG) staining, and the
percentage of phagacytic rnacraphages being quantified for instance by
microscopic analysis,
- they have the property of stimulating fhe proliferation of allagenic
30 lymphocytes such as determined by the following test :
allogenic primary mixed lymphocytes reaction (MLR) was carried out in 96-
well microtiter plates by adding increasing numbers (2x103 to 2x105) in 100
~.1
mediumlwell of macrophages to 2x10 irr 100 ~.1 mediumlwell of allogenic T
cells purified from huffy coats and after 5 days incubation at 3'~°C,
cell
35 proliferation was assessed by a calorimetric method, such as the hydrolysis
of
tetrazolium salt WST-1 (Boehringer Mannheim" Germany), (slightly red) to
Formozan (dark red;l.

CA 02252505 2005-09-16
' ' 11534-15(S)
4
The invention relates to a monocyte derived
antigen presenting cell (MD-APC) having the following
properties:
presents on its surface:
antigen CD80 and CD86, wherein said antigen CD80
and CD86 are present in an amount which corresponds to a
measured mean fluorescence intensity of about 20 to about
200 measured using an antibody of a given isotype relative
to a reference value, wherein said reference value is
derived from a parallel measurement using a non-specific
antibody of the same isotype,
antigen CD40 and mannose receptor, wherein said
antigen CD40 and mannose receptor are present in an amount
which corresponds to a measured mean fluorescence intensity
of 50 to 500 measured using an antibody of a given isotype
relative to a reference value, wherein said reference value
is derived from a parallel measurement using a non-specific
antibody of the same isotype,
is substantially devoid of the surface antigens CDla
and CDlc;
is capable of phagocytosis, and
is capable of stimulating proliferation of allogenic
lymphocytes.
According to one aspect of the invention, there is
provided a population of cells comprising monocyte derived
antigen presenting cells (MD-APCs) wherein about 30% to
about 100% of the MD-APCs present antigens CD80 and CD86 on
their surface: about 80% to about 100% of the MD-APCs
present antigen MHC-II on their surface: about 70% to about
100% of the MD-APCs present adherent properties; and about

CA 02252505 2005-09-16
' ' 1534-15(S)
4a
30~ to about 100 of the MD-APCs present a phagocytosis
property: wherein antigens CD80 and CD86 are expressed
according to the intensities described herein and wherein
antigen MHC-II is expressed according to the intensities
described herein.
According to one aspect of the invention, there is
provided a process for preparing a composition comprising
monocyte-derived antigen presenting cells (MD-APCs), said
process comprising:
starting from a composition containing leukocytes
derived from healthy donors or from patients and obtained
from peripheral blood by apheresis:
removal of platelets and anticoagulant from the
apheresis product, such as by centrifugation of the
apheresis products:
isolation of mononuclear cells (monocytes +
lymphocytes) from red cells and granulocytes in order to
have less than 10°s granulocytes and less than 5% red cells:
and
culture of mononuclear cells obtained at the previous
stage by placing them in hydrophobic bags in an appropriate
culture medium containing, as sole chemical ligands having
receptors on the membrane of mononuclear cells;
histamine or agonist of histamine receptor (H1 in
action) and a H2 antagonist
or IZ-13
in combination with "additional" GM-CSF, for a time
sufficient to obtain differentiated MD-APCs; preferably for
about 5 to 15 days, and possibly separating the MD-APCs from

CA 02252505 2005-09-16
' ' 11534-15(S)
4b
the lymphocytes, and recovering the MD-APCs and lymphocytes.
According to another aspect of the present
invention, there is provided monocyte-derived antigen
presenting cells (MD-APCs) such as obtained as described
herein.
According to still another aspect of the present
invention, there is provided monocyte-derived antigen
presenting cells (MD-APCs) which have the following
properties:
they present on their surface:
antigen CD14 and CD64 with a mean intensity of
about 20 to about 200,
antigen CD80 and CD86 with a mean intensity of
about 20 to about 200,
antigen CD40 and mannose receptor with a mean
intensity of 50 to 500,
antigens CDla and CDlc, with a mean intensity
lower than 20, the presence and mean intensities
respectively of CD14, CD64, CD80, CD86,CD40, mannose
receptor, CDla and CDlc being determined by
immunofluorescence staining and flow cytometry analysis,
they present a phagocytosis property such as determined
by the following test:
said phagocytosis capacity being evaluated by an uptake
of formalin fixed yeast, by culturing macrophages for 2
hours, adding yeast in 1/10 macrophages/yeast ratio and
incubating at 37°C, 5~ C02 atmosphere for 2-3 hours fixing by
the May-Grunwalk-Giemsa (MGG) staining, and the percentage

CA 02252505 2005-09-16
' ' 1534-15(S)
4c
of phagocytic MD-APCs being quantified for instance by
microscopic analysis,
they have the property of stimulating the proliferation
of allogenic lymphocytes such as determined by the following
test:
allogenic primary mixed lymphocytes reaction (MLR) was
carried out in 96-well microtiter plates by adding different
numbers (2x103 to 2x105 in 100 u1 medium/well) of MD-APCs to
2x105 in 100 u1 medium/well of allogenic T cells purified
from huffy coats and after 5 days incubation at 37°C, cell
proliferation was assessed by a colorimetric method, such as
the hydrolysis of tetrazolium salt WST-1 (Boehringer
Mannheim, Germany), (slightly red) to Formozan (dark red).
According to yet another aspect of the present
invention, there is provided pharmaceutical compositions
containing as active substance, MD-APCs as described herein.
According to a further aspect of the present
invention, there is provided cellular vaccine compositions
containing as active substance, MD-APCs as described herein.
According to yet a further aspect of the present
invention, there is provided medium containing elements
necessary for the growth and differentiation of monocytes
into MD-as described herein, containing as sole chemical
ligands having receptors on the membrane of mononuclear
cells:
histamine or agonist of histamine receptor (H1 in
action), and a H2 antagonist such as cimetidine,
or IL-13,
in combination with additional GM-CSF.

