GRANULYSIN, METHOD OF OBTAINING SAME, AND USES

Abstract

The present invention relates to granulysin, method of obtaining same, and uses, specifically to the granulysin polypeptide for the use thereof as a medicinal product via the systemic route and to a chimeric molecule comprising a recombinant antibody targeting a tumor antigen and the granulysin polypeptide.

Claims

1. (canceled)

2. A pharmaceutical composition comprising a molecule comprising: a) a recombinant antibody targeting a tumor antigen, wherein the recombinant antibody comprises anti-CEA scFv MFE23 of SEQ ID No: 2; and b) a granulysin polypeptide comprising SEQ ID NO:1, wherein said recombinant antibody and said granulysin polypeptide are part of a single polypeptide chain; and wherein the recombinant antibody is located at an N-terminal end of the single polypeptide chain and is physically bound by a peptide connector to the granulysin polypeptide.

3-8. (canceled)

9. The pharmaceutical composition according to claim 2, wherein said peptide connector has a length of 5 to 40 amino acids.

10. The pharmaceutical composition according to claim 2, wherein said peptide connector comprises 2 or more amino acids selected from the group consisting of Gly, Ser, Ala, and Thr.

11. The pharmaceutical composition according to claim 2, wherein said peptide connector comprises SEQ ID NO: 3.

12. The pharmaceutical composition according to claim 2, wherein said recombinant antibody consists of anti-CEA scFv MFE23 of SEQ ID NO: 2, wherein said granulysin polypeptide consists of the sequence SEQ ID NO: 1, and wherein said peptide connector consists of the sequence SEQ ID NO: 3.

13. The pharmaceutical composition according to claim 2, wherein said molecule comprises or consists of the polypeptide of sequence SEQ ID NO: 4.

14-15. (canceled)

16. A nucleic acid sequence encoding said molecule of claim 2.

17. The nucleic acid sequence according to claim 16, wherein said nucleic acid sequence further comprises a sequence encoding a signal peptide.

18. A recombinant expression vector comprising said nucleic acid sequence according to claim 16.

19. A host cell comprising the recombinant expression vector according to claim 18.

20. (canceled)

21. A production process for producing the molecule according to claim 2, comprising the steps of: a) introducing a recombinant expression vector comprising a nucleic acid sequence encoding the molecule in a host cell; and b) culturing the host cell under conditions which allow expression of the nucleic acid sequence to obtain an expressed polypeptide, and c) optionally isolating or purifying, or both, the expressed polypeptide.

22. The production process according to claim 21, wherein step b) is performed at a pH between 4.9 and 5.2 and at a temperature between 15° C. and 21° C.

23. The method of claim 22, wherein step b) is performed at pH 5 and at a temperature of 18° C.

24. The method of claim 21, wherein the nucleic acid sequence further comprises a sequence encoding a signal peptide, wherein the signal peptide is a factor alpha.

25. The pharmaceutical composition of claim 2, wherein the peptide connector has a length of 10 to 30 amino acids.

26. The pharmaceutical composition of claim 2, wherein the peptide connector has a length of about 20 amino acids.

27. The pharmaceutical composition of claim 2, wherein the pharmaceutical composition is formulated for systemic administration.

28. The pharmaceutical composition of claim 2, wherein the recombinant molecule is expressed in Pichia pastoris.

29. The pharmaceutical composition of claim 2, wherein the granulysin polypeptide is bound to a polyhistidine sequence.

30. A method of treating hematological or solid tumors, the method comprising administering the pharmaceutical composition of claim 2 to a patient in need thereof.

31. The nucleic acid sequence of claim 17, wherein said signal peptide is a factor alpha.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0068] FIG. 1. GRNLY structure (9 kDa). The a helices are numbered with roman numbers (image on the left). The image on the right shows the electrostatic potential of the molecular surface in the same orientation as in the image on the left (positive potential shown in blue and negative potential in red) [Kolter, T., et al., J Biol Chem, 2005. 280(50): p. 41125-8].

[0069] FIG. 2. pPICZα C map.

[0070] FIG. 3. Coomassie blue staining of a 12% acrylamide gel of the supernatants obtained in the small-scale expression test of A) GRNLY, where the positive control (GalNacT2WT) is a recombinant protein that is well expressed in P. pastoris, and B) the chimera.

[0071] FIG. 4. Coomassie blue staining of a 12% acrylamide gel of the small-scale expression test at different pH and temperature conditions of A) colony 2 transformed with pPICZαC-GRNLY and B) colony 8 transformed with pPICZαC-Chimera.

[0072] FIG. 5. Different purification phases of the pPICZα MFE23-GRNLY-transfected P. Pastoris supernatant which include the filtered supernatant, the permeate and concentrate resulting from concentrating with Pellicom, the dialysate, the dialysate supernatant after the addition of the resin and centrifugation, and the supernatant from washes performed on the resin. Pooled elution fractions obtained by means of affinity chromatography are also included. A) Coomassie blue staining of a 12% acrylamide gel, B) Immunoblot with anti-GRNLY polyclonal antibody.

[0073] FIG. 6. Elution fractions (differentiated into A and B because they come from different columns) and pooled eluate obtained during chimera purification, after affinity chromatography. A) Coomassie blue staining of a 12% acrylamide gel, B) Immunoblot with anti-GRNLY polyclonal antibody.

