U.S. patent application number 10/622727 was filed with the patent office on 2004-01-29 for combined preparation for the treatment of neoplasic diseases or of infectious diseases.
This patent application is currently assigned to I.D.M. IMMUNO-DESIGNED MOLECULES. Invention is credited to Bartholeyns, Jacques, Fouron, Yves, Romet-Lemonne, Jean-Loup.
Application Number | 20040018184 10/622727 |
Document ID | / |
Family ID | 8235320 |
Filed Date | 2004-01-29 |
United States Patent
Application |
20040018184 |
Kind Code |
A1 |
Bartholeyns, Jacques ; et
al. |
January 29, 2004 |
Combined preparation for the treatment of neoplasic diseases or of
infectious diseases
Abstract
The present invention relates to combined preparation containing
as active substance the following individual components, in the
form of a kit-of-parts: monocyte derived cells, particularly
cytotoxic macrophages, chemotherapy or immunotherapy drugs, for the
simultaneous, separate or sequential use, for the treatment of
cancer or infectious diseases.
Inventors: |
Bartholeyns, Jacques;
(Bures-Sur-Yvette, FR) ; Fouron, Yves;
(Marlborough, MA) ; Romet-Lemonne, Jean-Loup;
(Paris, FR) |
Correspondence
Address: |
YOUNG & THOMPSON
745 SOUTH 23RD STREET 2ND FLOOR
ARLINGTON
VA
22202
|
Assignee: |
I.D.M. IMMUNO-DESIGNED
MOLECULES
PARIS
FR
|
Family ID: |
8235320 |
Appl. No.: |
10/622727 |
Filed: |
July 21, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10622727 |
Jul 21, 2003 |
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09647529 |
Nov 29, 2000 |
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09647529 |
Nov 29, 2000 |
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PCT/EP99/02105 |
Mar 29, 1999 |
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Current U.S.
Class: |
424/93.21 ;
424/649; 514/192; 514/263.31; 514/283; 514/29; 514/291; 514/34;
514/410; 514/42; 514/50; 514/54 |
Current CPC
Class: |
C12N 5/0645 20130101;
A61K 31/655 20130101; A61P 35/00 20180101; A61K 45/06 20130101;
A61P 31/00 20180101; A61K 31/232 20130101; A61K 31/505 20130101;
A61K 35/15 20130101; A61K 31/282 20130101; A61K 2039/5154 20130101;
A61K 31/336 20130101; A61K 31/407 20130101; A61K 31/704 20130101;
A61K 35/15 20130101; A61K 2300/00 20130101; A61K 31/282 20130101;
A61K 2300/00 20130101; A61K 31/704 20130101; A61K 2300/00 20130101;
A61K 31/232 20130101; A61K 2300/00 20130101; A61K 31/336 20130101;
A61K 2300/00 20130101; A61K 31/505 20130101; A61K 2300/00 20130101;
A61K 31/407 20130101; A61K 2300/00 20130101; A61K 31/655 20130101;
A61K 2300/00 20130101 |
Class at
Publication: |
424/93.21 ;
514/34; 514/283; 514/263.31; 514/50; 424/649; 514/192; 514/410;
514/54; 514/42; 514/29; 514/291 |
International
Class: |
A61K 048/00; A61K
031/715; A61K 031/7008; A61K 031/43; A61K 031/522 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 2, 1998 |
EP |
98400783.1 |
Claims
What is claimed is:
1. A method for treating a patient suffering from a neoplasic or
infectious disease, comprising: administering an effective amount
of monocyte derived cells and an effective amount of chemotherapy
drugs to said patient.
2. The method according to claim 1, wherein said monocyte derived
cells and chemotherapy drugs are administered simultaneously.
3. The method according to claim 1, wherein the chemotherapy drug
is selected from the group consisting of anthracyclins,
daunorubicin, adriamycin, taxoter derivatives, vinca alcaloids,
vincristine, taxol, carmustine, cisplatin, fluorouracils, polyamine
inhibitors, topoisomerase inhibitors, tamoxifene, prodasone,
sandostatine, sodium butyrate, mitomycin C, penicillins,
.beta.-lactamines, cephalosporines, cyclines, aminoglucosides,
macrolides, sulfamides, AZT, protease inhibitors, acyclovir,
retrovir and foscarnet.
