TOXOPLASMA PLATFORM FOR TREATING CANCER
20220233607 · 2022-07-28
Assignee
- UNIVERSITÉ DE TOURS (Tours, FR)
- KYMERIS THERAPEUTICS INC. (Toronto, CA)
- INSTITUT NATIONAL DE RECHERCHE POUR L'AGRICULTURE, L' ALIMENTATION ET L'ENVIRONNEMENT (Paris, FR)
Inventors
- Isabelle Dimier-Poisson (Tours, FR)
- Marie-Noëlle MEVELEC (Tours, FR)
- Anne DI TOMMASO (La Riche, FR)
- Nicolas AUBREY (Truyes, FR)
- Louis LANTIER (Notre-Dame-d'Oé, FR)
- Gordon LEE (Miami, FL, US)
Cpc classification
C07K16/2851
CHEMISTRY; METALLURGY
C12N2800/22
CHEMISTRY; METALLURGY
C07K2319/912
CHEMISTRY; METALLURGY
A61K35/68
HUMAN NECESSITIES
International classification
A61K35/68
HUMAN NECESSITIES
C07K14/715
CHEMISTRY; METALLURGY
Abstract
A strain of an Apicomplexa of the family Sarcocystidae, wherein the strain is replicative and expresses one or more heterologous protein(s) selected from the group including therapeutic proteins, antigens, recombinant surface receptor or combinations thereof, and wherein the strain is selected from the group including Toxoplasma gondii and Neospora caninium. Also, the use of the strain for preventing or treating cancers or infectious diseases in a subject in need thereof.
Claims
1-16. (canceled)
17. A strain of an Apicomplexa of the family Sarcocystidae, wherein said strain is replicative and expresses at least one heterologous gene or protein.
18. The strain according to claim 17, wherein the strain is Toxoplasma gondii or Neospora caninum.
19. The strain according to claim 17, wherein the strain expresses and/or secretes one or more heterologous protein(s) selected from the group comprising therapeutic molecules, antigens, recombinant surface receptors, or combinations thereof.
20. The strain according to claim 19, wherein the therapeutic molecule is a cytokine.
21. The strain according to claim 20, wherein the cytokine is a human IL15Rα sushi.
22. The strain according to claim 19, wherein the antigen is a cancer antigen or a neoantigen.
23. The strain according to claim 19, wherein the recombinant surface receptor comprises at least one extracellular-binding domain.
24. The strain according to claim 23, wherein the at least one extracellular-binding domain is an antigen-binding fragment or an antibody selected from the group comprising whole antibody, humanized antibody, single chain antibody, dimeric single chain antibody, Fv, scFv, Fab, F(ab)′2, defucosylated antibody, bi-specific antibody, diabody, triabody, tetrabody surface-exposed binding domain.
25. The strain according to claim 24, wherein the antigen-binding fragment or antibody is a scFV directed to DEC205.
26. The strain according to claim 17, wherein the strain is at a tachyzoite stage.
27. A composition or a pharmaceutical composition comprising the strain according to claim 17, wherein said pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.
28. The composition according to claim 27, in combination with a therapeutic protein or molecule.
29. The composition according to claim 27, wherein said composition is a vaccine composition.
30. The composition according to claim 29, wherein said vaccine composition comprises an adjuvant.
31. A method of preventing and/or treating cancer or a chronic infectious disease in a subject in need thereof, wherein said chronic infectious disease is selected from chronic virus infection and chronic bacterial infection, said method comprising administering to said subject a therapeutically effective amount of the strain according to claim 17, or a composition, pharmaceutical composition or vaccine composition comprising said strain.
32. The method according to claim 31, wherein said cancer is a solid tumor.
33. The method according to claim 31, wherein said cancer is an ovarian cancer, pancreatic cancer, lung cancer, melanoma or glioblastoma.
34. The method according to claim 31, wherein said chronic infectious disease is associated with or induces an immunosuppression, and is selected from the group consisting of tuberculosis and HIV.
35. A method of producing at least one heterologous protein by a strain according to claim 17, said method comprising: a) infecting a cell with the strain, wherein the strain secretes said at least one heterologous protein, b) cultivating the infected cell of a) in a culture medium, and c) recovering the least one heterologous protein.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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EXAMPLES
[0268] The present invention is further illustrated by the following examples.
Example 1: Specific Expression of Cancer Antigen by the RH Strain and In Vivo Effects
[0269] Materials and Methods
[0270] Parasites
[0271] T. gondii strain RH tachyzoites were produced in human fibroblasts (HFFs) cultured in Dulbecco's minimal medium (DMEM) supplemented with 10% of fetal calf serum, 2 mM glutamine, 50 U/ml of penicillin and 50 μ/ml of streptomycin. They were harvested during lysis of the host cells.
[0272] Plasmid Construction of the RH-OVA
[0273] The plasmid pUC8.4 CAT/GFP-SAG1/OVA/GPI was used to construct the recombinant RH-OVA.
[0274] pUC8.4 CAT/GFP-SAG1/OVA/GPI is a pUC8 plasmid in which the sequence encoding the fusion protein SAG1-OVA including the N terminal signal sequence of SAG1 and the anchor SAG1 signal motif (GPI) is cloned in the expression cassette between PmeI and NotI sites.
[0275] pUC8 contains two expression cassettes. One was designed to express a CAT-GFP fusion protein to allow drug selection of stably transfected parasites (cassette CAT-GFP), the second was designed to express proteins of interest. The sequence encoding the protein of interest must include an ATG and a stop codon. The expression of CAT-GFP is driven by the promoter of the T. gondii α-tubulin gene (αTUB5) and the 3′ untranslated region of the T. gondii SAG1 gene. This expression cassette is bordered in the 3′ position and 5′ position by LoxP sites. These LoxP sites were added to suppress the cassette CAT-GFP from the DNA genome of the parasite by the use of a Cre recombinase which recognizes specifically these sites.
[0276] The expression of the protein of interest is driven by the promoter of the T. gondii α-tubulin gene (αTUB5) in which a five-repeat element was inserted upstream of the transcriptional start site (leading to promoter αTUB8) for high-level expression of the protein of interest (Soldati et al., 1995). The sequence of the protein of interest is cloned in PmeI/NotI sites.
[0277] Obtention of pUC8 and pUC5
[0278] Generation of the plasmid pCN1, containing a cassette to express the fusion protein SAG1-GFP driven by the promoter of the T. gondii α-tubulin gene (αTUB5) and the 3′ untranslated region of the T. gondii SAG1 gene (3′UTR SAG1):
[0279] The sequence encoding SAG1 (including the signal sequence, without the GPI anchor signal sequence) was amplified by PCR by using plasmid pcDNA3-SAG1 as the template (Mévélec et al., 2005) and the primer sequences GGTTTTGACGTCACCATGTTTCCGAAGGCAGTG (SEQ ID NO: 34) (AaTII, underlined) and TTGCTCACCATCCTAGGTGCAGCCCCGGCAAA (SEQ ID NO: 35) (AvrII, underlined). The sequence encoding GFP was amplified by PCR by using plasmid pmic3-GFP (Striepen et al., 2001) and the primer sequences TTTGCCGGGGCTGCACCTAGGATGGTGAGCAA (SEQ ID NO: 36) (AvrII, underlined) and
TABLE-US-00001 (SEQ ID NO: 37) CGGTGATTAATTAATCGAGCGGGTCCTGGTTCG
(PacI, underlined). SAG1 and GFP PCR products were digested by AaTII/AvrII and AvRII/PacI respectively and ligated into plasmid pT230TUB/Ble (Kim et al., 1993) previously digested with AaTII/PacI. In the resulting plasmid (pCN1), the sequence encoding BLE is replaced by the sequence encoding the secreted fusion protein SAG1-GFP under the control of promoter αTUB5.
[0280] Generation of the plasmid pCN5, containing a cassette expressing CAT under the promoter αTUB5, bordered by the Loxp sites in the 3′ position and 5′ position: The sequence of the cassette expressing CAT, driven by the promoter of the T. gondii a-tubulin gene (α-TUB5) and the 3′ untranslated region of the T. gondii SAG1 gene (3′UTR SAG1), was amplified by PCR by using pmic3KO-2 (Cérède et al., 2005) as the template and the primer sequences GCGGCCAAGCTTATAACTTCGTATAATGTATGCTATACGAAGTTATGATATGCAT GTCCGCgttcgtgaaatctctgatcaagcgg (SEQ ID NO: 38) (including HindIII and Loxp sequences, underlined and in italic respectively) and cgacgcacgctgtcactcaacttgctGCTAGAACTAGTGGATCCATAACTTCGTATAGCATAC ATTATACGAAGTTATCCCTCGG (SEQ ID NO: 39) (including SpeI and LoxP sequences, underlined and in italic respectively).
[0281] The PCR fragment was digested by HindIII/SpeI and cloned into pCN1 which has been previously digested by the same enzymes HindIII/SpeI. In the resulting plasmid (pCN5), the cassette expressing the fusion protein SAG1-GFP under the control of promoter αTUB5 is replaced by the cassette expressing CAT under the promoter αTUB5, bordered by the Loxp sites in the 3′ position and 5′ position.
[0282] Generation of the plasmid pUC18 CAT-GFP containing a cassette to express the fusion protein CAT-GFP driven by the promoter of the T. gondii α-tubulin gene (αTUB5) and the 3′ untranslated region of the T. gondii SAG1 gene (3′UTR SAG1). The cassette is bordered on both sides by Loxp sites:
[0283] To clone the sequence encoding GFP in fusion with the sequence encoding CAT, a fragment including LoxP (5′ position), promoter αTUB5 and the sequence encoding CAT without stop codon but with an AvrII site to clone GFP in fusion with CAT, was amplified by PCR by using pCN5 as the template and the primer sequences GTATCGATAAGCTTATAACTC (SEQ ID NO: 40) (HindIII underlined) and CACAACGGTGATTAACCTAGGAGCCCCGCCCTG (SEQ ID NO: 41) (AvrII, underlined). The amplified fragment was digested by HindIII/AvrII. The sequence encoding GFP was obtained from pCN1 by digestion with AvrII/PacI. The plasmid pCN5 was digested by HindIII/PacI to eliminate the HindIII/PacI fragment corresponding to LoxP (5′ position), promoter αTUB5 and the sequence encoding CAT. Finally the three fragments were ligated to obtain a recombinant plasmid containing the cassette expressing CAT-GFP under the promoter αTUB5, bordered by the Loxp sites in the 3′ position and 5′ position. This cassette was further cloned into pUC18 using HindIII and XbaI to obtain pUC18 CAT-GFP.