[0074] FIG. 7. ELISA performed with 200 ng of CEA per well (left overnight at 4° C.), PBS (negative control), 500 ng of recombinant scFv MFE23 (positive control), and the P. pastoris supernatant transformed with pPICZαC-Chimera were then added after inducing expression with methanol. To develop the binding of scFvs to the CEA antigen, an anti-histidine tag antibody and a peroxidase-conjugated goat anti-mouse IgG secondary antibody were used. The peroxidase substrate was OPD, producing a yellow-orange product that can be detected at 492 nm.

[0075] FIG. 8. Histograms obtained after analyzing the binding of the chimera to the CEA antigen by means of flow cytometry: A) experiment on Jurkat cells and B) experiment on HT29 cells. The histograms have different colors and correspond to the fluorescence detected in unlabeled cells (red), cells with only the anti-mouse IgG secondary antibody (blue), cells with the chimera and anti-mouse IgG (orange), cells with anti-histidine tag and anti-mouse IgG (light green), or cells with the chimera, anti-histidine tag, and anti-mouse IgG (dark green).

[0076] FIG. 9. Fluorescence microscopy image of HT29 cells with Hoechst 33342 staining (blue fluorescence) and with the chimera and FITC-labeled secondary antibody (green fluorescence) where the cells are incubated with anti-histidine tag and FITC-labeled anti-mouse IgG antibodies in A) and with the chimera and anti-histidine tag and FITC-labeled anti-mouse IgG antibodies in B).

[0077] FIG. 10. Experiment for determining the cytotoxicity of the chimera on Jurkat cells by adding different concentrations of said chimera. A volume of PBS equivalent to the added volume of the chimera was used as negative control. Furthermore, another control involved adding only the cells to the well in an RPMI culture medium. The percentage of annexin-positive cells after 24 hours of incubation is measured by means of flow cytometry. The results are the mean±SD of two different experiments.

[0078] FIG. 11. Experiment for determining the cytotoxicity of GRNLY and the chimera on HT29 cells by adding different concentrations. After 24 hours of incubation, a cell count was performed to determine the percentage of growth of the cells to which GNLY or chimera has been added with respect to the controls. A volume of PBS equivalent to the added volume of GRNLY or chimera was used as a control in each case.

[0079] FIG. 12. Flow cytometry diagrams of HT29 cells treated with different concentrations of chimera as indicated after 24 hours of incubation. After incubation, the cells were labeled with Alexa-46-conjugated annexin-V and 7AAD and analyzed by means of flow cytometry. The numbers shown in the diagrams correspond to the percentage of cells in each quadrant.

[0080] FIG. 13. In vivo results in a HeLa cell-CEA tumor development model in nude mice are shown and it is observed that if the control group is compared with the MFE (chimera) group, there are significant differences after the 7.sup.th injection, the difference being very significant in the last injections. The manner in which tumor growth in treated mice is somehow contained or attenuated can be seen. The X-axis shows treatments (injections) and the Y-axis shows tumor volume (mm.sup.3).

[0081] FIG. 14. In vivo results are shown and it is observed that if the control group is compared with the (non-chimeric) granulysin group, there are no significant differences, although the granulysin curve is below the control curve for all the points. The X-axis shows treatments (injections) and the Y-axis shows tumor volume (mm.sup.3).

[0082] FIG. 15. In vivo results are shown and the set of curves shown in FIG. 13 and FIG. 14 is observed. The X-axis shows treatments (injections) and the Y-axis shows tumor volume (mm.sup.3).

[0083] FIG. 16. In vivo results are shown and the means ±SD of the sizes of the tumors once removed and subjected to different treatments are observed. The Y-axis shows the sizes of the tumors once removed (mm.sup.3).

[0084] FIG. 17. In vivo results are shown and the means ±SD of the weights of the tumors once removed and subjected to different treatments are observed. The Y-axis shows the weight of the tumors once removed (g).

[0085] FIG. 18. In vivo results are shown and the results are observed in the form of a Kaplan-Meier curve. The time it each mouse takes to reach a tumor size of 800 mm.sup.3 under the three experimental conditions is shown.

[0086] FIG. 19. In vivo results are shown and the control and MFE curves are observed to enable calculating the statistical significance which renders a P of 0.027.

[0087] FIG. 20. Histochemistry and immunohistochemistry in tissue sections of HeLa-CEA tumors.

A) The images show hematoxylin-eosin staining of tissue sections of HeLa-CEA tumors in the control group and of the groups treated with granulisin (GRNLY) or treated with the MFE23-GRNLY chimera.
B) The sections of the tumor were incubated with an antibody against caspase-3 and revealed by DAB staining (Agilent, Madrid).
C) The nuclei were stained using DAPI (EMS, Madrid) and photographed in a fluorescence microscope. The arrows indicate apoptosis (fragmented nuclei) and the circles indicate marginated chromatin nuclear phenotype.

DETAILED DESCRIPTION OF THE INVENTION

Examples

Example 1

Materials and Methods

Cell Cultures

[0088] Cell lines. The following cell lines of human origin were used: [0089] 1. Jurkat, T-cell acute lymphoblastic leukemia cell line, clone E6-1, negative for CEA expression and positive for Tn expression. [0090] 2. HT-29, colon adenocarcinoma cell line, positive for CEA expression. [0091] 3. HeLa-CEA, cervical cancer cell line transfected with a cDNA construct encoding CEA and thereby expressing high levels of this antigen. The transfection was carried out in the laboratory of Dr. Laura Sanz, Hospital Puerta de Hierro, Madrid. [0092] 4. MDA-MB-231, breast adenocarcinoma cell line, negative for Tn expression. [0093] 5. MCF-7, metastatic breast adenocarcinoma cell line, positive for Tn expression. [0094] 6. Capan-2, pancreatic carcinoma cell line, positive for Tn expression. All the cell lines were originally obtained from the American Type Culture Collection (ATCC, USA).

