WO2004024171A1 - Traitement combine d'une leucemie lymphoide chronique (cellules de leucemie lymphoide chronique et cytokine traitees) - Google Patents

Traitement combine d'une leucemie lymphoide chronique (cellules de leucemie lymphoide chronique et cytokine traitees) Download PDF

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WO2004024171A1
WO2004024171A1 PCT/CA2003/001408 CA0301408W WO2004024171A1 WO 2004024171 A1 WO2004024171 A1 WO 2004024171A1 CA 0301408 W CA0301408 W CA 0301408W WO 2004024171 A1 WO2004024171 A1 WO 2004024171A1
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cll
cells
patient
cll cells
blood
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PCT/CA2003/001408
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David E. Spaner
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Vasogen Ireland Limited
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C12N5/0694—Cells of blood, e.g. leukemia cells, myeloma cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/40—Peroxides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/13—Tumour cells, irrespective of tissue of origin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/10—Inactivation or decontamination of a medicinal preparation prior to administration to an animal or a person
    • A61K41/17—Inactivation or decontamination of a medicinal preparation prior to administration to an animal or a person by ultraviolet [UV] or infrared [IR] light, X-rays or gamma rays
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/24—Interferons [IFN]

Definitions

  • This invention relates to leukemia alleviation, and to processes and cellular compositions useful therein. More specifically, it relates to compositions and processes for alleviating chronic lymphocytic leukemia in mammalian patients, especially humans.
  • CLL Chronic lymphocytic leukemia
  • acute lymphocytic leukemia acute myeioid leukemia
  • chronic myeloid leukemia chronic myeloid leukemia.
  • CLL is most commonly encountered in patients over the age of sixty. It has a gradual onset, and may not cause the patient discomfort or pain for several years. It is characterized by a large number of cancerous mature lymphocytes and enlarged lymph nodes. Cancerous cells crowd out the normal cells in the bone marrow and lymph nodes. Anemia develops in the patient and the number of normal white cells and platelets in the patient's blood decreases, whereas the total white cell count increases due to the proliferation of abnormal white cells. The level and activity of antibodies also decrease.
  • CLL cancer-derived neoplasm originating from a B cell leukemia
  • CD5+ B cell i.e. a B cell expressing the marker CD5.
  • CLL is the commonest leukemia in the Western world. Although often considered an indolent disease of the elderly, patients with advanced disease survive only about 1.5 years and cannot be cured with conventional chemotherapy such as fludarabine or monoclonal antibodies. A number of clinical observations, such as spontaneous remissions associated with increased immune activity during our infections and responses to immunomodulatory cytokines or autologous CLL vaccines suggest that T- cell mediated immunotherapies may be able to improve disease control.
  • CLL originates from transformed anergic B cells. It is believed that anergic B cells are killed by T-cells that recognize them. T-cells able to recognize autologous CLL cells exist in most patients, especially in early-stage disease. If CLL cells represent anergic B cells, they must evade killing by autoreactiveT-cells at some point in the evolution of the disease. This all suggests that it should be possible to develop a vaccine against CLL based upon autologous CLL cells, treated to enhance their immunogenicity so that they can stimulate autologous T-cells to eliminate circulating CLL cells from the patient.
  • Clinical stage of CLL characterized in the staging systems of Rai (stages 0-IV) and Binet (stages A-C), remains the strongest predictor of survival in CLL patients. Both systems are based on the amount of involved lymphoid tissue and the presence of anemia and/or thrombocytopenia. In general, patients with later stages have a significantly worse prognosis and a shorter survival. Patients with Rai stage IV or Binet stage C have a median survival of only 1.5 to 2 years.
  • Chemotherapy is the standard treatment for CLL. A patient diagnosed with CLL is normally monitored by tracking the white cell count in the blood.
  • Chemotherapy is not instituted until the patient starts to suffer symptoms such as fatigue, weight loss, fevers or pain as a result of the progression of the CLL.
  • CLL is not curable with conventional methods of chemotherapy, even though initial response rates are high.
  • the toxicities associated with the use of chemotherapy are well known and include nausea and myelosuppression with a risk of developing serious infections.
  • subsequent responses become inexorably short-lived, likely because drug-resistant tumor cells are selected by the use of cytotoxic agents.
  • interferon- ⁇ 2b interferon- ⁇ 2b
  • interferon IFN- ⁇ interferon- ⁇ 2b
