WO2004024171A1

WO2004024171A1 - Chronic lymphocytic leukemia combination treatment (treated cll cells and cytokine)

Info

Publication number: WO2004024171A1
Application number: PCT/CA2003/001408
Authority: WO
Inventors: David E. Spaner
Original assignee: Vasogen Ireland Ltd
Current assignee: Vasogen Ireland Ltd
Priority date: 2002-09-16
Filing date: 2003-09-15
Publication date: 2004-03-25
Anticipated expiration: 2005-03-16
Also published as: AU2003266086A1

Abstract

Chronic lymphocytic leukemia (CLL) in a patient is treated by administering to the patient oxidatively stressed CLL cells and at least one cytokine selected from IL-2 and IFN-α. The CLL cells are oxidatively stressed extracorporeally, e.g. by subjection to oxygen/ozone mixtures, and preferably are simultaneously subjected to other stressors such as UV light. Preferably also, the CLL cells are autologous, and are contained in an aliquot of the patient's blood at the time of subjection to stressing. The cytokine(s) is preferably administered in high dose, subsequent to the administration of the stressed CLL cells.

Description

CHRONIC LYMPHOCYTIC LEUKEMIA COMBINATION TREATMENT (TREATED CLL CELLS AND CYTOKINE)

FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

Chronic lymphocytic leukemia (hereinafter CLL) is one of the four major types of leukemia encountered by humans, the others being acute lymphocytic leukemia, acute myeioid leukemia and 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. As a result, the patient's immune system becomes compromised. It is more common for CLL sufferers to die from consequences of the compromised immune system, e.g. infections, than from the CLL itself. The most common type of CLL is a B cell leukemia, and the malignant cell of origin is a 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.

The importance of T-cell interactions in the pathogenesis of CLL is also suggested by the cellular origin of CLL. It is believed that 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 (initially with alkylating agents such as chlorambucil and subsequently with fludarabine) 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. However, 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. Moreover, subsequent responses become inexorably short-lived, likely because drug-resistant tumor cells are selected by the use of cytotoxic agents.

One example of a previously proposed chemotherapeutic method, for treatment of melanoma, is the administration of relatively high dosages of interferon-α2b (hereinafter interferon IFN-α). According to Kirkwood J.M., et.al., J. Clin. Oncol. 18, 2444-2458 (2000), and Balch CM., et.al., J. Clin. Oncol., 19, 3635-3648 (2002), IFN-α administered intravenously (IV) at high doses (20x10⁶U/m² , 20 times over 4 weeks) and then subcutaneously (SC) at low doses (10x10⁶U/m² , 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-α

International patent application publication number WO 02/34888 Vasogen Ireland Limited discloses a process of alleviating CLL in a mammalian patient, in which autologous CLL malignant cells are extracted from the patient, subjected extracorporeally to oxidative stress and electromagnetic radiation, and re-administered to the patient. Whilst useful and successful, this treatment has shown a tendency to be of limited effective duration.

Accordingly, it is an object of the present invention to provide novel procedures and compositions for alleviation of CLL in mammalian patients.

SUMMARY OF THE INVENTION

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, whereas 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.

In accordance with this invention, clinically relevant immunomodulatory agents which promote type 1 immunity and further increase the amount of CD 80 expression and the CD 80: CD 86 expression ratio, such as IFN a and IL-2, are used to manipulate costimulatory molecule expression on the CLL cells, before, during or after vaccination with oxidatively stressed CLL cells, to improve the therapeutic effects of the vaccination. Thus according to the present invention in its broad aspects, 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-α. 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. 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.

According to a preferred aspect of the present invention, 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-α. 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. Thus from one aspect, 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- α.

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-α.

Another aspect of this invention is a composition comprising stressed CLL cells and at least one cytokine selected from the group consisting of IL- 2 and IFN-α.The cells may be oxidatively stressed and may further be autologous CLL cells.