4. The method according to claim 1, wherein the monocyte derived
cells and the chemotherapy drugs are in the form of an injectable
solution.
5. The method according to claim 1, further comprising: a)
recovering blood derived mononuclear cells directly from blood
apheresis or from blood bag collection, followed by centrifugation,
to eliminate red blood cell granulocytes and platelets, and to
collect peripheral blood leukocytes; b) washing peripheral blood
leukocytes obtained at the preceding steps by centrifugation to
remove platelets, red blood cells and debris to obtain mononuclear
cells; c) resuspending the mononuclear cells obtained in the
preceding step in a culture medium, and d) culturing said cells of
preceding step for 5 to 10 days to obtain monocyte derived cells
and contaminating lymphocytes.
6. The method according to claim 1, wherein said monocyte derived
cells have been cultured for 5 to 10 days.
7. A method for the simultaneous, separate, or sequential
administration of a preparation for the treatment of cancer or
infectious disease in a patient, comprising: administering to said
patient an effective amount of said preparation, wherein said
preparation comprises the following components: monocyte derived
cells, and chemotherapy drugs.
8. The method according to claim 7, wherein said monocyte derived
cells and chemotherapy drugs are administered simultaneously.
9. The method according to claim 8, wherein the monocyte derived
cells are administered repeatedly up to ten times, with an interval
between each administration being between three days to two
months.
10. The method according to claim 7, wherein the chemotherapy drug
is administered at a dose of 0.1 to 1000 mg/day.
11. The method according to claim 8, wherein said drug is selected
from the group consisting of cytotoxic compounds, cytostatic
compounds, and compounds inducing apoptosis or cytokines, wherein
said drug is administered at a dose of 0.1 to 100 mg/day.
12. The process according to claim 11, wherein the chemotherapy
drug is administered repeatedly up to 10 times, with interval
between each administration being between one day to two
months.
13. The method according to claim 7, wherein the chemotherapy drug
and the monocyte derived cells are administered sequentially with
said chemotherapy drug being administered before the monocyte
derived cells.
14. The method according to claim 13, wherein the interval of time
between the administration of the monocyte derived cells and the
administration of the chemotherapy drugs is one day to two
months.
15. The method according to claim 7, wherein the monocyte derived
cells and the chemotherapy drug are administered sequentially with
the monocyte derived cells being administered before the
chemotherapy drug.
16. The method according to claim 7, wherein the monocyte derived
cells are administered before the administration of a vaccine
directed to a tumor or infectious antigen.
17. The method according to claim 16, wherein the administration of
the monocyte derived cells is preceded by chemotherapy.
18. The method according to claim 15, wherein the interval of time
between the administration of the chemotherapy drug and the
administration of the monocyte derived cells is one day to two
months.
19. The method according to claim 7, further comprising: a)
recovering blood derived mononuclear cells directly from blood
apheresis or from blood bag collection, followed by centrifugation,
to eliminate red blood cell granulocytes and platelets, and to
collect peripheral blood leukocytes; b) washing peripheral blood
leukocytes obtained at the preceding steps by centrifugation to
remove platelets, red blood cells and debris to obtain mononuclear
cells; c) resuspending the mononuclear cells obtained in the
preceding step in a culture medium, and d) culturing said cells of
preceding step for 5 to 10 days to obtain monocyte derived cells
and contaminating lymphocytes.
20. The method according to claim 7, wherein said monocyte derived
cells have been cultured for 5 to 10 days.
Description
[0001] The present invention relates to a new combined preparation
for the treatment of neoplasic diseases or of infectious
diseases.
[0002] The present invention describes sequences of conventional
treatments of cancer or infections and of immunotherapies reversing
or preventing chemoresistance and allowing long lasting therapeutic
responses.