CA 02252505 2005-09-16
' ~ 11534-15(S)
4d
According to still a further aspect of the present
invention, there is provided cell processor or kit
containing:
means for the recovery of lymphocytes and monocytes
free of contaminants,
appropriate buffer and wash solutions and possibly
appropriate means for the conservation of monocyte-derived
antigen presenting cells (MD-APCs),
means for preparing.a culture for the monocytes and
possibly the lymphocytes and containing as sole chemical
ligands having receptors on the membrane of mononuclear
cells,
histamine or an agonist of histamine receptor (H1 in
action), and a H2 antagonist such as cimetidine,
or IL-13,
in combination with GM-CSF,
possibly means for transfection of cultured cells and
means for targeting antigens to MD-APCs.
According to another aspect of the present
invention, there is provided products containing MD-APCs as
described herein, and lymphocytes, as a combined preparation
for simultaneous, separate or sequential use in cell
therapy.
According to yet another aspect of the present
invention, there is provided use of MD-APCs as described
herein, for the preparation of a drug, for the treatment of
cancer and of infections by pathogens.
According to yet another aspect of the present

CA 02252505 2005-09-16
11534-15(S)
4e
invention, there is provided use of MD-APCs as described
herein for the preparation of a drug, for the treatment of a
disease involving phagocytosis, and the stimulation of the
proliferation of T-lymphocytes.
According to yet another aspect of the present
invention, there is provided use as described herein
characterized in that said drug contains from about 10$ to
about 5x109 MD-APCs.
According to yet another aspect of the present
invention, there is provided use as described herein
characterized in that said drug also contains lymphocytes.
According to yet another aspect of the present
invention, there is provided use of histamine or an agonist
of histamine receptor (H1 in action), and a H2 antagonist, in
particular cimetidine, or of IL-13, in combination with GM-
CSF, for the preparation. of monocyte-derived antigen
presenting cells (MD-APCs) having the following properties:
they present on their surface:
antigen CD14 and CD64 with a mean intensity of
about 20 to about 200,
antigen CD80 and CD86 with a mean intensity of
about 20 to about 200,
antigen CD40 and mannose receptor with a mean
intensity of 50 to 500,
CDla and CDlc with a mean intensity larger than
20, the presence and mean intensities respectively of CD14,
CD64, CD80, CD86, CD40, mannose receptor, CDla and CDlc
being determined by immunofluorescence staining and flow
cytometry analysis,

CA 02252505 2005-09-16
11534-15(S)
4f
they present high phagocytosis property such as
determined by the following test:
said phagocytosis capacity being evaluated by an
uptake of formalin fixed yeast, by culturing MD-APCs for 2
hours to select adherent cells, adding yeast in 1/10 mMD-
APCs/yeast ratio and incubating at 37°C, 5% C02 atmosphere
for 2-3 hours fixing by the May-Grunwald-Giemsa (MGG)
staining, and the percentage of phagocytic MD-APCs being
quantified for instance by microscopic analysis,
they have the property of stimulating the proliferation
of allogenic lymphocytes such as determined by the following
test:
allogenic primary mixed lymphocytes reaction (MLR)
was carried out in 96-well microtiter plates by adding
different numbers (2x103 to 2x105 in 100 u1 medium/well) of
MD-APCs to 2x105 in 100 u1 medium/well of allogenic T cells
purified from huffy coats and after 5 days incubation at
37°C, cell proliferation was assessed by a colorimetric
method, such as the cleavage of tetrazolium salt WST-1
(slightly red) to Formozan (dark red) or such as Brdu
incorporation during DNA synthesis.
According to another aspect of the present
invention, there is provided a process for preparing MD-APCs
described herein, said process comprising: (i) culturing
mononuclear cells in a culture medium comprising a chemical
ligand of mononuclear cells, wherein said chemical ligand is
selected from the group consisting of: (a) a combination of
histamine and cimetidine; (b) a combination of histamine
agonists and histamine H2 receptor (H2) antagonists; and (c)
interleukin 13 (IL-13) wherein said chemical ligand is in
combination or not with exogenously added Granulocyte

CA 02252505 2005-09-16
' ~ 11534-~15 (S)
4g
Macrophage Colony Stimulating Factor (GM-CSF); and (ii)
allowing differentiation into MD-APCs.
According to still another aspect of the present
invention, there is provided a MD-APC obtained according to
the process described herein.
According to yet another aspect of the present
invention, there is provided a pharmaceutical composition
containing as active substance, MD-APC described herein or
the population of cells described herein diluted in a
pharmaceutically acceptable diluent or carrier.
According to a further aspect of the present
invention, there is provided a cellular vaccine composition
containing as active substance, MD-APC described herein or
the population of cells described herein and a
pharmaceutically acceptable vehicle.
According to yet a further aspect of the present
invention, there is provided a use of a culture medium
comprising chemical ligands of mononuclear cells, for
preparation of an MD-APC described herein, wherein said
chemical ligands are selected from the group consisting of:
histamine agonists and histamine H2 receptor antagonists;
histamine and cimetidine; and interleukin 13(IL-13); in
combination or not with GM-CSF.
According to still a further aspect of the present
invention, there is provided a cell processor or kit
comprising: (i) a culture medium comprising a chemical
ligand of mononuclear cells, wherein said chemical ligand is
selected from the group consisting of: histamine at a
concentration of at least 10-6 M and cimetidine; histamine
agonists and histamine H2 receptor (H2) antagonists; and
interleukin 13(IL-13); wherein said chemical ligand is in

CA 02252505 2005-09-16
' 1534=15(S)
4h
combination or not with exogenously added GM-CSF; and (ii)
instructions for culturing mononuclear cells in said medium
and for allowing differentiation into an MD-APC described
herein.
According to another aspect of the present
invention, there is provided a combined preparation for
simultaneous, separate or sequential use in cell therapy,
said preparation comprising: MD-APCs described herein, and
lymphocytes.
According to yet another aspect of the present
invention, there is provided a use of a combined preparation
in cell therapy, wherein said combined preparation comprises
MD-APCs described herein and lymphocytes.
According to another aspect of the present
invention, there is provided a use of MD-APCs described
herein for clinical treatment by adoptive immunotherapy.
According to still another aspect of the present
invention, there is provided a use of mononuclear cells in a
culture medium containing ligands selected from the group
consisting of: a combination of histamine and cimetidine; a
combination of histamine agonists and histamine H2 receptors
antagonists; and interleukin 13(IZ-13); in the presence or
absence of GM-CSF, for the preparation of a MD-APC having
the following properties: presents on its surface: antigen
CD80 and CD86, wherein said antigen CD80 and CD86 are
present in an amount which corresponds to a measured mean
fluorescence intensity of about 20 to about 200 measured
using an antibody of a given isotype relative to a reference
value, wherein said reference value is derived from a
parallel measurement using a non-specific antibody of the
same isotype; antigen CD40 and mannose receptor, wherein
said antigen CD40 and mannose receptor are present in an