Cell Culture Maintenance

[0095] The cells were incubated in a temperature-controlled incubator at 37° C. in moist air and with 5% CO.sub.2. The filter cap culture flasks, pipettes, and all the material for that purpose were used under sterile conditions in the laminar flow hood of the Biochemistry and Molecular Biology Department of UNIZAR.

[0096] The Jurkat cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin, 100 ug/ml of streptomycin, and 2 mM GlutaMAX. The culture was maintained with a density of up to one million cells per ml, which corresponds to one passage every 3 or 4 days.

[0097] The rest of the cell lines were cultured in Dulbecco's Modified Eagle's Medium (DMEM), Catalog No. 30-2002. This medium was supplemented with 10% FBS, 100 U/ml of penicillin, 100 ug/ml of streptomycin, and 2 mM GlutaMAX. For the passages, considerations were given to ATCC recommendations on the concentration of cells in the medium, i.e., about one million cells per ml before trypsinization and the subculture ratios depending on each cell type, in that sense CAPAN-2 required ratios of 1:2 to 1:4, MIA PACA-2 required ratios of 1:3 to 1:8, etc. These considerations were used for expanding the cells for in vivo assays.

[0098] Likewise, according to ATCC recommendations, cryopreservation of the cell lines was performed in 95% FBS and 5% dimethylsulfoxide (DMSO) at a cell density of 5×10.sup.6/ml. First, the cells were frozen for 24 to 48 hours in a freezer at −86° C. and then transferred to a liquid nitrogen container.

[0099] For the process of thawing the cells, a wash was performed with 10 ml of culture medium and the cells were then seeded at a density that was twice the density of that used for the passages.

Cell Count and Viability

[0100] Cell viability was determined by means of a 0.4% trypan blue solution in 0.15 M of NaCl (Sigma, Madrid), this dye being capable of entering the cell cytoplasm with a loss of cell membrane integrity. After mixing the dye with the cell suspension (50 ul of each), the cells were counted in a Neubauer chamber (hemocytometer) and observed under a microscope (Optiphot, Nikon). The formula used for calculating cell density is as follows:

[00001]$\mathrm{Concentration}=\frac{\mathrm{Total}\mathrm{cells}\mathrm{counted}\times 10,000}{\mathrm{Number}\mathrm{of}\mathrm{squares}}$

[0101] The percentage of cell viability was calculated from this formula so that assays are always performed with a viability greater than 90%.

Anti-Tn Chimera Cloning

[0102] Starting genetic material: the pPICZα integrative plasmid was used, with this construct having the following characteristics: [0103] pUC ori: allows plasmid replication in E. coli [0104] AOX1 promoter: induced by methanol and directs plasmid integration in the AOX1 locus of Pichia by means of homologous recombination. [0105] Factor α: allows efficient protein secretion [0106] Multiple cloning site: allows DNA insertion in the expression vector [0107] c-myc epitope: allows detection with anti-myc antibody [0108] Polyhistidine tag: facilitates recombinant protein purification [0109] AOX1 transcription terminator: increases mRNA stability by allowing efficient mRNA 3′ end processing, including polyadenylation [0110] Zeocin resistance gene (zeocin being a broad-spectrum antibiotic): serves as a selection marker. It is preceded by the TEF1 and EM7 promoters and followed by the CYC1 transcription terminator.

[0111] The cDNA of human GRNLY and the cDNA of human GRNLY-conjugated SM3 and Ar20.5 are directionally integrated between the cleaving sites of restriction enzymes Cla I and Xba I found at the multiple cloning site of pPICZαC and pPICZαA, within the reading frame.

[0112] The plasmid pPICZαC-GRNLY was synthesized by Dr. Laura Sanz (Hospital Puerta de Hierro, Madrid) from the human GRNLY DNA sequence kindly donated to the group by Dr. Alan Krensky (Northwestern University, Chicago). The plasmids pPICZαA-SM3 13° and pPICZαA-Ar20.5 were kindly donated by Dr. Ramón Hurtado (Institute for Biocomputation and Physics of Complex Systems BIFI of the University of Zaragoza).

Competent Cell Preparation

[0113] DH5α E. coli was made “competent” for transformation by means of opening the pores in the membrane with CaCl.sub.2 at a low temperature. The method consisted of seeding p DH5α E. coli in a 50 ml tube with 10 ml of liquid LB medium under stirring (180 rpm) at 37° C. overnight. 200 ul of that product were used for diluting in 20 ml of fresh LB medium and for repeating incubation until reaching an absorbance of about 0.3-0.4 at 600 nm, which is equivalent to a concentration of 5-10×10.sup.7 bacteria/ml). It was then incubated on ice for 30 minutes and centrifuged for 8 minutes at 4° C. and 8000×g. It was washed once with sterile water, incubated in 10 ml of 50 mM cold CaCl.sub.2 for 15 minutes and centrifuged again for 8 minutes at 3000×g. Finally, the decant was resuspended in 4 ml of a 50 mM CaCl.sub.2 solution with 15% glycerol and frozen at −80° C. until use.