[0284] Generation of pUC8 and pUC5, containing one cassette, bordered by LoxP sites, to express the fusion protein CAT-GFP driven by the promoter of the T. gondii α-tubulin gene (αTUB5) and the 3′ untranslated region of the T. gondii SAG1 gene (3′UTR SAG1) and another one to express a protein of interest driven by a modified promoter of the T. gondii α-tubulin gene (αTUB8) and the 3′ untranslated region of the T. gondii SAG1 gene (3′UTR SAG1). In pUC5 the two cassettes are in the same orientation, in pUC8 they are in opposite side:
[0285] The expression cassette with a modified promoter of the T. gondii α-tubulin gene (αTUB8) was obtained by addition of five repeat sequences (Soldati et al., 1995), 70 bases upstream the major transcription start site in the T. gondii α-tubulin gene (αTUB5). Plasmid pT230TUB/Ble was used to obtain the promoter αTUB8 and the 3′UTR SAG1 sequences. Two enzymes restriction sites PmeI and NotI were included between the αTUB8 and 3′UTR SAG1 sequences to allow insertion of the sequence encoding the protein of interest. The XbaI restriction sites located at both end of the expression cassette were used to clone this expression cassette in pUC18 CAT-GFP in both orientations. The resulting plasmids were pUC5 with the two cassettes in the same orientation and pUC8 with the two cassettes in opposite side.
[0286] Generation of Plasmid pUC8.4 CAT/GFP-SAG1/OVA/GPI
[0287] pUC8.4 CAT/GFP-SAG1/OVA/GPI is a pUC8 plasmid in which the sequence encoding the fusion protein SAG1-OVA including the N terminal signal sequence of SAG1 and the anchor SAG1 signal motif (GPI) is cloned in the expression cassette between PmeI and NotI sites. The fusion protein is expressed under the control of αTUB8. The anchor motif was added to the C terminus of OVA to achieve the retention of the fusion protein SAG1-OVA in the plasma membrane of the parasite.
[0288] The OVA fragment encoding amino acids 140 to 386 of chicken ovalbumin was amplified by PCR from an OVA containing plasmid template (Tagliani et al., 2008) using the primer sequences CAAACACCTAGGGATCAAGCCAGAGAGC (SEQ ID NO: 42) (AvrII, underlined) and GTTCCCTAGGGGAAACACATCTGCC (SEQ ID NO: 43) (AvrII, underlined). The amplified fragment was digested with AvrII and cloned into pCN1 previously linearized with AvrII and dephosphorylated. In the resulting plasmid (pCN1-OVA), the sequence encoding OVA is inserted in frame between the sequence encoding SAG1 and the sequence encoding GFP. pCN1-OVA expresses the fusion protein SAG1-OVA-GFP driven by the promoter of the T. gondii α-tubulin gene (αTUB5) and the 3′ untranslated region of the T. gondii SAG1 gene (3′UTR SAG1).
[0289] The sequence encoding the fusion protein SAG1-OVA (including the Kozak sequence, the start ATG codon and the N terminal signal sequence of SAG1, without the stop codon) was amplified by PCR using pCN1-OVA as the template and the primer sequences GGTGCTCACCGGTTTAAACGTCGAAAATGTTTCCG (SEQ ID NO: 44) (PmeI, underlined) and CTCACCATTCTAGAGGAAACACATCTGC (SEQ ID NO: 45) (XbaI, underlined). The sequence encoding the anchor signal (GPI), including the stop codon, was amplified by PCR using pcDNA3-SAG1 (Mevelec et al., 2005) and the primer sequences CTCATCTCTAGAGAGGATCTGGCTGCGGG (SEQ ID NO: 46) (XbaI, underlined) and
TABLE-US-00002 (SEQ ID NO: 47) ACCATGGAAGCGGCCGCTTACGCGACA
(NotI, underlined). SAG1-OVA and GPI PCR products were digested with XbaI and ligated. The ligated product was amplified with GGTGCTCACCGGTTTAAACGTCGAAAATGTTTCCG (SEQ ID NO: 44) (PmeI, underlined) and
TABLE-US-00003 (SEQ ID NO: 47) ACCATGGAAGCGGCCGCTTACGCGACA
(NotI, underlined) and cloned into plasmid pGEMT (Promega). The resulting plasmid pGEMT-SAG1-OVA-GPI was then digested with PmeI/NotI to get the fragment encoding SAG1-OVA-GPI. The SAG1-OVA-GPI fragment was cloned in pUC8 previously opened by PmeI/NotI.
[0290] The resulting plasmid pUC8.4 CAT/GFP-SAG1/OVA/GPI expresses the membrane anchored fusion protein SAG1-OVA under the control of promoter αTUB8 and the fusion protein CAT-GFP under the control of promoter αTUB5 to allow drug selection of stably transfected parasites.
[0291] Recombinant Toxoplasma gondii Strain
[0292] Transfections were performed with 10.sup.7 parasites in a volume of 800 μl of cytomix (Van den Hoff et al., 1992) containing 3 mM ATP and 3 mM glutathione and 20 ρg of purified plasmid DNA (the plasmids were purified using the Qiagen Kit®), linearized with PciI. Electroporations were performed in disposable cuvettes (4 mm gap) with an electroporator Biorad (electroporation settings: 2000 V, 50 ohms, 25 mF). After electroporation, the parasites are kept in the hood, for 15 min at room temperature and then transferred to a fresh culture of fibroblast monolayers (25 cm.sup.2 flask). After 24 hours the parasite are subjected to 20 mM chloramphenicol selection. After 10 to 15 days of selection, the parasites are cloned by limiting dilution, in the wells of a 96-well plate, of HFF cells, in the presence of selection agent, and the clones selected are amplified.
[0293] Immunofluorescence Assays
[0294] T. gondii tachyzoites filtered through a 5 μM Nucleopore filter, pelleted by centrifugation, washed 2 times with PBS, were air dried on standard IFA slides (10.sup.5 parasites/well) and were stored at −20° C. Immunofluorescence assays were performed at 37° C. after 2 min fixation in cold acetone (−20° C.). Fixed tachyzoites were washed in PBS and incubated 60 mM with polyclonal anti-chicken egg antibodies produced in rabbit (Sigma, 1:50). Slides were then washed with PBS and incubated 60 min with a 1:1000 dilution in PBS of Alexa Fluor 568-conjugated goat anti-rabbit IgG antibodies. After washes in PBS, the visualization was carried out using a Leica microscope.
[0295] Mice
[0296] Twenty-four week-old female C57BL/6 mice were purchased from CER Janvier (Le Genest Saint Isle, France) and maintained under pathogen-free conditions in the animal house of the University of Tours. Experiments were carried out in accordance with the guideline for animal experimentation (EU Directive 2010/63/EU) and the protocol was approved by the local ethics committee (CEEA VdL).
[0297] Toxoplasma gondii Strains (RH-OVA)
[0298] Strain RH-OVA tachyzoites were produced in HFFs cultured in DMEM (Pan Biotech GmbH) supplemented with 10% of heat-inactivated FCS (Dutscher), 2 mM glutamine (Pan Biotech GmbH), 50 U/ml of penicillin and 50 μ/ml of streptomycin (Pan Biotech GmbH) at 37° C. in 5% CO2 atmosphere. They were harvested during lysis of the host cells by centrifugation at 600 g for 10 min.
[0299] Tumor Cells and Tumor Cell Inoculation (EG7)
[0300] EG7 cells (EL4-OVA thymoma cells transfected with chicken albumin cDNA) are cultured in Roswell Park Memorial Institute medium (RPMI, Pan Biotech GmbH) with 5×10.sup.5 M of 2-mercaptoethanol, 50 UI/mL of penicillin and 50 mg/mL of streptomycin. 5×10.sup.5 live EG7 cells are inoculated subcutaneously in the right flank of mice. Tumor diameters are measured 3 times weekly, and mice are euthanized when tumor diameters reached 25,000 mm.sup.3.
[0301] T. gondii Administration
[0302] Mice are injected subcutaneously in the right flank at day 4 and again at day 7 with 5×10.sup.2 freshly isolated tachyzoites of RH ctrl and RH-OVA strain of T. gondii.
[0303] Spleens and Lymph Nodes Cytokine Measurements
[0304] Single cell suspensions were obtained from spleens and lymph nodes first pressed and then filtered through a nylon mesh. Hypotonic shock was used to remove erythrocytes. The cells were then resuspended in RPMI medium supplemented with 5% FBS, 25 mM HEPES, 2 mM glutamine, 1 mM sodium pyruvate, 50 μM 2 β-mercaptoethanol and 1 mM penicillin-streptomycin. Cells were dispensed, in triplicate, into 96-well culture plates (5 10.sup.5 cells/well) and cultured for 72 hours with or without 10 μg/mL OVA. Concanavalin A (5 μg/mL) was used as positive control of proliferation. Cytokine productions in supernatants from restimulated splenocytes and lymph nodes cells were evaluated using cytometric bead array for IFN-γ, TNF-α, Il-2, Il-4, Il-5, Il-10, Il-17A, IL-23 and GM-CSF (MACSPlex Cytokine 10 kit mouse, Miltenyi Biotec GmbH, Germany), commercial ELISA kits for TGF-β and IL-15 (Invitrogen, Thermo Fisher Scientific, U.S.A.).
[0305] Tumor Cytokine Measurements
[0306] Tumors were cut into small pieces of 2-4 mm and dissociated with Tumor Dissociation Kit and gentleMACS Dissociator (Miltenyi Biotech GmbH, Germany) Following dissociation, cells were pelleted and supernatant were collected for cytokine analysis. Cytokines were quantified in the supernatant using cytometric bead array for IFN-γ, TNF-α, Il-2, Il-4, Il-5, Il-10, Il-17A, IL-23 and GM-CSF (MACSPlex Cytokine 10 kit mouse, Miltenyi Biotec GmbH, Germany), commercial ELISA kits for TGF-β and IL-15 (Invitrogen, Thermo Fisher Scientific, U.S.A.).
[0307] Cell Populations Phenotyping
[0308] Spleen and tumor cell phenotypes were analyzed by flow cytometry. Spleen cells were obtained as described for cytokine measurements. Spleen cells were then pelleted and resuspended in PBS, 2 mM EDTA, 1% FBS for staining and cytometry analysis. Red blood cells from tumor cells obtained as described for cytokine measurements were lysed using red blood cell lysis buffer and cells were resuspended in PBS, 2 mM EDTA, 1% FBS for staining and cytometry analysis. Cells were stained with monoclonal antibodies purchased from Miltenyi Biotec GmbH. The following antibodies were used: REA clones of anti-mouse CD3-APC-Vio770 (REA606), CD4-Vioblue (REA 604), CD8a-PE-Vio770 (REA 601), Foxp3-Vio515 (REA 788), NKp46-APC (REA 815), CD11c-PE (REA 754), CD11b-APC-Vio770 (REA 592), Ly6C-Vioblue (REA 796), Ly6G-PE-Vio770 (REA 526). Foxp3 staining was performed using Foxp3 Staining Buffer Set (Miltenyi Biotech GmbH, Germany) FACS analysis was performed using a Miltenyi 8-color MACSQuant, and data were analyzed using Flowlogic (Milteni Biotech GmbH, Germany).