  • IFN- ⁇ administered intravenously (IV) at high doses (20x10 6 U/m 2 , 20 times over 4 weeks) and then subcutaneously (SC) at low doses (10x10 6 U/m 2 , 3 times per week for 48 weeks) has been shown to significantly prolong relapse-free survival and overall survival compared to observation alone, in high risk melanoma patients (Stage MB and Stage III). Since trials that used only SC injections failed to show such a survival benefit, it is a reasonable hypothesis that the high dose component provides the most important therapeutic element of IFN- ⁇
  • the present invention is based, at least in part, on the discovery of the role played by costimulatory molecule CD 80 in immunogenicity of CLL cells.
  • CLL patients whose disease appears to respond, or did not progress, after administration of oxidatively stressed CLL cells tended to have a relative overexpression of CD 80, compared with CD 86, on their CLL cells.
  • CD 80 signals are associated with type 1 immune responses
  • CD 86 signals [especially at the time of initiation of immune response] are associated with type 2 immunity.
  • the relative overexpression of CD 80 on oxidatively stressed, injected CLL cells appears to lead to more effective type 1 immune responses against CLL cells and, consequently, to better disease control.
  • CLL in a mammalian patient is alleviated by administering to the patient oxidatively stressed, compatible CLL malignant cells, and at least one cytokine selected from the group consisting of IL-2 and IFN- ⁇ .
  • the source of the CLL malignant cells may be the mammalian patient himself or herself ( e.g.
  • a withdrawn blood sample from the patient a compatible mammalian donor (e.g. a withdrawn blood sample from another, compatible CLL-suffering patient), or a cultured cell line of CLL malignant cells.
  • a compatible mammalian donor e.g. a withdrawn blood sample from another, compatible CLL-suffering patient
  • a cultured cell line of CLL malignant cells e.g. a withdrawn blood sample from another, compatible CLL-suffering patient
  • Subjection of the CLL malignant cells to oxidative stress takes place in vitro.
  • the oxidatively stressed CLL cells thus obtained are administered to the patient, prior to, along with, or following administration of an appropriate dose or series of doses of cytokine(s), to result in an alleviation of the patient's CLL.
  • CLL in a mammalian patient suffering therefrom is significantly alleviated by administering to the patient oxidatively stressed blood cells, including oxidatively stressed CLL malignant cells, obtained from the patient and subjected to oxidative stress in vitro and then reintroduced into the patient, prior to, along with, or following administration of an appropriate dose or series of doses of at least one cytokine selected from IL-2 and IFN- ⁇ .
  • oxidatively stressed blood cells including oxidatively stressed CLL malignant cells
  • This preferred procedure thus involves extracting an appropriate quantity of blood containing CLL cells from the CLL patient, treating the blood or a selected portion of it extracorporeally with an oxidative stressbr, and reintroducing it into the same patient, prior to, along with, or following administration of an appropriate dose or series of doses of said at least one cytokine.
  • the result after one or more of such treatments, is a significant alleviation of the patient's CLL condition, as indicated in a reduced white blood cell proliferation and a reduced swelling of lymph nodes of the patient.
  • the present invention provides a process for treating a CLL suffering patient for alleviation of CLL, which comprises extracting an aliquot of blood containing CLL cells from the patient, subjecting at least a portion of the extracted blood cells extracorporeally to appropriate oxidative stress, and re-introducing the oxidatively-stressed material into the patient, and administering to the patient an effective dosage of at least one cytokine selected from the group consisting of IL-2 and IFN- ⁇ .
  • a further aspect of the present invention is the use in preparation of a medicament active against CLL in a mammalian patient, of oxidatively stressed autologous blood or blood fractions, including oxidatively stressed autologous malignant CLL cells, and at least one cytokine selected from the group consisting of IL-2 and IFN- ⁇ .
  • Another aspect of this invention is a composition
  • a composition comprising stressed CLL cells and at least one cytokine selected from the group consisting of IL- 2 and IFN- ⁇ .
  • the cells may be oxidatively stressed and may further be autologous CLL cells.
  • Figure 1 is a graphical presentation of results obtained according to Example 3, namely the CD 80/86 ratio of CLL cells from patients given the autologous oxidatively stressed CLL cell vaccine;
  • Figure 2 is a graphical presentation of results obtained in Example 5, namely a plot of a relative CD 80 expression from cells cultured in different concentrations of IL-2. DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • compositions of the present invention may be viewed as comprising two parts, for separate or combined administration to a CLL patient.