Bacterial Transformation and Expansion

[0114] The transformation of competent p DH5α E. coli with pPICZαC-Ar20.5-GRNLY or pPICZαC-SM3-GRNLY was performed by heat shock. Two aliquots of about 200 ul were thawed by incubating them on ice and 50 ng of plasmid DNA were added in each aliquot (these methods were performed by blazing in a flame) for 20 minutes, it was introduced in a 42° C. bath for a minute and a half and again for two minutes on ice. 1 ml of liquid LB medium was added in each aliquot and it was incubated at 37° C. under stirring at 200 rpm for two hours. The transformed bacteria were then seeded in solid LB medium (1.5%) with zeocin (25 μg/ml) at 37° C. overnight, such that the bacteria incorporating the plasmid grew. From the colonies that developed, four colonies were randomly selected for each protein and cultured in 50 ml tubes with 10 ml of liquid LB medium and 25 ug/ml of zeocin (one colony per tube) under stirring at 180 rpm at 37° C. overnight.

Selection of Bacteria with a Higher Granulysin Expression Level

[0115] The plasmid was isolated by means of minipreparation with the “Nucleospin® Plasmid Easypure” kit. To check if plasmids with the suitable molecular weight were isolated, electrophoresis was performed in 1% agarose gel made up of 0.3 g of agarose (Scharlau), 30 ml of 1× TAE buffer (Tris-Acetate-EDTA buffer, Invitrogen, pH 8.3), and for DNA staining 3 μl of SYBR® Safe (Invitrogen). The gels were viewed in a Gel Doc 2000 transilluminator (BioRad). To expand the plasmids to a larger amount, transformed E. Coli colonies were cultured in LB medium with zeocin (25 μg/ml) using the “Quantum Prep™ Plasmid Midiprep” kit. In both cases, the concentration and purity of the obtained plasmids were determined by means of a NanoDrop® (NanoVue) apparatus.

Starting Genetic Material: Ppiczαc-GRNLY And Ppiczαc-Chimera

[0116] pPICZαC is an commercially available integrative plasmid [pPICZα A, B, and C: Pichlan expression vectors for selection on Zeocin™ and purification of secreted, recombinant proteins, MAN 0000035, Invitrogen, Corporate Headquarters, Rev. Date: Jul. 7, 2010] containing, as shown in FIG. 2, the following characteristics: [0117] pUC ori: allows plasmid replication within E. coli [0118] AOX1 promoter: induced by methanol and directs plasmid integration in the AOX1 locus of Pichia by means of homologous recombination. [0119] Factor α: allows efficient protein secretion [0120] Multiple cloning site: allows DNA insertion in the expression vector [0121] c-myc epitope: allows detection with anti-myc antibody [0122] Polyhistidine tag: allows easier recombinant protein purification [0123] AOX1 transcription terminator: increases mRNA stability by allowing efficient mRNA 3′ end processing, including polyadenylation [0124] Zeocin resistance gene (zeocin being a broad-spectrum antibiotic): serves as a selection marker. It is preceded by the TEF1 and EM7 promoters and followed by the CYC1 transcription terminator.

[0125] The cDNA of human GRNLY and the cDNA of human GRNLY-bound MFE23 are directionally integrated between the cleaving sites of restriction enzymes Cla I and Xba I found at the multiple cloning site of pPICZαC (framed in FIG. 2), within the reading frame. The human granulysin cDNA sequence (SEQ ID No. 1) was kindly donated to the group by Dr. Alan Krensky (Northwestern University, Chicago).

Chimera Structure and Parameters

[0126] In generic terms, the fusion protein that is obtained has the following structure:

MFE23-LINKER-GRANULYSIN (9 kDa)-HISTIDINE TAG

TABLE-US-00005 TABLE 1 Chimera parameters obtained with the ProtParam tool (Expasy) based on the amino acids thereof CHIMERA (MFE23-GRNLY) Extinction coefficient at 280 nm (M.sup.−1 cm.sup.−1) Extinction coefficient at Number of Theoretical assuming that all Cys 280 nm (M.sup.−1 cm.sup.−1) amino Molecular isoelectric point pairs form disulphide assuming that all Cys acids weight (kDa) (pI) bridges residues are reduced 347 37.1069 9.03 62840 62340
Pichia pastoris Transfection by Means of Electroporation

[0127] The plasmids must remain linear so that they can be more easily integrated in the genome of Pichia pastoris. To that end, digestion is performed with the enzyme Sacl, which has a single cleaving site within pPICZαC and does not cleave within the inserts to be introduced. Next, to check that the plasmid has been properly digested, electrophoresis is carried out in 1% agarose gel. After checking that the digestion was satisfactory, digestion residues (salts and residual agarose gel) are cleaned away and the plasmid is purified using the AccuPrep® Gel Purification kit.

