WO1999053027A1 - Use of monocytes derived cells and antibodies in a synergic way for optimal induction of immunotherapeutic efficiency - Google Patents

Use of monocytes derived cells and antibodies in a synergic way for optimal induction of immunotherapeutic efficiency Download PDF

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Publication number
WO1999053027A1
WO1999053027A1 PCT/EP1999/002104 EP9902104W WO9953027A1 WO 1999053027 A1 WO1999053027 A1 WO 1999053027A1 EP 9902104 W EP9902104 W EP 9902104W WO 9953027 A1 WO9953027 A1 WO 9953027A1
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antibodies
monocyte derived
cells
derived cells
binary complex
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PCT/EP1999/002104
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French (fr)
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Jacques Bartholeyns
Yves Fouron
Mohamed Chokri
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I.D.M. Immuno-Designed Molecules
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Priority to AU36012/99A priority Critical patent/AU3601299A/en
Publication of WO1999053027A1 publication Critical patent/WO1999053027A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4614—Monocytes; Macrophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464499—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/47—Brain; Nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to the use of monocytes derived cells and antibodies in a synergic way for optimal induction of immunotherapeutic efficiency.
  • the invention relates more particularly to the use of macrophages and antibodies in a synergic way for optimal induction of immunotherapeutic efficiency.
  • Macrophages play a major role in the anti-tumoral response, and they are able to be activated by immunological activators against cancer cells (Adams D. and Hamilton T. : "Activation of macrophages for tumor cell kill : effector mechanism and regulation” ; in Heppner & Fulton (eds), Macrophages and cancer. CRC Press, 1988, p. 27 ; Fidler M. Macrophages and metastases. A biological approach to cancer therapy. Cancer Res. 45_: 4714, 1985).
  • macrophages or other cells derived from monocytes or from their precursors, with their strong capacity for endocytosis, digestion, and surface antigen presentation, are capable of inducing a specific immune response.
  • Macrophages represent the first natural line of defence against infectious agents (bacteria, virus) which are normally killed.
  • infectious agents bacteria, virus
  • resistant pathogens can develop ways to escape recognition and killing by macrophages. Protection cannot be very effectively achieved with the attenuated infectious agents or with their recombinant peptidic components.
  • Monocytes derived cells are immune cells such as obtained by culture of blood mononuclear cells in non adherent gas permeable plastic or Teflon bags for
  • Monocytes derived cells can either be killer macrophages, phagocytozing cells, growth factors and cytokines releasing cells, or immature dendritic cells according to their conditions of differentiation.
  • Dendritic cells can for example be obtained as described in "Dendritic cells as adjuvants for immune- mediated resistance to tumors" (Schuler G. and Steinman R. M.; J. Exp. Med., 186: 1183-1187, 1997), and EP n° 97/02703.
  • Dendritic cells are very potent antigen presenting cells to initiate an immune response.
  • the dendritic cells can be characterized by the induction of T cell proliferation and by their phenotype (presence of CD80, CD86, CD83, MHC-I,
  • IgG, IgA and IgM antibodies directed against infectious agents can be isolated in human plasma an are prepared as monoclonal antibodies. These antibodies do very seldom neutralize the infection as such ; a cellular immune response is required.
  • Therapeutic vaccines are therefore difficult to develop with attenuated infectious agents or with their recombinant peptidic components.
  • Pan tumor antigens can be targeted, at least in vitro by specific human monoclonal antibodies. These pan anti-tumor antibodies do not react with normal cells and tissues such as epithelium, fibroblasts, neuroectodermal or muscle cells. However, these antibodies bind through their Fc part to reticuloendothelial cells such as macrophages bearing Fc receptors.
  • An advantageous property of pan tumor antibodies is that they can be used to target several tumor types.
  • One of the aims of the invention is to provide a binary complex between a monocyte derived cell and at least one antibody .
  • Another aim of the invention is to provide a process for the preparation of a binary complex between a monocyte derived cell and at least one antibody.
  • Another aim of the invention is to provide pharmaceutical compositions.
  • Another aim of the invention is to provide a method for inducing or increasing in vivo an immune response.
  • Another aim of the invention is to provide an improved method for the treatment of cancer. 4
  • Another aim of the invention is to provide a new method for the treatment of infectious diseases.
  • the invention relates to a binary complex, in a purified form and substantially free of contaminants, between a monocyte derived cell and at least one antibody directed against a tumor or an infectious agent, with said antibody being bound to the surface of said monocyte derived cell through a non covalent link between the Fc part of the antibody and the Fc receptors present on the monocyte derived cell membrane.
  • the present invention describes an effective method which relies on the interaction between ex-vivo isolated autologous macrophages or monocytes derived antigen presenting cells and anti-tumor antibodies, in vitro or after reinjection to the patient.
  • the resulting interaction allows a synergestic enhancement of the relevant biological activities and, as a result, induces in vivo cellular an humoral anti-tumor immune response.
  • the present invention represents an alternative approach to induce effective immunotherapy by injecting a binary complex formed by anti-infectious antibodies and monocyte derived cells, to force recognition and killing of the infectious agent.