BRIEF REFERENCE TO THE DRAWINGS

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. Optionally also, 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. Either the whole blood is subjected to the stressor(s), or an appropriate cellular fraction thereof containing the CLL malignant B cell fraction is separated by known methods and subjected to the aforementioned stressor(s). The stressed cells are then reintroduced into the CLL patient from whom the original aliquot was drawn. The term "aliquot" as used herein 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. Accordingly, the composition may optionally include a pharmaceutically acceptable excipient, such as sterile physiological saline.

Preferably also, the aliquot of blood is in addition subjected to mechanical stress. Such 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. Preferably, 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.

In some preferred embodiments of the invention, 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. Preferably, 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. Suitably, the gas stream has an ozone content of up to about 300 μg/ml, preferably up to about 100 μg/ml, more preferably about 30μg/ml, even more preferably up to about 20 μg/ml, particularly preferably from about 10 μg/ml to about 20 μg/ml, and most preferably about 14.5 ± 1.0 μg/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. Preferably the amount of ozone introduced to the blood does not exceed about 300 μg/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. As in the case of the oxidative stressor, 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. For example, 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/cm². Up to eight such lamps, surrounding the sample container holding the aliquot, with a combined output at 253.7nm of 10 to 30 watts, operated at an intensity to deliver a total UV light energy at the surface of the blood of from about 0.025 to about 10 joules/cm², preferably from about 0.1 to about 3.0 joules/cm² may advantageously be used. Such a treatment, applied in combination with the oxidative environment stressor, provides a modified blood aliquot which is ready for injection into the subject.

It is preferred to subject the aliquot to the oxidative environment stressor, the UV light stressor and the temperature stressor simultaneously, following the subjection of the aliquot to the mechanical stress, e.g. by extraction of the blood from the patient. Thus, 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. When UV light is used, the intensity of the UV light may affect the preferred time. The chosen temperature level may also affect the preferred time. When the oxidative environment in the form of a gaseous mixture of oxygen and ozone applied to the aliquot is chosen as one of the two stressors, the concentration of the oxidizing agent and the rate at which it is supplied to the aliquot may affect the preferred temperature. Some experimentation to establish optimum times may be necessary on the part of the operator, once the other stressor levels have been set. Under most stressor conditions, 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.

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-

ln the practice of the preferred process of the present invention, 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. When a temperature stressor is used in the combination, 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.

In the preferred method of the invention, 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.

Although it may be sufficient to administer only one course of treatment as described above to the subject, it may be preferred in some circumstances to administer more than one treatment or course of treatment, or to follow the above-described course of treatment by periodic "booster" treatments, if necessary, to maintain the desired effects of the present invention. There may be a substantial interval between individual treatments or courses of treatment. For example, it may be preferred to administer booster treatments at intervals of 1 week, 1 month, 3 months or 6 months or other appropriate periods following the initial treatment or course of treatment, depending upon the condition of the individual patient and the progression or remission of the CLL. Regular periodic monitoring of the patients undergoing the treatment according to the invention is contemplated, with repeats of the treatment or course of treatment as indicated by the patient's condition and as determined by the attending physician.

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-α.

IFN-α 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-α is administered to the patients in high dose, e.g. 1 - 50 x10⁶U/m² , 5 - 50 times over 4 weeks, preferably 10 - 30 and most preferably about 20 x 0⁶U/m² , 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-α subcutaneously at low doses, e.g. 10x10⁶U/m² , 3 times per week for 48 weeks), to improve the therapeutic result.

Alternatively, lFN-α 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. In another alternative, the IFN-α may be administered, at similar or reduced dosages, in combination with the vaccine, e.g. as a combination composition for intramuscular injection.

When IL-2 is chosen as the cytokine for use as the second component in the present invention, administration doses and frequencies 44- are generally the same as in the case of IFN-α. 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-α 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.

The compositions of the present invention, and subcomponents thereof may be supplied in unit dosage or kit form. 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-α 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-

Normally, however, progression of CLL after diagnosis is simply monitored by determining the white blood cell count of a patient. A normal healthy patient has a white blood cell count of about 10 (i.e. 10 x 10⁶ white cells per ml of blood), whereas a CLL patient has elevated white blood cell counts. As the CLL condition progresses, the patient's white blood cell count increases, mainly due to the proliferation of the malignant CD5+ B cells. When the patient's white blood cell count reaches the approximate range 30 x 10⁶ to 100 x 10⁶, institution of chemotherapy may be indicated. This is the indicator for the institution of the process of the present invention.