CA 02252505 2005-09-16
' ' 11534-15(S)
4i
amount which corresponds to a measured mean fluorescence
intensity of 50 to 500 measured using an antibody of a given
isotype relative to a reference value, wherein said
reference value is derived from a parallel measurement using
a non-specific antibody of the same isotype~ is
substantially devoid of surface antigens CDla and CDlc; is
capable of phagocytosis, and is capable of stimulating
proliferation of allogenic lymphocytes.
The invention relates to monocytes derived antigen
presenting cells (MD-APCs), particularly macrophages which
have the following properties:
they present on their surface:
* antigens CD14 and CD64 with a measured mean
fluorescence intensity of about 5 to about 200 relative to a
reference value,
* antigen CD80 and CD86 with a measured mean
fluorescence intensity of about 20 to about 200 relative to
a reference value,
* antigen CD40 and mannose receptor with a
measured mean fluorescence intensity of 50 to 500 relative
to a reference value,
they are substantially devoid of the surface antigens
CDla and CDc, the presence and mean intensities respectively
of CD14, CD64, CD80 and CD86, and the absence of CDla and
CDlc being for instance determined by immunofluorescence
staining and flow cytometry analysis,
they present a phagocytosis property such as determined
by the following test:

CA 02252505 2005-09-16
' ' 11534-~15 (S)
4j
said phagocytosis capacity being evaluated by an uptake of
formalin fixed yeast, for example by culturing MD-APCs for 2
hours, adding yeast in 1/10 MD-APCs/yeast ratio and
incubating at 37°C, 5g C02 atmosphere for 2-3 hours fixing by
the May-Grunwald-Giemsa (MGG) staining, and the percentage
of phagocytic MD-APCs being quantified for instance by
microscopic analysis,
they have the property of stimulating the proliferation
of allogenic lymphocytes such as determined by the following
test:

CA 02252505 2001-03-19
allogenic primary mixed lymphocytes reaction (MLR) was
carried out in 96-well microtiter plates by adding
increasing numbers (2x103 to 2x105) in 100 ~1 medium/well
of MD-APCs to 2x105 in 100 ~tl medium/well of allogenic T
5 cells purified from buffy coats and after 5 days
incubation at 37°C, cell proliferation was assessed by
measurement of Brdu incorporation during DNA synthesis by
ELISA method (Boehringer Mannheim, Germany).
In the context of the present invention, the
expression "macrophages" designates not only macrophages
but antigen presenting cells which derive from monocytes
and which will be hereafter designated by MD-APC.
The expression "substantially devoid of surface
antigens CDla and CDlc" means either that there is no
such surface antigens or that there is only a dim
intensity for these surface antigens, with said dim
intensity corresponding to about 10 times less than the
intensity obtained in the presence of such surface
antigens, as determined in immunofluorescence analysis,
or with said intensity being lower than 20.
In the following of the text, it will be often
referred to "the absence of surface antigen CDla and
CDlc", which is to be understood as "substantially devoid
of such antigens" as explained above (intensity lower
than 20).
The invention also relates to MD-APCs which
present, on their surface, antigen MHC-II with a mean
intensity of about 100 to about 600, such as determined
by immunofluorescence staining and flow cytometry
analysis.
The invention also relates to macrophages which
are substantially devoid of surface antigen CD83, such as

° CA 02252505 2001-11-29
6
determined by immunofluorescence staining and flow cytometry analysis.
The expression "substantially devoid of surface antigen CD83" corresponds to
an intensity lower than 20.
The MD-APCs of the invention present adherent properties such as
determined by the following test:
the MD-APCs are cultured for 2 h in culture medium (LM.D.M. or R.P.M.L) in
plastic flasks and the percentage (%) of adherent cells is quantified for
instance by
microscopic analysis.
The culture media LM.D.M. and R.P.M.I. are commercially available.
The invention relates to a population of cells comprising monocyte derived
antigen presenting cells (MD-APCs) wherein:
- about 30% to about 100% of the MD-APCs present antigens CD80 and CD86 on
their surface;
- about 80% to about 100% of the MD-APCs present antigen MHC-II on their
surface;
- about 70% to about 100% of the MD-APCs present adherent properties; and
- about 30% to about 100% of the MD-APCs present a phagocytosis property;
wherein antigens CD80, CD86 and MHC-II are expressed according to the above-
mentioned intensities.
The invention relates to a culture comprising MD-APCs wherein:
- about 10% to about 50% of the MD-APCs present antigen CD14 on their surface,
about 10% to about 50% of the MD-APCs present antigen CD64 on their surface,
- about 80% to about 100% of the MD-APCs present antigen MHC-II on their
surface,
- about 70% to about 100% of the MD-APCs present adherent properties,
- about 30% to about 100% of the MD-APCs present antigens CD80 and CD86 on
their surface,
- about 30% to about 100% of the MD-APCs present high phagocytosis property,
each macrophage having the above-mentioned properties being such that said
properties are expressed according to the intensities as specified above.
The invention also relates to a process for preparing a composition of
macrophages which comprises the culture of mononuclear cells in a culture
medium

CA 02252505 2003-08-O1
r
containing hi~tamine or histamine agonise (1-l~ icy action) and a H2
antagonist in
combination or not with "additional" GM-CSF.
The invention also relates to a process fox preparing a composition of MD-
APCs which comprises the culture of waonoruuclear cells in a culture medium
containing a chemical ligand having receptors on the membrane of mononuclear
cells,
for example histamine or histamine agoc7ist (1i a in action) and a H2
antagonist in
combination or not with "additional" ~iM-CSl"', with the proviso that if said
culture
medium comprises GM-CSF, it does not comprise 1,25~dihydroxy D3 vitamin.
'fhe invention also relates to a process for preparing a composition
comprising
MD-APCs ctescribe~ herein, said process cc~mprisir~g:
(i) culriiring mononuclear cc~Ils in ~~ culture medium comprising a
chemical ligand of mononuclear cells, wherein said chemical ~ligand is
selected
from the group consisting of:
a) histamine;
b) histamine agonists;
c) histamine H2 receptor (:H2) antagonists;
d) detoxif ed lipopolysaccharide (detoxified LPS)
e) ligands of complement receptors;
f) taxols;
g) oxydoreductoxs;
h) ligands to CD X13;
i) ligands to tumour necrosis factor (TNF) receptors;
j) ligands to vitamin I:~:3 receptors;
k) ligands to interleukin 13 (IL-13) receptors; and
1) any corrabination c>f (a) to (k);
wherein said chemical ligand is iz~ combinatic:>n or not with exogenously
added
Granulocyte Macrophage Colony Stimulating Factor {GM-CSF); with the proviso
that
if said culture medium comprises GM-CSF, it does roc>t comprise 1,25-dihydroxy
D3
vitamin; and
3Q {ii) allowing differentiation into MD-APCs, to obtain a culture comprising
MD-APCs.
An example of histamine aganist is 2 methyl-histamine.