  • the first part comprises the stressed CLL cells, and is sometimes referred to herein as a vaccine.
  • the second part comprises the selected cytokine(s). Both parts are suitably used along with an excipient for ease of administration.
  • a preferred embodiment of the present invention prepares the first composition by subjecting the blood cells, or the appropriate fraction of them including the CLL cells, to electromagnetic emission radiation as well as oxidative stress, either simultaneously or sequentially.
  • a temperature stressor may be applied to the cells, simultaneously or sequentially with the oxidative stressor and the electromagnetic emission stressor, i.e. a temperature at, above or below body temperature.
  • An aliquot of blood is drawn from the CLL patient, of volume up to about 400 ml, preferably from about 0.1 to 100 ml, more preferably from about 1 to about 15 ml, even more preferably from about 8 to about 12 ml.
  • aliquot refers to the sample subjected to the stressors; and embraces both the originally extracted whole blood and any fraction thereof subjected to stressors, before or after separation.
  • the modified aliquot comprising the first composition is re-introduced into the patient's body by any suitable method, most preferably intramuscular injection, but also including subcutaneous injection, intraperitoneal injection, intra-arterial injection, intravenous injection and oral administration.
  • the composition may optionally include a pharmaceutically acceptable excipient, such as sterile physiological saline.
  • the aliquot of blood is in addition subjected to mechanical stress.
  • mechanical stress is suitably that applied to the aliquot of blood by extraction of the blood aliquot through a conventional blood extraction needle, or a substantially equivalent mechanical stress, applied shortly before the other chosen stressors are applied to the blood aliquot.
  • This mechanical stress may be supplemented by the mechanical stress exerted on the blood aliquot by bubbling gases through it, such as ozone/oxygen mixtures, as described below.
  • the optionally applied temperature stressor either warms the aliquot being treated to a temperature above normal body temperature or cools the aliquot below normal body temperature.
  • the temperature is selected so that the temperature stressor does not cause excessive hemolysis in the blood contained in the aliquot and so that, when the treated aliquot is injected into a subject, the desired effect will be achieved.
  • the temperature stressor is applied so that the temperature of all or a part of the aliquot is up to about 55 °C, and more preferably in the range of from about -5 to about 55 °C.
  • the temperature of the aliquot is raised above normal body temperature, such that the mean temperature of the aliquot does not exceed a temperature of about 55°C, more preferably from about 4°C to about 50°C, even more preferably from about 40° to about 44° C, and most preferably about 42.5 i 1 °C. ln other preferred embodiments, the aliquot is cooled below normal body temperature such that the mean temperature of the aliquot is within the range of from about 4 °C to about 36.5 °C, more preferably from about 10 °C to about 30 °C, and even more preferably from about 15 to about 25°C.
  • the oxidative environment stressor can be the application to the aliquot of solid, liquid or gaseous oxidizing agents, including peroxides such as hydrogen peroxide.
  • it involves exposing the aliquot to a mixture of medical grade oxygen and ozone gas, most preferably by applying to the aliquot medical grade oxygen gas having ozone as a component therein.
  • the ozone content of the gas stream and the flow rate of the gas stream are preferably selected such that the amount of ozone introduced to the blood aliquot, either on its own or in combination with one of the other stressors, does not give rise to excessive levels of cell damage, and so that, when the treated aliquot is injected into a subject, the desired effect will be achieved.
  • the gas stream has an ozone content of up to about 300 ⁇ g/ml, preferably up to about 100 ⁇ g/ml, more preferably about 30 ⁇ g/ml, even more preferably up to about 20 ⁇ g/ml, particularly preferably from about 10 ⁇ g/ml to about 20 ⁇ g/ml, and most preferably about 14.5 ⁇ 1.0 ⁇ g/ml.
  • the gas stream is suitably supplied to the aliquot at a rate of up to about 2.0 litres/min, preferably up to about 0.5 litres/min, more preferably up to about 0.4 litres/min, even more preferably up to about 0.33 litres/min, and most preferably about 0.2 ⁇ 0.025 litres/min.
  • the lower limit of the flow rate of the gas stream is preferably not lower than 0.01 litres/min, more preferably not lower than 0.1 litres/min.
  • the amount of ozone introduced to the blood does not exceed about 300 ⁇ g/ml of blood in the aliquot.
  • the electromagnetic emission stressor is suitably applied by irradiating the aliquot under treatment from a source of electromagnetic emission while the aliquot is maintained at the aforementioned temperature and while the oxygen/ozone gaseous mixture is being bubbled through the aliquot.
