MODIFIED VSV-G AND VACCINES THEREOF

Abstract

A modified vesicular stomatitis virus glycoprotein (VSV-G) that includes at least one peptide, preferably an antigen or fragment thereof, nucleic acid sequence coding therefor, and vectors containing the nucleic acid sequence. Also vaccines and methods for the treatment of a disease or condition, in particular a cancer or an infectious disease.

Claims

1-15. (canceled)

16. An isolated nucleic acid sequence coding for a modified vesicular stomatitis virus glycoprotein (VSV-G) comprising at least one tumor antigen or fragment thereof.

17. The isolated nucleic acid sequence coding for a modified VSV-G according to claim 16, wherein said at least one tumor antigen or fragment thereof comprises at least one epitope.

18. The isolated nucleic acid sequence coding for a modified VSV-G according to claim 16, wherein said at least one tumor antigen or fragment thereof is a neoantigen.

19. The isolated nucleic acid sequence coding for a modified VSV-G according to claim 16, wherein said at least one antigen or fragment thereof is inserted into VSV-G at an amino acid position selected from the group consisting of positions 18, 51, 55, 191, 196, 217, 368 and C-terminal, and combinations thereof, wherein position numbering is with respect to vesicular stomatitis Indiana virus (VSIV) glycoprotein amino acid sequence.

20. The isolated nucleic acid sequence coding for a modified VSV-G according to claim 16, wherein said at least one antigen or fragment thereof is inserted into VSV-G at amino acid position 18 or 191 and combinations thereof, wherein position numbering is with respect to vesicular stomatitis Indiana virus (VSIV) glycoprotein amino acid sequence.

21. The isolated nucleic acid sequence coding for a modified VSV-G according to claim 16, wherein said VSV-G is from vesicular stomatitis Indiana virus (VSIV).

22. The isolated nucleic acid sequence coding for a modified VSV-G according to claim 16, wherein said VSV-G has a sequence identity of at least 70% with SEQ ID NO: 1.

23. The isolated nucleic acid sequence coding for a modified VSV-G according to claim 16, wherein said VSV-G comprises or consists of SEQ ID NO: 1.

24. A modified vesicular stomatitis virus glycoprotein (VSV-G) comprising at least one tumor antigen or fragment thereof.

25. A vaccine comprising a modified vesicular stomatitis virus glycoprotein (VSV-G) comprising at least one antigen or fragment thereof, a nucleic acid sequence coding therefor, a vector containing a nucleic acid sequence coding therefor, or a dendritic cell population transfected by a nucleic acid sequence coding therefor.

26. The vaccine according to claim 25 comprising at least one adjuvant.

27. The vaccine according to claim 25, wherein said vaccine is a polynucleotide vaccine.

28. The vaccine according to claim 25, wherein said vaccine is a protein vaccine.

29. A method for preventing and/or treating a disease or condition in a subject in need thereof comprising administering to said subject a modified vesicular stomatitis virus glycoprotein (VSV-G) comprising at least one antigen or fragment thereof, a nucleic acid sequence coding therefor, a vector containing a nucleic acid sequence coding therefor, a dendritic cell population transfected by a nucleic acid sequence coding therefor, or a vaccine comprising said modified VSV-G, nucleic acid sequence, vector or dendritic cell population and optionally at least one adjuvant.

30. The method according to claim 29, wherein said vaccine is a polynucleotide vaccine.

31. The method according to claim 29, wherein said vaccine is a protein vaccine.

32. The method according to claim 29, wherein said disease is a cancer.

33. The method according to claim 29, wherein said disease is an infectious disease.

34. The method according to claim 29, wherein said modified VSV-G, nucleic acid sequence, vector, dendritic cell population or vaccine is administered to the subject by intramuscular injection, intradermal injection, intratumoral injection, peritumoral injection, gene gun, electroporation or sonoporation.

35. The method according to claim 29, wherein said modified VSV-G, nucleic acid sequence, vector, dendritic cell population or vaccine is administered before, concomitantly or after one or more checkpoint blockade antibodies.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0561] FIG. 1A and FIG. 1B are graphs showing the effect of pTOP-OVA_CD8 prophylactic intramuscular immunization on the anti-tumor activity. FIG. 1A shows tumor growth follow-up after challenge. The tumor size was measured three times a week with an electronic digital caliper. Tumor volume was calculated as the lengthwidthheight (in mm.sup.3). FIG. 1B shows survival rates monitoring after challenge. The asterisks indicate significant differences compared with naive mice (**P<0.01) (n=6) (Comparison of survival curves, Mantel-Cox test).

[0562] FIGS. 2A and 2B are graphs showing the effect of pTOP-OVA_CD8 therapeutic intratumoral immunization on the anti-tumor activity. FIG. 2A shows tumor growth follow-up after challenge. The tumor size was measured three times a week with an electronic digital caliper. Tumor volume was calculated as the lengthwidthheight (in mm.sup.3). FIG. 2B shows survival rates monitoring after challenge. The asterisks indicate significant differences compared with naive mice (***P<0.001) (n=6) (Comparison of survival curves, Mantel-Cox test).