[0128] The prior treatments described in the manual [Pichia Expression Kit, Invitrogen, Rev. Date: Oct. 1, 2014], first with 100 mM LiAc solution, 10 mM DTT, 0.6 M sorbitol, and 10 mM Tris-HCL, pH 7.5, and then with another 1 M sorbitol solution, are necessary before proceeding to the electroporation of Pichia pastoris SMD1168 with the plasmids. The electroporator (BIORAD MicroPulser™) is set to the program for electroporating Pichia. Furthermore, Pichia pastoris SMD1168 cells electroporated in the absence and presence of the linearized plasmid are plated on plates containing zeocin (cells which do not contain the plasmid would not grow in plates with zeocin, and this accordingly indicates that the electroporation was satisfactory) and the plates are left in the oven at 30° C. throughout the entire electroporation process by way of control. The transfected colonies are selected, plating them on a YPDS plate (10 g/L of yeast extract, 20 g/L of peptone, 2% dextrose, 182.2 g/L of sorbitol, pH 6) with zeocin (200 μg/ml) and the plates are incubated for 3-10 days at 30° C. until colonies emerge. Finally, some well isolated colonies which have grown in the plate with zeocin are chosen and plated again in a new plate so as to later choose the best strain.

Recombinant Protein Production in Pichia pastoris Cultures

[0129] The selected colonies are inoculated in BMGY medium (10 g/L of yeast extract, 20 g/L of peptone, 100 mM phosphate buffer, pH 6, 13.4 g/L of nitrogenated yeast base without amino acids or ammonium sulfate, 1 ml/L of glycerol, and 0.4 mg/L of biotin), being cultured at 30° C. for a day for the yeast to grow. A change of medium to BMMY (10 g/L of yeast extract, 20 g/L of peptone, 100 mM sodium acetate buffer, pH 5, 13.4 g/L of nitrogenated yeast base without amino acids or ammonium sulfate, 0.5 ml/L methanol, and 0.4 mg/L of biotin) is then performed, maintaining the culture at 18° C. under stirring for a day for the induction of the expression of the recombinant protein to begin. After the first day of induction and every 24 hours for 2 more days, methanol is added at a final concentration of 1% in the culture medium and it is left under stirring until the next day at 18° C. First, a small-scale production of the selected colonies was performed. Once the colonies producing the most recombinant protein were chosen, different pH and temperature conditions were tested to find the optimum production conditions. Finally, large-scale recombinant protein production was performed.

Recombinant Protein Purification

[0130] The yeast supernatant is filtered by means of a vacuum filtration system first with a 0.45 μm filter and then with a 0.22 μm filter. It is then concentrated in a Pellicon XL Ultracel 5 kDa 0.005 m.sup.2 concentrator (Millipore) from 1 L to about 50 ml. Next, in the case of the chimera, dialysis is performed using a dialysis bag (Millipore), and in the case of the recombinant GRNLY, dialysis is performed using a “Slide-A-Lyzer™” dialysis cassette (Thermo Scientific Pierce) with a membrane having a pore size of 3.5 kDa due to the small molecular weight thereof. The dialysis bag or membrane is immersed in 5 L of washing buffer (300 mM NaCl, 50 mM Tris-HCl, and 20 mM imidazol, pH 7.4) and left to dialyze overnight. Dialysis is performed to change the medium originating from the yeast supernatant in which GRNLY can be found by the buffer that will be used later in nickel affinity chromatography. Imidazol is found at a low concentration in the buffer such that it competes with molecules that do not specifically bind to the nickel column.

Affinity Chromatography

[0131] Recombinant GRNLY and chimera are expressed with a histidine tag for the purpose of purifying them by means of nickel affinity chromatography since the imidazol rings of histidines have a high affinity for Ni.sup.2+ cation.

[0132] To that end, the Ni-NTA agarose resin (Qiagen) is mixed with washing buffer (300 mM NaCl, 50 mM Tris-HCl, and 20 mM imidazol, pH 7.4), centrifuged at 2500 rpm for 2 minutes, and the supernatant is removed. Washing buffer is then added and it is centrifuged again at 2500 rpm for 2 minutes to remove the supernatant. The resin is then resuspended in washing buffer and mixed with the resulting solution after dialysis. It is then placed in a rotating end-over-end shaker at 4° C. for about an hour and a half. It is then centrifuged at 2500 rpm for 5 minutes. The precipitate is washed three times with washing buffer, rotated in an end-over-end shaker for 15 minutes, and centrifuged at 2500 rpm for 5 minutes. The precipitate is placed in a column with washing buffer, and the resin is left to settle. The column is eluted with elution buffer (500 mM imidazol, 300 mM NaCl, and 50 mM Tris-HCl, pH 7,4). The amount of protein in the eluded fractions originating from affinity chromatography is then quantified by means of a NanoDrop® apparatus (NanoVue). The elution fractions containing an acceptable amount of protein are then pooled.

Buffer and Concentration Change

[0133] To change the elution buffer for PBS, the chimera in elution buffer is passed through a column with Sephadex G-25 (Thermo-Fisher) previously equilibrated with PBS and concentrated with an Amicon filter having a membrane pore size of 15 kDa (Millipore), or the buffer is directly changed and concentrated with said Amicon filter. In the case of the recombinant GRNLY, the buffer is changed and the elution is concentrated at the same time by means of an Amicon filter having a membrane pore size of 3 kDa. Finally, with a NanoDrop® apparatus (NanoVue), the protein concentration in the final concentrate is measured and it is sterilized by filtration through a 0.22 μm filter.

Coomassie Staining and Immunoblot

[0134] To perform the expression test, denaturing electrophoresis is performed in 12% acrylamide gel made up of two types of gels having the same composition but in different proportions (stacking gel and resolving gel), loading the supernatant obtained from each colony together with a molecular weight marker. Said gels are then stained with Coomassie Blue to find out which colony produced the highest amount of recombinant protein.

