Therapeutic cancer vaccine based on stress proteins rendered immunogenic

Abstract

The invention describes a method for preparing a composition comprising tumour cell stress proteins, said method comprising the following steps: providing tumour cells in a culture medium; subjecting the tumour cells under i) to a stress with the result that these cells produce stress proteins in response to the stress; obtaining or recovering stressed tumour cells and/or stress proteins; treating the stressed tumour cells and/or the stress proteins obtained with a molecule or a process capable of rendering the stress proteins immunogenic, preferably a hapten or haptenization. The invention also describes a pharmaceutical composition comprising tumour cell stress proteins and/or tumour cells comprising stress proteins, these stress proteins being rendered immunogenic, and are in particular haptenized, and a pharmaceutically acceptable excipient.

Claims

1. A pharmaceutical composition or vaccine comprising tumor cells, a plurality of different immunogenic stress proteins, and a pharmaceutically acceptable excipient, wherein said tumor cells developed a viable resistance mechanism in response to a stress applied in vitro thereto, said plurality of different immunogenic stress proteins also produced by said stress applied in vitro to said tumor cells, and wherein said stress applied in vitro to said tumor cells is selected from the group consisting of irradiation, heat shock, chemical shock, metabolic stress, and combinations thereof.

2. The pharmaceutical composition or vaccine according to claim 1, wherein the tumor cells are bearing or containing haptenized stress proteins.

3. The pharmaceutical composition or vaccine according to claim 1, wherein the tumor cells are selected from the group consisting of cell line cells, a combination of cells from allogeneic cell lines, cells from the patient, and a combination of cells from allogeneic cell lines and patient's cells, said tumor cells bearing or containing haptenized stress proteins.

4. The pharmaceutical composition or vaccine according to claim 1, wherein the stress proteins in said plurality of different immunogenic stress proteins are selected from the group consisting of heat shock proteins, radioresistant proteins, chemotherapy resistant proteins, metabolic stress resistant proteins.

5. The pharmaceutical composition or vaccine according to claim 1, further comprising an adjuvant.

6. The pharmaceutical composition or vaccine according to claim 1, wherein the stress proteins are haptenized.

7. The pharmaceutical composition or vaccine according to claim 1, wherein said stress proteins are distributed in the composition inside the tumor cells, bound to a tumor cell surface or fragment thereof, and/or outside the tumor cells.

8. The pharmaceutical composition or vaccine according to claim 1, wherein tumor cells are cells from one or more tumor cell lines.

9. The pharmaceutical composition or vaccine according to claim 1, wherein the stress proteins are selected from the group consisting of stress proteins caused by a chemotherapeutic drug, stress proteins caused by irradiation, stress proteins caused by heat, stress proteins caused by metabolic stress, and stress proteins caused by irradiation and heat.

10. The pharmaceutical composition or vaccine according to claim 1, wherein the composition is formed from at least two allogeneic line tumor cells that have been subject in vitro to a stress selected from the group consisting of irradiation, heat, chemical, metabolic stress, and combinations thereof, during the culturing thereof, and each one of the at least two allogeneic line tumor cells has undergone treatment capable of rendering its stress proteins immunogenic.

11. The pharmaceutical composition or vaccine of claim 10, wherein the at least two stressed allogeneic line cells are formulated as a kit of parts.

12. The pharmaceutical composition or vaccine according to claim 1, wherein said tumor cells were rendered non-proliferative after having produced the plurality of stress proteins.

Description

BRIEF DESCRIPTION OF THE DRAWING FIGURES

(1) The present invention will now be described in greater detail with the aid of examples provided by way of non-limiting examples and with reference being made to the Figures.

(2) FIG. 1 is a Western Blot showing the induction of the expression of HSP 70 by a thermal stress. In order to ensure ease of reading, the main calibration spots and HSP 70 spots have been encircled, with the latter being identified by the reference numeral 1.

(3) FIG. 2 is a Western Blot showing the induction of HSP 70 by heat stress and the haptenisation of HSP 70, with in parallel a Western Blot performed on a β-actin control. In order to ensure ease of reading, the main spots have been encircled. Identifying reference numerals: HSP 70 (2); HSP 70 haptenised (3); β-actin (4); β-actin haptenised (5); Molecular Weight Marker (6); CT26WT Cells treated for 1 hour at 37° C. (7); CT26WT Cells treated for 1 hour at 42° C. (8;) vaccine dose stressed, haptenised and irradiated (9); Molecular Weight Marker (10); CT26WT cells treated for 1 hour at 37° C.+14 hours at 37° C. (11); CT26WT cells treated for 1 hour at 42° C.+14 hours at 37° C. (12); vaccine dose stressed, haptenised and irradiated (13).