  • Preferred electromagnetic emissions are selected from photonic radiation, more preferably UV, visible and infrared light, and even more preferably UV light.
  • the most preferred UV sources are UV lamps emitting UV-C band wavelengths, i.e. at wavelengths shorter than about 280 nm.
  • Ultraviolet light corresponding to standard UV-A (wavelengths from about 315 to about 400 nm) and UV-B (wavelengths from about 280 to about 315) sources can also be used.
  • the UV dose should be selected, on its own or in combination with the other chosen stressor(s), so that excessive amounts of cell damage do not occur, and so that, when the treated aliquot is injected into a subject, the desired effect will be achieved.
  • an appropriate dosage of such UV light can be obtained from lamps with a power output of from about 10 to about 30 watts arranged to surround the sample container holding the aliquot, each lamp providing an intensity, at a distance of 16 mm, of from about 5 to 20 mW/cm 2 .
  • Such a treatment, applied in combination with the oxidative environment stressor, provides a modified blood aliquot which is ready for injection into the subject.
  • the aliquot may be maintained at a predetermined temperature above or below body 41- temperature while the oxygen/ozone gas mixture is applied thereto and while it is irradiated with ultraviolet light.
  • the time for which the aliquot is subjected to the stressors is normally within the time range of from about 0.5 minutes up to about 60 minutes. The time depends to some extent upon the chosen combination of stressors.
  • the intensity of the UV light may affect the preferred time.
  • the chosen temperature level may also affect the preferred time.
  • the concentration of the oxidizing agent and the rate at which it is supplied to the aliquot may affect the preferred temperature.
  • preferred times will be in the approximate range of from about 2 to about 5 minutes, more preferably about 3 minutes.
  • the starting aliquot temperature, and the rate at which it can be warmed or cooled to a predetermined temperature tends to vary from subject to subject. Warming is suitably by use of one or more infrared lamps placed adjacent to the aliquot container. Other methods of warming can also be adopted.
  • a mechanical stressor As noted, it is preferred to subject the aliquot of blood to a mechanical stressor, as well as the chosen stressor(s) discussed above. Extraction of the blood aliquot from the patient through a hypodermic needle constitutes the most convenient way of obtaining the aliquot for further extracorporeal treatment, and this extraction procedure imparts a suitable mechanical stress to the blood aliquot.
  • the mechanical stressor may be supplemented by subsequent processing, for example the additional mechanical shear stress caused by bubbling as the oxidative stressor is applied. 42-
  • the aliquot may be treated with the heat, UV light and oxidative environment stressors using an apparatus of the type described in U.S. Patent No. 4,968,483 to Mueller.
  • the aliquot is placed in a suitable, sterile container, which is fitted into the machine.
  • a UV-permeable container is used and the UV lamps are switched on for a fixed period before the other stressor is applied, to allow the output of the UV lamps to stabilize.
  • the UV lamps are typically on while the temperature of the aliquot is adjusted to the predetermined value, e.g. 42.5 ⁇ 1 C.
  • Four UV lamps are suitably used, placed around the container.
  • a mammalian patient is given one or more courses of treatments with the first composition prepared as described herein, each course of treatment comprising the administration to a mammalian subject of one or more (e.g. one to six) blood originating aliquots modified as disclosed above.
  • the treatment may be administered daily, but no more than one treatment should be administered to the subject per day.
  • the second composition used in the present invention is an effective dose of at least one cytokine selected from the group consisting of IL-2 and IFN- ⁇ .
  • IFN- ⁇ is a known chemotherapeutic agent . It is preferably administered to the patient after the conclusion of the course of treatments with the first composition as described above. Preferably, IFN- ⁇ is administered to the patients in high dose, e.g. 1 - 50 x10 6 U/m 2 , 5 - 50 times over 4 weeks, preferably 10 - 30 and most preferably about 20 x 0 6 U/m 2 , 20 times over 4 weeks, intravenously. Depending upon the patients response and condition at the end of the treatment, the clinician may elect to continue with administration of IFN- ⁇ subcutaneously at low doses, e.g. 10x10 6 U/m 2 , 3 times per week for 48 weeks), to improve the therapeutic result.
  • low doses e.g. 10x10 6 U/m 2 , 3 times per week for 48 weeks
  • lFN- ⁇ may be given to the patient, at similar dosage levels, prior to commencing a course of autologous vaccine administration, effectively to potentiate the patient's immune system towards the vaccine.