  • purifed form means that the binary complex formed by specific anti-tumor or anti-infections antibodies associated to macrophages is segregated to 90 % from other cell types.
  • substantially free of contaminants means that macrophages are 90 % pure and that no contaminants other than the antibodies are present in the sterile solution.
  • the expression "bound to the surface through a non covalent link” means that antibodies are associated to the cellular membranes through their Fc part of the antibody and the high affinity Fc receptors present in the cell membrane. 5
  • the invention also relates to a binary complex as above defined, wherein the Fc receptors involved are the Fc ⁇ receptors for IgG, Fc ⁇ receptors for IgA or Fc ⁇ receptors for IgM, or the oligosaccharide receptors bindfing the saccharides present on the Fc segment of immunoglobulins.
  • the invention also relates to a binary complex as above defined, wherein the antibody is directed against pan tumor antigens present on different tumor types.
  • the invention also relates to a process for the preparation of a binary complex between a monocyte derived cell and at least one antibody, comprising the following steps : a) preparation of the monocytes derived cells according to the following method:
  • no intracellular interiorization of the antibodies means that there is less than 20 % of antibodies which are interiorized in the monocyte derived cells.
  • the monocyte derived cells can be prepared as described in patents n° PCT/EP93/01232, n° WO94/26875 or EP 97/02703 or in the articles mentioned below :
  • the temperature is such that it prevents interiorization of said antibodies by said monocyte derived cells.
  • the temperature is below 10°C.
  • the step of addition of the antibodies is preceded by
  • the step of addition of antibodies is preceded by the step of freezing at a temperature below or equal to -80°C, aliquots of the monocyte derived cells with the addition of a cryopreservative, to obtain frozen aliquots of monocyte derived cells, and the preceeding step is possibly followed by melting said above frozen aliquots at a temperature enabling to obtain a suspension of monocyte derived cells, for instance at about 4°C, washing said suspension and resuspending, for instance in an isotonic medium, to obtain a suspension of monocyte derived cells.
  • the step of addition of antibodies is followed by the step of freezing at a temperature below or equal to -80°C aliquots of the binary complex, with the addition of a cryopreservative to obtain frozen aliquots of binary complex, and the preceeding step is followed by melting said above frozen aliquots at a temperature enabling to obtain a suspension of binary complex, for instance at 4°C, washing said suspension and resuspending it, for instance in an isotonic medium, to obtain a suspension of binary complex.
  • the invention also relates to a binary complex between monocyte derived cells and antibodies such as obtained by the process of the invention.
  • the invention also relates to a pharmaceutical composition containing as active substance a binary complex as described above, in association with a pharmaceutically acceptable vehicle.
  • the invention also relates to a pharmaceutical composition in the form of sterile injectable solutions.
  • the active substance present in an amount such that it corresponds from about 10 6 to about 10 9 cells/kg of body weight, particularly from about 10 7 to about 10 8 cells/kg of body weight.
  • the binary complex is injected repeatedly at total doses of 10 7 to 5.10 9 at intervals of 3 days to 6 months.
  • the injections can eventually be first local (subcutaneous, intramuscular, mucosal, in cavities or in tissues) and then systemic (intravenous or intralymphatic). 8
  • the invention also relates to the use of a binary complex as described above for the preparation of a drug for treating cancer or infectious diseases.
  • the invention also relates to a combined preparation containing, as active substance, the following individual components in the form of a kit-of-parts : - a solution of monocyte derived cells, and
  • the monocyte derived cells are administered at a dose of about 10 6 to about 10 9 cells/kg of body weight, particularly from about 10 7 to about 10 8 cells/kg of body weight, and the antibodies are administered at a dose of about 1 ⁇ g to about 1 mg/kg of body weight.
  • the antibodies are injected before the monocyte derived cells.
  • the two active ingredients of the combined preparation have never been used for a new joint effect and are unknown as a composition.
  • the active ingredients which are administered either at the same time, or separately, or sequentially, according to the invention do not represent a mere aggregate of known agents, but a new combination with the surprising valuable property that the binary complex formed ex vivo or in vivo presents higher anti- tumoral or anti-infections efficiency than would be expected by simply adding the effects of antibodies and of macrophages.
  • kit-of- parts means that the components of the combined preparation are not necessarily present as a union e.g. in composition, in order to be available for separate or sequential application.
  • kit-of-parts means that it is not necessarily a true combination, in view of the physical separation of the components.
  • the invention can be practised according to two separated and complementary approaches : 1) For tumors or virus or bacteria which are readily accessible to circulating antibodies, the monocyte derived cells, particularly the autologous macrophages, are advantageously injected separately from the monoclonal antibody ;
  • the monoclonal antibodies have thus preferably been contacted with monocyte derived cells, particularly macrophages prepared from the patient's blood. These macrophages bearing anti-tumor antibodies reinjected to the patient can leave the blood circulation due to their plasticity and migrate towards the tumors releasing or attracting chemokines. The tumor cells are killed locally by direct contact by a mechanism of antibody dependent cell cytotoxicity.
  • the invention also relates to a combined preparation wherein said monocyte derived cells and said antibodies form a binary complex as described above, when they are in vitro in the same solution or when they are administered in vivo.
