PHARMACEUTICAL COMPOSITION AND METHOD FOR INDUCING AN IMMUNE RESPONSE

Abstract

The present invention relates to a method for inducing an immune response in a human or animal subject, as well as to a pharmaceutical composition for inducing an immune response, furthermore to a method for producing the pharmaceutical composition in vitro and the use of cytotoxic CD8+ T-lymphocytes activated to recognize an antigenic peptide in a pharmaceutical composition or in a method for inducing an immune response.

Claims

1. A pharmaceutical composition for inducing an immune response in a human or animal subject suffering from a pathologic disease or disorder, comprising autologous activated cytotoxic CD8+ T-lymphocytes (CTL), activated by autologous primed mature dendritic cells of the human or animal subject, said autologous primed mature dendritic cells presenting an antigenic peptide, wherein the activated CTL are derived from an autologous population of peripheral blood mononuclear cells (PBMCs) isolated from peripheral blood of the same human or animal subject; and wherein the activated CTL are able to recognize the antigenic peptide; wherein the CTL have been activated by autologous dendritic cells primed with an antigenic peptide, wherein the dendritic cells have been isolated from the same human or animal subject as the CTL, wherein the CTL have been activated by co-culturing CD8+ T-cells derived from the population of PBMC, with the antigen-presenting dendritic cells; and wherein the dendritic cells prior to their use in the activation of CD8+ T cells have been cultured ex vivo and subsequently have been primed (loaded, pulsed) with the antigenic peptide, and have been matured in the presence of a cytokine cocktail prior to their use in activation of the CD8+ T cells by co-culturing, wherein the antigenic peptide is a tumor antigenic MUC1-peptide.

2. Pharmaceutical composition of claim 1, wherein the antigenic peptide is a MUC1-peptide of a length of at least 9 amino acids, preferably of exactly 9 amino acids, and more preferably a MUC (79-87) TLAPATEPA peptide (Seq. ID 1).

3. Pharmaceutical composition according to claim 1, wherein the CTL have been activated by co-culturing with the loaded mature dendritic cells a population of all non-adhering immune cells derived from the same population of PBMC as the population of which the dendritic cells have been derived, said population of non-adherent immune cells including the CD8+ T cells to be activated, as well as at least one of the following group of cells: CD4+ T-lymphocytes, B lymphocytes, platelets, neutrophils, basophils and eosinophils.

4. A method for obtaining human or animal autologous dendritic cells presenting a MUC (79-87) TLAPATEPA peptide (Seq. ID 1), for the preparation of a pharmaceutical composition according to claim 1, comprising the following steps: a.) culturing monocytes isolated from PBMCs of the human or animal subject suffering from a pathologic disease or disorder ; b.) culturing of adhering monocytes of step a.) with granulocyte-monocyte colony-stimulating factor and IL-4, resulting in a population of immature dendritic cells; c.) pulsing of the immature dendritic cells of step b.), resulting in a population of loaded dendritic cells presenting the antigenic peptide; d.) maturing of the loaded dendritic cells presenting the antigenic peptide of step c.) with a cytokine cocktail, and incubation .

5. A method for producing a pharmaceutical composition according to claim 1, for inducing an immune response in a human or animal subject in the treatment of a pathologic disease, comprising the following steps: A.) providing a population of autologous antigen-presenting mature dendritic cells which have been isolated from a population of immune cells, including monocytes, from peripheral blood of the human or animal subject, and subsequently cultured, differentiated and primed with an antigenic peptide related to a specific pathogenic disease or disorder of which the human or animal subject suffers; B.) providing a population of autologous (generated and expanded) cytotoxic CD8+ T cells isolated from a population of immune cells from peripheral blood of the same human or animal subject ; C.) co-culturing the primed autologous antigen-presenting mature dendritic cells with non-adherent immune cells, including the CD8+ T cells to be activated, wherein the non-adherent cells include at least one of the following: T-lymphocytes, B-lymphocytes, platelets, neutrophils, basophils, eosinophils; wherein said non-adherent cells have been isolated from a population of PBMCs from peripheral blood of the same human or animal subject, preferably from the same population of PBMCs from which the dendritic cells were derived; ; D.) isolation of CD8+ CTLs from the non-adhering immune cells by positive selection using CD8-specific magnetic beads; E.) quality control of isolated CD8+ CTLs.

6. Method according to claim 5, wherein step A.) comprises isolation of peripheral blood from the human or animal subject, comprising PBMCs as a starting material for the isolation of the PBMCs.

7. Method according to claim 5, wherein step A.) comprises a step of separating monocytes from the PBMCs, wherein in a first step, PBMCs are left in culture for 2 hours at 37° C. and 5% CO.sub.2, and subsequently, collection of a supernatant after monocyte adhesion .

8. Method according to claim 5, wherein step A.) comprises at least the following steps: e.) culturing monocytes isolated from peripheral blood mononuclear cells of the human or animal subject; f) culturing of adhering monocytes, resulting in a population of differentiated, immature dendritic cells; g.) priming of the immature DCs with an antigenic peptide ; h.) maturing of the loaded (primed) immature dendritic cells with a cytokine cocktail, and incubation.