  • the IFN- ⁇ may be administered, at similar or reduced dosages, in combination with the vaccine, e.g. as a combination composition for intramuscular injection.
  • administration doses and frequencies 44- are generally the same as in the case of IFN- ⁇ . It may also be administered prior to a course of autologous vaccine administration or in combination with the vaccine.
  • a particularly preferred embodiment of the invention uses both IFN- ⁇ and IL-2, simultaneously or sequentially, prior to, along with, or subsequent to the vaccine administration.
  • Dosages of the cytokines, when used together in this manner, are generally the same as or of the order of half of the dosages given above for their individual administrations.
  • Kits of this invention may comprise various components of the first composition that are provided in separate containers.
  • the containers may separately contain the CLL cells treated as described herein or pharmaceutically acceptable excipient, such that when mixed togther they constitute a vaccine comprising the first composition of this invention in unit dosage or multiple dosage form.
  • the containers will also contain suitable dosage forms of the cytokine(s) IL-2 and/or IFN- ⁇ and/or excipient therefor, to form the second composition of the invention. They may also contain suitable devices, such as syringes and needles for delivering the compositions to a patient.
  • Packaged compositions and kits of this invention typically include instructions for storage, preparation and administration of the compositions.
  • the process of the present invention is particularly indicated for CLL patients whose condition shows signs of accelerated progression to the point where chemotherapy would normally be instituted.
  • Patients may be selected for treatment based upon several criteria. For example, patients having a CLL cell count in the blood of from about 20 million - 100 million CLL cells per millilitre of blood are preferred candidates for the treatment. 45-
  • Patients may be selected for treatment with the methods and processes of this invention.
  • An assessment by an attending clinician will determine their suitability, but normally it will be a patient who has previously tested positive for CLL, has been monitored for some time without evidencing an increase in white cell count, but who has, in the previous 1 - 2 months prior to test evidenced a white blood cell count increase into the 30 x 10 6 to 100 x 10 6 approximate range.
  • the patient was given a course of treatments as follows. Each treatment involved withdrawing a 10 ml aliquot of blood from the patient via venal puncture, subjecting the whole blood aliquot, in a sterile UV- transparent container and in the presence of anticoagulant, to simultaneous ozone-oxygen bubbling and UV radiation exposure at elevated temperature, in an apparatus essentially as described in aforementioned U.S. patent 4,968,483.
  • the treated blood was re-administered to the patient by injection into the gluteal muscle.
  • the temperature of the blood aliquot in the apparatus was initially raised to 42.5°C and held steady at that level.
  • the constitution of the gas mixture was 14-15 mcg/ml ozone/oxygen, fed through the aliquot at a rate of about 200 mis/minute, for three minutes.
  • the UV radiation had a wavelength of 253.7 nm.
  • CLL cells from patients at various stages of CLL disease progression were purified and cultured in different concentrations of IL-2.
  • the CLL ceils were isolated directly from fresh blood by negative selection (RosetteSep, StemCell Technologies, Vancouver B.C.) according to the manufacturer's 49- instructions. Briefly, 75% of the plasma was first removed to concentrate peripheral blood mononuclear cells, increase cell yield, and minimize the required antibodies.
  • CLL-B cells were isolated with antibodies against CD 2, CD 3, CD 14, CD 16, CD 56 and glycophorin A.
  • the purified CLL cells were cultured in different amounts of IL-2 (zero, 5, 50, 500 and 5000 U/ml) for two to three days. Cells (1.5 x 10 6 cells per ml) were cultured in serum free AIM-V medium (Gibco BRL) in 6 - or 24
  • the enhanced expression of costimulatory molecule CD 80 on the IL- 2 treated CLL cells indicates the use of IL-2 in combination with autologous, oxidatively stressed CLL cell based vaccine, which as shown in Example 3 is more effective in patients whose CLL cells exhibit enhanced CD 80 expression.
  • CLL cells were obtained from patients, purified and isolated as described in Example 4. They were cultured for 48 hours in serum free AIM
  • CD 80 expression on the cells was determined by flow cytometry.
  • the results (average of 19 experiments) showed an approximately threefold increase in the number of cells expressing CD 80, and an approximately twofold increase in the fluorescent intensity from CD 80 expression cells, attributable to culturing in IFN ⁇ . This is an indication for the use of IFN ⁇ in combination with an autologous, oxidatively stressed CLL cell based vaccine, which as demonstrated above is most effective in the presence of CD 80 costimulatory molecule.