(4) The FIGS. 3 and 4 are graphs each representing the evolving change over time of the volume of tumours induced in mice between the control mice injected with an excipient (“Veh” for “vehicle”) and the mice treated with vaccines according to the invention (“The” for “Therapeutic”).

(5) FIGS. 5 and 6 are graphs showing, for the same groups represented in the FIGS. 1 and 2, the weight of the tumour in grams induced in the mice at the time of resection (“Veh” for “vehicle” or excipient; “The” for Therapeutic).

EXAMPLE 1: METHOD FOR PRODUCING ALLOGENEIC DOSES

(6) Cell Lines

(7) The cell lines are derived from commercially available pre-established lines (ATCC type) or resulting from the constitution of the line derived from the patient sample, characterized, and tested. By way of example, the lines that are used include Caov, OVCAR-3, ES-2, and OV-3, for an ovarian cancer therapeutic target. They are cultured separately in the recommended conditions.

(8) Patient Cells

(9) The patient cells are isolated from the patient and cultured in a medium and under the appropriate conditions.

(10) The cells from the cell lines and patient are subjected during the course of their growth phase or their plateau phase to a stress so as to enable the overexpression of factors of resistance (stress proteins).

(11) Type of Stress Applied: Irradiation 2 gray Heat Shock: temperature rise up to 40° C.-43° C. with a plateau of 30 to 60 minutes Chemical shock with ethanol, cyclophosphamide, doxorubicin or cisplatin.

(12) The expression of one or more factor(s) of resistance is confirmed by means of flow cytometry, Elisa or Western Blot). In particular by cytometry using the anti-HSP antibodies (anti-human HSP 27-FITC; anti-human HSP60-PE, anti-human HSP 72-FITC; anti-human HSP 90-PE).

(13) The factors of resistance (stress proteins) expressed by the allogeneic cells are chemically labeled or tagged with Dinitrophelyl. By way of a variant use can be made of sulphanilic acid, N-iodoacetyl-N′-(5-sulfonic-naphthyl) ethylene diamine (EDA), aniline, or p-amino benzoic acid).

(14) The allogeneic cells comprising the haptenised stress proteins are then rendered non-proliferative by high dose irradiation (25 gray). By way of a variant use may be made of ethanol fixation between 10% and 50% v/v or any other method that provides for the inhibition of cell proliferation while maintaining the cell structure intact.

(15) The haptenised cellular stress proteins of allogeneic origin are distributed in the form of cell suspension in a formulation medium that is suitable for therapeutic use and allows for the preservation thereof at low temperature (−20° C., −80° C.), and then distributed in doses comprising from 1 to 5 10.sup.6 inactivated and haptenised cells per dose corresponding to a therapeutic dose expressed in protein amount “HSP positive” and/or in organic matter amount. An immunizing adjuvant may be present, for example BCG, GM-CSF, IL2.

EXAMPLE 2: METHOD FOR PRODUCING AUTOLOGOUS DOSES

(16) The cells are derived from the tumour of the patient after resection. The biological material is transported in a specific kit that makes possible its preservation while ensuring optimal viability of the cells.

(17) The cells of the biopsy are dissociated by a suitable mechanical method, and then placed in suspension in a nutrient medium that provides for their growth or viability only.

(18) The tumour cells are used as they are or following a selection by means of cell sorting.

(19) The tumour cells in the expansion phase or in the stationary phase are subjected to a stress as defined in the production of allogeneic cells.

(20) The expression of one or more factor(s) of resistance is confirmed according to an appropriate method of analysis, as in example 1.

(21) The factors of resistance (stress proteins) expressed by autologous cells are chemically tagged by a method similar to that described for the allogeneic cells in Example 1.

(22) The autologous cells including the haptenised stress proteins are rendered non-proliferative by the same method as that used for the allogeneic cells in Example 1.

(23) The haptenised stress proteins integrated in the autologous cells, are distributed in the form of a cell suspension in a formulation medium that is suitable for therapeutic use and provides for their preservation and then distributed in therapeutic doses.

EXAMPLE 3: METHOD OF TREATMENT

(24) The therapeutic treatment regimen includes the administration of: one or more allogeneic or autologous doses; or one or more allogeneic and autologous doses, administered together or in a sequential manner.

(25) The administration of the product is by way of an intradermal injection. It is also possible to use another route of administration and in particular per os.

(26) The product is administered alone or in synergy with any other therapy that allows for a potentiation of the treatment.

EXAMPLE 4: PRODUCTION OF HSPS

(27) The CT26-WT line is a mouse colon carcinoma line, available under ATCC® CRL-263 8™. The cells grow easily and rapidly (doubling time 22 hours).

(28) The HL60 line is a human cell line (Caucasian promyelocytic leukemia) available from Sigma-Aldrich®. The cells are frozen after the expansion.