  • the invention also concerns a method for the treatment of cancer or of infectious diseases comprising the use of a binary complex as described above.
  • the invention also relates to a method for the treatment of cancer or infectious diseases comprising the separate or sequential administration of monocyte derived cells and antibodies against the tumor or infectious diseases to be treated, said monocyte derived cells and antibodies being administered in any order at an interval of time no longer than 24 hours, and said monocyte derived cells and antibodies forming, in the human body, a binary complex as described above.
  • the invention also relates to a method for inducing or increasing an immune response, comprising the use of a binary complex as described above.
  • ADCC antibody dependant cell cytotoxicity assay
  • the curve with dark lozenges corresponds to the use of macrophages and the above defined monoclonal antibody
  • the curve with white square corresponds to the use of macrophages without monoclonal antibody.
  • Figure 2 shows that phagocytosis of leukemia cells by activated human macrophage is increased in the presence of an anti CD20 mAb.
  • the abscissae corresponds to PKH26 fluorescence and the ordinate corresponds to CD 14 fluorescence (arbitrary units).
  • Figure 2 shows phagocytosis by activated macrophages (MAK) of PKH26- labelled lymphoblastic leukemia cells in the presence of (B) (D) or absence (A) (C) of anti-CD20 mAb.
  • MAK activated macrophages
  • Tumor-labelled cells (3000 cells/well) are incubated with purified MAK ( 6000 cells/well) for 3h at 4°C (C) (D) or 37°C (A) (B).
  • MAK are stained with a fluorescent monoclonal antibody specific for CD 14.
  • Activated macrophages can be injected together with the antibody or sequentially depending of the mmor target.
  • Macrophages or monocyte derived cells of the following examples are prepared according to the methods described in the above- mentioned references.
  • Monocyte derived cells present in vitro a cytotoxic activity against mmor cell lines. This activity is markedly enhanced when the monocyte derived cells are suspended with 5 ⁇ g antibodies directed against a tumor antigen.
  • the following example concerns a neuroblasma tumor and antibodies recognizing gangliosides expressed on the membranes of this tumor cell line (see figure 1).
  • l.b The following preclinical results show specific increase in interaction and phagocytosis of leukemia tumor cells by activated human macrophages This activity is markedly enhanced when monocytes derived activated macrophages are supplemented with 16 ⁇ g/ml of anti CD20 monoclonal antibody (mAb).
  • mAb monoclonal antibody
  • Human macrophages are obtained according to described methods Isolation of tumor cells.
  • Lymphoblastic leukemia cells are obtained from volunteer patient's peripheral blood. Tumor cells are isolated using ficoll gradient. The mmor cells are washed tree times with PBS.
  • a two color flow cytometric assay is used to determine spontaneous and antibody mediated phagocytosis of CLL (chronic lymphoid leukemia ) tumor cells by purified activated macrophages. Tumor cells are labelled with PKH26 dye (Sigma) according the manufacturer's instructions and are cultured overnight in RPMI 1640 12
  • Double labelled activated macrophages are considered to be phagocytic.
  • Nude mice bearing solid human tumors e.a. S.C. neuroblastomas of 1 cm 3
  • Nude mice bearing solid human tumors e.a. S.C. neuroblastomas of 1 cm 3
  • the tumor volume is measured with calipets in two perpendicular dimensions to follow tumoi evolution.
  • Monocyte derived cells are prepared from new mothers, and they are either isolated from blood or preferentially from the mother milk collected during the two first weeks of lactation after delivery (colostrum contains high concentration of monocyte derived cells, about 1 million per ml, which can be processed to obtain activated macrophages). These monocyte derived cells are processed according to the invention and resuspended in isotonic solution in the presence of antibodies specific for haemophilus and/or pneumococci at a temperamre below 10°C.
  • mice The suspensions containing 10 7 to 10 8 macrophages and 10 to lOO ⁇ g of antibodies are injected locally to the new-born and its effect on the infection is documented.
  • Preclinical data in mice illustrate the activity of macrophages bound to antibodies according to the invention on lethal relevant infections previously induced in mice.

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Abstract

The invention relates to a binary complex, in a purified form and substantially free of contaminants, between a monocyte derived cell and at least one antibody directed against a tumor or an infectious agent, with said antibody being bound to the surface of said monocyte derived cell through a non covalent link between the Fc part of the antibody and the Fc receptors present on the monocyte derived cell membrane.

Description

USE OF MONOCYTES DERIVED CELLS AND ANTIBODIES IN A SYNERGIC WAY FOR OPTIMAL INDUCTION OF IMMUNOTHERAPEUTIC EFFICIENCY
The invention relates to the use of monocytes derived cells and antibodies in a synergic way for optimal induction of immunotherapeutic efficiency.
The invention relates more particularly to the use of macrophages and antibodies in a synergic way for optimal induction of immunotherapeutic efficiency. Macrophages play a major role in the anti-tumoral response, and they are able to be activated by immunological activators against cancer cells (Adams D. and Hamilton T. : "Activation of macrophages for tumor cell kill : effector mechanism and regulation" ; in Heppner & Fulton (eds), Macrophages and cancer. CRC Press, 1988, p. 27 ; Fidler M. Macrophages and metastases. A biological approach to cancer therapy. Cancer Res. 45_: 4714, 1985).