9. Method according to claim 5, wherein in step C.), the activation of CD8+ T cells is performed by three separate steps of activation, wherein in each step of activation one aliquot of mature DCs pulsed with the antigenic peptide is used to activate the CD8+ T cells, wherein in a first step, non-adherent immune cells containing CD8+ T cells are co-cultivated with a first aliquot of mature DCs pulsed with the antigenic peptide ; and wherein-in a second step, a second aliquot of the pulsed mature DCs is added to the non-adherent immune cells, and wherein in a third step, a third aliquot of the pulsed mature DCs is added to the non-adherent immune cells .

10. Method according to claim 5, wherein the method comprises at least one of the following steps: determination of CTL proliferation; determination of cytotoxicity of CTLs.

11. Method of treatment of a pathologic disease or disorder in a human or animal subject, comprising the step of administering to said subject a pharmaceutical composition according to claim 1, said pharmaceutical composition comprising a therapeutically effective amount (dosage, concentration) of autologous activated CTLs capable of specifically recognizing an antigenic peptide related to said disease or disorder .

12. Method of treating of cancer, in a human or animal subject, comprising the step of administering to said subject a pharmaceutical composition produced by the method according to claim 5 and comprising a therapeutically effective amount of autologous activated CTL capable of specifically recognizing a MUC (79-87) TLAPATEPA peptide (Seq. ID 1).

13. Pharmaceutical composition for use as a medicament in the treatment of cancer of a human or animal subject, comprising as an active ingredient a therapeutically effective amount of activated cytotoxic CTLs capable of recognizing an antigenic MUC (79-87) TLAPATEPA peptide (Seq. ID 1), said pharmaceutical composition further comprising at least one of the following substances: a pharmaceutically acceptable additive, a carrier, an excipient, a stabilizer, wherein the activated CTLs have been obtained by the method defined in claim 5.

14. Pharmaceutical composition according to claim 1, for use as a medicament in the treatment of a cancer of a human or animal.

15. Method for using activated autologous CTLs in a pharmaceutical composition or a vaccine for the treatment or prevention of cancer in a human or animal subject suffering from cancer, wherein the activated CTLs are capable of recognizing an antigenic MUC (79-87) TLAPATEPA peptide (Seq. ID 1), following the activation of CD8+ T-cells by autologous dendritic cells primed with and presenting said antigenic peptide.

16. Pharmaceutical composition, for use as a medicament in the treatment of cancer of a human or animal subject or as a vaccine for the prevention of cancer of a human or animal subject, wherein the pharmaceutical composition comprises a therapeutically effective dosage of autologous primed dendritic cells each presenting on their cell surface a tumor antigenic MUC1 peptide.

17. Method for using autologous primed dendritic cells in a pharmaceutical composition or a vaccine for the treatment of cancer in a human or animal subject suffering from cancer, wherein the autologous primed dendritic cells each present on their cell surface an antigenic MUC1 peptide.

18. Method according to claim 5 for producing a pharmaceutical composition, wherein in step A.) the antigenic peptide is a MUC (79-87) TLAPATEPA peptide (Seq. ID 1).

19. Method according to claim 5 for producing a pharmaceutical composition, wherein in step B.) the population of autologous cytotoxic CD8+ T cells are isolated from the same population of immune cells as of which the dendritic cells were derived.

20. Method according to claim 5 for producing a pharmaceutical composition, wherein in step C.) the primed autologous antigen-presenting mature dendritic cells non-adherent cells are co-cultured with all the non-adherent immune cells derived from the same population of PBMCs from peripheral blood of the same human or animal subject.

21. Method according to claim 5 for producing a pharmaceutical composition, wherein in step C.) a ratio of non-adherent immune cells to autologous antigen-presenting mature dendritic cells is 30:1.

22. Method according to claim 5 for producing a pharmaceutical composition, wherein in step C.) the co-culturing is carried out in a co-culturing medium for 1 week at 37° C. and 5% CO.sub.2, wherein the co-culturing medium is supplemented with one or more interleukins that support cell survival and expansion.

23. Method according to claim 7, further comprising cryopreservation of non-adherent cells for future use in activation of CD8+ T cells in step C.) of claim 5.

24. Method according to claim 7, further comprising treatment of monocytes with 100 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) and 50 ng/ml IL-4 for 7 days for proliferation of monocytes and differentiation into dendritic cells.

25. Method according to claim 8, wherein in step g.) the immature DCs are primed with a MUC (79-87) TLAPATEPA peptide (Seq. ID 1).

26. Method according to claim 8, wherein in step e.) the monocytes are isolated by Ficoll-separation; and wherein in step f.), the adhering monocytes are cultured with granulocyte-monocyte colony-stimulating factor and IL-4; and wherein in step g.) the immature DCs are primed on day 6 of culture; and wherein in step h.), the loaded immature dendritic cells are matured with a cytokine cocktail including IL-6, IL-1β, TNF-α, and PGE2.