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 10⁶ to 100 x 10⁶ approximate range.

The beneficial effects of the process of the present invention, as with most leukemia treatments, vary widely in efficacy between individual patients. Some patients show an immediate and long lasting effect. Others show an immediate effect which wears off over time, but which can be re- effected by undertaking a further course of treatments according to the invention. In others, the immediate effect wears off, and apparently is not so re-effected.

The invention will be further described, for illustrative purposes, with reference to specific examples of clinical application of the processes and products of specifically preferred embodiments of the invention. EXAMPLE 1

An elderly male patient had been diagnosed with CLL three years earlier, and had been continuously monitored by physicians since that time, with a view to instituting chemotherapy treatments when the condition deteriorated to the appropriate extent. In the eight-month period leading up to the treatments described herein, the patient's white blood cell count had increased from 30 to 70, indicating an imminent need to institute chemotherapy. The patient also exhibited significantly swollen lymph nodes.

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.

After a course of 6 such treatments, administered over three weeks with a two or three-day interval between each treatment, a favorable response was noted. Instead of a continuing increase in white cell count, the patient exhibited a decrease, from 70 to 61. There was also a 50% 47- decrease in peripheral adenopathy. The treatments were well tolerated and no significant side effects have been reported by the patient.

Maintenance of this white cell count, or even further reduction in it, can be obtained by subjecting the patient subsequently to a course of high dose IFN-α or IL-2 in accordance with the invention, e,g, about 20 x10⁶U/m² 20 times over 4 weeks

EXAMPLE 2

Following the protocol described in Example 1 , the following patients have been treated.

A 55-year old man suffering from CLL and diabetes was treated. The increase in his white cell count was arrested, an effect which has lasted one month since the end of treatment.

• A 50-year old man suffering from CLL was treated. His white blood cell count dropped from 30 to 15. This lower level has been maintained for at least one month after the conclusion of treatment.

A 50-year old woman suffering from CLL was treated. She had been treated previously for CLL by chemotherapy using Chlorambucil. Her condition had relapsed and the Chlorambucil was no longer effective. After treatment with the above protocol her white blood ceil count was stabilized.

A 60-year old man with CLL and heart problems was treated. His white cell count of 30 was stabilized by the course of treatment. The improved condition of each of these patients can be maintained, or even further improved, by subsequent administration of IFN-α and/or IL-2 accordance with the invention, e,g, about 20 x10⁶U/m² , 20 times over 4 weeks.

EXAMPLE 3

A total of 18 CLL patients, equal numbers of males and females, who had been suffering from the disease for at least a year, were given the treatment described in the previous examples, mainly extra corporeal oxidative stressing autologous CLL cells and readministration thereof by intramuscular injection. Of the 18 patients, the treatment was effective (regression or stabilization of the disease, as opposed to the natural progression of it, seven weeks after the initial treatment) in 11 of them, but continued to progress in the other seven. The percentages of CD 80+ and CD 86+ circulating cells were determined for each patient, by flow cytometry, prior to extracting the CLL cells for stressing. The average CD 80/CD 86 for the patients on which the treatment was effective was 2.08, as compared with 0.35 for the seven nonresponding patients. These results are presented on Figure 1 , a bar graph of the average CD 80/CD 86 ratios of cell expressions of each group of patients, in arbitrary units. These results suggest that patients whose disease responded, received vaccines that expressed relatively more CD 80 than CD 86.

EXAMPLE 4

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 10⁶ cells per ml) were cultured in serum free AIM-V medium (Gibco BRL) in 6 - or 24

- well plates at 37° C. in 5% carbon dioxide. The cells were recovered and washed, and CD 80 surface expression was measured by flow cytometry.