Furthermore, macrophages, or other cells derived from monocytes or from their precursors, with their strong capacity for endocytosis, digestion, and surface antigen presentation, are capable of inducing a specific immune response.
Macrophages represent the first natural line of defence against infectious agents (bacteria, virus) which are normally killed. However, resistant pathogens can develop ways to escape recognition and killing by macrophages. Protection cannot be very effectively achieved with the attenuated infectious agents or with their recombinant peptidic components.
Monocytes derived cells (MDCs) are immune cells such as obtained by culture of blood mononuclear cells in non adherent gas permeable plastic or Teflon bags for
5 to 10 days at 37°C in O2/CO2 atmosphere. They can also be isolated from milk colostrum. Their culture medium (RPMI, IMDM, AIM5 (Gibco) or X-VIVO (Biowhittaker) contains eventually cytokines or iigands as defined in patents n° PCT/EP93/01232, n° WO94/26875 or EP 97/02703 or in the articles mentioned below : 2
. "Autologous lymphocytes prevent the death of monocytes in culture and promote, as do GM-CSF, IL-3 and M-CSF, their differentiation into macrophages". (Lopez M., Martinache Ch., Canepa S., Chokri M., Scotto F., Bartholeyns J. ; J. of Immunological Methods, 159: 29- 38, 1993) ;
. "Immune therapy with macrophages : Present status and critical requirements for implementation" (Bartholeyns J., Romet-Lemonne J- L., Chokri M., Lopez M. ; Immunobiol., 195: 550-562, 1996) ; . "Dendritic cells as adjuvants for immune-mediated resistance to tumors" (Schuler G. and Steinman R. M. ; J. Exp. Med., 186: 1183-
1187, 1997).
All these patents applications and articles are included herein for references. They can be activated by INF-γ at the end of culture to obtain in particular cytotoxic macrophages. They can be centrifuged to be concentrated and purified before resuspension in isotonic solution.
Monocytes derived cells (MDCs) can either be killer macrophages, phagocytozing cells, growth factors and cytokines releasing cells, or immature dendritic cells according to their conditions of differentiation. Dendritic cells can for example be obtained as described in "Dendritic cells as adjuvants for immune- mediated resistance to tumors" (Schuler G. and Steinman R. M.; J. Exp. Med., 186: 1183-1187, 1997), and EP n° 97/02703.
Dendritic cells are very potent antigen presenting cells to initiate an immune response. The dendritic cells can be characterized by the induction of T cell proliferation and by their phenotype (presence of CD80, CD86, CD83, MHC-I,
MHC-II on their membranes).
Human IgM or IgG anti-tumoral antibodies are developed to target tumors or metastases. Clinical anti-cancer responses achieved with these monoclonal human antibodies are not impressive due to unsolved problems of tissue distribution and disseminated sites, limited access to the tumor or to low local concentration, 3
inadequate pharmacokinetics or ineffective dosage, and mainly to the lack of effector cells impairing cellular and humoral immune responses against the relevant tumor.
IgG, IgA and IgM antibodies directed against infectious agents can be isolated in human plasma an are prepared as monoclonal antibodies. These antibodies do very seldom neutralize the infection as such ; a cellular immune response is required.
Therapeutic vaccines are therefore difficult to develop with attenuated infectious agents or with their recombinant peptidic components.
Pan tumor antigens (present in breast, prostate, lung, melanoma, glioma, neuroblastoma and colorectal tumors for example) can be targeted, at least in vitro by specific human monoclonal antibodies. These pan anti-tumor antibodies do not react with normal cells and tissues such as epithelium, fibroblasts, neuroectodermal or muscle cells. However, these antibodies bind through their Fc part to reticuloendothelial cells such as macrophages bearing Fc receptors. An advantageous property of pan tumor antibodies is that they can be used to target several tumor types.
In the case where tumor surface antigens or viral or bacterial antigens are not readily accessible, most systemic antibodies do not succeed in reaching the tumor target or the bacterial or viral target.
In the case where tumors or virus or bacteria are readily accessible, the efficiency of the anti-tumoral, bacterial or viral antigen might be impaired due to the lack of effector cells.
These problems are solved by the invention.
One of the aims of the invention is to provide a binary complex between a monocyte derived cell and at least one antibody . Another aim of the invention is to provide a process for the preparation of a binary complex between a monocyte derived cell and at least one antibody. Another aim of the invention is to provide pharmaceutical compositions. Another aim of the invention is to provide a method for inducing or increasing in vivo an immune response. Another aim of the invention is to provide an improved method for the treatment of cancer. 4
Another aim of the invention is to provide a new method for the treatment of infectious diseases.
In a more specific way, the invention relates to a binary complex, in a purified form and substantially free of contaminants, between a monocyte derived cell and at least one antibody directed against a tumor or an infectious agent, with said antibody being bound to the surface of said monocyte derived cell through a non covalent link between the Fc part of the antibody and the Fc receptors present on the monocyte derived cell membrane.