27. Method according to claim 8, wherein in step g.) the immature DCs are primed in the presence of β2 microglobulin.

28. Method according to claim 8, further comprising i.) freezing aliquots of the mature loaded dendritic cells for a subsequent step of stimulation of CD8+ T cells.

29. Method according to claim 9, wherein the activation of CD8+ T cells is performed by three separate steps of activation at intervals of 7 days.

30. Method according to claim 9, wherein in the first step, a ratio of non-adherent immune cells to mature DCs is 30:1.

31. Method according to claim 9, wherein in each of the second and third step of activation IL-2 and IL-7 are added.

32. Method according to claim 9, wherein in each of the second and third step of activation the antigenic peptide is added at a final concentration of 10 .Math.g/ml.

33. Method according to claim 9, wherein in each of the second and third step of activation β2 microglobulin is added.

34. Method according to claim 10, wherein the determination of CTL proliferation is carried out by a CFSE assay (Carboxyfluorescin Diacetate Succinimidyl Ester), and wherein the determination of cytotoxicity of CTLs is carried out by an LDH cytotoxicity detection assay.

35. Method of treating cancer in a human or animal subject according to claim 11, comprising the step of administering to said subject a pharmaceutical composition according to claim 1, said pharmaceutical composition comprising a therapeutically effective amount (dosage, concentration) of autologous activated CTLs capable of specifically recognizing a MUC (79-87) TLAPATEPA peptide (Seq. ID 1), wherein the pharmaceutical composition is administered via one of the following pathways: intravenously; into a cavity adjacent to a location of a solid tumor, such as into the intraperitoneal cavity; directly infused into or adjacent to a solid tumor.

36. Pharmaceutical composition according to claim 16, for use as a medicament in the treatment of cancer of a human or animal subject or as a vaccine for the prevention of cancer of a human or animal subject, wherein the autologous primed dendritic cells each present on their cell surface a MUC (79-87) TLAPATEPA peptide (Seq. ID 1).

37. Method according to claim 17 for using autologous primed dendritic cells in a pharmaceutical composition or a vaccine for the treatment of cancer in a human or animal subject suffering from cancer, wherein the autologous primed dendritic cells each present on their cell surface a MUC (79-87) TLAPATEPA peptide (Seq. ID 1).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings,

[0059] FIG. 1 shows an image of phase-contrast microscopy of immature dendritic cells at a magnification of X40;

[0060] FIG. 2 shows an image of phase-contrast microscopy of mature dendritic cells at a magnification of X40;

[0061] FIG. 3 shows an image of phase-contrast microscopy of all non-adherent cells co-cultured with mature dendritic cells at a magnification of X10;

[0062] FIG. 4 shows the results of a CFSE proliferation assay;

[0063] FIGS. 5A,B show the results of an LDH cytotoxicity detection assay, wherein in FIG. 5A, the results are shown in a plate-format and in FIG. 5B, in table format;

[0064] FIG. 6 shows the results of a LDH cytotoxicity detection assay in table format.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0065] The present invention describes, in a first aspect, a novel approach of the development of autogenous antigen specific CTLs that can be administered for adoptive T cell therapy, as well as, in a second aspect, the priming of autogenous dendritic cells to present an antigenic peptide. The cells that are used herein are autologous, i.e. from the same species and same donor, as the recipient subject, i.e the patient suffering from a specific disease or disorder, which may or may not be pathogen related. The first aspect of this invention is the selection of an appropriate 9-mer peptide of the antigen that is related to the specific pathogen or disease of which the human subject, i.e. patient suffers or is at risk to suffer (in case of a vaccine). The selection can be performed from databases that are well known in the art such as the SYFPEITHI MHC databank, an epitope prediction database. The selection is based on comparison of the higher scores of immunogenicity and T cell epitope prediction derived from such databases. The next step is the isolation of immune cells from the peripheral blood. The peripheral blood contains monocytes, T lymphocytes, B lymphocytes, NK cells, platelets, neutrophils, eosinophils, basophils. Characterization of the immune cell populations can be performed with flow cytometry (forward scatter vs. side scatter analysis). DCs which in the method according to the invention serve as the antigen presenting cells, can be obtained from the patient’s peripheral blood following protocols known in the art for monocyte isolation and dendritic cell differentiation. Differentiation of monocytes to dendritic cells is achieved in the presence of cytokines well known in the art, such as the granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4). DCs that have been stimulated with GM-CSF and IL-4 express MHC class I and class II molecules. The synthetic selected antigenic peptide can be obtained from any qualified manufacturer. “Loading” of the dendritic cells with said peptide, a step also termed “priming” or “pulsing”, is preferably performed with the addition of β2 microglobulin. This step is followed by the maturation of DCs in the presence of a “cocktail” of cytokines. According to the first aspect of the invention, the autologous primed mature DCs are used for ex vivo activation of autologous CD8+ T cells, i.e. for triggering an immune response ex vivo. As an alternative, according to the second aspect of the invention, these autologous primed mature DCs can either be used directly in a pharmaceutical composition for administration to the human or animal subject for triggering an immune response, i.e. activation of CD8+ T cells in vivo following administration.