[0135] Furthermore, in all the purification steps aliquots are kept at 4° C. to enable analyzing them by means of electrophoresis of the different aliquots in 12% polyacrylamide gel, Coomassie Blue staining is performed, and immunoblot is also performed by transferring the proteins separated in the gel to nitrocellulose membranes according to the previously described method [Anel, A., et.al., J Biol Chem, 1993. 268(23): p. 17578-87] and incubating the membrane with a rabbit polyclonal primary antibody kindly donated by Dr. Carol Clayberger (Northwestern University, Chicago). After washing, a peroxidase-conjugated rabbit anti-IgG secondary antibody (Sigma) is then added. It is later washed with buffer B (PBS with 0.05% of Tween-20, pH 7.4) under stirring to remove excess antibodies.

[0136] The complexes are detected by means of chemiluminescence (ECL) development. This technique is based on the detection of the light emitted as a result of the oxidation of luminol, a chemiluminescent substrate, by peroxidase. This light is captured by photographic films (high-performance chemiluminescence film, GE HealthCare) in the dark and following membrane incubation with “Pierce® ECL Western Blotting Substrate” (Thermo Scientific). The films are exposed in a radiological developing cassette (Hypercassette™, Amersham Bioscience) and in a dark room with suitable lighting for photographic development. The films are developed after exposure by means of immersion in developer-distilled water-fixer solutions, varying the developer solution time according to the signal that is obtained.

Specificity Assay

[0137] An ELISA was performed using 200 ng of CEA per well, which was incubated overnight at 4° C., PBS (negative control), 500 ng of the recombinant scFv MFE23 (positive control), and the P. pastoris supernatant transformed with pPICZαC-Chimera after inducing expression with methanol were then added. To develop the binding of scFvs to the CEA antigen, an anti-histidine tag antibody and a peroxidase-conjugated goat anti-mouse IgG secondary antibody were used. The peroxidase substrate was OPD, producing a yellow-orange product that can be detected at 492 nm.

Flow Cytometry

[0138] For the purpose of studying the binding of the chimera to the CEA antigen, an experiment was performed with HT29 cells (ATCC) which express the CEA antigen on the surface, and with Jurkat cells (ATCC) which do not express the CEA antigen on the surface as a negative control.

[0139] These cells were deposited in a round bottom 96-well plate at a concentration of 100,000 cells per well, washed with PBS with 5% FBS, and chimera was added (10 μg/ml). After incubating for 1 hour at 4° C. and washing with PBS with 5% FBS, they were labeled with anti-HIS murine antibody (1:200), and after incubating for another hour at 4° C., an FITC-conjugated anti-mouse IgG antibody was added (1:200). Finally, it was incubated for another hour at 4° C. and fluorescence was analyzed by means of flow cytometry. In this manner, fluorescence is observed if the chimera binds to the surface of the cells. Several negative controls were carried out in the absence of chimera and/or antibodies to assure that the chimera binds specifically to CEA.

Fluorescence Microscopy

[0140] Furthermore, the same process was performed on a slip in a 24-well plate with HT29 cells, the chimera, and anti-His and FITC-conjugated anti-mouse IgG antibodies, after which Hoechst 33342 staining was performed to enable viewing the cell nuclei and performing analysis by means of fluorescence microscopy.

In Vitro Cytotoxicity Assay

[0141] The cytotoxicity of GRNLY or of the chimera was assayed on T-cell acute leukemia Jurkat which constitutes the standard for sensitivity to GRNLY in this laboratory, and on human colon carcinoma HT29. The Jurkat cells grow in suspension in RPMI 1640 culture medium (Gibco®) supplemented with 5% unsupplemented fetal bovine serum, glutamine, and antibiotics, and do not express the CEA antigen. The HT29 cells are adherent cells which grow in DMEM culture medium (Gibco®) supplemented in a similar manner and express the CEA antigen on their surface. Adherent cells must be trypsinized for handling and reseeding. In the control samples, a volume of PBS which is equivalent to the added volume of GRNLY or the chimera is added on the cells. In the case of the Jurkat cell line, cells at a concentration of 30000 cells per well, PBS, or the chimera/GRNLY are added, the cells are seeded in 96-well plates, and incubated at 37° C. with 5% CO.sub.2 for 24 hours. In the case of the HT29 cell line, first only the cells are seeded at a concentration of 30000 cells per well in DMEM culture medium and incubated for 24 hours at 37° C. in an incubator with 5% CO2 for the cells to adhere, and once adhered, the chimera or PBS is added and they are incubated at 37° C. for another 24 hours.

[0142] An important characteristic of the apoptotic phenotype is the exposure of phosphatidylserine in the outer layer of the plasma membrane [Martin, S. J., et al., J Exp Med, 1995. 182(5): p. 1545-56]. To measure this translocation, annexin V, a protein which binds specifically to phosphatidylserine, can be used. In late stages of apoptosis, when the plasma membrane has lost its integrity and the DNA becomes accessible, fluorophores such as 7-AAD, which act as intercalating agents in double-stranded nucleic acids, are used. In these experiments, the cells are incubated with annexin V conjugated with Alexa-46 fluorophore (Immunostep), and 7AAD (Immunostep) in the case of HT29 cells, in ABB buffer, 140 mM NaCl, 2.5 mM CaCl.sub.2, 10 mM Hepes (NaOH, pH 7.4, for 15 minutes in the dark. The percentage of apoptotic cells in each of the assay conditions can thereby be quantified by means of flow cytometry.