The present invention describes an effective method which relies on the interaction between ex-vivo isolated autologous macrophages or monocytes derived antigen presenting cells and anti-tumor antibodies, in vitro or after reinjection to the patient. The resulting interaction (tumor-anti-tumor Ab-effector macrophage) allows a synergestic enhancement of the relevant biological activities and, as a result, induces in vivo cellular an humoral anti-tumor immune response. The present invention represents an alternative approach to induce effective immunotherapy by injecting a binary complex formed by anti-infectious antibodies and monocyte derived cells, to force recognition and killing of the infectious agent.
The ex vivo fixation of anti-viral or anti-bacterial antibodies on monocyte derived cells allows the subsequent delivery of effector cells in optimal conditions to kill the infectious agents and to trigger a specific immune response.
The expression "purifed form" means that the binary complex formed by specific anti-tumor or anti-infections antibodies associated to macrophages is segregated to 90 % from other cell types.
The expression "substantially free of contaminants" means that macrophages are 90 % pure and that no contaminants other than the antibodies are present in the sterile solution.
The expression "bound to the surface through a non covalent link" means that antibodies are associated to the cellular membranes through their Fc part of the antibody and the high affinity Fc receptors present in the cell membrane. 5
It is to be stressed that the majority of the total antibodies are bound to the surface and that there is less than about 20 % of antibodies which are interiorized in the monocyte derived cells.
The invention also relates to a binary complex as above defined, wherein the Fc receptors involved are the Fcγ receptors for IgG, Fcα receptors for IgA or Fcμ receptors for IgM, or the oligosaccharide receptors bindfing the saccharides present on the Fc segment of immunoglobulins.
The invention also relates to a binary complex as above defined, wherein the antibody is directed against pan tumor antigens present on different tumor types. The invention also relates to a process for the preparation of a binary complex between a monocyte derived cell and at least one antibody, comprising the following steps : a) preparation of the monocytes derived cells according to the following method:
1) recovery of blood derived mononuclear cells directly from blood apheresis, from blood bag collection, or from colostrum, followed if necessary by centrifugation, to eliminate a substantial part of red blood cells granulocytes and platelets, and collection of peripheral blood leukocytes ;
2) washing peripheral blood leukocytes obtained at the preceeding steps for instance by centrifugation (to remove 90 % of platelets, red blood cells and debris) to obtain mononuclear cells ;
3) resuspension of the total mononuclear cells obtained at the preceeding step in culture medium (RPMI or IMDM type) at 106 to 2.107 cells/ml, possibly completed by cytokines and/or autologous serum, and culture for 5 to 10 days at 37°C under O2/CO2 atmosphere in hydrophobic gas permeable bags, to obtain monocyte derived cells and contaminating lymphocytes ; b) addition of the anti-tumor or anti-infectious agent antibodies in an amount sufficient to bind a majority of the membrane Fc receptors and in particular of about 1 μg/ml to about lmg/ml to the suspension of monocyte derived cells under conditions preventing interiorization of said antibodies by said monocyte derived cells, to obtain a binary complex and no intracellular interiorization of the antibodies.
The expression "no intracellular interiorization of the antibodies" means that there is less than 20 % of antibodies which are interiorized in the monocyte derived cells.
The monocyte derived cells can be prepared as described in patents n° PCT/EP93/01232, n° WO94/26875 or EP 97/02703 or in the articles mentioned below :
. "Autologous lymphocytes prevent the death of monocytes in culture and promote, as do GM-CSF, IL-3 and M-CSF, their differentiation into macrophages". (Lopez M., Martinache Ch., Canepa S., Chokri M., Scotto F., Bartholeyns J. ; J. of Immunological Methods, 159: 29-38, 1993) ;
. "Immune therapy with macrophages : Present status and critical requirements for implementation" (Bartholeyns J., Romet-Lemonne J-
L., Chokri M., Lopez M. ; Immunobiol., 125: 550-562, 1996) ; . "Dendritic cells as adjuvants for immune-mediated resistance to tumors" (Schuler G. and Steinman R. M. ; J. Exp. Med., 186: 183-1187, 1997). All these patents applications and articles are included herein for references.
According to an advantageous embodiment, in the process of the invention, the temperature is such that it prevents interiorization of said antibodies by said monocyte derived cells.
According to another advantageous embodiment, in the process of the invention, the temperature is below 10°C.
According to another advantageous embodiment, in the process of the invention, the step of addition of the antibodies is preceded by
- centrifugation of the monocyte derived cells and resuspension of said monocyte derived cells, for instance in isotonic medium, to obtain a suspension of monocyte derived cells. 7
According to an advantageous embodiment, in the process according to the invention, the step of addition of antibodies is preceded by the step of freezing at a temperature below or equal to -80°C, aliquots of the monocyte derived cells with the addition of a cryopreservative, to obtain frozen aliquots of monocyte derived cells, and the preceeding step is possibly followed by melting said above frozen aliquots at a temperature enabling to obtain a suspension of monocyte derived cells, for instance at about 4°C, washing said suspension and resuspending, for instance in an isotonic medium, to obtain a suspension of monocyte derived cells.