[0563] FIGS. 3A and 3B are graphs showing the effect of restriction sites addition around the inserted epitope sequence, for prophylactic intramuscular immunization. FIG. 3A shows tumor growth follow-up after challenge. The tumor size was measured three times a week with an electronic digital caliper. Tumor volume was calculated as the lengthwidthheight (in mm.sup.3). FIG. 3B shows survival rates monitoring after challenge. The asterisks indicate significant differences compared with naive mice (**P<0.01) (n=6) (Comparison of survival curves, Mantel-Cox test).

[0564] FIGS. 4A and 4B are graphs showing the effect of pTOP1-OVA_CD8 and pTOP1-OVA_CD4 prophylactic intramuscular immunization on the anti-tumor activity. FIG. 4A shows tumor growth follow-up after challenge. The tumor size was measured three times a week with an electronic digital caliper. Tumor volume was calculated as the lengthwidthheight (in mm.sup.3). FIG. 4B shows survival rates monitoring after challenge. The asterisks indicate significant differences compared with naive mice (***P<0.001) (n=6) (Comparison of survival curves, Mantel-Cox test).

[0565] FIG. 5A-D are graphs showing the effect of pTOP1-OVA_CD8 and pTOP1-OVA_CD4 therapeutic intramuscular immunization on the anti-tumor activity. FIG. 5A and FIG. 5C show tumor growth follow-up after challenge. FIG. 5B and FIG. 5D show survival rates monitoring after challenge. Survival curves were compared with a Mantel-Cox test. The asterisks indicate significant differences compared with naive mice (***P<0.001) (n=10 and n=6 respectively).

[0566] FIG. 6 is a graph showing the effect of co-delivery of pTOP1-OVA_CD4 with pTOP1-OVA_CD8 on the cytotoxic T cell response. Percentages of OVA target cell killing were compared and the asterisks indicate significant differences (***P<0.001) (n=5) (Student's T-test).

[0567] FIG. 7 is a graph showing an OTII proliferation assay and effect of immunization with MHC class II restricted epitope inserted in pTOP1. The percentages of cell division were compared by Student's T-test (***p<0.001) (n=5).

[0568] FIG. 8 is a set of graphs showing OTI proliferation assay and the effect of immunization with MHC class I restricted epitope inserted in pTOP1. The graph shows the percentages of cell division. The asterisks indicate significant differences (***P<0.001) (n=5) (Student's T-test).

[0569] FIGS. 9A and 9B are graphs showing the effect of pTOP1 intramuscular therapeutic immunization in combination with immune checkpoint blockade (ICB) therapy. FIG. 9A shows tumor growth follow-up after challenge. Tumor volume was calculated as the lengthwidthheight (in mm.sup.3). FIG. 9B shows survival rates monitoring after challenge. The asterisks indicate significant differences between curves (*P<0.05; ***P<0.001) (n=6) (Comparison of survival curves, Mantel-Cox test).

[0570] FIGS. 10A and 10B are graphs showing the effect of pTOP1-OVA_CD4(18)_OVA_CD8(191) and pTOP1_gp100_CD4(18)_TRP2_CD8(191) therapeutic intramuscular immunization on the anti-tumor activity. FIG. 10A shows tumor growth follow-up after challenge. The tumor size was measured three times a week with an electronic digital caliper. Tumor volume was calculated as the lengthwidthheight (in mm3). FIG. 10B shows survival rates monitoring after challenge. The asterisks indicate significant differences compared with naive mice (**P<0.01; ***P<0.001) (n=6) (Comparison of survival curves, Mantel-Cox test).

[0571] FIGS. 11A and 11B are graphs showing the effect of pTOP1-PADRE(18)_P1A_CD8(191) prophylactic intramuscular immunization on the anti-tumor activity. FIG. 11A shows tumor growth follow-up after challenge. The tumor size was measured three times a week with an electronic digital caliper. Tumor volume was calculated as the lengthwidthheight (in mm3). FIG. 11B shows survival rates monitoring after challenge. The asterisks indicate significant differences compared with naive mice (**P<0.01) (n=6) (Comparison of survival curves, Mantel-Cox test).

[0572] FIG. 12 is a graph showing the effect of pTOP1-PADRE(18)_P1A_CD8(191) therapeutic intramuscular immunization on the anti-tumor activity. It indicates survival rate monitoring after challenge. The asterisk indicates significant differences compared with naive mice (*P<0.05) (n=6) (Comparison of survival curves, Mantel-Cox test).

[0573] FIGS. 13A and 13B are graphs showing the effect of pTOP1-PADRE(18)_AH1A5_CD8(191) prophylactic intramuscular immunization on the anti-tumor activity. FIG. 13A shows tumor growth follow-up after challenge. The tumor size was measured three times a week with an electronic digital caliper. Tumor volume was calculated as the lengthwidthheight (in mm3). FIG. 13B shows survival rates monitoring after challenge. The asterisks indicate significant differences compared with naive mice (***P<0.001) (n=6) (Comparison of survival curves, Mantel-Cox test).

[0574] FIGS. 14A and 14B are graphs showing the effect of pTOP1-PADRE(18)_TRP2_CD8(191) prophylactic intramuscular immunization on the anti-tumor activity. FIG. 14A shows tumor growth follow-up after challenge. The tumor size was measured three times a week with an electronic digital caliper. Tumor volume was calculated as the lengthwidthheight (in mm3). FIG. 14B shows survival rates monitoring after challenge. The asterisks indicate significant differences compared with naive mice (***P<0.001) (n=6) (Comparison of survival curves, Mantel-Cox test).