(29) The original cells had been received in frozen form. The cells were thawed, after which they were put to culture in flasks, in an appropriate culture medium. The culture medium was changed on D+1. After expansion, a count of the live cells was performed, then the concentration was adjusted to about 2.Math.10.sup.6 living cells/ml of culture medium. A pool of 200.Math.10.sup.6 cells was recovered, and then washed after confirming that the cells were at approximately 50% confluence. The suspension was then divided into 50 ml tubes based on 25 ml per tube. The tubes were then immersed in a water bath heated to 42° C. The tubes were allowed to remain for about 1 hour in the water bath. The cells were then transferred into the flasks at a rate of 2.Math.10.sup.6 cells. Thereafter the cells were incubated for 14 hours at 37° C. The cell concentration was then adjusted to 5.Math.10.sup.6 living cells/ml.

(30) The cells were then haptenised with a solution of 2,4-dinitrofluorobenzene (DNFB) 0.07%. The haptenisation is revealed by labeling with FACS. FACS flow cytometry was chosen as the analytical technique, it provides the ability to give a percentage of cells haptenised in relation to the total number of cells.

(31) The cell suspension is adjusted to a concentration of 2 to 5.Math.10.sup.6 cells/ml with freezing medium and stored in a freezer at −80° C.±3° C. for a minimum of 24 hours before testing them.

(32) The frozen cells were then subjected to irradiation by X-rays at 25 grays, in order to inactivate the tumour cells.

(33) Two standard techniques were used to detect the expression of HSPs or haptenised HSPs; these same techniques can be used to detect any stress protein, by means of the antibody specific to the protein to be detected: Western blot using the antibodies specific to the HSP to be detected. Flow Cytometry (FACS, fluorescence-activated cell sorting) after tagging of the intracellular HSPs by using specific antibodies. By way of specific antibodies, use was made of the commercially available anti-HSP 27 antibodies, anti-HSP 70 antibodies, anti-HSP 90α/β antibodies. According to the technique that was used, further use was made, as this is known per se, of additional antibodies, mouse IgG1, FITC conjugated sheep anti-mouse IgG, control antibody (anti-KLH-FITC), antibody (anti-TNP FITC), DNFB specific. Commercially marketed antibodies that are usable are available from Santa Cruz Biotechnology Inc. and BD Biosciences.

(34) For the cytometry, the haptenised cells were thawed, they were subsequently washed and the concentration was adjusted from 1 to 2.Math.10.sup.6 cells/ml. The following were deposited in a V bottom 96-well plate: 5 μl of control antibody (anti-KLH FITC) in two wells, 5 μl of antibody (anti-TNP FITC) in two wells.

(35) Subsequently 100 μl of haptenised cell suspension was then added per well, and the plate was incubated for 15 minutes at +5° C.±3° C. Thereafter the cells were then washed, and the cell suspensions were transferred to tubes for passing over a “FACS Calibur”. The “DNP-FITC” tubes were passed through according to the parameters fixed in advance with the control.

(36) Results:

(37) 3. CT26-WT Line: Compared to the same line maintained at 37° C. for 1 hour, in Western blot a band is seen to appear at about 70 kD (FIG. 1) corresponding to HSP 70 in the stressed cells at 42° C. according to the protocol here above.

(38) 4. HL60 Line:

(39) The analysis of control cells (1 hour at 37° C.+14 hours recovery time at 37° C.) and stressed cells (1 hr at 42° C.+14 hr recovery time at 37° C.) was performed by means of flow cytometry after intracellular labeling of HSP 27, 70 and 90 by the specific antibodies mentioned here above.

(40) Results:

(41) TABLE-US-00001 % cells analysed Intensity Average of (window) labelled cells No stress Positive Control 92.07 59.42 HSP 27 92.37 37.09 HSP 70 92.14 7.85 HSP 90 91.78 31.19 Stress Positive Control 71.56 68.11 HSP 27 72.07 71.90 HSP 70 70.47 30.90 HSP90 70.32 57.03

(42) An overexpression of HSPs and more specifically of the HSP 27 and 70 in the stressed cells is noted.

EXAMPLE 4: ANIMAL TESTING

(43) The production of tumour doses consisted in bringing about expansion of the CT26.WT cells, followed thereafter by a freezing in the culture medium supplemented with 5% DMSO and at a rate of 6.25.Math.10.sup.4 cells in 25 μl final.

(44) The production of vaccine doses consisted in bringing about the expansion of the CT26.WT cells, then after 2 passages the cells were subjected to a heat stress for 1 hr at +42° C. followed by a recovery time of 14h at 37° C. Then, the cells were haptenised (by DNFB, dinitrofluorobenzene), then frozen at −80° C. based on 6.25.Math.10.sup.5 cells in 250 μl final and finally the vaccine doses were irradiated (25 Gray).