[0309] In Vitro Generation of Tolerogenic Dendritic Cells (DCs)
[0310] Bone marrow dendritic cells were obtained essentially as described previously (Madaan et al., Journal of Biological Methods, Vol. 1, 2014). Bone marrow cells collected from femur and tibias of mice were plated at 4×10.sup.6 cells/mL in low petri dishes and cultured at 37° C., 5% CO.sub.2 in 10 mL complete medium [RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum (FBS), 10 mM HEPES, 1 mM pyruvate, 2 mM 1-glutamine, 100 U/mL penicillin, streptomycin (All from PAN BioTech), 1% non-essential amino acids (GIBCO), and 50 μM 2β-mercaptoethanol], and 5 ng/mL of GMCSF from J558L cells supernatant to generate mouse bone marrow dendrtiic cells. Medium was replaced every 2 days. To promote tolerogenic dendritic cells, 1,25dihydroxyvitamin D3 (Sigma) was added on days 0, 2, 4, 6 at 10.sup.−8M as previously described (Ferreira et al., Journal of Immunology, 192(9):4210-20, 2014). Cells were recovered at day 7 and plated at 5.10.sup.5 cells/ml in 24-wells culture plates in a final volume of 1 ml of RPMI containing 2% FBS, 10 mM HEPES, 1 mM pyruvate, 2 mM 1-glutamine, 100 U/mL penicillin, streptomycin and 50 μM 2β-mercaptoethanol]. Parasites were added at multiplicity of infection (MOI) 1 (5.10.sup.5) or 2 (10.sup.6) and incubated for an additional 18 hours period. Supernatants were collected and 11-12 and IL-10 cytokine productions were evaluated using commercial ELISA kits according to the manufacturer's instructions (Invitrogen, Thermo Fisher).
[0311] Results
[0312] SAG1-OVA Plasmid Construct
[0313] The plasmid pUC8.4 was constructed as described in
[0314] Briefly, plasmid pUC8.4 is a pUC18 plasmid in which two cassettes have been cloned. One cassette encodes the fusion protein CAT-GFP to select stably transfected tachyzoites with chloramphenicol. The sequence encoding the fusion protein CAT-GFP is flanked by the promoter T. gondii tubulin gene (a TUB 5) and the 3′ untranslated region of the SAG1 gene (3′UTR SAG1). The second cassette expresses a fusion protein consisting of the complete T. gondii SAG1 fused to the amino acids 140-386 of chicken ovalbumin enchored in the plasma membrane. To achieve the retention of SAG1-OVA in the plasma membrane, the SAG1 GPI motif has been added to the C terminus of OVA. The sequence encoding the fusion protein SAG1-OVA and the GPI motif is flanked by a modified promoter T. gondii tubulin gene (αTUB8) and the 3′ untranslated region of the SAG1 gene (3′UTR SAG1). Then, the T. gondii strain RH tachyzoites were transfected with plasmid pUC8.4.
[0315] Expression of the Fusion Protein SAG1-OVA
[0316] An immunofluorescence assay was preformed to confirm that the transfected RH strains express SAG1-OVA on their cell surface. Briefly, air dried, free T. gondii tachyzoites non-transfected (RH) or transfected (RH-OVA) with the plasmid PUC8.4 were fixed on slides with acetone, washed with PBS and stained with rabbit anti-chicken ovalbumin followed by ALEXA 568-conjugated goat anti-rabbit antibodies. Slides were examined under a Leica microscope.
[0317] As shown in
[0318] RH-OVA Treatment Suppresses and/or Regresses an Established Solid Tumor Development
[0319] As shown in
[0320] Moreover, a significant reduction of tumor implantation was observed in mice treated with RH-OVA (
[0321] All these results suggest that a sub-cutaneous injection at peritumoral level of RH-OVA tachyzoites exhibited good efficacy against tumor development and seems to confirm the benefit to express specific tumor antigen by our construct.
[0322] RH-OVA Induces a Protective Immune Response Against Tumor Development
[0323] The administration of RH-OVA tachyzoites at the site of the tumor, D4 and D7 post EG-7 thymoma injection induce protective immune responses at splenic and tumor levels.
[0324] a) Systemic Compartment (i.e., Spleen)
[0325] As described in
[0326] Concerning IFN-γ, we observe an increase of secretion in spleen (
[0327] Concerning spleen myeloid cell sub-populations, we observe a significant increase of CD11c+/CMH II+ cells (dendritic cells) (
[0328] For spleen lymphoid cell sub-populations, as shown in
[0329] b) Tumor Compartment
[0330] As described in
[0331] Concerning tumor myeloid cell sub-populations, we observe a significant increase of Ly6C+Ly6G % (neutrophils) (
[0332] For tumor lymphoid cell sub-populations, as shown in
[0333] RH-OVA Reactivates Tumor-Altered Dendritic Cells
[0334] The active form of the vitamin D3, 1,25-Dihydroxyvitamin D3 (1.25-(OH).sub.2D.sub.3) can affect the function and development of monocytes and dendritic cells (DCs). Thus, DCs differentiated in the presence of 1,25-vitD3 share several features with tolerogenic DCs like low surface expression of MHC class II and costimulatory molecules (e.g., CD40, CD80, and CD86), decreased production of IL-12, and enhanced secretion of IL-10.
[0335] Indeed, as shown in
[0336] Interestingly, parasites adjunction on the TolDCs partially restored the IL12p40 secretion and decreased the IL10 levels. No significant difference was observed in cytokines secretion between type 1 (RH-OVA) and type 2 (Me49) parasites. No TGFβ secretion was detected and IL6 levels were not significantly different between the control DCs or TolDCs (data not shown).
[0337] These results suggest that parasites are able to reverse the tolerogenicity induced by 1.25(OH).sub.2D3.
Example 2: Specific Targeting of Dendritic Cells by the Strain and In Vivo Effects
[0338] Materials and Methods
[0339] Parasites
[0340] T. gondii strain RH tachyzoites were produced in human fibroblasts (HFFs) cultured in Dulbecco's minimal medium (DMEM) supplemented with 10% of fetal calf serum, 2 mM glutamine, 50 U/ml of penicillin and 50 μ/ml of streptomycin. They were harvested during lysis of the host cells
[0341] Plasmid Construction of the RH-DC2 and RH-DC2-SAG1
[0342] The plasmids pUC8SSDC2GPI and pUC8SSDC2SAG1GPI were used to construct the recombinant RH-DC2 and RH-DC2-SAG1 respectively.
[0343] pUC8SSDC2GPI is a pUC8 plasmid in which the sequence encoding the membrane anchored ScFv DEC 205 (DC2) including a HA tag, is cloned in the expression cassette between PmeI and NotI sites.
[0344] pUC8SSDC2SAG1GPI is a pUC8 plasmid in which the sequence encoding the membrane anchored SAG1 ScFv DEC 205 (DC2 SAG1) including a HA tag is cloned in the expression cassette between PmeI and NotI sites.
[0345] pUC8 contains two expression cassettes. One was designed to express a CAT-GFP fusion protein to allow drug selection of stably transfected parasites (cassette CAT-GFP), the second was designed to express proteins of interest. The sequence encoding the protein of interest must include an ATG and a stop codon. The expression of CAT-GFP is driven by the promoter of the T. gondii α-tubulin gene (αTUB5) and the 3′ untranslated region of the T. gondii SAG1 gene. This expression cassette is bordered in the 3′ position and 5′ position by LoxP sites. These LoxP sites were added to suppress the cassette CAT-GFP from the DNA genome of the parasite by the use of a Cre recombinase which recognizes specifically these sites.
[0346] The expression of the protein of interest is driven by the promoter of the T. gondii α-tubulin gene (αTUB5) in which a five-repeat element was inserted upstream of the transcriptional start site (leading to promoter αTUB8) for high-level expression of the protein of interest (Soldati et al., 1995). The sequence of the protein of interest is cloned in PmeI/NotI sites.
[0347] Obtention of pUC8 and pUC5
[0348] Generation of the plasmid pCN1, containing a cassette to express the fusion protein SAG1-GFP driven by the promoter of the T. gondii α-tubulin gene (αTUB5) and the 3′ untranslated region of the T. gondii SAG1 gene (3′UTR SAG1): The sequence encoding SAG1 (including the signal sequence, without the GPI anchor signal sequence) was amplified by PCR by using plasmid pcDNA3-SAG1 as the template (Mevelec et al., 2005) and the primer sequences GGTTTTGACGTCACCATGTTTCCGAAGGCAGTG (SEQ ID NO: 34) (AaTII, underlined) and TTGCTCACCATCCTAGGTGCAGCCCCGGCAAA (SEQ ID NO: 35) (AvrII, underlined). The sequence encoding GFP was amplified by PCR by using plasmid pmic3-GFP (Striepen et al., 2001) and the primer sequences TTTGCCGGGGCTGCACCTAGGATGGTGAGCAA (SEQ ID NO: 36) (AvrII, underlined) and
TABLE-US-00004 (SEQ ID NO: 37) CGGTGATTAATTAATCGAGCGGGTCCTGGTTCG
(PacI, underlined). SAG1 and GFP PCR products were digested by AaTII/AvrII and AvRII/PacI respectively and ligated into plasmid pT230TUB/Ble (Kim et al., 1993) previously digested with AaTII/PacI. In the resulting plasmid (pCN1), the sequence encoding BLE is replaced by the sequence encoding the secreted fusion protein SAG1-GFP under the control of promoter αTUB5.
[0349] Generation of the plasmid pCN5, containing a cassette expressing CAT under the promoter αTUB5, bordered by the Loxp sites in the 3′ position and 5′ position: The sequence of the cassette expressing CAT, driven by the promoter of the T. gondii a-tubulin gene (α-TUB5) and the 3′ untranslated region of the T. gondii SAG1 gene (3′UTR SAG1), was amplified by PCR by using pmic3KO-2 (Cérède et al., 2005) as the template and the primer sequences GCGGCCAAGCTTATAACTTCGTATAATGTATGCTATACGAAGTTATGATATGCAT GTCCGCgttcgtgaaatctctgatcaagcgg (SEQ ID NO: 38) (including HindIII and Loxp sequences, underlined and in italic respectively) and cgacgcacgctgtcactcaacttgctGCTAGAACTAGTGGATCCATAACTTCGTATAGCATAC ATTATACGAAGTTATCCCTCGG (SEQ ID NO: 39) (including SpeI and LoxP sequences, underlined and in italic respectively).
[0350] The PCR fragment was digested by HindIII/SpeI and cloned into pCN1 which has been previously digested by the same enzymes HindIII/SpeI. In the resulting plasmid (pCN5), the cassette expressing the fusion protein SAG1-GFP under the control of promoter αTUB5 is replaced by the cassette expressing CAT under the promoter αTUB5, bordered by the Loxp sites in the 3′ position and 5′ position.
[0351] Generation of the plasmid pUC18 CAT-GFP containing a cassette to express the fusion protein CAT-GFP driven by the promoter of the T. gondii α-tubulin gene (αTUB5) and the 3′ untranslated region of the T. gondii SAG1 gene (3′UTR SAG1). The cassette is bordered on both sides by Loxp sites:
[0352] To clone the sequence encoding GFP in fusion with the sequence encoding CAT, a fragment including LoxP (5′ position), promoter αTUB5 and the sequence encoding CAT without stop codon but with an AvrII site to clone GFP in fusion with CAT, was amplified by PCR by using pCN5 as the template and the primer sequences GTATCGATAAGCTTATAACTC (SEQ ID NO: 40) (HindIII underlined) and CACAACGGTGATTAACCTAGGAGCCCCGCCCTG (SEQ ID NO: 41) (AvrII, underlined). The amplified fragment was digested by HindIII/AvrII. The sequence encoding GFP was obtained from pCN1 by digestion with AvrII/PacI. The plasmid pCN5 was digested by HindIII/PacI to eliminate the HindIII/PacI fragment corresponding to LoxP (5′ position), promoter αTUB5 and the sequence encoding CAT. Finally the three fragments were ligated to obtain a recombinant plasmid containing the cassette expressing CAT-GFP under the promoter αTUB5, bordered by the Loxp sites in the 3′ position and 5′ position. This cassette was further cloned into pUC18 using HindIII and XbaI to obtain pUC18 CAT-GFP.