[0143] The cytotoxicity of GRNLY had not yet been assayed on HT29 cells, such that the cytotoxicity of both GRNLY and the chimera were assayed by means of trypan blue staining. Trypan blue is a dye derived from toluidine having the capacity to stain only dead tissues and cells as it is not capable of going through the intact membranes of living cells. A microscope and a Neubauer chamber were used for the cell count. After the count, the number of cells of each well was compared with the control well which only contained cells and culture medium. The percentage of growth with respect to the control was thereby obtained.

Example 2

[0144] Recombinant Protein Production in Pichia astoris Cultures

[0145] After transfecting and culturing Pichia pastoris as indicated in the materials and methods section, the recombinant protein was successfully expressed and secreted into the medium as a result of factor-α.

[0146] There can be seen in FIG. 3A, in all the chosen and induced colonies, a band corresponding to a molecular weight close to 11 kDa which corresponds with the weight of recombinant GRNLY given that GRNLY, which has a weight of about 9 kDa, experiences a molecular weight increase to about 11 kDa since it is fused to a histidine tag. In reality, at least two bands with very similar molecular weights belonging to different recombinant GRNLY isoforms are seen. This is because GRNLY undergoes post-translational O-glycosylation modifications. Similar observations had previously been made by another research group which wrote a similar paper in which it was determined that the different bands obtained were due to O-glycosylation [Guo, Y., et al., Appl Microbel Biotechnol, 2013. 97(17): p. 7669-77].

[0147] Furthermore, there can be seen in FIG. 3B, in all the chosen and induced colonies, a band corresponding to a molecular weight close to 35 kDa which corresponds with the weight of recombinant MFE23-bound GRNLY (FIG. 3B). In view of these results (FIG. 3), the decision was made to perform large-scale expression with colony 8 transformed with pPICZαC-Chimera and with colony 2 transformed with pPICZαC-GRNLY. Another group has described large-scale recombinant GRNLY production as a result of a fermenter and obtained better expression results when induction was carried out at pH 5 and 30° C. [Guo, Y., et al., Appl Microbel Biotechnol, 2013. 97(17): p. 7669-77]. Accordingly, an expression test was performed in which induction was carried out at different pH and temperature conditions in order to check under which conditions the proteins of the present invention were best expressed. In both cases, the best results were achieved by carrying out induction at pH 5 and 18° C. (FIG. 9). By carrying out induction at 30° C. and pH 5, many Pichia pastoris proteins in addition to GRNLY are expressed, and this is not of interest as it may negatively affect protein purity during purification (FIG. 4A).

Example 3

Recombinant Protein Purification

[0148] FIG. 5 shows the steps of one of the purifications carried out on the chimera. In the case of GRNLY, this process was shown in an earlier paper [Ibáñez, R., University of Zaragoza. 2015]. It can be seen in FIG. 5A that the P. Pastoris supernatant obtained after induction (lane 1) contains rather diluted proteins. After concentrating same with Pellicom, protein bands are not seen in the permeate (lane 3), but proteins that are much more concentrated than in the supernatant are seen in the concentrate (lane 2). After dialysis (lane 4), the band profile remains similar to the concentrate. Furthermore, protein bands are not seen in the buffer in which the dialysis bag (lane 5) was introduced. Upon addition of the nickel resin, the chimera binds to said resin as it has a histidine tag. After adding the resin (lane 6), the intensity of a band corresponding to a protein of about 40 kDa decreases with respect to the concentrate and dialysate. This band may correspond to the chimera. The fact that this band does not altogether disappear may indicate that the nickel resin was saturated. In the washes performed on the resin, particularly in the first wash (lane 7), it can be seen how the residues of other proteins are removed. Finally, after the elution of the nickel column, a major protein with a molecular weight of about 40 kDa corresponding to the molecular weight of the chimera (lane 11) is clearly observed. As shown in FIG. 5B, it was confirmed by means of immunoblot that this band of about 40 kDa corresponds to the chimera (lane 11). It is also confirmed that the resin was saturated because a band appears in the post-resin dialysis phase (lane 6).

[0149] FIG. 6 shows different elution fractions and the pooling of all of them with the exception of elution fraction 1. FIG. 6A shows several bands in the different elution fractions and in the total eluate. The band with the highest intensity has a molecular weight corresponding to the chimera. Furthermore, other bands having intermediate molecular weights are observed, which means that the chimera undergoes partial proteolysis. The band with the second highest intensity has a molecular weight of about 10 kDa, which corresponds to 9-kDa GRNLY, as its molecular weight increases since it is bound to a histidine tag. In FIG. 6B, it was confirmed by means of immunoblot that these bands of about 40 and 10 kDa correspond to the chimeric recombinant protein and to recombinant GRNLY, respectively.

[0150] Once the chimera is generated, its functionality must be assured, that is, on one hand the scFv still recognizes the CEA antigen, and on the other hand GRNLY is still cytotoxic.

Example 4

Specificity Assay

Elisa

[0151] Part of the supernatant was used to check, by means of ELISA, that the chimera is capable of binding to the CEA antigen. The result showed that the chimera is capable of binding to the CEA antigen in a manner similar to scFv MFE23 (FIG. 7).