According to another advantageous embodiment, in the process according to the invention, the step of addition of antibodies is followed by the step of freezing at a temperature below or equal to -80°C aliquots of the binary complex, with the addition of a cryopreservative to obtain frozen aliquots of binary complex, and the preceeding step is followed by melting said above frozen aliquots at a temperature enabling to obtain a suspension of binary complex, for instance at 4°C, washing said suspension and resuspending it, for instance in an isotonic medium, to obtain a suspension of binary complex.
The invention also relates to a binary complex between monocyte derived cells and antibodies such as obtained by the process of the invention.
The invention also relates to a pharmaceutical composition containing as active substance a binary complex as described above, in association with a pharmaceutically acceptable vehicle.
The invention also relates to a pharmaceutical composition in the form of sterile injectable solutions.
In the injectable preparation, the active substance present in an amount such that it corresponds from about 106 to about 109 cells/kg of body weight, particularly from about 107 to about 108 cells/kg of body weight.
In particular embodiment, the binary complex is injected repeatedly at total doses of 107 to 5.109 at intervals of 3 days to 6 months.
The injections can eventually be first local (subcutaneous, intramuscular, mucosal, in cavities or in tissues) and then systemic (intravenous or intralymphatic). 8
The invention also relates to the use of a binary complex as described above for the preparation of a drug for treating cancer or infectious diseases.
The invention also relates to a combined preparation containing, as active substance, the following individual components in the form of a kit-of-parts : - a solution of monocyte derived cells, and
- a solution of antibodies directed against a tumor or an infectious disease, for the simultaneous, separate or sequential use, said monocyte derived cells and said antibodies forming a binary complex as described above.
In the combined preparation, the monocyte derived cells are administered at a dose of about 106 to about 109 cells/kg of body weight, particularly from about 107 to about 108 cells/kg of body weight, and the antibodies are administered at a dose of about 1 μg to about 1 mg/kg of body weight.
According to an advantageous embodiment of the invention, the antibodies are injected before the monocyte derived cells. The two active ingredients of the combined preparation have never been used for a new joint effect and are unknown as a composition.
The active ingredients which are administered either at the same time, or separately, or sequentially, according to the invention, do not represent a mere aggregate of known agents, but a new combination with the surprising valuable property that the binary complex formed ex vivo or in vivo presents higher anti- tumoral or anti-infections efficiency than would be expected by simply adding the effects of antibodies and of macrophages.
It is to be stressed that the combined preparation also designated by a kit-of- parts means that the components of the combined preparation are not necessarily present as a union e.g. in composition, in order to be available for separate or sequential application. Thus the expression kit-of-parts means that it is not necessarily a true combination, in view of the physical separation of the components.
Therefore, the invention can be practised according to two separated and complementary approaches : 1) For tumors or virus or bacteria which are readily accessible to circulating antibodies, the monocyte derived cells, particularly the autologous macrophages, are advantageously injected separately from the monoclonal antibody ;
2) In the case where tumor or viral or bacterial surface antigens are not readily accessible, most systemic antibodies do not effectively reach the tumor target. Ex vivo, the monoclonal antibodies have thus preferably been contacted with monocyte derived cells, particularly macrophages prepared from the patient's blood. These macrophages bearing anti-tumor antibodies reinjected to the patient can leave the blood circulation due to their plasticity and migrate towards the tumors releasing or attracting chemokines. The tumor cells are killed locally by direct contact by a mechanism of antibody dependent cell cytotoxicity.
The invention also relates to a combined preparation wherein said monocyte derived cells and said antibodies form a binary complex as described above, when they are in vitro in the same solution or when they are administered in vivo. The invention also concerns a method for the treatment of cancer or of infectious diseases comprising the use of a binary complex as described above.
The invention also relates to a method for the treatment of cancer or infectious diseases comprising the separate or sequential administration of monocyte derived cells and antibodies against the tumor or infectious diseases to be treated, said monocyte derived cells and antibodies being administered in any order at an interval of time no longer than 24 hours, and said monocyte derived cells and antibodies forming, in the human body, a binary complex as described above.
The invention also relates to a method for inducing or increasing an immune response, comprising the use of a binary complex as described above.
LEGEND TO THE FIGURE :
Figure 1 :
It represents the lysis of neuroblastoma cells by macrophages in antibody dependant cell cytotoxicity assay (ADCC). 10
It represents lysis (on the y axis, expressed as percentage of cytotoxicity) of GD2+ LAN1 neuroblastoma cells by activated macrophages (MAK as defined in PCT/EP93/01232) plotted against E:T (effector/target = macrophage/neuroblastoma cells) ratios ranging from 100: 1 to 12.5: 1 in the presence of 5 μg/ml of the mouse monoclonal antibody 7A4 MoAb, IgC3κ (an antidisialoganglios.de : GD2) (see Michon et al. ; Blood, Vol. 86, n° 3, 1995, p.1124-1130).
The curve with dark lozenges corresponds to the use of macrophages and the above defined monoclonal antibody, and the curve with white square corresponds to the use of macrophages without monoclonal antibody.
Figure 2 :
Figure 2 shows that phagocytosis of leukemia cells by activated human macrophage is increased in the presence of an anti CD20 mAb. The abscissae corresponds to PKH26 fluorescence and the ordinate corresponds to CD 14 fluorescence (arbitrary units).