CA 02252505 2001-11-29
7a
The expression "additional"corresponds to the fact that there is no GM-CSF
added to the culture in standard condition, but this does not exclude the fact
that
exogenous GM-CSF could be added in the culture to increase yield and functions
of
MD-APCs obtained.
As example of H2 antagonist one may cite cimetidine but also tiotidine,
burimamide, metiamide, ranitidine.
As example of other chemical ligands interacting with mononuclear cells and
allowing differentiation into MD-APCs, one may cite detoxified LPS such as
lipid A,
C3 and other ligands of complement receptors, taxols, oxydoreductors such as
flavenoids or polyphenols, ligands to CD40, to the TNF receptors or to vitamin
D3
receptors.
The invention also relates to a process wherein the culture medium contains
chemical ligands, such as histamine and cimetidine or a H2 antagonist without

CA 02252505 2001-03-19
8
"additional" GM-CSF, histamine being present at a
concentration of about 10-2 M to about 10-6 M, preferably
of about 10-4 M, and cimetidine or the H2 antagonist being
present at a concentration of about 10-4 M to about 10-9 M,
preferably of about 10-6 M.
When the concentration of histamine or
cimetidine or other chemical ligands for membrane
receptors is lower than the lower value of the given
range, there is substantially no effect, i.e. less than
loo difference with cells cultured in the absence of
histamine and cimetidine.
When the concentration of histamine or
cimetidine is higher than the higher value of the given
range, the culture medium becomes toxic.
When no additional GM-CSF is incorporated into
the culture medium, GM-CSF secreted in situ by the MD-
APCs can be present at a concentration of about 5 to
about 500 U/ml.
The invention also relates to a process wherein
the culture medium contains a chemical ligand, for
example histamine and cimetidine or a H2 antagonist in
combination with "additional" GM-CSF, histamine being
present at a concentration of about 10-2 M to about 10-6 M,
preferably of about 10-9 M, cimetidine or the HZ
antagonist being present at a concentration of about 10-4
M to about 10-9 M, preferably of about 10-6 M, and
additional GM-CSF being present at a concentration of
about 50 U/ml to about 1000 U/ml, preferably of about 500
U/ml.
When additional GM-CSF is incorporated into the
culture, then the total concentration of GM-CSF is from

CA 02252505 2001-03-19
9
about 50 U/ml to about 2000 U/ml, and preferably from
about 50 U/ml to about 500 U/ml.
According to a particular embodiment of the
invention, the culture medium does not comprise the
following elements: exogenous cytokines such as IL4,
IL10, TNF.
The invention also relates to a process
comprising:
- isolation of leukocytes, from healthy donors or from
patients, from peripheral blood by apheresis and removal
of platelets and anticoagulant from the apheresis
product,
- isolation of mononuclear cells (monocytes +
lymphocytes) from red cells and granulocytes in order to
have less than loo granulocytes and less than 5o red
cells,
- culture of the mononuclear cells obtained at the
previous stage by placing them in an appropriate culture
medium containing a chemical ligand of mononuclear cells,
such as histamine or an agonist of histamine, an H2
antagonist such as cimetidine, in combination or not with
GM-CSF, for a time sufficient to obtain differentiated
MD-APCs, preferably for about 5 to 15 days, and possibly
separating the MD-APCs from the lymphocytes, and
recovering the MD-APCs or the MD-APCs and lymphocytes.
The invention also relates to a process wherein
the culture medium of MD-APCs is added
- with crude antigens, for instance autologous tumor
membrane, killed tumoral cells bacterial capsides, viral
homogenates cleared from nucleic acids,
- specific peptides against which an immune response is
desired,

CA 02252505 2003-08-O1
9a
- cDNA or genetic material Braked to vectors (for example
gluconated polylysine) to allow transfection of the
macrophage with material coding for t:he relevant peptide
or protein to be presented on the MD-APCs membrane and
against which an immune response is desired,
- or bispecific antibodies targeting on the one side, a
surface antigen or a surface receptor of the MD--APCs and,
on the other side, a relevant antigen against which an
immune response is desired.
The invention also relates to MD-APCs liable to
be obtained according to the process of the invention
described hereabove.
The invention also relates to pharmaceutical
compositions containing as active substances MD-APCs
according to the invention, in admixture with a
pharmaceutically acceptable diluent or carrier,
The invention also relates to a Cellular
vaccine and cellular vaccine compositions containing as
active substance, MD-APCs according t~ the invention.
The invention also relates to the medium
containing elements necessary for the growth and
differentiation of monocytes into MD-APCs according to
the invention, and in addition containing chemical
ligands of mononuclear ,:;ells, such as histamine,
cimetidine in combination or not with GM-CSF, with the
proviso that if said meda.um comprises GM-C3F, it does not
comprise 1,25-dihydroxy f~~ vitamin;ranc~ its usP in the
preparation of 1~~7-~PCS.
As c~xamplc~~ c~f other chemical ligands
interacting wa_th rnonc~r~uc.vear cell's and allowing
differentiation int<:~ MD-AI?C~M , one. ma~~ c.:ite detoxified 7~PS
such as lipid A, C'3 <~xz.c~ other :Ligands of complement
receptors, ta~col;~, c>:xydo:r-educ.vt:ors such as flaver~oids or