[0575] FIG. 15 is graph showing the effect of pTOP1-gp100_CD4(18)_OVA_CD8(191) and pTOP1_gp100_LP (18)_OVA_CD8(191) therapeutic intramuscular immunization on the anti-tumor activity. It indicates survival rates monitoring after challenge. The asterisks indicate significant differences compared with naive mice (*P<0.05; **P<0.01) (n=6) (Comparison of survival curves, Mantel-Cox test).

EXAMPLES

[0576] The present invention is further illustrated by the following examples.

[0577] Materials and Methods

[0578] Material

[0579] Plasmids

[0580] Codon-optimized gene sequences of VSV-G (pTOP), VSV-G-OVA_CD8 (pTOP-OVA_CD8) and VSV-G-RS (with restriction sites, pTOP1) were designed using GeneOptimizer and obtained by standard gene synthesis from GeneArt (Thermo Fisher Scientific, Waltham, Mass., US). These sequences were subcloned in the pVAX2 vector using cohesive-ends cloning. The pVAX2 vector consists of a pVAX1 plasmid (Invitrogen, Carlsbad, Calif.) in which the promoter was replaced by the pCMV plasmid promoter (Clontech, Palo Alto, Calif.). The plasmids were prepared using the EndoFree Plasmid Giga Kit (Qiagen, Venlo, Netherlands) according to the manufacturer's protocol. Plasmid dilutions were performed in Dulbecco's Phosphate Buffered Saline (1) (PBS) (Life Technologies, Carlsbad, Calif., US). The quality of the purified plasmid was assessed by the ratio of optical densities (260 nm/280 nm) and by 0.5% agarose gel electrophoresis. DNA concentration was determined by optical density at 260 nm. The plasmids were stored at 20 C.

[0581] VSV-G Sequences Cloned in pVAX2 [0582] Vesicular stomatitis Indiana virus glycoprotein G (VSV-G) (SEQ ID NO: 1, encoded by SEQ ID NO: 10). [0583] Plasmid nomenclature: pVAX2-VSVG (pTOP).

TABLE-US-00003 MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHN DLIGTAIQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITQSIRS FTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHV LVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISM DITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSG VWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLC QETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVD IAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPL YMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNP IELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQI YTDIEMNRLGK. [0584] VSV-G (SEQ ID NO: 1) containing SIINFEKL sequence (OVA_CD8, SEQ ID NO: 11) at position 191 (SEQ ID NO: 8). [0585] Plasmid nomenclature: pVAX2-VSVG-OVA_CD8 (pTOP-OVA_CD8).

TABLE-US-00004 MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHN DLIGTAIQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITQSIRS FTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHV LVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKSIINFEKLGL CDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKH WGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVE RILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYF ETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRT SSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFG DTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKL KHTKKRQIYTDIEMNRLGK.
(in bold underlined is the OVA_CD8 sequence, SEQ ID NO: 11), [0586] VSV-G (SEQ ID NO: 1) containing restriction sites (RS) at position 191 (SEQ ID NO: 9). [0587] Plasmid nomenclature: pVAX2-VSVG-RS (pTOP1).

TABLE-US-00005 MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHN DLIGTAIQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITQSIRS FTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHV LVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKTSEFGLCDSN LISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVR LPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILD YSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRY IRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGY KFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGL SKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTK KRQIYTDIEMNRLGK.
(In bold underlined are the SpeI/EcoRI restriction sites).

[0588] Peptide Insertion in pTOP1

[0589] To insert epitopes in position 191 of VSV-G (SEQ ID NO: 1), into the pTOP1 vector, cohesive-ends cloning was used. pVAX2-VSVG-RS was opened using SpeI and EcoRI and two complementary and overlapping phosphorylated oligonucleotides were incorporated. Multiple plasmids were obtained by varying the sequence of the oligonucleotides which were ordered from Eurogentec (Seraing, Belgium) or IDT-DNA (Leuven, Belgium). For peptide insertion in position 18 of pTOP1, Gibson Assembly Cloning Kit (New England BioLabs Inc.) with gBlocks gene fragments was used according to the manufacturer instructions. A HindIII restriction site was added for allowing easy peptide modification at the position 18. Plasmids were then purified, characterized and stored as explained here above.

TABLE-US-00006 TABLE3 PeptidesinsertedinpTOP-1bycohesive- endscloningatposition191ofSEQIDNO:1. Peptidesequence,nameandfunctionaredescribed. SEQ ID NO: Peptide Name Function 11 SIINFEKL OVA_CD8 CD8Tcell epitopeagainst ovalbumin 12 ISQAVHAAHAEINEAGR OVA_CD4 CD4Tcell epitopeagainst ovalbumin 13 LPYLGWLVF P1A_CD8 CD8Tcell epitopeagainst P1A 14 ELAGIGILTV MELANA_CD8 CD8Tcell epitopeagainst MART-1 15 IMDQVPFSV GP100_CD8 CD8Tcell epitopeagainst gp100 16 YMDGTMSQV TYR_CD8 CD8Tcell epitopeagainst tyrosinase 133 SPSYAYHQF AH1A5_CD8 CD8Tcell epitopeagainst gp70 134 SVYDFFVWL TRP2_CD8 CD8Tcell epitopeagainst TRP2