Figure 2 shows phagocytosis by activated macrophages (MAK) of PKH26- labelled lymphoblastic leukemia cells in the presence of (B) (D) or absence (A) (C) of anti-CD20 mAb. Tumor-labelled cells (3000 cells/well) are incubated with purified MAK ( 6000 cells/well) for 3h at 4°C (C) (D) or 37°C (A) (B). MAK are stained with a fluorescent monoclonal antibody specific for CD 14.
After 3h of contact at 37°C, a relatively small percentage of human macrophages show spontaneous phagocytosis of mmor leukemia cells (7.6%) (A). In contrast, in the presence of anti CD20 mAb, (Rituximab, roche) the capacity of macrophages to phagocytose tumor cells increases dramatically (33.6% of macrophages labelled) (B). At 4°C, spontaneous phagocytosis (C) as well as antibody induced phagocytosis (D) of tumor cells decreases significantly (to 1 % and 13 % respectively). 11
EXAMPLES
Practical applications of the invention are described hereafter as examples. Activated macrophages can be injected together with the antibody or sequentially depending of the mmor target. Macrophages or monocyte derived cells of the following examples are prepared according to the methods described in the above- mentioned references.
1) Preclinical results showing induction of specific immunity are obtained in vitro on tumor cell lines.
Monocyte derived cells present in vitro a cytotoxic activity against mmor cell lines. This activity is markedly enhanced when the monocyte derived cells are suspended with 5μg antibodies directed against a tumor antigen. The following example concerns a neuroblasma tumor and antibodies recognizing gangliosides expressed on the membranes of this tumor cell line (see figure 1). l.b) The following preclinical results show specific increase in interaction and phagocytosis of leukemia tumor cells by activated human macrophages This activity is markedly enhanced when monocytes derived activated macrophages are supplemented with 16μg/ml of anti CD20 monoclonal antibody (mAb).
Materials and methods :
Human macrophages are obtained according to described methods Isolation of tumor cells.
Lymphoblastic leukemia cells are obtained from volunteer patient's peripheral blood. Tumor cells are isolated using ficoll gradient. The mmor cells are washed tree times with PBS.
Tumor cell phagocytosis assay.
A two color flow cytometric assay is used to determine spontaneous and antibody mediated phagocytosis of CLL (chronic lymphoid leukemia ) tumor cells by purified activated macrophages. Tumor cells are labelled with PKH26 dye (Sigma) according the manufacturer's instructions and are cultured overnight in RPMI 1640 12
culture medium to eliminate non incorporated dye and are incubated with 3x 105 effector macrophages in lOOμl solution in polypropylene round bottom capped tubes. Macrophages are incubated in the presence or absence of 16μg/ml of the anti-CD20 antiboby for 30 min before adding labelled mmor cells.
Cell mixture is incubated for 30 min to 3h at 37 °C or at 4°C in a final volume of 500μl. After various periods of time, MAK cells are stained with CD14- or CD64 specific mAbs. Flow cytometry cell analysis for double staining is performed using FACSCalibur (Becton Dickinson).
Double labelled activated macrophages are considered to be phagocytic.
2) Nude mice bearing solid human tumors (e.a. S.C. neuroblastomas of 1 cm3 ) are injected i.v. with about 106 monocyte derived cells activated with γ-IFN and resuspended in the presence of 2 μg anti-tumor antibodies. The tumor volume is measured with calipets in two perpendicular dimensions to follow tumoi evolution.
3) Patients with chronic lymphoid leukemia, in remission after chemotherapy but with residual disease in blood are injected intravenously with about 5.108 activated monocyte derived cells previously resuspended in saline in the presence of 5 mg anti- CD20 IgG antibodies. Evolution of residual diseases (number of leukemic cells) or regression are measured concomitantly with the detection of anti-leukemia antibodies measured in blood.
4) Patients with residual brain neuroblastoma or glyoblastoma tumor resistant to chemotherapy are hospitalized progressing after conventional therapy. Mononuclear cells are collected by apheresis, differentiated into macrophages activated by interferon gamma (MAK cells). These MAK are purified, mixed with the monoclonal anti-tumor antibodies which bind tightly through high affinity/avidity receptors the tumor cells and the macrophages (about 109 MAK cells prepared according to previously described method) are resuspended in sterile isotonic solution for injection in the presence of 1 mg anti-glyoblastoma antibodies at a temperature of 4-10°C until infusion. The preparation is reinfused locally in the CNS and then intravenously to the 13
patient. Tumor evolution regression, partial responses are documented by imaging. Cellular T cytotoxic anti-tumoral immune response is measured in peripheral blood.
5) New-born children, in particular premature infants, have no mature immune system and are particularly sensitive to viral or bacterial infections ; some of these such as these caused by haemophilus or pneumococci are difficult to treat and induce severe diseases. Monocyte derived cells (MDCs) are prepared from new mothers, and they are either isolated from blood or preferentially from the mother milk collected during the two first weeks of lactation after delivery (colostrum contains high concentration of monocyte derived cells, about 1 million per ml, which can be processed to obtain activated macrophages). These monocyte derived cells are processed according to the invention and resuspended in isotonic solution in the presence of antibodies specific for haemophilus and/or pneumococci at a temperamre below 10°C. The suspensions containing 107 to 108 macrophages and 10 to lOOμg of antibodies are injected locally to the new-born and its effect on the infection is documented. Preclinical data in mice illustrate the activity of macrophages bound to antibodies according to the invention on lethal relevant infections previously induced in mice.