[0353] Generation of pUC8 and pUC5, containing one cassette, bordered by LoxP sites, to express the fusion protein CAT-GFP driven by the promoter of the T. gondii α-tubulin gene (αTUB5) and the 3′ untranslated region of the T. gondii SAG1 gene (3′UTR SAG1) and another one to express a protein of interest driven by a modified promoter of the T. gondii α-tubulin gene (αTUB8) and the 3′ untranslated region of the T. gondii SAG1 gene (3′UTR SAG1). In pUC5 the two cassettes are in the same orientation, in pUC8 they are in opposite side:
[0354] The expression cassette with a modified promoter of the T. gondii α-tubulin gene (αTUB8) was obtained by addition of five repeat sequences (Soldati et al., 1995), 70 bases upstream the major transcription start site in the T. gondii α-tubulin gene (αTUB5). Plasmid pT230TUB/Ble was used to obtain the promoter αTUB8 and the 3′UTR SAG1 sequences. Two enzymes restriction sites PmeI and NotI were included between the αTUB8 and 3′UTR SAG1 sequences to allow insertion of the sequence encoding the protein of interest. The XbaI restriction sites located at both end of the expression cassette were used to clone this expression cassette in pUC18 CAT-GFP in both orientations. The resulting plasmids were pUC5 with the two cassettes in the same orientation and pUC8 with the two cassettes in opposite side.
[0355] Generation of the Plasmid pUC8SSDC2GPI
[0356] pUC8SSDC2GPI expresses the membrane anchored ScFv DEC 205 (DC2) protein, including a HA tag, under the control of promoter αTUB8 and CAT-GFP under the control of promoter αTUB5. The sequence encoding the membrane anchored ScFv DEC 205 (DC2) including a HA tag, is cloned in the PmeI and NotI sites of pUC18.
[0357] The sequence encoding the N terminal signal sequence of SAG1 (SEQ ID NO: 21), including the Kozak sequence and the start ATG was amplified by PCR using pGEMT-SAG1-OVA-GPI as the template and the primer sequences G GTG CTCA CCG GTT TAA ACG TCG AAA ATG TTT CCG (SEQ ID NO: 44) (PmeI, underlined) and GGC AAC ACT AGT GGG ATC CGA TGC (SEQ ID NO: 48) (SpeI, underlined). The sequence encoding the ScFv anti-DEC 205 including an N-terminal HA (hemagglutinin) tag (SEQ ID NO: 33), and a linker at the C terminal end (SEQ ID NO: 28) was amplified by PCR using pMT DA2.2 (from our laboratory: this plasmid contains the VH and VL regions of monoclonal antibody NLDC 145, obtained by RT-PCR from total RNA of hybridome NLDC 145 ATCC 1996) as the template with the primer sequences CTCGGGACTAGTTACCCATACGATGTTCCAGATTACGCTCAGGTGCAGCTGC AGGAGAG (SEQ ID NO: 49) (SpeI and HA sequences, underlined and in italic respectively) and ATTGGCCACTCTAGAGCTAGCGCCTCCGCCCTTCAGC (SEQ ID NO: 50) (XbaI and liker, underlined and in italic respectively). SAG1 N terminal signal sequence and ScFv PCR products were digested with SpeI and ligated. The ligated product was amplified with
TABLE-US-00005 (SEQ ID NO: 44) G GTG CTCA CCG GTT TAA ACG TCG AAA ATG TTT CCG
(PmeI, underlined) and ATTGGCCACTCTAGAGCTAGCGCCTCCGCCCTTCAGC (XbaI and linker, underlined and in italic respectively) and cloned into plasmid pGEMT (Promega). The resulting plasmid pGEMT-SS SAG1-HA-ScFv-linker was then digested with PmeI/XbaI to get the fragment encoding SS SAG1-HA-ScFv-linker. The sequence encoding the SAG1 anchor signal (GPI) (SEQ ID NO: 18), including the stop codon, was amplified by PCR using pcDNA3-SAG1 (Mévélec et al., 2005) and the primer sequences CTCATCTCTAGAGAGGATCTGGCTGCGGG (SEQ ID NO: 46) (XbaI, underlined) and
TABLE-US-00006 (SEQ ID NO: 47) ACCATGGAAGCGGCCGCTTACGCGACA
(NotI, underlined). The fragment encoding SS SAG1-HA-ScFv-linker obtained from pGEMT-SS SAG1-HA-ScFv-linker and the GPI PCR product were digested with XbaI, ligated, then amplified by PCR with the sequence primers G GTG CTCA CCG GTT TAA ACG TCG AAA ATG TTT CCG (SEQ ID NO: 44) (PmeI, underlined) and ACCATGGAAGCGGCCGCTTACGCGACA (SEQ ID NO: 47) (NotI, underlined). The amplified fragment was cloned into pGEMT (Promega) to obtain pGEMT-SS SAG1-HA-ScFv-linker-GPI. Finally, pGEMT-SS SAG1-HA-ScFv-linker-GPI was digested with PmeI/NotI to get the fragment encoding SS SAG1-HA-ScFv-linker-GPI (DC2) and the DC2 fragment was inserted into pUC8, previously opened by PmeI/NotI. The resulting plasmid pUC8SSDC2GPI expresses the membrane anchored ScFv DEC 205 (DC2) including a HA tag under the control of promoter αTUB8 and CAT-GFP under the control of promoter αTUB5.
[0358] Generation of the Plasmid pUC8SSDC2SAG1GPI
[0359] pUC8SSDC2SAG1GPI expresses the membrane anchored SAG1 ScFv DEC 205 (DC2) protein, including a HA tag, under the control of promoter αTUB8 and CAT-GFP under the control of promoter αTUB5. The sequence encoding the membrane anchored SAG1 ScFv DEC 205 (DC2) including a HA tag, is cloned in the PmeI and NotI sites of pUC18. To obtain this plasmid, the sequence encoding SAG1 without the N terminal signal sequence and without the stop codon was cloned in frame at the C terminus of the linker (located at the C terminus of the sequence encoding ScFv DEC 205) using the XbaI site.
[0360] The sequence encoding SAG1 without the N terminal signal sequence and without a stop codon was amplified by PCR using pGEMT-SAG1-OVA-GPI as the template and the primer sequences ATGGCATCGTCTAGACCTCTTGTTGCCAAT (SEQ ID NO: 51) (XbaI underlined) and CCTAGGTGCTCTAGAGGCAAACTCCAG (SEQ ID NO: 52) (XbaI underlined). The PCR product was cloned into pGEMT (Promega). The resulting plasmid pGEMT-SAG1t was digested with XbaI to get the fragment encoding the truncated SAG1 protein (SEQ ID NO: 23). This fragment was then cloned in pGEMT SS DC2 GPI previously opened with XbaI. pGEMT SS DC2 SAG1 GPI with SAG1 in the proper orientation was selected by restriction mapping. Finally, pGEMT SS DC2 SAG1 GPI with SAG1 in the proper orientation was digested by PmeI/NotI to get the fragment encoding SS SAG1-HA-ScFv-SAG1-linker-GPI (DC2 SAG1) and this fragment was inserted into pUC8, previously opened by PmeI/NotI. The resulting plasmid pUC8SSDC2SAG1GPI expresses the membrane anchored ScFv DEC 205 (DC2 SAG1) including a HA tag under the control of promoter αTUB8 and CAT-GFP under the control of promoter αTUB5.
[0361] Western Blot
[0362] Electrophoresis (SDS-PAGE) was performed according to Laemmli. The parasites were heated before electrophoresis in sample buffer with SDS and 0.1 M DTT (reduced conditions). The lysates (2 10.sup.5 or 5 10.sup.5 tachyzoites/well) were separated on 10% acrylamide gels by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose and were probed as described previously (Chardes et al., Infection and Immunity, 1990) with rabbit anti-HA polyclonal antibodies (1:200, ThermoFisher Scientific) followed by a mouse monoclonal anti-rabbit IgG (γ-chain specific) alkaline phosphatase conjugate (1:4000, Sigma). Alkaline phosphatase activity was detected using the 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) liquid substrate system (Sigma). Molecular mass standards ProSieveQuadColor (Lonza) were used.
[0363] ELISA on T. gondii Parasites
[0364] ELISA was performed on whole tachyzoites, essentially as described previously (Chardes et al., Infection and immunity, 1990). Flat bottomed wells (96-well plate, NUNC) were coated with 2 10.sup.5 parasites/well in PBS. After centrifugation at 200×g and 4° C. for 5 min, 25 μL of 0.5% of glutaraldehyde in cold PBS was added to each well and left for 8 min at room temperature. The plates were washed twice in PBS and saturated with PBS-4% BSA for 1 h at 37° C. Rabbit anti-HA polyclonal antibodies (1:400, ThermoFisher Scientific) or serum from infected T. gondii rabbit (1:2000) in PBS-1% BSA were incubated for 1 h at 37° C. After 3 washes in PBS-0.05% Tween, mouse monoclonal anti-rabbit IgG (γ-chain specific) alkaline phosphatase conjugate (1:4000 in PBS-BSA 1%, Sigma) was added to each well and incubated for 1 h at 37° C. After 3 washes with PBS-0.05% Tween, bound phosphatase activity was measured with p-nitrophenylphosphate (Sigma) (1 mg/ml in DEA-HCl 1 M bu.er pH 9.8).
[0365] ELISA on Recombinant Murine DEC205 Protein
[0366] Coding sequence for the N-Terminal part of the murine DEC205, which binds NLDC145 (Srimpton et al., Mol. Immunol., 2009) was purchased from Geneart. The synthetic gene was inserted in the plasmid vector pMT/BiP/V5 (Invitrogen) using BglII and NheI restriction enzymes and a Tween StrepTag was introduced at the C-Terminal end using NheI and XhoI restriction enzymes. The N-Terminal part of murine DEC205 was named (CF14), produced in the Drosophila Scheinder 2 cell line and purified with the (Twin)-Strep-tag kit according to the manufacturer's instructions (Iba Solutions For Life Sciences).
[0367] ELISA was performed by standards procedures. Briefly, the 96 flat-bottom wells of microtiter plates (Maxisorp; Nunc, Roskilde, Danemark) were coated overnight at 4° C. with recombinant CF14) at 5 μg/ml in PBS. The plates were washed with PBS, nonspecific binding sites were blocked with PBS containing 4% bovine serum albumin (PBS-4% BSA) for 1 h at 37° C. After 3 washes with PBS, tachyzoites (5 10.sup.5/well) in PBS were added, centrifuged at 200×g for 5 min and incubated for 1 h30 at 37° C. After 6 washes with PBS-Tween 0.05%, serum from infected T. gondii rabbit (1:400) in PBS-1% BSA was added in each \veil and incubated for 1 h at 37° C. After 3 washes with PBS-0.05% Tween, mouse monoclonal anti-rabbit IgG (7-chain specific) alkaline phosphatase conjugate (1:4000 in PBS-BSA 1%, Sigma) was added to each well and incubated for h at 37° C. After 3 washes with PBS-0.05% Tween, bound phosphatase activity was measured with p-nitrophenylphosphate (Sigma) (1 mg/ml in DEA-HCl 1 M buffer pH 9.8).