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Abstract

La présente invention concerne un procédé pour traiter un patient atteint d'une leucémie lymphoïde chronique (CLL). Ce procédé consiste à administrer à ce patient des cellules de CLL soumises à un stress oxydatif et au moins une cytokine choisie parmi IL-2 et IFN-α. Les cellules de CLL sont soumises à un stress oxydatif de manière extracorporelle, par exemple en les soumettant à des mélanges d'oxygène et d'ozone, et sont de préférence soumises simultanément à d'autres facteurs de stress comme de la lumière ultraviolette. Les cellules de CLL sont de préférence autologues et sont contenues dans une aliquote du sang du patient au moment où elles sont soumises à un stress. La/les cytokine(s) est/sont de préférence administrée(s) à haute dose, après administration des cellules de CLL stressées.
PCT/CA2003/001408 2002-09-16 2003-09-15 Traitement combine d'une leucemie lymphoide chronique (cellules de leucemie lymphoide chronique et cytokine traitees) WO2004024171A1 (fr)

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Citations (2)

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EP0426521A1 (fr) * 1989-10-17 1991-05-08 Roussel-Uclaf L'utilisation de l'interleukine 2 pour le traitement des leucémies
WO2002034888A1 (fr) * 2000-10-25 2002-05-02 Vasogen Ireland Limited Cellules llc soumises ex vivo a un stress oxydatif pour traitement de la leucemie lymphoide chronique (llc)

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0426521A1 (fr) * 1989-10-17 1991-05-08 Roussel-Uclaf L'utilisation de l'interleukine 2 pour le traitement des leucémies
WO2002034888A1 (fr) * 2000-10-25 2002-05-02 Vasogen Ireland Limited Cellules llc soumises ex vivo a un stress oxydatif pour traitement de la leucemie lymphoide chronique (llc)

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Title
BELARDELLI F ET AL: "Cytokines as a link between innate and adaptive antitumor immunity", TRENDS IN IMMUNOLOGY, ELSEVIER, CAMBRIDGE, GB, vol. 23, no. 4, 1 April 2002 (2002-04-01), pages 201 - 208, XP004345055, ISSN: 1471-4906 *

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