TABLE-US-00007 TABLE4 PeptidesinsertedinpTOP1bygBlocks cloningatposition18ofSEQIDNO:1. Peptidesequence,nameandfunctionaredescribed. SEQ ID NO: Peptide Name Function 12 ISQAVHAAH OVA_CD4 CD4Tcell AEINEAGR epitopeagainst ovalbumin 17 AKFVAAW PADRE Universalantigenic TLKAAA CD4Tcell epitopeagainst pan-HLADR 18 VQGEESNDK VIL1 Universalantigenic CD4Tcell epitopefromIL1 19 QYIKANSK TT Universalantigenic FIGITEL CD4Tcell epitopefromTetanus toxoid 20 WNRQLYPE GP100_CD4 CD4Tcell WTEAQRLD epitopeagainst gp100 21 DPNAPKRPP HP91 Universalantigenic SAFFLFCSE CD4Tcellepitope againstHMGB1- derived immunostimulatory peptidehp91 22 KVPRNQDWL GP100_LP Longpeptide GVSRQLRTK containingaCD8 AWNRQLYPE (underlined)and WTEAQRLD potentialCD4 (italic)Tcell epitopesagainst gp100 23 NLLHRYSLE P1A_LP Longpeptide EILPYLGWL containingaCD8 VFAVVTTSF (underlined)and LALQMFIDA potentialCD4 LYEE Tcellepitopes againstP1A

[0590] List of Constructs

TABLE-US-00008 TABLE 5 List of chimeric VSV-G used in the present invention. Given are their amino acid sequence ID and nucleic acid sequence ID. Nucleic Protein Acid SEQ ID SEQ ID Name Function 38 24 Modified VSV-G in CD8 T cell epitope against ovalbumin in pTOP1-OVA_CD4(18)- position 191 and CD4 T cell epitope against OVA_CD8(191) ovalbumin in position 18 of VSV-G (SEQ ID NO: 1) in pTOP1 39 25 Modified VSV-G in CD8 T cell epitope against ovalbumin in pTOP1-OVA_CD8(191) position 191 of VSV-G (SEQ ID NO: 1) in pTOP1 40 26 Modified VSV-G in CD4 T cell epitope against ovalbumin in pTOP1-OVA_CD4(191) position 191 of VSV-G (SEQ ID NO: 1) in pTOP1 41 27 Modified VSV-G in CD8 T cell epitope against MART-1 in position pTOP1- 191 of VSV-G (SEQ ID NO: 1) in pTOP1 MELANA_CD8(191) 42 28 Modified VSV-G in CD8 T cell epitope against gp100 in position pTOP1- 191 of VSV-G (SEQ ID NO: 1) in pTOP1 GP100_CD8(191) 43 29 Modified VSV-G in CD8 T cell epitope against P1A in position 191 pTOP1-P1A_CD8(191) of VSV-G (SEQ ID NO: 1) in pTOP1 44 30 Modified VSV-G in CD8 T cell epitope against tyrosinase in pTOP1-TYR_CD8(191) position 191 of VSV-G (SEQ ID NO: 1) in pTOP1 45 31 Modified VSV-G in Universal antigenic CD4 T cell epitope against pTOP1-PADRE(18)- pan-HLA DR in position 18 and CD8 T cell OVA_CD8(191) epitope against ovalbumin in position 191 of VSV-G (SEQ ID NO: 1) in pTOP1 46 32 Modified VSV-G in Universal antigenic CD4 T cell epitope from pTOP1-VIL1(18)- IL1 in position 18 and CD8 T cell epitope OVA_CD8(191) against ovalbumin in position 191 of VSV-G (SEQ ID NO: 1) in pTOP1 47 33 Modified VSV-G in Universal antigenic CD4 T cell epitope from pTOP1-TT(18)- Tetanus toxoid in position 18 and CD8 T cell OVA_CD8(191) epitope against ovalbumin in position 191 of VSV-G (SEQ ID NO: 1) in pTOP1 48 34 Modified VSV-G in CD4 T cell epitope against gp100 in position 18 pTOP1-GP100_CD4(18)- and CD8 T cell epitope against ovalbumin in OVA_CD8(191) position 191 of VSV-G (SEQ ID NO: 1) in pTOP1 49 35 Modified VSV-G in HMGB1-derived immunostimulatory peptide pTOP1-HP91(18)- hp91 in position 18 and CD8 T cell epitope OVA_CD8(191) against ovalbumin in position 191 of VSV-G (SEQ ID NO: 1) in pTOP1 50 36 Modified VSV-G in Long peptide containing a CD8 and potential pTOP1-P1A_LP(18)- CD4 T cell epitopes against P1A in position 18 OVA_CD8(191) and CD8 T cell epitope against ovalbumin in position 191 of VSV-G (SEQ ID NO: 1) in pTOP1 51 37 Modified VSV-G in Long peptide containing a CD8 and potential pTOP1-GP100_LP(18)- CD4 T cell epitopes against gp100 in position OVA_CD8(191) 18 and CD8 T cell epitope against ovalbumin in position 191 of VSV-G (SEQ ID NO: 1) in pTOP1 135 139 Modified VSV-G in CD4 T cell epitope against gp100 in position 18 pTOP1-GP100_CD4(18)- and CD8 T cell epitope against TRP2 in TRP2_CD8(191) position 191 of VSV-G (SEQ ID NO: 1) in pTOP1 136 140 Modified VSV-G in Universal antigenic CD4 T cell epitope against pTOP1-PADRE(18)- pan-HLA DR in position 18 and CD8 T cell P1A_CD8(191) epitope against P1A in position 191 of VSV-G (SEQ ID NO: 1) in pTOP1 137 141 Modified VSV-G in Universal antigenic CD4 T cell epitope against pTOP1-PADRE(18)- pan-HLA DR in position 18 and CD8 T cell AH1A5_CD8(191) epitope against gp70 in position 191 of VSV-G (SEQ ID NO: 1) in pTOP1 138 142 Modified VSV-G in Universal antigenic CD4 T cell epitope against pTOP1-PADRE(18)- pan-HLA DR in position 18 and CD8 T cell TRP2_CD8(191) epitope against TRP2 in position 191 of VSV-G (SEQ ID NO: 1) in pTOP1