[0368] Flow Cytometry Analysis
[0369] Binding of RH strains to mutuDC cells was determined by flow cytometry. MutuDC 1950 cells (a murine dendritic cell line, DEC205.sup.+) were cultured as previously described (Fuertes Marraco et al., 2012). Aliquots of 10.sup.6 mutuDC cells in cold PBS 5% FBS were mixed with 2×10.sup.6 parasites (MOI 2) and incubated for 1 hour on ice. Unbound parasites were removed by three washes at 100xg for 5 min. The cells transferred in a 96-well plate round bottom, were incubated with an anti-FcγR mAb (clone 2.4G2, eBioscience) to block nonspecific binding and further incubated for 30 min on ice with monoclonal antibody T4 2E12, specific for T. gondii tachyzoite surface glycoprotein, gp23 (Tomavo et al., 1993). Cells were washed with cold PBS 5% FBS and then stained with APC-conjugated anti-mouse IgG for 30 min on ice. The fluorescence intensity of the cells was determined by flow cytometry (MACS Quant, Miltenyi Biotec).
[0370] Immunofluorescence Assays
[0371] For immunofluorescence assays, MutuDC 1950 cells (a murine dendritic cell line, DEC205+) were cultured as previously described (Fuertes Marraco et al., 2012). MutuDC cells were seeded on coverslips overnight. After washes with PBS, cells were fixed with a 3% paraformaldehyde solution for 30 min Cells were washed 3 times with PBS and saturated during 1 h in PBS-10% FBS, and then incubated with 106 tachyzoites for 30 min at room temperature. Cells were washed 3 times with PBS to eliminate unspecific tachyzoite fixation and incubated with serum from T. gondii infected rabbit (1:100). After washes with PBS, bound tachyzoites were detected using Alexa Fluor 568-conjugated goat anti-rabbit IgG antibodies (1:1,000, Molecular Probes). After washes in PBS, the visualization was carried out using a Leica microscope.
[0372] Mice
[0373] Twenty-four-week-old female C57BL/6 mice were purchased from CER Janvier (Le Genest Saint Isle, France) and maintained under pathogen-free conditions in the animal house of the University of Tours. Experiments were carried out in accordance with the guideline for animal experimentation (EU Directive 2010/63/EU) and the protocol was approved by the local ethics committee (CEEA VdL).
[0374] Toxoplasma gondii Strains (RH-DC2 and RH-DC2-SAG1)
[0375] T. gondii strain RH control, RH-DC2 and RH-DC2-SAG1 tachyzoites were produced in HFFs cultured in DMEM (Pan Biotech GmbH) supplemented with 10% of heat-inactivated FCS (Dutscher), 2 mM glutamine (Pan Biotech GmbH), 50 U/ml of penicillin and 50 μ/ml of streptomycin (Pan Biotech GmbH) at 37° C. in 5% CO2 atmosphere. They were harvested during lysis of the host cells by centrifugation at 600 g for 10 min.
[0376] Tumor Cells and Tumor Cell Inoculation (EG7)
[0377] EG7 cells (EL4-OVA thymoma cells transfected with chicken albumin cDNA) are cultured in Roswell Park Memorial Institute medium (RPMI, Pan Biotech GmbH) with 5×10.sup.5 M of 2-mercaptoethanol, 50 UI/mL of penicillin and 50 mg/mL of streptomycin. 5×10.sup.5 live EG7 cells are inoculated subcutaneously in the right flank of mice. Tumor diameters are measured 3 times weekly, and mice are euthanized when tumor diameters reached 25,000 mm.sup.3.
[0378] T. gondii Administration
[0379] Mice are injected subcutaneously in the right flank at day 4 and again at day 7 with 5×10.sup.2 freshly isolated tachyzoites of RH ctrl and RH-DC2-SAG1 strain of T. gondii.
[0380] Tumor Cytokine Measurements
[0381] Tumors were cut into small pieces of 2-4 mm and dissociated with Tumor Dissociation Kit and gentleMACS Dissociator (Miltenyi Biotech GmbH, Germany) Following dissociation, cells were pelleted and supernatant were collected for cytokine analysis. Cytokines were quantified in the supernatant using cytometric bead array for IFN-γ, TNF-α, Il-2, Il-4, Il-5, Il-10, Il-17A, IL-23 and GM-CSF (MACSPlex Cytokine 10 kit mouse, Miltenyi Biotec GmbH, Germany), commercial ELISA kits for TGF-β and IL-15 (Invitrogen, Thermo Fisher Scientific, USA) and for IL12p40 and IL-6 (eBioscience).
[0382] Cell Populations Phenotyping
[0383] Spleen and tumor cell phenotypes were analyzed by flow cytometry. Spleen cells were obtained as described in Example 1. Spleen cells were then pelleted and resuspended in PBS, 2 mM EDTA, 1% FBS for staining and cytometry analysis. Red blood cells from tumor cells obtained as described for cytokine measurements were lysed using red blood cell lysis buffer and cells were resuspended in PBS, 2 mM EDTA, 1% FBS for staining and cytometry analysis. Cells were stained with monoclonal antibodies purchased from Miltenyi Biotec GmbH. The following antibodies were used: REA clones of anti-mouse CD3-APC-Vio770 (REA606), CD4-Vioblue (REA 604), CD8a-PE-Vio770 (REA 601), Foxp3-Vio515 (REA 788), NKp46-APC (REA 815), CD11c-PE (REA 754), CD11b-APC-Vio770 (REA 592), Ly6C-Vioblue (REA 796), Ly6G-PE-Vio770 (REA 526). Foxp3 staining was performed using Foxp3 Staining Buffer Set (Miltenyi Biotech GmbH, Germany) FACS analysis was performed using a Miltenyi 8-color MACS Quant, and data were analyzed using Flowlogic (Milteni Biotech GmbH, Germany).
[0384] Results
[0385] ScFv-DEC205 Plasmid Constructs
[0386] The two constructions of the scFv anti-DEC205 are described in
[0387] As shown in
[0388] As shown in
[0389] Cell Surface Expression of scFv-DEC205
[0390] A Western-blot analysis was performed to analyze the expression of the scFv anti-DEC205 (DC2) and the scFv anti-DEC205 fused to SAG1 (DC2 SAG1) proteins in various selected clones of T. gondii (
[0391] As shown in
[0392] An ELISA analysis was further performed on various selected clones of RH-DC2-SAG1 (
[0393] As shown in
[0394] RH-DEC205 Targets the DEC205 Receptor
[0395] The ability of RH-DC2 and RH-DC2-SAG1 strains to bind to DEC205 receptor was analyzed by ELISA. The N-Terminal part of the murine DEC205 receptor, which binds NLDC145 (Srimpton et al., Mol. Immunol., 2009) was produced in the Drosophila Scheinder 2 cell line (CF14 recombinant protein) and used to coat flat bottomed wells. Following incubation and washing steps, the bound parasites were probed with a serum from a T. gondii infected rabbit. Results are expressed as optical density (OD).
[0396] As shown in
[0397] RH-DEC205 Targets Dendritic Cells
[0398] The ability of RH-DC2 and RH-DC2 SAG1 strains to bind to dendritic cells was analyzed by flow cytometry (
[0399] As shown in
[0400] To visualize parasite binding to mutuDC cells, immunofluorescence assays were performed. Briefly, MutuDC 1950 cells (murine dendritic cell line, DEC205±) were seeded on coverslips overnight in culture medium. After washes with PBS, cells were fixed with a 3% paraformaldehyde solution, washed with PBS washed, saturated in PBS-10% FBS, and then incubated with 10.sup.6 tachyzoites (RH, RH-DC2, RH-DC2-SAG1). After washes to eliminate unspecific tachyzoite fixation, bound tachyzoites were detected using serum from T. gondii infected rabbit followed by Alexa Fluor 568-conjugated goat anti-rabbit IgG. Slides were examined under a Leica microscope.
[0401] As shown in
[0402] RH-DEC-205 Treatment Suppresses and/or Regresses an Established Solid Tumor Development
[0403] As shown in
[0404] Interestingly, a significant reduction of tumor implantation was observed in mice treated with RH-DC2-SAG1 (
[0405] All these results suggest that a sub-cutaneous injection of RH-DC2-SAG1 tachyzoites exhibited good efficacy against tumor development.
[0406] RH-DC2 SAG1 Induces a Protective Immune Response Against Tumor Development
[0407] MIC1-3 KO is a mutant strain of T. gondii RH lacking the mic1 and mic3 genes. Disruption of these two genes impairs virulence in mice.
[0408] a) Tumor Compartment
[0409] As described in
[0410] Concerning tumor myeloid cell sub-populations, a significant increase of Ly6C+Ly6G % (PMN) and CD11b+(monocytes) was observed for the four T. gondii treated groups compared to the tumor EG7 mice (
[0411] For tumor lymphoid cell sub-populations, an increase of NK cell population (NKp46+ cells) was observed for RH, MIC1-3KO and RH-OVA treated groups (
[0412] b) Systemic Compartment (i.e., Spleen)
[0413] Concerning spleen myeloid cell sub-populations, a significant increase of CD11c+/CMH II+ cells (dendritic cells) was observed for the RH K01-3 treated group, a significant decrease for the RH DC2 SAG1 treated group, while no significant differences were observed for both RH and RH-OVA treated groups, compared to the tumor EG7 mice (
[0414] For spleen lymphoid cell sub-populations, a significant decrease of CD4+ and CD8+ T cells was observed for the four T. gondii treated groups compared to the tumor EG7 mice (
[0415] A decrease of Treg cells (Foxp3+) was observed for RH-DC2 SAG1 T. gondii treated group, compared to tumor EG7 mice. No significant differences were observed for RH, RH K01-3 and RH-OVA treated groups, compared to the tumor EG7 mice (
Example 3: Specific Secretion of Immunotherapeutic Cytokine by the Strain and in Vivo Effects
[0416] Materials and Methods
[0417] Parasites
[0418] N. caninum (NC-1 strain) tachyzoites were grown by continuous passage in confluent human foreskin fibroblasts (HFFs) in Dulbecco's minimal medium (DMEM, Pan Biotech GmbH) supplemented with 10% of heat-inactivated fetal calf serum (FCS, Dutscher), 2 mM glutamine (Pan Biotech GmbH), 50 U/ml of penicillin/50 μ/ml of streptomycin (Pan Biotech GmbH) and 1% HEPES (Invitrogen) at 37° C. in 5% CO2 atmosphere. For subsequent experiments, infected cultures at the stationary growth phase were scraped and then passed several times through a 27-gauge needle (Millipore, Billerica, USA). The N. caninum tachyzoites were collected, during lysis of the host cells, by centrifugation at 600 g for 10 min.