[0066] The effector cells (CD8+ T cells) used in the method according to the first aspect of the invention can be generated and expanded in vitro in accordance with known techniques (including but not limited to those described in Ranieri, Cytotoxic T-Cells: Methods & Protocols, ISBN: 978-1-4939-1157-8, Humana Press, New York, NY, 2014) or variations thereof that are well known to those skilled in the art.

[0067] The activation of CD8+ T cells can be achieved by culturing a population of cells containing CD8+ T cells with an aliquot of mature DCs pulsed with the selected antigenic peptide. Some protocols first select only the CD8+ T lymphocytes from the population of PBMCs (of which the monocytes have already been separated for the purpose of generation of DCs). However, according to a preferred embodiment of the present invention, the mature, pulsed DCs were co-cultured with all the non-adherent cells that have been isolated, including T lymphocytes, B lymphocytes, platelets, neutrophils, basophils, eosinophils. Preferably, in the method according to the present invention, for the purpose of activation of CTLs, the pulsed/loaded DCs were co-cultured with the majority of the immune cells, i.e. all immune cells isolated from the peripheral blood except for the monocytes. The reason for doing so is to mimic the processes in the immune system in vivo, i.e. to stimulate the interactions that physiologically happen in vivo. This leads to a stronger activation and thus a greater degree of cytotoxicity of CTL, as the cell-cell interactions, cell-signaling and differentiation pathways, i.e. reactions to cytokine release from various cells are increased.

[0068] The non-adherent cells obtained from peripheral blood, preferably from the same sample as used for the isolation of monocytes, are thawed using protocols known in art, and subsequently co-cultured with the mature DCs preferably for 1 week at 37° C. and 5 % CO.sub.2. Therein, the ratio of non-adherent cells to mature DCs preferably is 30:1. . The medium for the co-cultivation preferably is supplemented with interleukins which support cell survival and expansion. Isolation of activated CD8+ T cells from the bulk T cell cultures is preferably performed by positive selection using magnetic beads coated with an antibody specific for the CD8 membrane antigen. Magnetic beads can be removed from the positively isolated cells using protocols known in art.

[0069] After CD8+ T-cell selection, the activated CTLs are ready for quality control, such as proliferation and cytotoxic activity, and when they pass the assessment they can be administered back to the patient. Cells can furthermore be analyzed in terms of gene and protein expression. T cell proliferation can be determined by CFSE assay, where every generation of cells is presented with a different subset in flow cytometry. Thereby, distinct generations of proliferating cells can be monitored by dye dilution. Live cells are covalently labeled with a very bright, stable dye. Every generation of cells appears as a different peak on a flow cytometry histogram. Suitable means for evaluating the biologic activity of the cells may be the in vitro stimulation of CTLs with target cells for a period of time, usually 24 or 48h, and determination of cytokine production by a suitable protein-based detection assay, for example by flow cytometry, ELISPOT or ELISA assay. Moreover, measurement of the cytotoxic activity of CTLs can be performed by in vitro stimulation of CTLs with target cells and determination with suitable means, for example by an LDH cytotoxicity detection assay. Alternatively, the activated CTLs generated according to the method with respect to the first aspect of the invention may be evaluated using an in vivo animal model suitable for the targeting of the CTLs.

[0070] The CTLs produced according to the method of the present invention are intended for the induction of an immune response in the recipient human or animal subject. The cells administered to the subject are autologous cells, i.e. the donor is the same as the recipient. The pharmaceutical composition can be prepared by any of the methods well known in the art of pharmacy that are non-toxic to the recipients at the concentrations and dosages used. Preferably, at least 10.sup.6 activated CTL are used for infusion. Formulations typically require one active ingredient, being the population of activated CTLs, with one or more acceptable carriers. The pharmaceutical composition comprising the activated CTLs can then be directly administered to the subject to be treated. It can be administered intravenously, or into a cavity adjacent to the location of a solid tumor (for example, intraperitoneal cavity) or directly infused into or adjacent to a solid tumor. Sterile injectable solutions well known in the art can be used for resuspension of the CTLs and as carriers for the injection, such as for example 0.9% sodium chloride.

Example 1

[0071] CD8+ T cells from a breast cancer patient activated against MUC (79-87) TLAPATEPA (Seq. ID 1) efficiently kill MCF7 human breast adenocarcinoma cells

Epitope Selection

[0072] MUC1 is a single pass type I transmembrane protein with an extracellular domain that is heavily glycosylated and extends up to 200-500 nm from the cell surface and it is normally expressed in epithelial cells. Tumor-associated MUC1 is overexpressed and under glycosylated in most human epithelial cancers. It has been considered as a remarkable target for immunotherapies, although so far MUC1 targeting vaccines have not met significant benefits to the patients [Scheikl-Gatard T.,et al., J Transl Med. 15, 154 (2017)].