[0003] In conventional therapy, residual tumor cells or infectious
agents are left undamaged due to chemoresistance or due to the fact
that these cells are shaded in protected areas or located in
hypoxic areas poorly vascularized and not accessible to
conventional treatments. The genetic instability and heterogeneity
of tumors and micro-organisms indeed allow them to adapt and to
develop resistance to therapies.
[0004] The beneficial effects of chemotherapy can be compromised by
cellular mechanisms that allow infectious agents or neoplasic
tissue to evade the toxicity of drugs. In some cases, pleiotropic
resistance to a variety of unrelated drugs has been observed, and
this phenomenon has been called multidrug resistance.
[0005] Resistance to chemotherapy, whether it is intrinsic or
acquired, is a major cause of failure in the curative treatment of
chronic infections or neoplasic malignancies. Among the most active
anti-cancer agents used in the treatment of haematological
malignancies are some natural toxin-derived drugs, such as the
anthracycline daunorubicin or adriamicin, the epipodophyllotoxins,
taxoter derivatives, the vinca alkaloid vincristine, cisplatin,
fluorouracils.
[0006] Development of cross-resistance to these structurally and
functionally unrelated drugs, called multidrug resistance, is
frequently observed in second or third intention cytotoxic
treatment of cancer.
[0007] Multiple drug resistance of infectious agents and
particularly of bacteria to antibiotics such as penicilins,
.beta.-lactamines, cephalosporines, aminoglucosides, macrolides and
sulfamides, is more and more often seen in hospitals.
[0008] 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.degree. C.
in O.sub.2/CO.sub.2 atmosphere. Their culture medium (RPMI, IMDM,
AIM5 (Gibco) or X-VIVO (Biowhittaker)) contains eventually
cytokines or ligands as defined in patents n.degree.
PCT/EP93/01232, n.degree. WO94/26875 or EP 97/02703 or in the
articles mentioned below:
[0009] "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 C h.,
Canepa S., Chokri M., Scotto F., Bartholeyns J.; J. of
Immunological Methods, 159: 29-38, 1993);
[0010] "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);
[0011] "In vitro generation of CD83.sup.+ human blood dendritic
cells for active tumor immunotherapy" (Thurnher M., Papesh C.,
Ramoner R., Gastlt G. and al.; Experimental Hematology, 25:
232-237, 1997);
[0012] "Dendritic cells as adjuvants for immune-mediated resistance
to tumors" (Schuler G. and Steinman R. M.; J. Exp. Med., 186:
1183-1187, 1997).
[0013] All these patents applications and articles are included
herein for references.
[0014] They can be activated by INF-.gamma. 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.
[0015] Monocytes derived cells (MDCs) can either be killer
macrophages, phagocytozing cells, growth factors and cytokines
releasing cells, or dendritic cells according to their conditions
of differentiation. Dendritic cells can for example be obtained as
described in "In vitro generation of CD83.sup.+ human blood
dendritic cells for active tumor immunotherapy" (Thurnher M.,
Papesh C., Ramoner R., Gastlt G. and al.; Experimental Hematology,
25: 232-237, 1997) and "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.degree.
97/02703.
[0016] In addition, activated monocyte derived cells (macrophages)
can be used to deliver therapeutic agents to tumor or infectious
sites.
[0017] One of the aims of the invention is to provide a combined
preparation of active substances under the form of individual
components for the simultaneous separate or sequential use, in the
treatment of cancer or of infectious diseases.
[0018] Another aim of the invention is to provide a method for the
treatment of residual cancer resistant to chemotherapy.
[0019] Another aim of the invention is to provide a method for the
treatment of infectious diseases resistant to antibiotic
treatment.
[0020] The invention relates to a combined preparation containing,
as active substance, the following individual components, in the
form of a kit-of-parts:
[0021] monocyte derived cells, particularly cytotoxic
macrophages,
[0022] chemotherapy or immunotherapy drugs,
[0023] for the simultaneous, separate or sequential use, for the
treatment of cancer or infectious diseases.
[0024] The present treatment consists in the local or systemic
injection of autologous activated macrophages (MAK.RTM. killer
cells) or monocyte derived cells which have access to injured
areas, and in particular to hypoxic areas, where they tend to
concentrate.