[0419] T. gondii strain RH tachyzoites were produced in HFFs cultured in DMEM (Pan Biotech GmbH) supplemented with 10% of heat-inactivated FCS (Dutscher), 2 mM glutamine (Pan Biotech GmbH), 50 U/ml of penicillin and 50 μ/ml of streptomycin (Pan Biotech GmbH) at 37° C. in 5% CO2 atmosphere. They were harvested during lysis of the host cells by centrifugation at 600 g for 10 min.
[0420] Plasmid Construction of the RH-IL-15hRec
[0421] The plasmid pUC8 CAT/GFP-IL-15hRec (human IL-15/IL-15Rα sushi) was used to construct the recombinant RH-IL-15hRec and NC1-IL15hRec.
[0422] pUC8 CAT/GFP-IL-15hRec is a pUC8 plasmid in which the sequence encoding the complex human IL-15/IL-15Rα sushi (IL-15hRec) including the N-terminal signal sequence and the prodomain motif of SUB1 or MICS is cloned in the expression cassette between PmeI and NotI sites.
[0423] pUC8 contains two expression cassettes. One was designed to express a CAT-GFP fusion protein to allow drug selection of stably transfected parasites (cassette CAT-GFP), the second was designed to express proteins of interest. The sequence encoding the protein of interest must include an ATG and a stop codon. The expression of CAT-GFP is driven by the promoter of the T. gondii α-tubulin gene (αTUB5) and the 3′ untranslated region (3′UTR) of the T. gondii SAG1 gene. This expression cassette is bordered in the 3′ position and 5′ position by Loxp sites. These LoxP sites were added to suppress the cassette CAT-GFP from DNA genome of the parasite by the use of a Cre recombinase which recognizes specifically these sites.
[0424] The expression of the protein of interest is driven by the promoter of the T. gondii α-tubulin gene (αTUB5) in which a five-repeat element was inserted upstream of the transcriptional start site (leading to promoter αTUB8) for high-level expression of the protein (Soldati et al., 1995 PMCID: PMC231911). The sequence of the protein of interest is cloned in PmeI/NotI sites.
[0425] Obtention of pUC8
[0426] Generation of the plasmid pCN1, containing a cassette to express the fusion protein SAG1-GFP driven by the promoter of the T. gondii α-tubulin gene (αTUB5) and the 3′ untranslated region of the T. gondii SAG1 gene (3′UTR SAG1). The sequence encoding SAG1 (including the signal sequence, without the GPI anchor signal sequence) was amplified by PCR by using plasmid pcDNA3-SAG1 as the template (Mévélec et al., 2005 D0110.1016/j.vaccine.2005.04.025) and the primer sequences GGTTTTGACGTCACCATGTTTCCGAAGGCAGTG (SEQ ID NO: 53) (AaTII, underlined) and TTGCTCACCATCCTAGGTGCAGCCCCGGCAAA (SEQ ID NO: 54) (AvrII, underlined). The sequence encoding GFP was amplified by PCR by using plasmid pmic3-GFP (Striepen et al., 1998 D0110.1016/50166-6851(00)00379-0) and the primer sequences TTTGCCGGGGCTGCACCTAGGATGGTGAGCAA (SEQ ID NO: 36) (AvrII, underlined) and
TABLE-US-00007 (SEQ ID NO: 37) CGGTGATTAATTAATCGAGCGGGTCCTGGTTCG
(PacI, underlined). SAG1 and GFP PCR products were digested by AaTII/AvrII and AvRII/PacI respectively and ligated into plasmid pT230TUB/Ble (Kim et al., 1993 PMID: 8235614) previously digested with AaTII/PacI. In the resulting plasmid (pCN1), the sequence encoding BLE is replaced by the sequence encoding the secreted fusion protein SAG1-GFP under the control of promoter αTUB5.
[0427] Generation of the plasmid pCN5, containing a cassette expressing CAT under the promoter αTUB5, bordered by the Loxp sites in the 3′ position and 5′ position. The sequence of the cassette expressing CAT, driven by the promoter of the T. gondii a-tubulin gene (α-TUB5) and 3′ untranslated region of the T. gondii SAG1 gene (3′UTR SAG1), was amplified by PCR by using pmic3KO-2 (Cérède et al., 2005 DOI:10.1084/jem.20041672) as the template and the primer sequences GCGGCCAAGCTTATAACTTCGTATAATGTATGCTATACGAAGTTATGATATG CATGTCCGCgttcgtgaaatctctgatcaagcgg (SEQ ID NO: 38) (including HindIII and Loxp sequences, underlined and in italic respectively) and cgacgcacgctgtcactcaacttgctGCTAGAACTAGTGGATCCATAACTTCGTATAGCATA CATTATACGAAGTTATCCCTCGG (SEQ ID NO: 39) (including SpeI and Loxp sequences, underlined and in italic respectively).
[0428] The PCR fragment was digested by HindIII/SpeI and cloned into pCN1 which has been previously digested by the same enzymes HindIII/SpeI. In the resulting plasmid (pCN5), the cassette expressing the fusion protein SAG1-GFP under the control of promoter αTUB5 is replaced by the cassette expressing CAT under the promoter αTUB5, bordered by the Loxp sites in the 3′ position and 5′ position.
[0429] Generation of the plasmid pUC18 CAT-GFP containing a cassette to express the fusion protein CAT-GFP driven by the promoter of the T. gondii α-tubulin gene (αTUB5) and the 3′ untranslated region of the T T. gondii SAG1 gene (3′ UTR SAG1). The cassette is bordered on both sides by Loxp sites.
[0430] To clone the sequence encoding GFP in fusion with the sequence encoding CAT, a fragment including LoxP (5′ position), promoter αTUB5 and the sequence encoding CAT without stop codon but with an AvrII site to clone GFP in fusion with CAT, was amplified by PCR by using pCN5 as the template and the primer sequences GTATCGATAAGCTTATAACTC (SEQ ID NO: 40) (HindIII underlined) and CACAACGGTGATTAACCTAGGAGCCCCGCCCTG (SEQ ID NO: 41) (AvrII, underlined). The amplified fragment was digested by HindIII/AvrII. The sequence encoding GFP was obtained from pCN1 by digestion with AvrII/PacI. The plasmid pCN5 was digested by HindIII/PacI to eliminate the HindIII/PacI fragment corresponding to LoxP (5′ position), promoter αTUB5 and the sequence encoding CAT. Finally, the three fragments were ligated to obtain a recombinant plasmid containing the cassette expressing CAT-GFP under the promoter αTUB5, bordered by the Loxp sites in the 3′ position and 5′ position. This cassette was further cloned into pUC18 using HindIII and XbaI to obtain pUC18 CAT-GFP. Generation of pUC8, containing one cassette, bordered by LoxP sites, to express the fusion protein CAT-GFP driven by the promotor of the T. gondii α-tubulin gene (αTUB5) and the 3′ untranslated region of the T. gondii SAG1 gene (3′UTR SAG1) and another one to express a protein of interest driven by a modified promotor of the T. gondii α-tubulin gene (αTUB8) and the 3′ untranslated region of the T. gondii SAG1 gene (3′UTR SAG1). In pUC8 the two cassettes are in opposite orientation.
[0431] The expression cassette with a modified promoter of the T. gondii α-tubulin gene (αTUB8) was obtained by addition of five repeat sequences (Soldati et al., 1995), 70 bases upstream the major transcription start site in the T. gondii α-tubulin gene (αTUB5). Plasmid pT230TUB/Ble was used to obtain the promoter αTUB8 and the 3′UTR SAG1 sequences. Two enzymes restriction sites PmeI and NotI were included between the αTUB8 and 3′UTR SAG1 sequences to allow insertion of the sequence encoding the protein of interest. The XbaI restriction sites, located at both end of the expression cassette, were used to clone this expression cassette in pUC18 CAT-GFP in both orientations. The resulting plasmid was pUC8 with the two cassettes in the opposite orientation.
[0432] Generation of Plasmid pUC8 CAT/GFP-IL-15hRec
[0433] pUC8 CAT/GFP-IL-15hRec is a pUC8 plasmid in which the sequence encoding the complex IL-15/IL-15Rα sushi (IL-15hRec) including the N-terminal signal sequence and the prodomain motif of SUB1 or MICS is cloned in the expression cassette between PmeI and NotI sites. The complex is expressed under the control of αTUB8.
[0434] Engineering of Human Recombinant Cytokine IL-15 (IL-15hRec)
[0435] Il-15hRec results from the association of the human sushi domain of the human IL-15 receptor subunit alpha precursor (SEQ ID NO: 3 or 4) with the human mature protein IL-15 (SEQ ID NO: 5 or 6) via a peptide linker (SEQ ID NO: 7 or 8). All these sequences were also optimized for protozoan organism (https://eu.idtdna.com/CodonOpt). Because IL-15hRec is destined for regulated secretion by T. gondii or N. caninum, it is synthesized as preproprotein with an N-terminal signal peptide (SEQ ID NO: 9, 10, 13 or 14) and a separate, cleavable prosequence or prodomain (SEQ ID NO: 11, 12, 15 or 16) necessary for trafficking to the micronemes. We have engineered two possible secretion pathways with i) proDomain TgSUB1 (a micronemal subtilisin-like serine protease) or with ii) proDomain MICS (a small and soluble micronemal protein). In each case, the proDomain is critical for correct folding as well as progression in the secretory pathway. Kozak sequence comprising start codon ATG (Seeber et al., 1997 DOI10.1007/s004360050254) and stop codon (TAA) were included. Finally, in upstream and in downstream of the sequence, two restriction sites were added as respectively: PmeI (GTTTAAAC) and NotI (GCGGCCG). The synthetic gene SUB1IL-15hRec or MICSIL-15hRec was inserted into the pUC8 plasmid with restriction sites PmeI and NotI.
[0436] All basic molecular biology procedures were carried out as described by Sambrook and Russel (Sambrook J., R. D. W. (2001) in Molecular Cloning: A Laboratory Manual (Sambrook, J., and Russel, D. W., eds) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Taq polymerase, restriction enzymes, calf intestinal phosphatase and T4 DNA ligase were from Promega or New England Biolabs.
[0437] The resulting plasmid pUC8 CAT/GFP-IL-15hRec expresses the secreted complex IL-15/IL-15Rα sushi (IL-15hRec) under the control of promotor αTUB8 and the fusion protein CAT-GFP under the control of promotor αTUB5 to allow drug selection of stably transfected parasites.
[0438] Production of Human Recombinant Cytokine IL-15 (IL-15hRec) by N. caninum or T. gondii
[0439] Transfections were performed with 10.sup.7 N. caninum or T. gondii tachyzoites in a volume of 650 μl of cytomix (Van den Hoff et al., Nucleic Acids Res. 1992 Jun. 11; 20(11): 2902) containing 3 mM ATP and 3 mM gluthatione and 50 μg of purified plasmid DNA (the plasmids were purified using the Qiagen Kit®) linearized with PciI or 100 μg of purified plasmid DNA (the plasmids were purified using the Qiagen Kit®) non linearized. Electroporations were performed in disposable cuvettes (4 mm gap) with an electroporator Biorad (electroporation settings: 2000 V, 50 ohms, 25 mF). After electroporation, the parasites were kept in the hood for 15 min at room temperature and then transferred to a fresh culture of fibroblast monolayers (25 cm2 flask). After 24 hours the parasites N. caninum and T. gondii were respectively subjected to 80 μM and 40 μM of chloramphenicol selection. After 10 to 15 days of selection, the parasites were cloned by limiting dilution in the wells of a 96-well plate of HFF cells in the presence of selection agent and the clones were amplified.