The above-mentioned experiments have been performed with different subsets of monocyte derived cells expressing immunoglobulin Fc receptors, and in particular with macrophages.

Claims

14 CLAIMS
1. Binary complex, in a purified form and substantially free of contaminants, between a monocyte derived cell and at least one antibody directed against a mmor or an infectious agent, with said antibody being bound to the surface of said monocyte derived cell through a non covalent link between the Fc part of the antibody and the
Fc receptors present on the monocyte derived cell membrane.
2. Binary complex according to claim 1, wherein the Fc receptors involved are the Fc γ receptors for IgG, Fcα receptors for IgA or Fcμ receptors for IgM, or the oligosaccharide receptors binding the saccharides present on the Fc segment of immunoglobulins .
3. Binary complex according to claim 1, wherein the antibody is directed against pan tumor antigens present on different tumor types.
4. Process for the preparation of a binary complex between a monocyte derived cell and at least one antibody, comprising the following steps : a) preparation of the monocytes derived cells according to the following method:
1) recovery of blood derived mononuclear cells directly from apheresis from blood bag collection, or from colostrum, followed if necessary by centrifugation, to eliminate a substantial part of red blood cells granulocytes and platelets, and collection of peripheral blood leukocytes ;
2) washing peripheral blood leukocytes obtained at the preceeding steps for instance by centrifugation (to remove 90 % of platelets, red blood cells and debris) to obtain mononuclear cells ;
3) resuspension of the total mononuclear cells obtained at the preceeding step in culture medium (RPMI or IMDM type) at 106 to
2.107 cells/ml, possibly completed by cytokines and/or autologous 15
serum, and culture for 5 to 10 days at 37°C under O2/CO2 atmosphere in hydrophobic gas permeable bags, to obtain monocyte derived cells and contaminating lymphocytes ; b) addition of the anti-tumor or anti-infectious agent antibodies in an amount sufficient to bind a majority of the membrane Fc receptors and in particular of about lμg/ml to about lmg/ml to the suspension of monocyte derived cells under conditions preventing interiorization of said antibodies by said monocyte derived cells, to obtain a binary complex and no intracellular interiorization of the antibodies.
5. Process according to claim 4, wherein the temperature is such that it prevents interiorization of said antibodies by said monocyte derived cells.
6. Process according to claim 5, wherein the temperature is below 10°C.
7. Process according to anyone of claims 4 to 6, wherein the step of addition of the antibodies is preceded by
- centrifugation of the monocyte derived cells and resuspension of said monocyte derived cells, for instance in isotonic medium, to obtain a suspension of monocyte derived cells.
8. Process according to anyone of claims 4 to 7, wherein the step of addition of antibodies is preceeded by the step of freezing at a temperature below or equal to -80°C, aliquots of the monocyte derived cells with the addition of a cryopreservative, to obtain frozen aliquots of monocyte derived cells, and the preceeding step is possibly followed by melting said above frozen aliquots at a temperature enabling to obtain a suspension of monocyte derived cells, for instance at about 4°C, washing said suspension and resuspending, for instance in an isotonic medium, to obtain a suspension of monocyte derived cells.
9. Process according to anyone of claims 4 to 7, wherein the step of addition of antibodies is followed by the step of freezing at a temperature below or equal to 16
-80 °C aliquots of the binary complex, with the addition of a cryopreservative to obtain frozen aliquots of binary complex, and the preceeding step is followed by melting said above frozen aliquots at a temperature enabling to obtain a suspension of binary complex, for instance at 4°C, washing said suspension and resuspending it, for instance in an isotonic medium, to obtain a suspension of binary complex.
10. Binary complex between monocyte derived cells and antibodies such as obtained by the process according to anyone of claims 4 to 9.
11. Pharmaceutical composition containing as active substance a binary complex according to anyone of claims 1 to 3, in association with a pharmaceutically acceptable vehicle.
12. Pharmaceutical composition according to claim 11, in the form of sterile injectable solutions.
13. Use of a binary complex according to anyone of claims 1 to 3, for the preparation of a drug for treating cancer or infectious diseases.
14. Combined preparation containing, as active substance, the following individual components in the form of a kit of parts :
- a solution of monocyte derived cells, and
- a solution of antibodies directed against a mmor or an infectious disease, for the simultaneous, separate or sequential use, said monocyte derived cells and said antibodies forming a binary complex according to anyone of claims 1 to 3.
15. Combined preparation of claim 14, wherein said monocyte derived cells and said antibodies form a binary complex according to claims 1 to 3, when they are in vitro in the same solution or when they are administered in vivo.
PCT/EP1999/002104 1998-04-09 1999-03-29 Use of monocytes derived cells and antibodies in a synergic way for optimal induction of immunotherapeutic efficiency WO1999053027A1 (en)

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