[0025] This treatment can be conducted after first failure and
relapse following chemotherapies, or before chemotherapy, to
prevent chemoresistance. Local treatment with chemotherapy drugs
causes cell necrosis and release of chemokines which call and
actively recrute macrophages and monocytes derived cells.
Therefore, combining the chemotherapy with macrophage immunotherapy
can in synergy increase cytotoxicity and increase immune response
at the same time as preventing the establishment of resistance.
Additionally to a first treatment combining conventional approach
with immunotherapy, macrophage adoptive therapy can be proposed
after failure and relapse.
[0026] It is shown through the invention that the local or systemic
injection of activated monocyte derived cells, or macrophages,
restores clinical responses to cytotoxic drugs for which resistance
was previously demonstrated, or prevents the apparition of
chemoresistance.
[0027] The present invention also shows that activated monocyte
derived cells can overcome this resistance and synergize for
therapy.
[0028] The two active ingredients of the combined preparation have
never been used for a new joint effect and are unknown as a
composition.
[0029] 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
immunotherapy with monocyte derived cells modifies the
chemoresistance/chemosensitivity and allows a new effective
treatment (partial or complete response) with similar chemotherapy
protocole. Furthermore, synergy is observed between monocyte
derived cells immunotherapy and chemotherapy.
[0030] 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.
[0031] In an advantageous combined preparation of the invention,
the monocyte derived cells contain chemotherapy or immunotherapy
drugs.
[0032] In another advantageous combined preparation of the
invention, the monocyte derived cells are such as prepared
according to the method comprising the following steps:
[0033] 1) recovery of blood derived mononuclear cells directly from
blood apheresis or from blood bag collection, followed if necessary
by centrifugation, to eliminate a substantial part of red blood
cells, granulocytes and platelets, and collection of peripheral
blood leukocytes;
[0034] 2) washing peripheral blood leukocytes obtained at the
proceeding steps for instance by centrifugation (to remove 90% of
platelets, red blood cells and debris) to obtain mononuclear
cells;
[0035] 3) resuspension of the total mononuclear cells
(monocytes+lymphocytes) obtained at the preceeding step in culture
medium (RPMI or IMDM type) at 10.sup.6 to 2.10.sup.7 cells/ml,
possibly completed by cytokines and/or autologous serum, and
culture for 5 to 10 days at 37.degree. C. under O.sub.2/CO.sub.2
atmosphere in hydrophobic gas permeable bags, to obtain monocyte
derived cells and contaminating lymphocytes.
[0036] According to an advantageous combined preparation, the
chemotherapy drug is selected among cytotoxic compounds such as
anthracyclins, daunorubicin, adriamycin, taxoter derivatives, vinca
alcalods, vincristine, carmustine, cisplatin, fluorouracils,
cytostatic compounds such as polyamine inhibitors, topoisomerase
inhibitors, tamoxifene, prodasone, or sandostatine, or compounds
inducing apoptosis such as sodium butyrate or mitomycin C,
antibiotics such as penicilins, .beta.-lactamines, cephalosporines,
cyclines, aminoglucosides, macrolides or sulfamides, or antiviral
drugs such as AZT, protease inhibitors or acyclovir, retrovir or
foscarnet.
[0037] According to an advantageous embodiment, the combined
preparation of the invention is such that the immunotherapy drug is
selected among cytokines such as cyclosporine, azathioprine,
cyclophosphamide, IFN.gamma., IL-12, IL-2, GM-CSF, G-CSF, adjuvants
such as murapeptides or BCG, or vaccines with or without substances
with adjuvant effect. The vaccines can be constituted by tumor or
infectious antigens which are of natural, recombinant or gene
transfer origin, formulated in the presence or not of an adjuvant
for the administration to humans. The vaccine can also be a nucleic
acid coding for the antigen or a fragment of antigen, or a virus
expressing the antigen.
[0038] According to an advantageous embodiment, in the combined
preparation of the invention, the monocyte derived cells and the
chemotherapy or immunotherapy drugs are in the form in injectable
solutions.