Cell Isolation

[0073] Peripheral blood mononuclear cells (PBMC) were isolated from the blood of a cancer patient with Ficoll-separation (Biocoll separating solution-Catalog#1077, Biochrom, Berlin, Germany).

Generation of DCs

[0074] Peripheral blood contains monocytes or immature dendritic cells (DC) that can differentiate in the presence of cytokines. Separation of monocytes from the other types of cells is carried out, as monocytes adhere to the flask during incubation. For this purpose, PBMCs were cultured for 2 h at 37° C. and 5% CO.sub.2. After monocyte adhesion, the supernatant was collected and non-adherent cells, which included T-lymphocytes, B-lymphocytes, platelets, neutrophils, basophils, and eosinophils, were cryopreserved for later use in the “activation” step. Proliferation of isolated monocytes and differentiation into DC was performed by treatment of the isolated monocytes with 100 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) and 50 ng/ml interleukin 4 (IL-4) for 7 days. This resulted in a population of differentiated, but still immature DC (see FIG. 1. The generation of immature DCs and the progress of the development of mature DCs was reviewed by phase-contrast inverted microscopy. At the beginning of the culture, monocytes were spherical and adherent to the surface of the flask. At day 5, immature DCs were found forming clusters suspending in culture medium.

Pulsing of DCs

[0075] Pulsing/priming of immature DCs was carried out with the addition of the MUC(79-87) TLAPATEPA peptide (Seq. ID 1) at the final concentration of 10 .Math.g/ml and β2 microglobulin at the final concentration of 3 .Math.g/ml and at incubation time 2-4 h at 37° C. and 5 % CO.sub.2, with occasional agitation. After pulsing of the immature DCs with the peptide, supernatant was removed from the flask, centrifuged, discarded and the cell pellet was resuspended in fresh medium (RPMI 1640) containing IL-10 at 25 ng/ml, TNF-α at 50 ng/ml, IL-6 at 10 ng/ml, PGE2 at 10.sup.-6 M and 20 .Math.l Pen/Strep. Cells were left in culture for 48 h at 37° C. and 5 % CO.sub.2 for maturation. Maturation was verified by phase-contrast inverted microscopy. In FIG. 2, it is shown that upon maturation, DCs were transformed into large irregular cells with cytoplasmic projections. Mature DCs were distributed in three aliquots and two aliquots thereof were cryopreserved for further use.

Activation and Expansion of CTLs

[0076] For the activation and expansion of CTLs, an aliquot of non-adherent cells (from the PBMC population of which the adherent monocytes were isolated and removed) were thawed and co-cultivated with an aliquot of mature DCs pulsed with the MUC(79-87) TLAPATEPA peptide (Seq. ID 1) in OPTMIZER T CELL EXPANSION SFM containing 1% L-Glutamine, IL-2 at 10 ng/ml and 10% Pen/Strep. On day 7, the second aliquot of mature DCs was thawed and the stimulation of T cells was repeated with the addition of IL-2 at 10 ng/ml and IL-7 at 5 ng/ml, at a final concentration of the MUC(79-87) TLAPATEPA peptide (Seq. ID 1) of 10 .Math.g/ml and a final concentration of β2 microglobulin of 3 .Math.g/ml. In FIG. 3, an image of all non-adherent cells, including T-cells co-cultured with dendritic cells is shown.

[0077] On day 14, the third aliquot of mature DCs was thawed and the stimulation of T cells was repeated in the same way as the first and second stimulation with the addition of IL-2 at 10 ng/ml and IL-7 at 5 ng/ml, at a final concentration of the MUC(79-87) TLAPATEPA (Seq. ID 1) peptide of 10 .Math.g/ml and a final concentration of β2 microglobulin of 3 .Math.g/ml..

[0078] On day 21, CD8+ T cells were separated and isolated from the bulk T cell cultures by positive selection using magnetic beads specific for CD8.

CFSE Assay

[0079] T cell proliferation was assessed with CellTraceTM CFSE Cell Proliferation Kit Catalog#C34554 (ThermoFischer Scientific, Darmstadt, Germany) according to the manufacturer’s suggestions. After five days of co-culture with DCs, live T cells were analysed for Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) dilution by flow cytometry. Every generation of cells is presented with a different subset on flow cytometry. As controls, unstimulated T cells cultured alone, showed the CFSE intensity of non-divided cells, while non-labeled cells showed the auto-fluorescence of the cells and the limits of detectable cell divisions. As shown in FIG. 4, a data set was analysed using the proliferation plot on FCS Express to determine the ability of mature DCs to stimulate T cell proliferation. The software was able to recognize the first generation (undivided cells) and counted a total of 9 generations of divided cells of the CD8+ T cells that had been stimulated with mature DCs that were pulsed with MUC(79-87) TLAPATEPA peptide (Seq. ID 1).