[0591] Cell Culture

[0592] B16F10-OVA, a melanoma cell line from C57BL/6 mice that stably expresses ovalbumin, was cultured in MEM medium supplemented with GlutaMAX with 10% FBS, 100 g/mL streptomycin and 100 U/mL penicillin (Life Technologies, Carlsbad, Calif., US).

[0593] B16F10, a melanoma cell line from C57BL/6 mice, was cultured in MEM medium supplemented with GlutaMAX with 10% FBS, 100 g/mL streptomycin and 100 U/mL penicillin (Life Technologies, Carlsbad, Calif., US).

[0594] CT26, a colon carcinoma cell line from BALB/C mice, was cultured in DMEM with 10% FBS, 100 g/mL streptomycin and 100 U/mL penicillin, and supplemented with L-glutamate and pyruvate (Life Technologies, Carlsbad, Calif., US).

[0595] P815, a mastocytoma cell line from DBA/2 mice, was cultured in DMEM with 10% FBS, 100 g/mL streptomycin and 100 U/mL penicillin (Life Technologies, Carlsbad, Calif., US).

[0596] Animals

[0597] Six to eight-week-old C57BL/6, BALB/C and DBA/2 female mice were obtained from Janvier Labs (Le Genest Saint Isle, FR) and housed in a minimal disease facility with ad libitum access to food and water.

[0598] For tumor implantation and electroporation, the mice were anaesthetized by intraperitoneal (ip) injection of 150 L of a solution of 10 mg/mL ketamine and 1 mg/mL xylazine. The ethical committee for Animal Care and Use of the Medical Sector of the Universit Catholique de Louvain approved our experimental protocols (UCL/MD/2011/007 and UCL/MD/2016/001).

[0599] Methods

[0600] Immunization

[0601] After removing the hair using a rodent shaver (AgnTho's, Liding, Sweden), 1 g or 50 g of plasmid were injected, diluted in 30 L of PBS, into the left tibial cranial muscle. Immediately after injection, the leg was placed between 4-mm-spaced plate electrodes (BTX Caliper Electrodes), and 8 square-wave electric pulses (80 V, 20 ms, 2 Hz) were delivered by a Gemini System generator (BTX; both from VWR International, Leuven, Belgium). A conductive gel was used to ensure electrical contact with the skin (Aquasonic 100; Parker Laboratories, Inc., Fairfield, N.J., USA).

[0602] For prophylactic vaccination experiments, two boosts (i.e., second and third administrations of the vaccine) were similarly applied two and four weeks after the priming.

[0603] For therapeutic vaccination experiments, the treatment started two days after the injection of the tumor cells and the two boosts were delivered every week.

[0604] Alternatively, plasmids were injected and electroporated into the tumors when they reached a size in-between 30 and 50 mm.sup.3. This treatment was then repeated after two days.

[0605] For the study of the OT-I and OT-II proliferation, plasmids were injected into ears and 2-mm-spaced electrodes were applied to deliver 10 square-wave electric pulses (100 V, 20 ms, 1 Hz).

[0606] Tumor Implantation

[0607] 110.sup.5 B16F10-OVA or B16F10 cells, diluted in 100 L PBS, were injected subcutaneously into the right flank of each C57BL/6.

[0608] 110.sup.6 CT26 cells, diluted in 100 L PBS, were injected subcutaneously into the right flank of each BALB/C.

[0609] 110.sup.6 P815 cells, diluted in 100 L PBS, were injected subcutaneously into the right flank of each DBA/2.

[0610] Tumor cells were implanted two days before the first plasmid administration or two weeks after the last administration for therapeutic and prophylactic DNA immunization studies, respectively. The tumor size was measured three times a week with an electronic digital caliper. Tumor volume was calculated as the lengthwidthheight (in mm.sup.3). The mice were sacrificed when the volume of the tumor reached 1500 mm.sup.3 or when they were in poor condition and expected to die shortly.

[0611] Administration of Immune Checkpoint Blockade (ICB) Antibodies

[0612] For administration of ICB, mice received 100 g of InVivoMAb anti-mouse CTLA-4 (CD152) clone 9D9 and 100 g of InVivoMAb anti-mouse PD-1 (CD279) clone 29F.1A12, both from BioXcell (CT, US) by intraperitoneal injection in 200 L of PBS at day 3, 6 and 9 following implantation of the B16F10-OVA cells.