[0039] In another advantageous embodiment of the invention, in the
combined preparation, the injectable solutions are in the form of
locally injectable solutions.
[0040] In another advantageous embodiment in the combined
preparation of the invention, the injectable solutions are in the
form of systemically injectable solutions.
[0041] In another advantageous combined preparation of the
invention, the monocyte derived cells are administered at a dose of
about 10.sup.7 to about 10.sup.10 monocyte derived cells per
injection.
[0042] In another advantageous combined preparation of the
invention, the monocyte derived cells are administrated at a dose
of about 10.sup.8 to about 10.sup.9.
[0043] In another advantageous combined preparation of the
invention, the monocyte derived cells are administered in a
repeated way up to ten times, the interval between each
administration being between three days to two months.
[0044] In another advantageous combined preparation of the
invention, the immunotherapy or chemotherapy drug is administered
at a dose of about 0.1 to about 1000 mg/day.
[0045] In another advantageous combined preparation of the
invention, in the case of administration of a drug chosen among
immunotherapy drug, antiviral drug, cytotoxic drugs, or
antibiotics, said drug is administered at a dose of about 10 to
about 1000 mg/day.
[0046] More specifically, in the case of cytotoxic compounds such
as vincristine, taxol, carmustine, daunorubicin, adryamicin,
cisplatin, fluorouracil, they are administered at a dose of about
10 to about 500 mg/day.
[0047] In the case of antiviral drugs such as retrovir, aciclovir,
foscarnet, said drug is administered at a dose of about 20 to about
500 mg/day.
[0048] In the case of antibiotics such as penicilins,
cephalosporine, sulfamides, cyclines, said drug is administered at
a dose of about 10 to about 1000 mg/day.
[0049] In the case of immunotherapy drugs such as cyclosporine,
azathioprine, cyclophosphamide, said drug is administered at a dose
of about 10 to about 1000 mg/day.
[0050] In another advantageous combined preparation of the
invention, in the case of administration of a drug chosen among
cytostatic compounds, apoptosis inducing compounds or cytokines,
said drug is administered at a dose of about 0.1 to about 100
mg/day.
[0051] In the case of cytostatic compounds such as amoxifene,
prodasone, sandostatine, polyamine inhibitors or apoptosis inducing
compounds such as sodium butyrate or mitomycin C, said drug is
administered at a dose of about 0.1 to about 100 mg/day.
[0052] In another advantageous combined preparation of the
invention, the immunotherapy or chemotherapy drug is administered
in a repeated way up to 10 times, the interval between each
administration being between one day to two months.
[0053] In another advantageous combined preparation of the
invention, the chemotherapy or immunotherapy drug and the monocyte
derived cells are injected simultaneously.
[0054] In another advantageous combined preparation of the
invention, the chemotherapy or immunotherapy drug and the monocyte
derived cells are administered in sequential way, the immunotherapy
or chemotherapy drug being administered before the monocyte derived
cells.
[0055] In another advantageous combined preparation of the
invention, the interval of time between the administration of the
monocyte derived cells and the administration of the immunotherapy
or chemotherapy drugs is of one day to two months.
[0056] In another advantageous combined preparation of the
invention, the monocyte derived cells and the chemotherapy or
immunotherapy drug are administered seqentially, the monocytes
derived cells being administered before the immunotherapy or
chemotherapy drug.
[0057] In another advantageous combined preparation of the
invention, the monocyte derived cells are administered before the
administration of a vaccine directed to tumor or infectious
antigens, the monocyte derived cells administration being possibly
preceded by a chemotherapy treatment.
[0058] In an advantageous embodiment of the invention, the monocyte
derived cells are administered before the administration of the
vaccine, the time internal between the respective administrations
being for example of one week to three months. The vaccine
administration can be repeated several times for optimal
immunisation, according to classical procedures. In this case, the
monocyte derived cells administration can be considered as a
priming for the reaction to the antigen and the administration(s)
of the vaccine as a boost of the immune responses.