(45) Controls were performed for the tumour and vaccine doses:

(46) Tumour doses production of the tumour in animals):

(47) Sterility by blood cultures.fwdarw.negative blood cultures

(48) Control of endotoxins..fwdarw.endotoxins ≤200 IU/ml

(49) Control of cell number and viability of tumour doses..fwdarw.amount comprised between 5.5 and 6.95.Math.10.sup.5 cells, viability ≥95%.

(50) Vaccine doses:

(51) Sterility by blood cultures:.fwdarw.negative blood cultures.

(52) Control of endotoxins: 2.fwdarw.endotoxins ≤200 IU/ml

(53) Control of the haptenisation by FACS labeling with the use of the anti-KLH-TNP antibody: .fwdarw.haptenisation %≥96%

(54) Control of the cellular quantity of tumour doses: .fwdarw.amount between 5.96 and 6.10.Math.10.sup.5 cells),

(55) Control of the expression of HSP by cytometry using the specific antibodies mentioned here above: .fwdarw.the expression of HSP 27, 70 and 90 was detected.

(56) Control of detection of HSP by Western blot using the specific antibodies mentioned here above: .fwdarw.the FIG. 2 shows a HSP 70 spot after thermal stress and a haptenised HSP 70 spot.

(57) Viability test in order to ensure that the cells of the vaccine composition are not proliferative: addition of propidium iodide+putting to culture; the lack of proliferative capacity of the cells that make up the vaccine doses was confirmed

EXAMPLE 5: IN VIVO STUDY IN MICE

(58) BALB/c male and female mice aged 6 to 8 weeks, obtained from Charles River, were used. 5 mice were placed per 16×19×35 cm cage under controlled temperature (22±2° C., in alternating lighting conditions (12 h cycles of day and 12 hours of dark) and supplied with water and food ad libitum. The mice were acclimated for at least 1 week before starting the experiments.

(59) The tumour model is the subcutaneous CT26 carcinoma (CT26WT, ATCC ° CRL-2638™), which is a syngeneic tumour model commonly used for the study of therapeutic applications against cancer in animals, in particular for testing immunotherapy protocols and study the immune response.

(60) 50 female mice were divided into 5 groups of 10. Each mouse was injected subcutaneously (SC) on day 0 (DO) with 5.Math.10.sup.4 CT26WT tumour cells. The treatment protocol was as follows:

(61) Group 1: excipient

(62) Group 2: therapeutic treatment 1

(63) Group 3: therapeutic treatment 2

(64) Cyclophosphamide was administered by injection at 15 mg/kg via the intraperitoneal route (IP) on D+2 to the groups 2 and 3.

(65) Treatment 1 (BCG IL-2/vaccine) for the group 2:

(66) BCG: 2.Math.10.sup.6 CFU (colony forming units) per SC injection

(67) Recombinant murine IL-2: 4 000 IU per SC injection

(68) Vaccine: 5.Math.10.sup.5 CT26WT cells (irradiated and haptenised) by SC injection.

(69) Treatment 2 (BCG/GM-CSF/vaccine) for the group 3:

(70) BCG: 2.Math.10.sup.6 UFC by SC injection

(71) Recombinant murine GM-CSF: 25 000 IU (international unit) via SC route

(72) Vaccine: 5.Math.10.sup.5 CT26WT cells (irradiated and haptenised) by SC injection

(73) Protocols:

(74) TABLE-US-00002 Day Treatment/route Group 1 Group 2 Group 3 D 0 SC CT26WT X X X D + 2 IP X X D + 7 Cyclophosphamide Excipient T1 T2 D + 14 SC Treatment Excipient T1 T2 D + 21 SC Treatment Excipient T1 T2 D + 28 SC Treatment Excipient T1 T2 SC Treatment

(75) Parameters measured during the course of the study, and other specifications:

(76) Daily monitoring of the behaviour of the mice.

(77) Monitoring 3 times per week of the body weight and tumour volume. The tumour volume in mm.sup.3 was calculated using the formula: volume=[(width).sup.2×length]/2. Measure dimensions with the help of a caliper.

(78) The mice are sacrificed in case of signs of unexpected distress.

(79) The mice were sacrificed when the tumour reached 1000 m.sup.3 and in any case no later than 40 days after implantation of the tumour. The tumours were excised and measured.

(80) The results of measurement of the tumour volume in mm.sup.3 over time show a favorable impact of both the two therapeutic vaccines T1 and T2 as compared to the control mice: See FIGS. 3 and 4.

(81) The results of measuring of the weight of the tumours after resection also showed a favorable impact of the vaccines T1 and T2 as compared to the control mice: see FIGS. 5 and 6.