[0613] OT-I and OT-II Proliferation

[0614] T cells were isolated from spleen and lymph nodes of transgenic OT-I and OT-II mice using CD8+ and CD4+ T cell isolation kit II mouse (Miltenyi Biotec, The Netherlands). Subsequently the T cells were labeled with CFSE (carboxyfluorescein diacetate succinimidyl ester; Molecular probes) by incubating 5010.sup.6 cells/mL with 5 M CFSE for 7 minutes at 37 C. The reaction was blocked by adding ice-cold PBS (Lonza, Belgium)+10% serum. 210.sup.6 OT-I or OT-II cells were injected into the tail vein of C57BL/6 mice. They were treated 2 days later by plasmid injection and electroporation. Mice were sacrificed 4 days later to collect the draining lymph nodes for single cell suspension preparation. Flow cytometric measurement was performed after staining with aqua live dead (Invitrogen, Belgium), CD19 APC-Cy7, CD8 PerCP (all BD Biosciences), dextramer SIINFEKL H-2kb PE (Immudex, Denmark).

[0615] In Vivo Killing Assay

[0616] Splenocytes from naive mice were pulsed with SIINFEKL peptide or with an irrelevant peptide (40 g in 40 mL PBS) for one hour at 37 C. Subsequently, these pulsed splenocytes were washed and respectively stained with high (5 M, hi) or low (0.5 M, low) CFSE concentration. The two populations of splenocytes were mixed in a 1:1 ratio, and 10.sup.7 splenocytes were intravenously injected into immunized mice two weeks after the last booster immunization. Two days after transfer, the spleens of the host mice were isolated and analyzed by flow cytometry after staining with -F4/80 (BD Biosciences, San Diego, Calif., USA) to exclude auto-fluorescent macrophages. The percentage antigen-specific killing was determined using the following formula:

[00001]$\%.Math..Math.\mathrm{antigen}.Math..Math.\mathrm{specific}.Math..Math.\mathrm{killing}=100-\left(100\frac{{\left[\frac{\%.Math..Math.{\mathrm{CFSE}}^{\mathrm{hi}}.Math..Math.\mathrm{cells}}{\%.Math..Math.{\mathrm{CFSE}}^{\mathrm{low}}.Math..Math.\mathrm{cells}}\right]}^{\mathrm{immunized}.Math..Math.\mathrm{mice}}}{{\left[\frac{\%.Math..Math.{\mathrm{CFSE}}^{\mathrm{hi}}.Math..Math.\mathrm{cells}}{\%.Math..Math.{\mathrm{CFSE}}^{\mathrm{low}}.Math..Math.\mathrm{cells}}\right]}^{\mathrm{non}.Math.\text{-}.Math.\mathrm{immunized}.Math..Math.\mathrm{mice}}}\right)$

Example 1: The Effect of pTOP-OVA_CD8(191) Prophylactic Intramuscular Immunization on the Anti-Tumor Activity

[0617] B16 melanoma is a spontaneous melanoma derived from C57BL/6 mice. The most commonly used variant is B16F10, which is highly aggressive and will metastasize from a primary subcutaneous site to the lungs, as well as colonize lungs upon intravenous (iv) injection.

[0618] C57BL/6 mice were immunized in a regimen of one prime and two boosts at a 2-week interval with the pTOP-OVA_CD8(191) plasmid (1 g). Two weeks after the last vaccination, they were challenged with B16F10-OVA cells. This B16F10-OVA cell line is a stable transfectant derived from B16F10 melanoma that stably expresses chicken ovalbumin.

[0619] Tumor growth and mouse survival were assessed for three months.

[0620] Inoculation of B16F10-OVA cells induced tumors that grow rapidly and killed nave mice. However, prophylactic immunization by intramuscular electroporation of a plasmid encoding VSV-G containing a tumor model CD8 T cell epitope delayed tumor growth and improved mice survival (FIGS. 1A and 1B).

Example 2: The Effect of pTOP-OVA_CD8(191) Therapeutic Intratumoral Immunization on the Anti-Tumor Activity

[0621] C57BL/6 mice were challenged with B16F10-OVA cells. When tumor reached between 30 and 50 mm.sup.3, mice were immunized twice with a two-day interval with the pTOP-OVA_CD8(191) plasmid, the pTOP control plasmid (expressing VSV-G of SEQ ID NO: 1 without inserted peptide) or the empty pVAX2 (pEmpty) plasmid (50 g each).

[0622] Therapeutic immunization by intratumoral electroporation of a plasmid encoding VSV-G containing a tumor model CD8 T cell epitope delays tumor growth (FIGS. 2A and 2B).

Example 3: The Effect of Restriction Sites Addition Around the Inserted Epitope Sequence on Vaccine Efficacy

[0623] C57BL/6 mice were immunized in a regimen of one prime and two boosts at a 2-week interval with the pTOP-OVA_CD8(191) plasmid or the pTOP1-OVA_CD8(191) plasmid (1 g each). Two weeks after the last vaccination, they were challenged with B16F10-OVA cells. Tumor growth and mouse survival were assessed.