[0059] In another advantageous embodiment of the invention, the
patients are first treated by conventional chemotherapy drugs, this
treatment being followed sequentially by administration of monocyte
derived cells and then by the vaccine administration as a boost, to
induce optimal immunization against cancer or infectious
disease.
[0060] In another advantageous combined preparation of the
invention, the interval of time between the administration of the
immunotherapy or chemotherapy drug and the administration of the
monocyte derived cells is of one day to two months.
[0061] In another advantageous combined preparation of the
invention, the administration of monocyte derived cells is followed
by an administration of chemotherapy or immunotherapy drug.
[0062] In another advantageous combined preparation of the
invention, the interval of time between the administration of
monocyte derived cells and the administration of chemotherapy or
immunotherapy drugs is of one day to two months.
[0063] The invention also relates to a method for the treatment of
residual cancer resistant to chemotherapy or of infectious diseases
resistant to chemotherapy comprising the use of a combined
preparation of the invention.
[0064] The invention also relates to a method for the treatment of
infectious diseases resistant to antibiotic treatment comprising
the use of a combined preparation of the invention.
[0065] The monocytes derived cells which are involved in the
invention, can be activated macrophages and/or monocytes derived
antigen presenting cells.
[0066] According to an advantageous embodiment, the combined
preparation of the invention comprises monocyte derived cells,
loaded with a complex mixture of antigens, and a vaccine containing
purified antigens.
[0067] The monocyte derived cells loaded with a complex mixture of
antigens will be also referred to as "the cellular vaccine".
[0068] The monocyte derived cells can be macrophages or dendritic
cells derived from blood monocytes, preferably cultured in the
presence of lymphocytes.
[0069] The expression "a complex mixture of antigens" designates
antigens with a large spectrum of specificity for the infectious
agent or the tumor cells.
[0070] As to the vaccine containing purified antigens, its
specificity is restricted to one or a few epitopes of the target
agent (infectious agent or tumor cells).
[0071] According to an advantageous embodiment, when the
macrophages or dendritic cells derived from blood moriocytes are
cultured in the presence of lymphocytes, said lymphocytes are T
lymphocytes (CD4.sup.+ and CD8.sup.+ types) and natural killer
cells (NK cells) generated during the coculture. These lymphocytes
and NK cells can be recovered and possibly expanded ex vivo, for
simultaneous or sequential injection with said monocyte derived
cells (loaded with a complex mixture of antigens).
[0072] In a particular embodiment, immunomonitoring of patients
treated with vaccines containing purified antigens, allows
detection and identification of the specific cellular response to
conventional antigens. The useful specific T cell response can be
amplified ex vivo. Monocyte derived cells presenting a mixture of
complementary antigens can then be designed to avoid the
development of immunoresistant viruses or tumors.
[0073] In the optimal sequence of said cellular vaccine and said
vaccine containing purified antigens, said vaccine containing
purified antigens is used as a boost to achieve immune memory
against the targeted infectious or tumor disease.
[0074] The above defined combined preparation of the invention
induces cellular and humoral responses to cancer and viral
antigens.
[0075] According to another advantageous embodiment, the vaccine
containing purified antigens is injected first, followed by the
injection of the cellular vaccine.
[0076] FIGS. 1 and 2 represent the in vitro synergy between
chemotherapy (use of cisplatin) and macrophages (MAK) cytotoxicity
on human ovary carcinoma tumor (IGR-OV1).
[0077] FIG. 1 corresponds to chemosensitive tumor and FIG. 2
corresponds to chemoresistant tumor.
[0078] Tumor cells have been grown for 3 days of cocuitured at
37.degree. C., 5% CO.sub.2 from an initial seeding of 10.sup.5
cells under the following conditions:
[0079] presence of cisplatin,
[0080] presence of human macrophages,
[0081] presence of cisplatin and human macrophages.
[0082] The percentage of tumor cell survival is measured according
to the method described in "Feasibility of drug screening with
panels of human tumor cell lines using a microculture tetrazolium
assay" (Alley M. C., Scudiero D. A., Monks A., et al.; Cancer Res.,
1988, 48: 489-501), and is plotted against the dose of cisplatin
(abscissa) in the test tube (molar concentration).