LDH Cytotoxicity Detection Assay

[0080] The assay was performed by following the manufacturer’s advice. Briefly, 500 cancer cells were seeded in a U-bottom 96-well plate Catalog#4430200 (Orange Scientific, Braine-IAlleud, Belgium) and were left overnight at 37° C. to adhere. On the next day, pre-activated T cells were added at a ratio of 1:10 and co-cultured for 20 hr at a final volume of 100 .Math.l. Plates were centrifuged at 2000 rpm for 10 minutes and 50 .Math.l of supernatants were transferred into corresponding wells of an optically clear 96-well flat bottom microplate Catalog#781722 (Brand, Wertheim, Germany). The level of cytotoxicity was measured with Cytotoxicity Detection Kit (LDH) Catalog#11644793001 (Roche, Darmstadt, Germany). 50 .Math.l of substrate were added to the corresponding wells and microplate was left to incubate for 30 minutes at room temperature in the dark. Absorbance was measured at 490 nm using an ELISA reader at a reference wavelength of 605 nm. The percentage of cytotoxicity was calculated as (experimental value- spontaneous effector cell release -spontaneous target cell release) / (maximum target cell release - spontaneous target cell release) × 100%. The background value was subtracted from all the above values. In FIG. 5A, activated CTLs targeting MCF7 and MDA-MB-231 human breast adenocarcinoma cell lines are shown. The darker color represents higher cell death. As shown in FIG. 5B, “low” controls represent regular death rate of cells, while “high” controls represent death rate of cells with Triton-X 100. The background Control is RPMI + 1% FBS.

Results

[0081] The in vitro generation of antigen-specific CTL from a breast patient’s blood was successfully achieved. The process required careful handling of the samples as primary cells were very sensitive. It seems that activation triggered the CD8+ T cells to start proliferating as there were nine generations of new T cells. Results from the LDH Cytotoxicity Detection Assay demonstrated that MUC1-specific CTLs had the ability to induce cytotoxicity in human breast adenocarcinoma cell lines. MCF7 and MDA-MB-231 were two cell lines that had different characteristics, in that MCF7 was a population from a patient suffering from luminal type breast cancer and MDA-MB-231 was a population from a patient suffering from triple negative breast cancer. The two cell lines resulted in different resistance to CTLs. CTLs which had been activated to recognize the MUC(79-87) TLAPATEPA peptide (Seq. ID 1), demonstrated greater cytotoxic effect in the MCF7 cell line (106%) than in the MDA-MB-231 cell line (9.8%). The percentage of cytotoxicity was calculated as (experimental value - spontaneous effector cell release - spontaneous target cell release) / (maximum target cell release - spontaneous target cell release) × 100%. The background value was subtracted from all the above values. A value above 100% is to be interpreted as a very high cytotoxicity, i.e. the whole population of target cells was killed. The absorbance coming from the sample in this colorimetric assay was higher than the “high” control. These results suggest that the activated CTLs killed almost the whole population of MCF7 target cells.

Example 2

[0082] CD8+ T cells from healthy donors activated against SARS-CoV2 S-protein (84-92) peptide LPFNDGVYF (Seq. ID 2)

Epitope Selection

[0083] Similar to other coronaviruses, the spike (S) protein of SARS-CoV, the severe acute respiratory syndrome coronavirus 2, is a large type I transmembrane glycoprotein with multiple biological functions. The predicted S1 subunit corresponding to the region of amino acids (aa) 13 to 680 contains the minimal receptor-binding domain (RBD) and mediates binding of the S protein to angiotensin-converting enzyme 2 (ACE2), a functional receptor on susceptible cells. The predicted S2 subunit (aa 681 to 1255) contains two heptad repeat regions (HR1 and HR2) and is responsible for fusion between viral and cellular membranes. A second major feature of coronavirus S protein is its capacity to induce neutralizing antibodies and protective immunity, and it is thereby considered a major target for vaccine development (He et al., 2006).

[0084] In example 2, immature dendritic cells are primed with SARS-CoV2 S-protein (84-92) peptide LPFNDGVYF (Seq. ID 2), a peptide of the surface glycoprotein, the so-called “spike protein” of SARS-CoV2 and then matured. The primed DCs were then either co-cultured with non-adherent immune cells including CD8+ T cells as described in example 1 for inducing an immune response ex vivo, or directly used in a pharmaceutical composition for triggering an immune response in vivo. -The activation of T cells can be verified by extracting a blood sample from the subjects and confirming the presence of activated CTLs against the specific antigen.

Example 3

[0085] CD8+ T cells from human patients or from healthy donors, respectively, activated ex vivo or in vivo against SARS-CoV2 S-protein (1185-1200) RLNEVAKNLNESLIDL (Seq. ID 3)

Epitope Selection

[0086] In example 3, immature dendritic cells are also primed with a SARS-CoV2 S-protein peptide of the surface glycoprotein, the so-called “spike protein”, i.e. S-protein of SARS-CoV2, as in example 2. However, this time with SARS-CoV2 S-protein (1185-1200) peptide RLNEVAKNLNESLIDL (Seq. ID 3) and then matured. This antigentic peptide of the SARS-CoV-2 has a length of 16 amino acids. The recognition also happens at a sequence of nine amino acids, but the full length peptide is more immunogenic. The primed DCs can then either be co-cultured with non-adherent immune cells including CD8+ T cells as described in example 1 for inducing an immune response ex vivo, or directly used in a pharmaceutical composition for triggering an immune response in vivo. -The activation of T cells can be verified by extracting a blood sample from the subjects and confirming the presence of activated CTLs against the specific antigen.