[0624] The addition of SpeI and EcoRI restriction sites introduce amino acids TS and EF around the inserted epitope. This result showed that adding these amino acids around the T cell epitope does not alter vaccine efficacy (FIGS. 3A and 3B).

Example 4: The Effect of pTOP1-OVA_CD8(191) and pTOP1-OVA_CD4(191) Prophylactic Intramuscular Immunization on the Anti-Tumor Activity

[0625] Insertion of a CD8 T cell epitope in VSV-G is necessary to observe anti-tumor efficacy. There is no anti-tumor effect following pTOP and pTOP1-OVA_CD4(191) delivery. Prophylactic immunization by intramuscular electroporation of two pTOP1 plasmids containing respectively OVA_CD8 and OVA_CD4 T cell epitopes improve protection against tumor challenge as compared to pTOP1-OVA_CD8(191) alone. The tumor growth delay and mice survival are improved when the helper epitope is co-delivered with the MHC class I restricted epitope (FIGS. 4A and 4B).

Example 5: The Effect of pTOP1-OVA_CD8(191) and pTOP1-OVA_CD4(191) Therapeutic Intramuscular Immunization on the Anti-Tumor Activity

[0626] C57BL/6 mice were challenged with B16F10-OVA cells. Two days later, they were immunized in a regimen of one prime and two boosts at a 1-week interval with 1 g of the pTOP1-OVA_CD8(191) alone or combined with 1 g of the pTOP1-OVA_CD4(191) plasmid. Tumor growth and mouse survival were assessed.

[0627] Therapeutic immunization by intramuscular electroporation of two pTOP1 plasmids containing respectively CD8 and CD4 T cell epitopes improves protection against tumor challenge. Two separate experiments have been performed. First, it was shown that therapeutic immunization with pTOP1-OVA_CD8(191) tends to improve protection against challenge (but the effect is not significant). Second, the combination of pTOP1-OVA_CD4(191) and pTOP1-OVA_CD8(191) drastically improved mice survival and delayed tumor growth (FIG. 5A-D).

Example 6: The Effect of Co-Delivery of pTOP1-OVA_CD4(191) with pTOP-OVA_CD8(191) on the Cytotoxic T Cell Response

[0628] C57BL/6 mice were immunized in a regimen of one prime and two boosts at a 2-week interval with 1 g of the pTOP1-OVA_CD8(191) plasmid alone or combined with 1 g of the pTOP1-OVA_CD4(191) plasmid. The percentage of antigen specific killing was analyzed by in vivo cytotoxic assay. Immunized mice were adoptively transferred with two populations of labelled splenocytes: MHC-I OVA peptide-pulsed-target cells and a MHC-I irrelevant-peptide-pulsed cells. Two days after transfer, the specific killing of target cells was obtained by comparing the relative decrease of the two populations.

[0629] An in vivo killing assay demonstrated that co-delivery of pTOP1-OVA_CD8(191) and pTOP1-OVA_CD4(191) improves the cytotoxic T cell response to the vaccine antigen as compared to delivery of pTOP1-OVA_CD8(191) alone (FIG. 6).

Example 7: OT-II Proliferation Assay

[0630] The effect of immunization with MHC class II-restricted epitope inserted in pTOP1 on the CD4+ T cell response has been demonstrated using OT-II cells. T cells were isolated from spleen and lymph nodes of transgenic OT-II mice, labeled with CFSE and adoptively transferred to C57BL/6 mice. Mice were immunized two days later with 1 g of pTOP1-OVA_CD4(191) or 1 g of pTOP1-OVA_CD8(191). Mice were sacrificed four days later and labelled T cell proliferation was assessed.

[0631] The insertion of MHC class II-restricted epitopes in VSV-G-induced CD4+ T cell response, whereas MHC class I-restricted epitopes are unable to induce helper response (FIG. 7).

Example 8: OT-I Proliferation Assay

[0632] The effect of immunization with MHC class I-restricted epitope inserted in pTOP1 on the CD8+ T cell response has been demonstrated using OT-I cells. T cells were isolated from spleen and lymph nodes of transgenic OT-I mice, labeled with CFSE and adoptively transferred to receptor C57BL/6 mice. Mice were immunized two days later by electroporation of pTOP1-OVA_CD4(191) (1 g) or pTOP1-OVA_CD8(191) (1 g). Mice were sacrificed four days later and labelled T cell proliferation was assessed.

[0633] The insertion of MHC class I-restricted epitopes in VSV-G induced CD8+ T cell response, whereas MHC class II-restricted epitopes are unable to induce CD8+ T cell response (FIG. 8).

Example 9: The Effect of pTOP1 Immunization in Combination with Immune Checkpoint Blockade (ICB) Therapy

[0634] C57BL/6 mice were challenged with B16F10-OVA cells. Two days later, they were immunized in a regimen of one prime and two boosts at a 1-week interval. On day 3, 6 and 9 following challenge, the ICB treatments were given. Mice received either [0635] (1) both pTOP1-OVA_CD8(191) (1 g) and pTOP1-OVA_CD4(191) (1 g) plasmids; [0636] (2) a cocktail of anti-PD-1 and anti-CTLA-4 antibodies [ICB group]; or [0637] (3) a combination of the two plasmids (1 g each) and the antibodies cocktail [combination group].