[0083] The amount of macrophages used in the experiment is
constant.
[0084] The initial ratio between macrophage and tumor cells is 4/1
for FIG. 1, and 1/1 in FIG. 2.
[0085] The dotted line corresponds to the addition of macrophages
alone.
[0086] The open circle curve corresponds to the addition of
cisplatin alone.
[0087] The dark square curve corresponds to the addition of
macrophages and cisplatin.
[0088] The open triangle curve corresponds to the theoretical
addition of the effects of macrophages alone, plus cisplatin
alone.
[0089] On FIG. 2, the open circle curve and the open triangle curve
are superimposed.
[0090] Additive effects of macrophages and cisplatin are seen on
chemosensitive tumor cells. Synergy or potentiation of macrophages
and cisplatin is observed for the chemoresistant tumor.
[0091] FIG. 3 represents the percentage of cellular survival as a
function of cisplatin concentration (M).
[0092] The curve with hollow circles corresponds to the use of
cisplatin alone.
[0093] The plain curve corresponds to the use of MAK alone.
[0094] The curve with black squares corresponds to the use of the
combination of MAK followed by cisplatin.
[0095] The curve with hollow triangles corresponds to the
theoretical additive curve of a treatment with MAK alone and a
treatment with cisplatin alone.
EXAMPLES
[0096] The following examples describe some applications of the
invention:
[0097] 1) The synergy between macrophage immunotherapy and
chemotherapy has been demonstrated in vitro on a carcinoma tumor
cell line. Relative sensitivity of a human ovary tumor cell line
and a derived line resistant to cisplatinum is documented, as well
as the cytotoxicity of activated macrophages on these lines.
Additive antitumoral effects for macrophages and cisplatinum is
documented, allowing an effective dose response with lower levels
of the drug, as demonstrated in FIG. 1 and FIG. 2.
[0098] I6R-OV1/DDP human ovary cancer cell line was rendered
resistant to cisplatinum by continuous exposure to increasing
concentrations of cisplatin (Fajac A., et al. Cisplatin induced
apoptosis and p.53 gene status in a cisplatin resistant human
ovarian carcinoma cell line. Int. J. Cancer 68, 67-74, 1996). The
cells are grown at 37.degree. C., 5% CO.sub.2 in RPMI 1640 medium
in the presence of activated human macrophages (MAK) during 24
hours and then with cisplatin at increasing concentrations.
Cisplatin alone (0) inhibited survival by 35% at maximal dose
(10.sup.-3M, see FIG. 3). Activated macrophages inhibited survival
by 10% (effector/tumor ratio 4, basal line). The combination of MAK
and of a low dose cisplatin (10.sup.-6M) inhibited survival by 40%
(.box-solid.). The sequential combination of MAK and cisplatin (MAK
followed by cisplatin) acted in synergy since the cytotoxicity was
much higher at low cisplatin dose than the theoretical additive
curve of both treatments (.DELTA., see FIG. 3).
[0099] 2) Nude mice inoculated with human carcinoma solid tumors
are initially treated with cytotoxic drugs (Adriarnycin,
Etretinate, Taxotere), used alone or in combination. After a first
response, the tumors grow again and the animals are treated
systemically, or locally by injection of 1 million activated human
macrophages which allow tumor stabilization. A second treatment
with the same cytotoxic drugs used initially allow further
antitumoral effect documented by measurement of subcutaneous tumor
size.
[0100] 3) Three patients with colorectal cancer and four patients
with lung mesothelioma became resistant to 5-fluorouracil+Cisplatin
(or its oxaliplatin derivative) chemotherapy. They then have been
injected with autologous activated macrophages and they have
presented tumor stabilization or partial response illustrated by
radiography. After a few months, the tumor relapsed and cancer
evolution was reported. A second chemotherapy treatment, with
similar cocktail of cytotoxic drugs, induced complete responses or
major partial responses. This indicates a modification of the
chemoresistance caused by immunotherapy.
[0101] 4) Patients with prostate cancer treated by radio and
chemotherapy present a 50% relapse rate of their cancer within 2
years. A treatment with activated macrophages is proposed after the
conventional therapy. The time of relapse within 2 years as well as
the evolution of the tumor are documented.
