CA2252505C - New antigen presenting cells, a process for preparing the same and their use as cellular vaccines - Google Patents

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 mannos e 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.

Description

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.

~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 ~°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.

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.

' ' 11534-15(S) 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 ' ' 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 ' ' 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 ' ' 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.

' ~ 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 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, 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 ' ~ 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 ' 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 ' ' 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:

' ' 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:

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 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 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 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.

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 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 "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 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, 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 ~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, 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.

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 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 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 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 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 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.

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).

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~

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.

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.

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

is CD 64 43 35 91 46 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.

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, 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 + + + + +

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 CD56 3,8 CD25 1, 7 CDI6 1,8 31 Io CDIa 31 216 CDlc ~ 58 505 CD83 9 1g HLA-I 99,6 582 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

Claims (64)

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.

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).

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 - 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.

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 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, 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 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 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:

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 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.

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 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 action), and - a H2 antagonist, in combination with GM-CSF.

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.

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 (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 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.