[0638] Tumor growth and mice survival were assessed following challenge.

[0639] Efficacy of pTOP1 is further enhanced by combination with immune checkpoint blockade therapy. These results demonstrated that the combinatory treatment has a synergic effect compared to treatments alone. Indeed, survival, tumor growth and tumor volume observed after the combinatory treatment are better than the sum of effects obtained after separate treatments (FIGS. 9A and 9B).

Example 10: The Effect of pTOP1-OVA_CD4(18)_OVA_CD8(191) and pTOP1-Gp100_CD4(18)_TRP2_CD8(191) Therapeutic Intramuscular Immunization on the Anti-Tumor Activity

[0640] C57BL/6 mice were challenged with B16F10-OVA cells. Two days later, they were immunized in a regimen of one prime and two boosts at a 1-week interval with 1 g of the pTOP1-OVA_CD4(18)_OVA_CD8(191) plasmid or 1 g of the pTOP1-gp100_CD4(18)_TRP2_CD8(191) plasmid. Tumor growth and mouse survival were assessed.

[0641] Therapeutic immunization by intramuscular electroporation of pTOP1-OVA_CD4(18)_OVA_CD8(191) plasmid or pTOP1-gp100_CD4(18)_TRP2_CD8(191) was able to significantly delay tumor growth. There was no statistical difference between the two vaccines (FIGS. 10A and 10B).

Example 11: The Effect of pTOP1-PADRE(18)_P1A_CD8(191) Prophylactic Intramuscular Immunization on the Anti-Tumor Activity

[0642] DBA/2 mice were immunized in a regimen of one prime and two boosts at a 2-week interval with the pTOP1-PADRE(18)_P1A_CD8(191) plasmid (1 g). Two weeks after the last vaccination, they were challenged with P815 cells. Tumor growth and mouse survival were assessed for two months.

[0643] Inoculation of P815 cells induced tumors that grow rapidly and killed nave mice. However, prophylactic immunization by intramuscular electroporation of a plasmid encoding VSV-G containing a tumor model CD8 T cell epitope and a universal antigenic CD4 T cell epitope delayed tumor growth and improved mice survival (FIGS. 11A and 11B).

Example 12: The Effect of pTOP1-PADRE(18)_P1A_CD8(191) Therapeutic Intramuscular Immunization on the Anti-Tumor Activity

[0644] DBA/2 mice were challenged with P815 cells. Two days later, they were immunized in a regimen of one prime and two boosts one and two weeks later with the pTOP1-PADRE(18)_P1A_CD8(191) plasmid (1 g). Mice survival was assessed for two months.

[0645] Therapeutic immunization by intramuscular electroporation of pTOP1-PADRE(18)_P1A_CD8(191) plasmid was able to significantly delay tumor growth. (FIG. 12).

Example 13: The Effect of pTOP1-PADRE(18)_AH1A5_CD8(191) Prophylactic Intramuscular Immunization on the Anti-Tumor Activity

[0646] BALB/C mice were immunized in a regimen of one prime and two boosts at a 2-week interval with the pTOP1-PADRE(18)_AH1A5_CD8(191) plasmid (1 g). Two weeks after the last vaccination, they were challenged with CT26 cells. Tumor growth and mouse survival were assessed for two months.

[0647] Inoculation of CT26 cells induced tumors that grow rapidly and killed nave mice. However, prophylactic immunization by intramuscular electroporation of a plasmid encoding VSV-G containing a tumor model CD8 T cell epitope and a universal antigenic CD4 T cell epitope delayed tumor growth (FIGS. 13A and 13B).

Example 14: The Effect of pTOP1-PADRE(18)_TRP2_CD8(191) Prophylactic Intramuscular Immunization on the Anti-Tumor Activity

[0648] BALB/C mice were immunized in a regimen of one prime and two boosts at a 2-week interval with the pTOP1-PADRE(18)_TRP2_CD8(191) plasmid (1 g). Two weeks after the last vaccination, they were challenged with B16F10 cells. Tumor growth and mouse survival were assessed for two months.

[0649] Inoculation of B16F10 cells induced tumors that grow rapidly and killed nave mice. However, prophylactic immunization by intramuscular electroporation of a plasmid encoding VSV-G containing a tumor model CD8 T cell epitope and a universal antigenic CD4 T cell epitope delayed tumor growth and improved mice survival (FIGS. 14A and 14B).

Example 15: The Effect of pTOP1-Gp100_CD4(18)_OVA_CD8(191) and pTOP1-Gp100_LP(18)_OVA_CD8(191) Therapeutic Intramuscular Immunization on the Anti-Tumor Activity

[0650] C57BL/6 mice were challenged with B16F10-OVA cells. Two days later, they were immunized in a regimen of one prime and two boosts at a 1-week interval with 1 g of the pTOP1-gp100_CD4(18)_OVA_CD8(191) plasmid or 1 g of the pTOP1-gp100_LP(18)_OVA_CD8(191) plasmid. Tumor growth and mouse survival were assessed.

[0651] Therapeutic immunization by intramuscular electroporation of pTOP1-gp100_CD4(18)_OVA_CD8(191) plasmid or pTOP1-gp100_LP(18)_OVA_CD8(191) was able to significantly delay tumor growth. There was no statistical difference between the two vaccines (FIG. 15).