CA 02252505 2003-08-O1
~b
polyphenols, ligands to CD~O, to the TNF receptors or to
vitamin D3 receptors.
The invention also relates to a cell processor
or a kit containing:
- means for the re~rovery af: lymphocytes arid monocytes
free of contaminants,
- appropriate buffer and wash solutions and possibly
appropriate means far the conservation of macrophages,
- means for preparing a cu:l.ture far the monocyt.es and
possibly the lymphocytes and containing chemical ligands
of mononuclear cells, for e:~ample histamine, c:imetidine
or a H2 antagonist in combination or not with GM-CSF,
- possibly means for transfectian of cultured cells and
means for targeting antigens 'to MD-APCs.
In an embodiment, t:h~e above-mentioned cell processor or
kit further comprises instructions for: the preparation of
MD-APCs.
Regarding the conservat.ian of MD-APCS, it can
include freezing means fox example in 10~ glycerol or
D.M.S.O. (dimethyl sulfaxyde) in the presence of
autologous or A~31 serum'.
The invention salsa r~.:lates to a cell
processor or a kit as described abcwe which contains:
- means for recovering and centrifuging blood to obtain a
leukocyte concentrate, means for separating lymphocytes
and monocytes from the ether whx.te cells and for
eliminating the contaminating red c::elis,
- culture medium far MD-AP~"s and possibly lymphocytes
with complements and particularly chemical ligands of
mononuclear cells, such as r~istam~..ne and cimetidine or a
H2 antagonist in cambiraatiorx or nc~t:. with GM-CSF,
- appropriate means f:ar the c;ar~serro~rat~on of macrophages,

CA 02252505 2001-11-29
9c
- appropriate buffer and wash solution.
The invention also relates to a cell processor or kit comprising:
(i) a culture medium comprising a chemical ligand of mononuclear cells,
wherein said chemical ligand is selected from the group consisting of.
a) histamine;
b) histamine agonists;
c) histamine H2 receptor (H2) antagonists;
d) detoxified lipopolysaccharide (detoxified
LPS)
e) ligands of complement receptors;
f) taxols;
g) oxydoreductors;
h) ligands to CD 40;
i) ligands to tumour necrosis factor
(TNF) receptors;
j) ligands to vitamin D3 receptors;
k) ligands to interleukin 13 (IL-13)
receptors; and
1) any combination of (a) to (k);
wherein said chemical ligand is in combination or not with exogenously added
GM-
CSF; with the proviso that if said culture medium comprises GM-CSF, it does
not
comprise 1,25-dihydroxy D3 vitamin; and
(ii) instructions for culturing mononuclear cells in said medium and for
allowing differentiation into an MD-APC as defined above.
The invention also relates to a use of any of the above-mentioned cell
processors or kits for preparation of an MD-APC as defined above.
The invention also relates to products containing MD-APCs according to the
invention, and lymphocytes, as a combined preparation for simultaneous,
separate or
sequential use in cell therapy.
The invention also relates to a use of the above-mentioned combined
preparation in cell therapy, wherein said use is simultaneous, separate or
sequential.
The invention also relates to products as described hereabove which contain
the MD-APCs and the lymphocytes in a ratio of at least 20% to 50% of MD-APCs
expressed in cell number.

CA 02252505 2001-11-29
9d
The invention also relates to bispecific antibodies liable to recognize an
antigen of a MD-APCs of the invention and an antigen of a tumoral cell or of a
pathogen which is to be targetted to said MD-APCs.
The invention also relates to a method for the clinical treatment, comprising
the administration of an appropriate amount of MD-APCs according to the
invention,
and preferably in an amount of about 10g to about S x 109 MD-APCs.
The invention also relates to a use of MD-APCs according to the invention for
clinical treatment.
The invention also relates to a method for the treatment of any disorder,
comprising the administration of lymphocytes from the culture in an amount of
about
4x 1 O9 to about 1 Ox 1 O9 lymphocytes.
The invention also relates to the use of a chemical ligands of mononuclear
cells, such as an agonist of histamine receptor, in particular histamine, and
a H2
antagonist in particular cimetidine, in combination or

CA 02252505 2001-11-29
9e
not with GM-CSF, for the preparation of MD-APCs having
the following properties:
- they present on their surface:
* antigens CD14 and CD64 with a measured mean
fluorescence intensity of about 5 to about 200. relative
to a reference value,
* antigen CD80 and CDBfi with a measured mean
fluorescence intensity of about 20 to about 200 relative
to a reference value,
* antigen CD40 and mannose receptor with a
measured mean fluorescence intensity .of 50 to 500
relative to a reference value,
- they are substantially devoid of the surface antigens
CDla and CDlc, the presence and, mean intensities
respectively of CD14, CD64, CD80, CD86 and the absence of
CDla and CDlc being for instance determiwed by
immunofluorescence staining.and flow cytometry analysis,
- they present high phagocytosis property such as
determined by the following test:
said phagocytosis capacity being evaluated by an uptake
of formalin fixed yeast, for example by culturing MD-APCs
for 2 hours to select adherent cells, adding yeast in
1/10 macrophages to yeast ratio and incubating at 37°C,
5% ~C02 atmosphere for 2-3 hours fixing by the May-
Griinwald- .Giemsa (MGG) staining, and the percentage of
phagocytic MD-APCs being quantified for instance by
microscopic analysis, -
- they have the property of stimulating the proliferation
of allogenic lymphocytes such as determined by the
following test:
allogenic primary mixed lymphocytes reaction (MLR) was.
carried. out in 96-well microtiter plates by adding

~ CA 02252505 2001-11-29
9f
different numbers 2x103 to 2x105 in 100 ~tl medium/well of
MD-APCs to 2x105 in 100 ~1 medium/well of allogenic T
cells purified from huffy coats and after 5 days
incubation at 37°C, cell proliferation