[0440] Immunofluorescence Assays
[0441] HFF monolayers on glass coverslips were infected with NC-IL-15hRec or RH-IL-15hRec for 24 to 48 h. The cells were then fixed, permeabilized and blocked. Infected cells were first washed two times in PBS and fixed in PBS plus 4% paraformaldehyde for 30 min at room temperature. Then the coverslips were washed thrice in PBS and were permeabilized in PBS supplemented with 0.2% Triton X-100 for 20 min at room temperature. At this concentration and incubation time, triton X-100 permeabilizes membranes of the infected host cell, including the membrane of the parasitophorous vacuole and the parasite plasma membrane. After three washes the coverslips were blocked in PBS plus 1% bovine serum albumin (BSA) for 1 h at room temperature. Fixed cells were labeled with IL-15 polyclonal antibody (PA5-34511 ThermoFischer Scientific, 10 μg/mL in PBS/BSA 0.2%) over night at 4° C. in humid chamber. After three washes in PBS, the samples were incubated for 45 min with secondary Alexa Fluor 568-conjugated goat anti-rabbit IgG antibody (A-11036 ThermoFisher Scientific, 1/1000 dilution in PBS/BSA 0.2%) at room temperature and in a humid chamber. Microscope slides were mounted on glass slides using Immu-Mount (Thermo Scientific) and the visualization was carried out using a Leica microscope. Image analysis was performed with NIH ImageJ software.
[0442] ELISA: IL-15hRec
[0443] The secretion in the culture supernatant of human recombinant cytokine IL-15 has been assessed using enzyme-linked immunosorbent assay (ELISA) method. Briefly and according to the manufacturer's instructions (DY6924 RD SYSTEMS), Capture Antibody was coated in a 96-well plates at 4 μg/mL and incubated overnight at 4° C. The wells were saturated with Reagent Diluent for a minimum of 60 min at room temperature and washed with PBS-Tween 0.05% prior to incubation with standard at increasing concentrations (ranging from 0 to 4000 pg/mL) and diluted samples ( 1/10 and 1/50 in Reagent Diluent) overnight at 4° C. Wells were then washed, which was repeated after each of the following incubations: Detection Antibody for 2 h at room temperature, Streptavidin-HRP for 20 min at room temperature and finally Substrate Solution for 20 at room temperature avoiding placing the plate in direct light. Enzymatic reactions were stopped with the Stop Solution and the absorbance was measured at 450 nm using a microplate reader (Biotek). Wells coloration correlated to the presence of secreted IL-15hRec in the culture supernatant and the absorbance at 450 nm was then proportional to IL-15hRec content.
[0444] Cytokine Production
[0445] Splenocytes from naïve mice were recovered and purified as described (Rhode et al., 2016 DOI: 10.1158/2326-6066.CIR-15-0093-T). Briefly, single-cell splenocyte suspensions were obtained from spleen first pressed and then filtered through a nylon mesh. Hypotonic shock (0.155 M NH.sub.4Cl, pH 7.4) was used to remove splenic erythrocytes. The splenocytes were stimulated for 72 h with IL15hRec containing supernatants diluted twice. The cells (5×10.sup.5) were seeded into 24-well plates in 1 ml RPMI 1640 containing 5% FCS, and supernatants were collected 72 h after activation. IFNγ was quantified by ELISA using ready-set-go kit (ebioscience).
[0446] Cytokine Measurement and Cell Populations Phenotyping after NC1-IL15hRec Infection
[0447] Briefly, supernatants of culture of the engineered NC1-IL15hRec strain and NC1 strain were dosed for IL-15 48 h after infection (MOI 0.2). Mouse splenocytes were infected with NC1-IL15hRec (MOI 1) and IFN-γ was dosed in the supernatant after 48 h.
[0448] Human PBMCs were infected with NC1-IL15hRec or NC1 (MOI 1), and supernatants of culture were dosed by ELISA 24 h after infection. At the same time point, cells were analyzed by flow cytometry.
[0449] Mice
[0450] Eight week-old female inbred C57BL/6 mice were purchased from CER Janvier (Le Genest Saint Isle, France) and maintained under pathogen-free conditions in the animal house of the University of Tours. Experiments were carried out in accordance with the guideline for animal experimentation (EU Directive 2010/63/EU) and the protocol was approved by the local ethics committee (CEEA VdL).
[0451] Tumor Cells and Tumor Cell Inoculation (EG7)
[0452] EG7 cells (EL4-OVA thymoma cells transfected with chicken albumin cDNA) are cultured in Roswell Park Memorial Institute medium (RPMI, Pan Biotech GmbH) with 5×10.sup.5 M of 2-mercaptoethanol, 50 UI/mL of penicillin and 50 mg/mL of streptomycin.
[0453] 5×10.sup.5 live EG7 cells are inoculated subcutaneously in the right flank of mice. Tumor diameters are measured 3 times weekly, and mice are euthanized when tumor diameters reached 25,000 mm.sup.3.
[0454] N. caninum Administration
[0455] Mice are injected subcutaneously in the right flank at day 4 and again at day 7 with 5×10.sup.6 freshly isolated tachyzoites of NC1 ctrl and NC1-IL-15hRec strain of N. caninum.
[0456] Results
[0457] IL-15hRec Plasmid Constructs
[0458] Both T. gondii (RH) and N. caninum (NC) strains are engineered to express and secrete a human IL-15/IL-15Rα sushi (IL-15hRec) cytokine. IL-15hRec sushi stands for recombinant human interleukin 15 covalently linked to sushi domain of the human IL-15 receptor alpha. It is composed of the sushi domain (amino acids 117 to 182) of the human alpha receptor, a peptide linker and the human interleukin-15. The sushi domain plays a role of chaperone protein, stabilizes and increases IL-15 activity (Desbois et al., J Immunol. Jul. 1, 2016, 197 (1) 168-178). The complex formed by IL-15 and the sushi domain of IL-15Rα generates a more potent ligand compared to the cytokine alone.
[0459] The construction of IL-15hRec is described in
[0460] The IL-15hRec construction encodes for the expression cassettes CAT/GFP and human IL-15/IL-15Rα sushi complex. In particular, the sequence encoding said IL-15hRec protein included: the locations of the preprotein sequence (Signal sequence and Prodomain sequence of MICS or SUB1), human sushi domain of the human IL-15 receptor subunit alpha sequence (Sushi domain sequence (IL-15Rα), linker sequence (grey hatched diagram), and human mature IL-15 (human IL-15 sequence). The light and dark grey blocks represent respectively: restriction site PmeI and restriction site NotI. Then, the T. gondii (RH) and N. caninum (NC) strains tachyzoites were transfected with the IL-15hRec construction.
[0461] Of note, according to signal sequence and prodomain of T. gondii used for the plasmid construction (MICS or SUB1), two different strains RH-IL-15 MICS or RH-IL-15 SUB1 are respectively obtained.
[0462] IL-15hRec Expression by the Recombinant RH-IL-15 Strains
[0463] The expression of GFP and IL-15hRec into transfected RH parasites was analyzed by immunofluorescence assays (
[0464] As shown in
[0465] Finally, as shown in
[0466] The IL-15hRec constructions used for T. gondii were also used for N. caninium.
[0467] NC-IL-15 Treatment Suppresses and/or Regresses an Established Solid Tumor Development
[0468] As shown in
[0469] Interestingly, the reduction of the tumor volume was also observed in mice for which subcutaneous injection of NC-IL-15 tachyzoites was performed “at distance” of the tumor site.
[0470] These results suggest that sub-cutaneous injection of NC-IL-15 tachyzoites exhibited good efficacy against tumor development.
[0471] NC-IL-15 Induces a Protective Immune Response Against Tumor Development
[0472] The inventors demonstrated that N. caninum was able to induce tumor regression, notably through an immune cell activation and associated cytokine increased secretion, reprogramming of the tumor microenvironment and direct oncolytic effect. However, improving these protective effects seems essential in order to obtain a total protection in advanced or refractory tumors. To this aim, they engineered a N. caninum strain able to secrete the human IL-15, associated with its sushi domain (alpha subunit of the IL-15 receptor), increasing its stability, binding and biological abilities, to strongly induce the expansion of Th1 associated lymphocyte subsets and prevent their apoptosis in vivo.
[0473] They first assessed that the engineered clones were able to secrete a biologically active form of the cytokine. They showed that supernatant of culture of NC1-IL15hRec were able to induce IFN-γ secretion by mouse splenocytes in vitro (the human IL-15 being cross reactive with mice cells, or able to stimulate mouse cells), 4 to 5 times as compared to supernatant from WT Neospora. The NC1-IL15hRec strain was thus able to secrete a functional IL-15 in its environment.
[0474] The effect of the NC1-IL15hRec strain on human cells was then tested. Human PBMCs were infected (MOI 1) with NC-1 and NC1-IL15hRec. After 24 hours, the levels of IL-15 were measured in the supernatant, which was only detectable in the wells treated with NC1-IL15hRec. Then, as the recombinant IL-15, the NC1-IL15hRec strain was able to induce the proliferation of human NK cells as shown by the increase of Ki67 expression by human NK cells. Meanwhile, WT N. caninum displayed a much lower increase of proliferation by NK cells. Moreover, while recombinant IL-15 was not able to induce IFN-γ secretion by human PBMCs, but only their proliferation, NC1-IL15hRec induced a strong IFN-γ secretion by human PBMCs, much more importantly than WT N. caninum.
[0475] These data clearly demonstrated the plus-value potential of IL-15-armed strain of N. caninum that might strengthen the already important immunomodulatory properties of the WT protozoan parasite.
Example 4: Toxoplasma Gondii Treatment Suppresses and/or Regresses an Established Glioblastoma Development
[0476] Materials and Methods
[0477] Mice
[0478] Sixteen-week-old female inbred albino C57BL/6 mice were purchased from CER Janvier (Le Genest Saint Isle, France) and maintained under pathogen-free conditions in the animal house of the University of Tours. Experiments were carried out in accordance with the guideline for animal experimentation (EU Directive 2010/63/EU) and the protocol was approved by the local ethics committee (CEEA VdL).
[0479] Toxoplasma gondii Strains (Me49)
[0480] Tachyzoites of the Me49 strain of Toxoplasma gondii were harvested from infected human foreskin fibroblasts HFF (ATCC Hs27) cultured in monolayers in DMEM, supplemented with 10% heat-inactivated FCS, 50 U/ml penicillin/50 μg/ml 20 streptomycin, and 1% HEPES. Toxoplasma gondii tachyzoites are harvested when monolayers of HFF were completely lysed.
[0481] Tumor Cells (GL261)
[0482] GL261-Luc cell line was a generous gift of Stéphan Birklé (Institut de Recherche en Cancérologie Nantes-Atlantique UMR S 1232 Centre de Recherche en Cancérologie et Immunologic Nantes-Angers). Cells were grown in DMEM containing 10% fetal calf serum and 1% penicillin/streptomycin. The GL261-Luc murine model is one of the most extensively used for preclinical testing of immunotherapeutic approaches for GBM.