Example 4

[0087] CD8+ T cells from human patients or from healthy donors, respectively, activated ex vivo or in vivo against SARS-CoV2 S-protein (1185-1193) RLNEVAKNL (Seq. ID 4)

[0088] In example 4, immature dendritic cells are primed with SARS-CoV2 S-protein (1185-1193) RLNEVAKNL (Seq. ID 4), a peptide of the surface glycoprotein, the so-called “spike protein” of SARS-CoV2 and then matured. The primed DCs can then either be co-cultured with non-adherent immune cells including CD8+ T cells as described in example 1 for inducing an immune response ex vivo, or directly used in a pharmaceutical composition for triggering an immune response in vivo. The activation of T cells can be verified by extracting a blood sample from the treated subjects and confirming the presence of activated CTLs against the specific antigen.

Example 5

[0089] CD8+ T cells from human patients or from healthy donors, respectively, activated ex vivo or in vivo against SARS-CoV2 S-protein (1192-1200) NLNESLIDL (Seq. ID 5)

[0090] In example 5, immature dendritic cells are primed with SARS-CoV2 S-protein (1192-1200) NLNESLIDL (Seq. ID 5), a peptide of the surface glycoprotein, the so-called “spike protein” of SARS-CoV2 and then matured. The primed DCs can then either be co-cultured with non-adherent immune cells including CD8+ T cells as described in example 1 for inducing an immune response ex vivo, or directly used in a pharmaceutical composition for triggering an immune response in vivo. -The activation of T cells can be verified by extracting a blood sample from the subjects and confirming the presence of activated CTLs against the specific antigen.

Example 6

[0091] CD8+ T cells from two healthy individuals, activated against MUC (79-87) TLAPATEPA (Seq. ID 1), efficiently kill MCF7 human breast adenocarcinoma cells cultured as spheroids

Cell Isolation

[0092] Peripheral blood mononuclear cells (PBMC) were isolated from the blood of two healthy individuals with Ficoll-separation (Biocoll separating solution-Catalog#1077, Biochrom, Berlin, Germany).

Generation of DCs

[0093] Peripheral blood contains monocytes or immature dendritic cells (DCs) that can differentiate in the presence of cytokines. Separation of monocytes from the other types of cells was carried out, as monocytes adhere to the flask during incubation. For this purpose, PBMCs were cultured for 2 h at 37° C. and 5% CO.sub.2. After monocyte adhesion, the supernatant was collected and non-adherent cells, which included T-lymphocytes, B-lymphocytes, platelets, neutrophils, basophils, and eosinophils, were cryopreserved for later use in the “activation” step. Proliferation of isolated monocytes and differentiation into DCs was performed by treatment of the isolated monocytes with 100 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) and 50 ng/ml interleukin 4 (IL-4) for 7 days. This resulted in a population of differentiated, but still immature DCs. At the beginning of the culture, monocytes were spherical and adherent to the surface of the flask. At day 5, immature DCs were found forming clusters suspended in culture medium.

Pulsing of DCs

[0094] Pulsing/priming of immature DCs was carried out with the addition of the MUC(79-87) TLAPATEPA peptide at a final concentration of 10 .Math.g/ml and β2 microglobulin at a final concentration of 3 .Math.g/ml and an incubation time of 2-4 h at 37° C. and 5 % CO.sub.2, with occasional agitation. After pulsing of the immature DCs with the peptide, the supernatant was removed from the flask, centrifuged and discarded. The cell pellet was resuspended in fresh medium (RPMI 1640) containing IL-1β at 25 ng/ml, TNF-α at 50 ng/ml, IL-6 at 10 ng/ml, PGE2 at 10.sup.-6 M and 20 .Math.l Pen/Strep. Cells were left in culture for 48 h at 37° C. and 5% CO.sub.2 for maturation. Maturation was verified by phase-contrast inverted microscopy. Mature DCs were distributed in three aliquots and two aliquots thereof were cryopreserved for further use.