[0102] 5) Bacterial infections induced in nude mice are relatively
resistant to antibiotics. Effective therapy is achieved by
sequential injection of macrophages and of antibiotics at usually
ineffective doses. The additive effects of classical
anti-infections drugs and of macrophage immunotherapy are
documented.
[0103] 6) Patients with myeloid leukemia, or with multiple myeloma,
are treated with high dose chemotherapy. During the 6 weeks of
aplasia, they present multiple infections, in particular nosocomial
infections. Injections of activated macrophages during this period
is performed to prevent infections and to allow a cure at lower
doses of antibiotics.
[0104] 7) C57B16 mice bearing solid carcinoma are injected
intraperitonealy with a drug inducing apoptosis (1 mg mitomycin C
or 0.1 mg sodium butyrate). After 24 h, mice are injected with 0.1
million monocyte derived cells in tumor periphery. Tumor regression
and protection against further tumor challenge is observed only
after this combined treatment. In another protocol, carcinoma cells
are treated in vitro with 0.01 mM sodium butyrate, and then
submitted to phagocytosis by murine monocyte derived cells.
Injection of mice with 0.1 million of these monocyte derived cells
protects the animals against carcinoma challenge.
[0105] 8) Dendritic cells (DC) are obtained from bone marrow
precursors of Balb/c syngeneic mice after 7 days of culture in
medium supplemented with GM-CSF and IL-13. The dendritic cells are
loaded with the S protein of the hepatitis B virus at 20 .mu.g/ml
for 4 hours. One million cells are injected intravenously. 7 days
later, a mixture of HBS protein and adjuvant is injected in the
peritoneal cavity. At day 15, the immune response is assessed by
two different methods:
[0106] serum titer of antibody against HBS is measured by
ELISA,
[0107] the spleen from the mice is removed and T lymphocytes are
stimulated with irradiated isogenic splenocytes loaded with
peptides for 7 days. At day 22, the significant cytotoxic activity
of the T lymphocytes is measured using as targets p815 cells loaded
with the 28-39 peptide (an immunogenic peptide of the S
antigen).
[0108] 9) Patients, whose primary melanoma tumor was removed by
surgery, are treated with chemotherapy agent (DTIC) (dacarbazine)
after relapse. When their blood count is back to normal, blood is
drawn up through apheresis in order to prepare large amounts of
MD-APCs. These cells are then incubated for 4 hours with allogeneic
tumor extract. 3 weekly sub-cutaneous injections (at 4 different
sites) of 10.sup.7 cells are made. Two months later, a cocktail of
three antigens (MAGE-3, MELAN A and gp-100) (Boon et al. Immunology
today, June 1997, Vol. 18, n.degree. 6, 267) plus adjuvant is
injected to the patients in order to boost the immune system. The
increased immune response is monitored by measuring the number of
antigen specific CD8 T lymphocytes by ELISPOT technique (Herr. et
al. Detection and quantification of blood-derived CD8+ T
lymphocytes secreting tumor necrosis factor alpha in response to
HLA-A2.1-binding melanoma and viral peptide antigens. J Immunol
Methods 191, no. 2:131-42.) It is also assessed that the
relapse-free time is significantly increased.
[0109] In a particular embodiment of the invention, macrophages are
loaded ex vivo with a drug as promyxin (a bioreductive agent)
active in hypoxic areas. In this case, the macrophages having been
fed with the drug, concentrate in the necrotic/hypoxic area, kill
tumor cells in contact and release locally during several days the
cytotoxic drug killing the remaining cancer cells. A radiotherapy
enhancer (tirazone) is also loaded into macrophages which cause,
after reinjection, a potentiation of radiotherapy at specific tumor
sites.
[0110] In another embodiment of the invention, an antibiotic is
loaded into macrophages from patients with nosocomial infections
resistant to conventional antibiotics.
[0111] The proper sequence and timing of macrophages injections,
allowing maximum activity at the tumor or infectious site, are
disclosed.
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