[0483] Tumor Cell Inoculation
[0484] To establish syngeneic gliomas, GL261-Luc cells (10.sup.5 cells in 2.5 μL) were intracranially implanted in the brains of albino C57BL/6 mice.
[0485] All mice were given subcutaneous buprenorphine injections (0.1 mg/kg) as pre-emptive analgesia. Mice under deep anesthesia after inhalation of isoflurane gas in a nose cone adapted for the stereotaxic frame to maintain an appropriate level of anesthesia were prepared for surgery. After a midline scalp and periosteum incision with lidocaine local anesthesia, tumor cells were stereotactically implanted in the right frontal lobe with a 26 gauge needle into the following coordinates: at a depth of 3 mm, 2 mm lateral to midline and 0.5 mm anterior to bregma.
[0486] Animals were monitored daily for any neurological change, weight loss and for their ability to freely access food and water.
[0487] Tumor burden was monitored by luciferase imaging on day 7 after implantation. Mice were randomly allocated into treatment arms based on tumor radiance, so that the average tumor radiance in each group was roughly equivalent.
[0488] When mice showed predetermined signs of neurologic deficits (failure to ambulate, weight loss >20% mass, lethargy, hunched posture), they were killed.
[0489] Toxoplasma gondii Administration
[0490] Eleven days after GL261-Luc cell implantation mice received by sub-cutaneous route 500 tachyzoites of Me49 strain (type II) in 50μl.
[0491] Results
[0492] Evaluation of the number of metastatic sites by 3D in vivo bioluminescent imaging was performed at days 1, 22, 24, 28, 31 and 39.
[0493] As shown in
[0494] Moreover, in vivo bioluminescent imaging revealed at D28 that GL261 tumor mice exhibited highly tumor volume (11 mm.sup.3). On the other hand tumors from mice treated with T. gondii were less invasive (7 mm.sup.3) (
[0495] All these results suggest that a single sub-cutaneous injection of Toxoplasma gondii exhibited good efficacy against glioblastoma development.
Example 5: Recombinant RH-OVA Treatment Suppresses and/or Regresses an Established Lung Melanoma Development in Mice
[0496] Materials and Methods
[0497] Mice
[0498] Twenty-four-week-old female C57BL/6 mice were purchased from CER Janvier (Le Genest Saint Isle, France) and maintained under pathogen-free conditions in the animal house of the University of Tours. Experiments were carried out in accordance with the guideline for animal experimentation (EU Directive 2010/63/EU) and the protocol was approved by the local ethics committee (CEEA VdL).
[0499] Toxoplasma gondii Strains (RH-OVA)
[0500] T. gondii strain RH-OVA tachyzoites were produced in HFFs cultured in DMEM (Pan Biotech GmbH) supplemented with 10% of heat-inactivated FCS (Dutscher), 2 mM glutamine (Pan Biotech GmbH), 50 U/ml of penicillin and 50 μ/ml of streptomycin (Pan Biotech GmbH) at 37° C. in 5% CO2 atmosphere. They were harvested during lysis of the host cells by centrifugation at 600 g for 10 min.
[0501] Tumor Cells (B16F10)
[0502] B16F10 murine melanoma cell line were cultured at 37° C. in 5% CO2 atmosphere in complete media consisting of RPMI 1640, 2 mM 1-glutamine, 100 U ml.sup.−1 penicillin and 100 mg ml.sup.−1 streptomycin and 10% fetal bovine serum.
[0503] Tumor Cell Inoculation
[0504] For tumor inoculation, mice were challenged with 10.sup.6 B16/F10 melanoma cells by tail venous injection. Development of subcutaneous tumor was observed daily. Lung metastasis were observed on mice at day 19 and day 32 post implantation.
[0505] Toxoplasma gondii Administration
[0506] Mice are injected subcutaneously in the right flank at day 2 with 5×10.sup.2 freshly isolated tachyzoites of RH-OVA strain of T. gondii.
[0507] Results
[0508] As shown in
[0509] These results clearly demonstrate that Toxoplasma gondii injection inhibit not only growth of the tumor implanted into the dermis, but also the development of lung metastasis.
Example 6: Recombinant RH-OVA Strain for Treating Ovarian Cancer in Mice
[0510] Materials and Methods
[0511] Mice
[0512] Twenty-four-week-old female C57BL/6 mice were purchased from CER Janvier (Le Genest Saint Isle, France) and maintained under pathogen-free conditions in the animal house of the University of Tours. Experiments were carried out in accordance with the guideline for animal experimentation (EU Directive 2010/63/EU) and the protocol was approved by the local ethics committee (CEEA VdL). Toxoplasma gondii strains (RH-OVA)
[0513] Tumor Cells (ID8)
[0514] ID8-Luc ovarian carcinoma cell line was cultured at 37° C. in 5% CO2 atmosphere in complete media consisting of RPMI 1640, 2 mM 1-glutamine, 100 U ml.sup.−1 penicillin and 100 mg ml.sup.−1 streptomycin and 10% fetal bovine serum. Cells were grown in DMEM supplemented with 5% FBS and 1×insulin-transferrin-sodium selenite media supplement.
[0515] Tumor Cell Inoculation
[0516] For in vivo tumor development assays, 5×10.sup.6 subconfluent ID8-Luc cells in 200 μl of 1×PBS were injected i.p. in C57BL/6 female mice.
[0517] Toxoplasma gondii Administration
[0518] Mice are injected intravaginally at day 15 with 5×10.sup.6 freshly isolated tachyzoites of RH-OVA strain of T. gondii.
[0519] Results
[0520] As shown in
[0521] Moreover, at postmortem examination, tumor weight was significantly decreased on the surface on omentum (
Example 7: Specific Targeting of Dendritic Cells by the Strain and In Vivo Effects
[0522] Materials and Methods
[0523] Parasites
[0524] T. gondii strain RH tachyzoites were produced in human fibroblasts (HFFs) cultured in Dulbecco's minimal medium (DMEM) supplemented with 10% of fetal calf serum, 2 mM glutamine, 50 U/ml of penicillin and 50 μ/ml of streptomycin. They were harvested during lysis of the host cells
[0525] Plasmid Construction of the RH-hPDL1Rec and NC-hPDL1Rec (recombinant anti-human PD1 ligand)
[0526] The anti-hPDL1Rec scFv results from the association of the heavy (IMGT 9814_H) and light (IMGT 9814_L) variable domains of Atezolizumab via a (Gly4Ser)3 peptide linker (SEQ ID NO: 55) and from a spacer GGGAS (SEQ ID NO: 28) in the C-terminal and a peptide HA tag in the N-terminal. The nucleotide and amino acid sequences of the optimized anti-hPDL1Rec scFv are given below.
TABLE-US-00008 Optimized anti-hPDL1Rec scFv nucleotide sequence (SEQ ID NO: 56): GAGGTGCAACTCGTCGAAAGCGGGGGCGGTTTGGTGCAACCTGGGGGAAGC CTGCGGTTGTCTTGCGCCGCAAGCGGCTTTACGTTTTCCGATTCGTGGATT CATTGGGTGAGACAAGCCCCAGGTAAGGGGCTCGAATGGGTGGCGTGGATC AGTCCGTATGGTGGATCGACTTATTACGCGGACTCTGTGAAAGGAAGGTTT ACAATCTCCGCGGATACGTCCAAAAATACCGCATATTTGCAGATGAATAGC CTTCGCGCAGAGGACACAGCAGTTTATTACTGCGCCCGGAGACATTGGCCA GGCGGCTTCGATTACTGGGGGCAAGGTACGCTGGTTACAGTTAGCAGCGGG GGAGGAGGATCTGGGGGAGGTGGGTCGGGAGGGGGAGGTTCCGACATCCAA ATGACTCAGTCGCCATCTAGTCTTTCTGCCTCTGTGGGGGATCGTGTTACC ATCACGTGCCGTGCCAGCCAGGACGTTAGCACTGCTGTGGCCTGGTACCAG CAAAAGCCGGGGAAGGCACCCAAACTTCTGATCTATAGTGCGTCGTTCCTC TATAGTGGCGTTCCGTCGCGCTTCTCTGGTAGTGGCTCCGGCACCGACTTT ACCCTGACAATCAGTAGCCTGCAGCCTGAGGACTTCGCTACCTATTATTGC CAACAATACCTGTACCACCCGGCAACCTTCGGTCAAGGAACCAAAGTGGAG ATTAAAGGAGGGGGGGCCAGTTCCAGA Optimized VH anti-hPDL1Rec amino acid sequence (SEQ ID NO: 57): EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWI SPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWP GGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVT ITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDF TLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKGGGASSR
[0527] As described hereabove for other recombinants, the anti-hPDL1Rec scFv is fused to SAG1. More precisely, the sequence encoding anti-hPDL1Rec SAG1 protein includes: the kozak sequence (consensus sequence for the initiation of the translation), the ATG, the sequence encoding the N-terminal signal sequence of SAG1, the HA tag, the VH region followed by the VL region of the anti-hPDL1Rec, the linker GGGAS, the sequence encoding a truncated SAG1 (without the N-terminal signal sequence) and the sequence encoding the SAG1 anchor signal (GPI=glycosylphosphatidylinositol) with a stop codon. This sequence is flanked in 5′ by a PmeI site and in 3′ by a NotI site. Thus, the pUC5 CAT/GFP-anti-hPDL1Rec/SAG1/GPI expresses the membrane-anchored fusion protein anti-hPDL1Rec-SAG1 under the control of promoter αTUB8 and the fusion protein CAT-GFP under the control of promoter αTUB5 to allow drug selection of stably transfected parasites (T. gondii strain RH or N. caninum tachyzoites).
[0528] ELISA on T. gondii Parasites
[0529] ELISA was performed on whole tachyzoites, essentially as described previously (Chardes et al., Infection and immunity, 1990). Flat bottomed wells (96-well plate, NUNC) were coated with 2×10.sup.5, 5×10.sup.5 or 10×10.sup.5 parasites/well in PBS. After centrifugation at 200×g and 4° C. for 5 min, 25 μL of 0.5% of glutaraldehyde in cold PBS was added to each well and left for 8 min at room temperature. The plates were washed twice in PBS and saturated with PBS-4% BSA for 1 h at 37° C. Rabbit anti-HA polyclonal antibodies (1:400, ThermoFisher Scientific) in PBS-1% BSA were incubated for 1 h at 37° C. After 3 washes in PBS-0.05% Tween, mouse monoclonal anti-rabbit IgG (γ-chain specific) alkaline phosphatase conjugate (1:4000 in PBS-BSA 1%, Sigma) was added to each well and incubated for 1 h at 37° C. After 3 washes with PBS-0.05% Tween, bound phosphatase activity was measured with p-nitrophenylphosphate (Sigma) (1 mg/ml in DEA-HCl 1 M bu.er pH 9.8).
[0530] Results
[0531] Cell Surface Expression of scFv-hPDL1Rec
[0532] An ELISA analysis was performed on various selected clones of RH-anti-hPDL1Rec (
[0533] As shown in