The results are presented graphically on Figure 2, a plot of relative CD 80 expression against concentration of IL-2 in the culture medium. CD 80 expression was especially sensitive to IL-2. Although baseline expression of CD 80 could sometimes be found on circulating CLL cells, treatment with IL-2 increased both the number of CD 80 cells and the mean fluorescent intensity from them. As Figure 2 shows, CD 80 expression was directly proportional to the amount of IL-2 in the culture medium.

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.

EXAMPLE 5.

CLL cells were obtained from patients, purified and isolated as described in Example 4. They were cultured for 48 hours in serum free AIM

- V medium and IFN-α2B (500 U/ml) for 48 hours. 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 IFNα. This is an indication for the use of IFNα 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.

Claims

WHAT IS CLAIMED IS:

1. Use in manufacture of compositions for treating a CLL suffering patient for alleviation of CLL, of compatible CLL cells which have been ex vivo subjected to oxidative stress and electromagnetic radiation, and an effective dosage of at least one cytokine selected from the group consisting of IL-2 and INF-α.

2. Use as claimed in claim 1 wherein said CLL cells are autologous cells, obtained by 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 electromagnetic radiation.

3. Use as claimed in Claim 1 or Claim 2 wherein the oxidatively stressed material includes malignant CLL cells.

4. Use as claimed in any preceding claim wherein the electromagnetic radiation is UV radiation.

5. Use as claimed in any preceding claim wherein the aliquot of blood has a CLL cell content of from about 20 million to about 100 million cells per ml of blood.

6. Use as claimed in any preceding claim wherein the aliquot of blood has a white blood cell count of from about 30 x 10⁶ to about 100 x 10⁶.

7. Use as claimed in any preceding claim wherein the oxidative stress comprises applying an oxidizing agent to the CLL cells.

8. Use as claimed in claim 7 wherein the oxidizing agent comprises a mixture of ozone gas and medical grade oxygen, the ozone gas being contained in the mixture in a concentration of up to about 300 μg/ml.

9. Use as claimed in Claim 8, wherein the ozone gas is contained in the mixture in a concentration of up to about 30 μg/ml.

10. Use as claimed in Claim 9, wherein the ozone gas is contained in the mixture in a concentration of from about 13.5 μg/ml to about 15.5μg/ml.

11. Use as claimed in any of claims 8 - 10, wherein the mixture is applied to the CLL cells at a flow rate of up to about 0.33 litres/min.

12. Use as claimed in Claim 11 , wherein the mixture is applied to the CLL cells at a flow rate of from about 0.21 litres/min to about 0.27 litres/min.

13. Use according to claim 4, wherein the UV radiation comprises UV light having one or more UV-C band wavelengths.

14. Use according to any preceding claim, further comprising applying temperature stressor to the CLL cells such that the temperature of at least part of the CLL cells is in the range of from about -5° C to about 55° C.

15. Use according to claim 14, wherein the temperature of the CLL cells is in the range of from about 37° C to about 55° C.

16. Use according to any preceding claim, wherein the CLL cells are subjected to oxidative stress for a period of up to about 60 minutes.

17. Use according to any preceding claim wherein the cytokine is IFN-α, administered at a dose of 1 - 50 x10⁶U/m² , 5 - 50 times over 4 weeks.

18. Use according to claim 17 wherein the IFN-α administration commences after the conclusion of the administration of the first composition.

19. Use according to any of claims 1 - 16 wherein the cytokine is IL-2.

20. Use according to any of claims 1 - 16 wherein the cytokine is a combination of IL-2 and IFN-α

21. A kit of parts for use in the treatment of a mammalian patient suffering from chronic lymphocytic leukemia, said kit comprising: autologous CLL cells treated ex vivo with oxidative stress and electromagnetic radiation; a pharmaceutically acceptable excipient for said cells; means for administering said cells and the excipient to the patient; at least one cytokine selected from the group consisting of IL-2 and IFN-α; a pharmaceutically acceptable excipient for said selected cytokine(s); and means for administering the cytokine(s) and the excipient therefor to the patient.