Activation and Expansion of CTLs

[0095] For the activation and expansion of CTLs, an aliquot of non-adherent cells (from the PBMC population of which the adherent monocytes were isolated and removed) were thawed and co-cultivated with an aliquot of mature DCs pulsed with the MUC(79-87) TLAPATEPA peptide in an OpTmizer T CELL EXPANSION SFM containing 1% L-Glutamine, IL-2 at 10 ng/ml and 10% Pen/Strep. On day 7, the second aliquot of mature DCs was thawed and the stimulation of T cells was repeated with the addition of IL-2 at 10 ng/ml and IL-7 at 5 ng/ml, at a final concentration of the MUC(79-87) TLAPATEPA peptide of 10 .Math.g/ml and a final concentration of β2 microglobulin of 3 .Math.g/ml. On day 14, the third aliquot of mature DCs was thawed and the stimulation of T cells was repeated in the same way as the first and second stimulation with the addition of IL-2 at 10 ng/ml and IL-7 at 5 ng/ml, at a final concentration of the MUC(79-87) TLAPATEPA peptide of 10 .Math.g/ml and a final concentration of β2 microglobulin of 3 .Math.g/ml. On day 21, CD8+ T cells were separated and isolated from the bulk T cell cultures by positive selection using magnetic beads specific for CD8.

Tumor Spheroid Formation

[0096] Tumor spheroids were prepared by the hanging drop technique. Briefly, MCF7 cells were harvested from 2D culture and counted. For each drop, 10,000 cells were transferred in 30 .Math.l of RPMI 1640 medium containing 10% Methocel. Drops were placed on petri dishes and left at 37° C. and 5 % CO.sub.2 until spheroids were formed.

3D Cytotoxicity Assay

[0097] The assay was performed following the manufacturers advice. Briefly, a spheroid (10,000 cells) of MCF7 was seeded in a U-bottom 96-well plate Catalog#4430200 (Orange Scientific, Braine-IAlleud, Belgium) and pre-activated T cells (100000) were added at a ratio of 1:10 and co-cultured for 20 hr at a final volume of 100 .Math.l. Plates were centrifuged at 2000 rpm for 10 minutes and 50 .Math.l of supernatants were transferred into corresponding wells of an optically clear 96-well flat bottom microplate Catalog#781722 (Brand, Wertheim, Germany). The level of cytotoxicity was measured with Cytotoxicity Detection Kit (LDH) Catalog#11644793001 (Roche, Darmstadt, Germany). 50 .Math.l of substrate were added to the corresponding wells and the microplate was left to incubate for 30 minutes at room temperature in the dark. Absorbance was measured at 490 nm using an ELISA reader at a reference wavelength of 605 nm. The percentage of cytotoxicity was calculated as follows: (experimental value- spontaneous effector cell release) / (spontaneous target cell release) × 100%. The background value was subtracted from all the above values.

Results

[0098] The in vitro generation of antigen-specific CTLs from two human healthy individuals was successfully achieved. In previous experiments, the cytotoxic effect of antigen-specific CTLs against human breast adenocarcinoma cell lines was determined in 2D cell culture. In the present study, determination of cytotoxicity of antigen-specific CTLs was performed under 3D conditions. The pre-activated cells from subject A showed 86.8% cytotoxicity against MCF7 spheroids and pre-activated cells from subject B had 84.6% cytotoxicity against MCF7 spheroids. The table shown in FIG. 6 illustrates the results of the two populations of pre-activated cells along with the relative cytotoxicity of control cells. The control group was generated from DCs that were matured without prior pulsing with the respective peptide.

LIST OF REFERENCE SIGNS

[0099] none

REFERENCES

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[0109] Sant, A.J., 6 - Overview of T-Cell Recognition: Making Pathogens Visible to the Immune System, Editor(s): Robert R. Rich, Thomas A. Fleisher, William T. Shearer, Harry W. Schroeder, Anthony J. Frew, Cornelia M. Weyand, Clinical Immunology (Fifth Edition), Content Repository Only!, 2019, Pages 93-106.e1, ISBN 9780702068966, https://doi.org/10.1016/B978-0-7020-6896-6.00006-5.

SEQUENCE LISTING

[0110] TABLE-US-00001 <110> R.G.C.C. Holdings AG

TABLE-US-00002 <120> Antigenic Peptides for activation of CTL

TABLE-US-00003 <130> F06304

TABLE-US-00004 <160> 5

TABLE-US-00005 <170> BiSSAP 1.3.6 <210> 1 <211> 9 <212> PRT <213> Homo sapiens

TABLE-US-00006 <220> <223> synthetic peptide HLA-A*02:01-binding peptide MUC1 (79-87)        TLAPATEPA

TABLE-US-00007 <400> 1 Thr Leu Ala Pro Ala Thr Glu Pro Ala 1 5

TABLE-US-00008 <210> 2 <211> 9 <212> PRT <213> SARS coronavirus

TABLE-US-00009 <400> 2 Leu Pro Phe Asn Asp Gly Val Tyr Phe 1 5 <210> 3 <211> 16 <212> PRT <213> SARS coronavirus

TABLE-US-00010 <400> 3 Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu 1 5 10 15

TABLE-US-00011 <210> 4 <211> 9 <212> PRT <213> Coronaviridae

TABLE-US-00012 <400> 4 Arg Leu Asn Glu Val Ala Lys Asn Leu 1 5

TABLE-US-00013 <210> 5 <211> 9 <212> PRT <213> Coronaviridae

TABLE-US-00014 <400> 5 Asn Leu Asn Glu Ser Leu Ile Asp Leu 1 5