Flow Cytometry

[0152] An experiment was carried out by means of flow cytometry and fluorescence detection using HT29 colon carcinoma cells expressing CEA or T-cell leukemia Jurkat cells not expressing CEA as a negative control. To carry out this analysis, the chimera was added under cold conditions to the cells, followed by a mouse anti-histidine tag antibody, and an FITC-bound anti-mouse IgG antibody. Fluorescent labeling will therefore be observed if the chimera binds to CEA expressed on the surface of HT29 cells. Several negative controls were carried out in the absence of chimera and/or antibodies to assure that the chimera binds specifically to CEA.

[0153] As can be seen in FIG. 8, although a certain increase in fluorescence is detected in Jurkat cells when the chimera and the two antibodies are added (FIG. 8A), this increase is much lower than that observed when the same experiment is performed on HT29 cells (FIG. 8B). The data obtained in the controls seem to indicate that the combination of the anti-His antibody with the anti-mouse IgG antibody leads to unspecific labeling which is more prominent in the case of HT29 than in the case of Jurkat cells (Table 2). However, specific labeling in the presence of the chimera is in any case more prominent in the case of HT29 cells.

TABLE-US-00006 TABLE 2 Mean fluorescence intensity (MFI) obtained after analyzing the binding of the chimera to the CEA antigen by means of flow cytometry MFI JURKAT HT29 Cells 2.29 3.4 Cells + α-mouse 2.46 3.4 Cells + MFE23-GRNLY + α-mouse 2.46 4.22 Cells + α-His + α-mouse 2.64 6.04 Cells + MFE23-GRNLY + α-His + α-mouse 3.92 8.35

Fluorescence Microscopy

[0154] For the purpose of illustrating these results, fluorescence microscopy was performed, the results of which are shown in FIG. 9 which depicts the staining of HT29 cell nuclei with the fluorescent Hoechst 33342 molecule. FIG. 9B shows the cell nuclei surrounded by a green halo corresponding to CEA labeling by the chimera in the plasma membrane of the cells, whereas said halo is not seen in FIG. 9A, used as a negative control, in which no chimera was added.

Example 5

In Vitro Cytotoxicity Assay

[0155] Cells from the Jurkat cell line were treated with different doses of chimera. The result was that the chimera is toxic to Jurkat cells in a dose-dependent manner as can be seen in FIG. 10. Almost all the cells undergo apoptotic death with a chimera concentration of 6 μM.

[0156] HT29 cells were treated with different doses of GRNLY and chimera. The result was that both GRNLY and the chimera are toxic to HT29 cells in a dose-dependent manner as can be seen in

[0157] FIG. 11. GRNLY or chimera concentrations of 4 or 5 μM seem to have a similar effect, but the chimera is more cytolytic than GRNLY at a concentration of 6 μM, achieving a percentage of growth of 30% with respect to the control, i.e., 70% cytotoxicity. To match said effect, a GRNLY concentration of about 20 μM must be used. Furthermore, labeling was also performed with Alexa-46-conjugated annexin-V showing phosphatidylserine exposure and with 7AAD showing membrane integrity on HT29 cells treated with different concentrations of chimera for analyzing the type of induced cell death. By increasing the concentration of chimera, an increase in cells labeled with annexin which still have not lost membrane integrity is observed, indicating that cell death is caused by apoptosis (FIG. 12). Furthermore, a significant increase in cytotoxicity is observed when incubating the cells with a chimera concentration of 6 μM, as shown in FIG. 16. The maximum dose of chimera used was 6 μM, whereas in the case of GRNLY, a concentration of up to 20 μM was reached.

Example 5

[0158] In Vivo Assay with Hela-Cea Cells

[0159] Five mice per group (control group, granulysin group, and MFE group (with the chimera) were assayed. Although there was a mouse in the MFE group that died after the sixth injection, the other 4 mice, however, reached the end of the experiment in good conditions state. The tumor was subcutaneously injected with Matrigel at 2 million cells. Treatments began when the tumors reached a size of 150 mm.sup.3. The treatments were systemic intraperitoneal treatments performed every two days (injections): [0160] Control group, 500 ul of PBS. [0161] Granulysin group, 220 ul of a stock at 500 ug/ml (40 uM), i.e., 110 ug per injection, which yields a concentration of about 5 uM in 2 ml of total blood. [0162] MFE group, 500 ul of stocks of about 900 ug/ml (25 uM), i.e., 425 ug per injection, which yields a concentration of about 5 uM in 2 ml of total blood.

[0163] Ten injections were performed and the mice were sacrificed 2 days after the last injection.

[0164] The results are illustrated in FIGS. 13 to 19. FIG. 13 shows that if the control group is compared with MFE group (chimera), significant differences can be seen after the 7.sup.th injection, with the difference being very significant in the last injections. It can be seen how tumor growth in treated mice is somehow contained or attenuated. FIG. 14 shows that if the control group is compared with the (non-chimeric) granulysin group, there are no significant differences, although the granulysin curve is below the control curve for all the points. FIG. 15 shows all the results shown in FIG. 13 and FIG. 14. FIG. 16 shows the means±SD of the sizes of the tumors once removed and subjected to different treatments, a smaller tumor size with granulysin treatment, and an even smaller size when the chimera is used, being shown. FIG. 16 shows the means±SD of the weights of the tumors once removed and subjected to different treatments, a lower tumor weight with granulysin treatment, and an even lower weight when the chimera is used, being shown.