CA 02252505 2003-08-O1
1~
was assessed by a colorimetrie method, such as the cleavage of tetrazolium
salt
WST-1 (slightly red) to Formozan (dark red).
The monocytes derived antigens presenting cells of the invention and the
MD-APCs can be obtained as follows
a) Isolation of leukocytes from blood by apheresis, and elimination of
platelets, by centrifugation. If cells collected have <. lfl r°6
granulocyies and
haematocrit < S~, they can be seeded as such in culture. Otherwise
mononuclear cells have to be prepared first by centrifugation on Ficoll
f'a9ue~of
density 1,077.
to b) Mononuclear cells are then culntred for 5 to 15 days in hydrophobic
bags ethylene vinyl acetate (E.V.A., Stedim) or polypropylene (Life Cell,
Haxter), at 37°C, 596 Cf~. Cells are seeded at 5.1061m1 in
1.M.D.h~. (as
previous) or equivalent medium supplemented with indomethacin (SxlO'6?Vn
mercaptoethanol (3x10-SNl), non essential amino-acids (1'9~, Gibeo) and 2 to
is 59'0 of reconstituted autologaus orAB+ serum,
- with GM-CSF, 500 Ulml chemieal ligands interacting with mononuclear
cells and allowing differentiation into MD-Al'Cs, such as detoxifed Ll'S such
as lipid A, C3 and other ligands of complement receptors, taxols, .
oxydoreductors susch as flavenoids or polyphenols, ligands to CD4a, to tie
20 TNF receptors or to vitamin D3 receptors ; as exs~mple of ligands,
histamine
(10"4 M), cimetidine (10"'6 M) or another 13~ antagonast of histamine, or with
histamine, cimetidine in the absence of any exogenous cytokines. Endogenous
cytokines are released by mononuclear cells stimulated by ligands.
- and exogenous antigens, peptides or transfectants (cDNA + vector
25 coding for the relevant antigen.
c) Afler cultvie, the specific monocyte derived antigen presenting cells
.(MD-APCsI are centrifuged, washed and resuspended far injection in the
patient, to induce hutnoral and cellular immune response against the antigen.
The specificity of the cellular vaccine is achieved during the in vitro
3o culture according to one of the following items.
a) culture of MD-AFCs as explained above in b), in the presence of crock
antigens, for example autologous .tumor rraembrane, bacterial capsides, viral
homogenates cleared fi'orn nucleic acids
b) culture of MD-APCs as explained above in b), in the presence of
35 specific peptides against which in~mur~ response would be beneficial,
c) or culture of MD-APCs as explained above in b), in the presence of
cDNA or genetic material linked to vectors Cfor example gluconated
* Trade-mark

CA 02252505 2001-03-19
WO 97/44441 11 PCT/EP97/02703
polyphysine) to allow transfection of MD-APCs with material coding for the
relevant peptide or protein to be presented on the membrane.
In order to obtain specific cellular vaccine, it is also possible at the end
of
the differentiation stage of the macrophage culture to add bispecifie
antibodies
targeting a membrane antigen, or a surface receptor of MD-APCs on one side
and the relevant antigen on the other side.
According to a preferred embodiment, the process of the invention
comprises the following steps:
- isolation of leukocytes from blood of healthy subjects or patients by
to apheresis, to obtain the apheresis products (i.e. concentrated leukocytes),
- platelet -elimination, for instance by centrifugation of the apheresis
products, to obtain a leukocyte enriched product,
- separation, in the leukocyte enriched products, of the mononuclear cells
on one hand, and of the contaminating red blood cells and granulocytes on the
~ s other hand,
- culture of the mononuclear cells (monocytes + lymphocytes) in a
medium containing chemical ligands of mononuclear cells, such as histamine
and cimetidine and GM-CSF for about 5 to 15 days, to obtain differentiated
monocyte derived antigen presenting cells (MD-APCs).
20 The lymphocytes can be separated from the monocytes before the culture
step.
The lymphocytes can be separated from the MD-APCs after the culture.
In the process of the invention, chemical ligands for mononuclear cells,
such as histamine are used at a concentration of 10-2 M to ,about 10-6 M,
25 preferably of about 10'~ M.
In the process of the invention, GM-CSF is used at a concentration of
about 50 to about 1000 U/ml, particularly of about 100 to about 500 U/ml.
In the process of the invention, the culture medium is RPMI, IMDM,
MEM, or DMEM selected for very low endotoxin content.
3o These media are commercially available.
Advantageously, the culture medium contains indornethacin (or another
cyclo-oxygenase inhibitor) or/and cimetidine (an histamine H2 antagonist)
andlor at other chemical ligands of mononuclear cells.
An advantageous process for preparing the MD-APCs of the invention is
35 the following:
Apheresis
Leukocytes from healthy subjects or from patients are isolated from
peripheral blood by apheresis using the Cobe Spectra~~ontinous-flow blood cell
* Trade-mark

CA 02252505 2001-03-19
WO 97/44441 1~ PCT/EP97/02703
separators keeping granulocytes contamination very low (< 10% and less than
% red cells). The apheresis product is centrifuged for 10 min at 280 g in
order
to reduce platelet contamination. The platelet-enriched plasma is removed and
leukocyte pellet resuspended in a phosphate buffer solution (PBS) containing
5 0.1 % glucose, 0.17 % P03HNa2, 2H20, 0.27 % P03H2Na, 0.14 % NH4C1,
0.78 % NaCI (solution TS745 laboratoire Bruneau, France).
The enriched leukocyte pellet is obtained with an average of 7 to
1.5x1010 leukocytes (50% of mononuclear cells).
Isolation of mononuclear cells
to If the collected leukocytes have more than 10% granulocytes
contamination and/or 5 % hematroerite, human mononuclear cells are separated
from red blood cells and from contaminating granulocytes, by 15 min
centrifugation at 1000 g on a COBS 2991 or Stericelhcell processor using
Ficoll
Paque of density 1.077 {Pharrnacia). After 3 washings in phosphate buffered
saline solution without calcium and magnesium, the monocytes are obtained
with about 20% to 50% purity as shown by channelyser analysis (Coulter
Margency-France).
Culture
Differentiated human MD-APC are obtained by 5-15 days in culture of
2o mononuclear cells in hydrophobic bags in E.V.A. (Ethylene Vinyl Acetate,
STEDIM, Aubagne) or polypropylene (life cell-Baxter) at 37°C and S%
C02,
95 % humidified atmosphere. Total mononuclear cells are seeded at
5x106 cells/rnl in Iscove modified medium (LM.D.M., Gibco) or equivalent
medium supplemented by penicillin (100 UI/ml), streptomycine (100 teg/ml),
L-glutamine (2 mM, Gibco), pyruvic acid (2 mM, Gibco), Indomethacin
(5x10'6 M, Sigma), cimetidine (10-8 to 10''1 M), histamine (10-6 to 10-2 M or
other chemical ligands), mercaptoethanol (3x10'5 M, Gibco) non-essential
amino-acids (1 %, Gibco) and 2-5 % of autologous or AB serum. The addition of
GM-CSF (500 U/ml, SANDOZ) was done in comparative experiment.
3o According to a preferred embodiment, the process of the invention is such
that killed tumoral cells are added into the culture medium simultaneously
with
monocytes, both cells coming preferably from the same patient, preferably at
the ratio of about 1 million of killed tumoral cells/ml, with said killed
tumoral
cells being processed at the same time as macrophages.
The killed tumoral cells can then be processed simultaneously with the
leukocytes, in an amount of about 1x106/ml.
* Trade-mark

CA 02252505 1998-10-21
WO 97/44441 ~ ~ PCT/EP97/02703
This process allows to obtain MD-APCs and lymphocytes specific for the
tumor, inducing very efficiently in vivo an immune response to these specific
tumor cells.
The invention also relates to MD-APCs liable to be obtained according to
the above-defined process.
The invention also relates to pharmaceutical compositions containing, as
active substance, MD-APCs as defined above.
The invention also relates to a medium containing elements necessary for
the growth and differentiation of monocytes into MD-APCs of the invention,
and in addition chemical ligands for mononuclear cells, for example histamine
and GM-CSF.
The monocyte derived antigens presenting cells of the invention can be
part of a cell processor or a kit containing:
- means for the recovery of lymphocytes and monocytes free of
contaminants;
- appropriate buffer and wash solutions, and possibly appropriate means
for the conservation of MD-APCs ;
- means for preparing a culture medium for the monocytes and possibly
the lymphocytes and containing chemical ligands for mononuclear cells, for
2o example histamine and/or GM-CSF.
According to an advantageous embodiment of the invention, the cell
processor or kit contains
means for recovering and centrifuging blood to obtain a leukocyte
concentrate;
- means for separating lymphocytes and monocytes from the other white
cells and for eliminating the contaminating red cells;
- culture medium for MD-APCs and possibly lymphocytes with
complements and particularly and/or GM-CSF and possibly indomethacin
and/or cimetidine ;
- appropriate means for the conservation of the MD-APCs ;
- appropriate buffer and wash solutions ;
The invention also relates to products containing MD-APCs according to
the invention, and lymphocytes, as a combined preparation for simultaneous,
separate or sequential use in adoptive immunotherapy.
According to an advantageous embodiment, the products of the invention
as defined above are characterised in that they contain the MD-APCs and the
lymphocytes in a ratio of at least 20 % to 50 % of MD-APCs expressed in cell
number.

CA 02252505 1998-10-21
WO 97/44441 ~ ~ PCT/EP97/02703
In this embodiment, the MD-APCs and the lymphocytes are both injected
to a patient.
The invention also relates to bispecific antibodies liable to recognize an
antigen of a MD-APC of the invention, for example FCyRI (CD 64) and an
s antigen of a relevant antigen.
The bispecific antibodies can be prepared as described in Chokri et al.
Res. Immunol. ~ (1992).
The bispecific antibodies can be injected at the same time as the
MD-APCs of the invention, or can be pre-incubated with MD-APCs before
to injection.
The invention also relates to a method for the treatment of cancer and
pathogens comprising the administration of an appropriate amount of MD-APCs
according to the invention, and preferably in an amount of about 1 x 10g to
about
5x109 MD-APCs.
Descritztion of the Fi
Figure la represents the allogenic T cell proliferation induced by
MD-APCs of the invention recovered in the presence of histamine (10~ M) and
cimetidine (10-6 M) as adjuvant, with or without GM-CSF (S00 U/ml)
2o comparing to standard macrophages produced only in the presence of the
GM-CSF (500 U/ml).
Figure 1 b represents the stimulation by MD-APCs recovered in the
presence of GM-CSF or GM-CSF + IL-13.
In Figure la, the optical density (450-690 nm) has been plotted against the
ratio between the MD-APCs of the invention and the responder lymphocyte
cells.
The clear dotted bars correspond to the presence of GM-CSF at
S00 U/ml; the lighter grey bars correspond to the presence of histamine
( 10~ M) and cimetidine ( 10-6 M).
3o The darker grey bars correspond to the presence of GM-CSF (500 U/ml),
in combination with histamine (10~ M) and cimetidine (10-6 M).
The results show that the capacity of the MD-APCs to stimulate the
proliferation of allogenic lymphocytes is strongly increased. The stimulation
of
allogenic proliferation of lymphocytes by MD-APCs is very potent (at least
200% increase with respect to standard macrophages). The combination of
histamine/cimetidine effect or IL-13 effect with GM-CSF can potentiate this
activity {maximum of 300% increase with respect to the induction by
macrophages).

CA 02252505 2003-08-O1
In Figure 1b, the optical density (950-690 nia)
has been plotted against the ratio between the MD-APCs of
the invention and the responder lymphocyte cells.~The
curve with darl~ circles corresponds to the presence of
5 GM-CSF/IL-13 and the curve with open circles correspond
to the presence of GM-CSF.
EXAMPLE . Preparation of MD-APCs according to-
the invention:
1- Apheresis:
Z,eukocytes froia healthy donors or fro~a patients
are isolated from peripheral blood by apheresis using
CORE spectra blood cell separator keeping gzanulocytes v
contamination the lowest possible. The apheresis product
is diluted in a phosphate buffered solution (PHS~ (final
volume = 950 m1). The apheresis product is centrifuged
for 10 min at X80 ~ to remove platelets. and
anticoagulant.
zo
2- Isolation of mononuclear cells:
If the collected ce:~.~s have > 10% granulocytes
contamination and/or > 5 ~ haematocrit, they should be
centrifuged' over Ficoll*(density ~ 1.4?7) to remove~zed
cells and granulocytes,
3- Culture
The monocyte derived antigen presenting cells
(MD-APCs) are obtained after 5 to 15 days in culture of
mononuclear cells in hydrophobic bags in EVA (Ethylene
Vinyl Acetate /STEDIM) or equivalent bags (Tefla~) at
37°C and 5 % C02 in humidified atmosphere. The mononuclear
*Trade~~m~~rk~

CA 02252505 2001-03-19
15a
cells are seeded at 5x106 cells/ml in Iscove modified
medium (I.M.D.M. - Gibco) or equivalent supplemented by
(see WO 94/26875, published November 24, 1994, page 7)
the addition of ligands for mononuclear cells, such as
histamine (10-9 M) or analogues and cimetidine (10-6 M) or
similar H2 antagonist, in the presence or not of GM-CSF
(500 U/ml).
As example of other chemical ligands
interacting with mononuclear cells and allowing
differentiation into MD-APCs, one may cite detoxified LPS
such as lipid A, C3 and other ligands of complement
receptors, taxols, oxydoreductors such as flavenoids or
polyphenols, ligands to CD40, to the TNF receptors or to
vitamin D3 receptors.

CA 02252505 1998-10-21
WO 97/44441 ' b PCT/EP97/02703
4- Characterization and functionality
a- Flow cytometry
After the maturation of MD-APCs, the phenotype analysis of the obtained
cells was performed by flow cytometry using murine FITC or P.E labelled
s monoclonal antibodies directed against membrane proteins.
b- Mixed lymphocytes reactions (MLR)
To evaluate the induction of T cell response to MD-APCs, allogenic
primary MLR was carried out in 96-Well microtiter plates by adding different
numbers of MD-APCs to 2x105 allogenic T cells purified from buffy coats.
to After 5 days at 37°C, cell proliferation was assessed by a
colorimetric methods
such as WST-1.
c- Phagocytosis
The phagocytic capacity of the different cells obtained was evaluated by
uptake of formalin fixed yeast. Briefly the cells were cultured for 2 hours to
~5 select adherent cells. The yeast was added at 1/10 macrophage/yeast ratio
and
incubated in 37°C, 5% C02 atmosphere for 2-3 h and then fixed by the
May-
Grunwald-Giemsa (MGG) staining. The percentage of phagocytic cells was
quantified by microscopic analysis.
The results are gathered in table 1, table 2 and table 3.
Table 1
Yield (% of cells) differentiated in culture
Time of culture (a) (b)
2s Hist/Cim Hist.Cim/GM
Day 4 71 % 7g
Day 7 49 % 48 %
Day 11 19 % 31 %
Table 1 shows that there is a recovery of a large quantity of MD-APCs
after 5 to 11 days of culture of mononuclear cells in hydrophobic bags, in the
presence of histamine (10''l M) and cimetidine (10-6 M) (a) and additional
GM-CSF (S00 U/ml) (b) as adjuvant. In comparison with production of mature
macrophages in standard conditions, the recovery of MD-APC cells is in the
same order.

CA 02252505 1998-10-21
WO 97/44441 ~~ PCT/EP97/02703
Table 2
Phenotype of MD-APCs
produced under various conditions after 6 days of culture
(a) (b)
Surface Ag Hist/Cim Hist.Cim/GM-CSF
% MIF % MIF
CDla N N
to CDlc N N
CD83 N N
CD 14 95 114 95 92
CD 54 96 40 96 40
CD 58 97 41 97 31
is CD 64 43 35 91 46
HLA-DR 92 350 96 400
MIF = Mean Intensity Fluorescence.
= percentage of positive cells.
2o N = Negative or weak signal.
This table corresponds to the immunofluorescence profile analysis by flow
cytometry of MD-APCs generated in culture (6 days) under different
conditions. The cells obtained under histamine (10~ M) 'and cimetidine
25 (10-6 M) conditions (a) are positive for macrophage markers (CD14, CD64 and
HLA-DR). The combination with GM-CSF (b) does not change the phenotypic
profile of the MD-APCs. They also clearly express CD54 and CD58. The
CD80 (B7.1) and CD86 (B7.2) are alsa expressed on their membrane but in
lower level. In contrast, CDla, CDIc and CD83 which are positive markers of
3o dendritic cells, are weakly expressed by the MD-APCs.
From these data, it can be confirmed that the antigen presenting cells
generated in the culture system of the invention are different from dendritic
cells.

CA 02252505 1998-10-21
WO 97/44441 f $ PCT/EP97/02703
Table 3
% of phagocytic cells after 3 h contact with fixed yeast
Number of (a) (b)
intracellular Yeast Hist/cim Hist.Cim/GM-CSF
to 0 42% 20%
1-5 43 .6 % 43 %
6-10 11 % 27.5 %
~5
> 10 3.4 % 9.5 %
The MD-APCs are tested for the phagocytic activity using formalin fixed
2o yeast. After 3 h incubation and MGG (May Grumwald Giemsa) staining, the
intracellular particles are quantified by microscopic analysis. The MD-APCs
generated in the presence of histamine/cimetidine (a) present an important
capacity of phagocytosis (60%) and this capacity is increased in the presence
of
GM-CSF in the culture (80 %a ) (b).
25 From the above-mentioned results, it is shown that human mononuclear
cells cultured in the presence of histamine and cimetidine, in combination or
not
with GM-CSF mainly fox one week, differentiate into mature antigen presenting
cells. During the culture, these cells acquire a high level of accessory
functions
like.
3o The results of the invention indicate that the MD-APCs obtained in the
culture system of the invention are different from the dendritic cells (DC),
since
DC have been described as FC (CD64) receptor negative, poorly adherent and
non-phagocytic cells, possessing only a small number of lysosomes.
In contrast the MD-APCs of the invention
3s (a) show a high adherence capacity,
(b) show an important phagocytic and processing activity,
(c) express high level HLA-DR membrane antigens and a low level of
CDIa and CDlc, which is strongly expressed by dendritic cells,

CA 02252505 1998-10-21
WO 97144441 ,~ PCT/EP97/02703
(d) are also positive for CD54, CD58, CD80 and CD86 membrane
antigens,
(e) stimulate T-cell proliferation in allogenic MLR reaction, and this is in
a far better way than macrophages obtained according to techniques of the
prior
art.
Table 4 hereafter gathers the comparative characterization of MD-APC of
the invention on the one hand, and of dendritic cells on the other hand.
Table 4
1o Comparative characterization of Antigen Presenting Cells:
Dendritic Cells MD-APC
cells Intensity
1 s Phenotype
CD 1 a -l- _ 0
CDlc + - 0
CD 83 + _ 0
CD 14 + /- + 10 to 100 5 to 200
2s CD64 - + 10 to 100 5 to 200
CMH-II + + + + + + 80 to 100 I00 to 400
Functions:
Adherence - + + 70 to 90
Phagocytosis - + + 30 to 80
3s Stimulation of MLR + + + + +

CA 02252505 1998-10-21
WO 97/44441 ~ PCT/EP97/02703
Table 5
It gathers a complete phenotypic characterization of MD-APCs recovered after
6 days of culture according to the invention (analysis by flow cytometry).
s
Phenotype ~ % Cells ~ Mean fluo
intensity
CD45 97,6
CD14 6,8 172
CD3 13
CD19 15
CD56 3,8
CD4-PE 95
CD25 1, 7
CD45R0 99
CDI6 1,8 31
CD32 63 163
CD64 4 12
Io
CDIa 31 216
CDlc ~ 58 505
CD83 9 1g
HLA-DR 99 266
HLA-I 99,6 582
CD40 98 991
CD80 78 64
CD86 99
IgG I -FITC 6,7 11
IgGl-PE 20 ( 16)55
IgGI-Cy5 19 (29)50
IgG2a-FITC 5 , 3 19
IgGl i 1,6 75
IgG2a i 3,8 21
IgG2b i 3,2 15