An mRNA cancer vaccine encoding human GM-CSF fused to multiple tandem epitopes

Abstract

The present invention provides an mRNA cancer vaccine encoding human GM-CSF fused to multiple tandem epitopes. pVec-GM-CSF-hTes encoding human GM-CSF fused to three tandem hTERT epitopes, pVec-GMKE encoding human GM-CSF fused to three tandem epitopes respectively from MUC1, Kras and EGFR, pVec-hIL-12 encoding human interleukin-12 are respectively constructed, and used as templates for generating the corresponding in vitro transcribed mRNAs, which are mixed together as an mRNA cancer vaccine. This mRNA cancer vaccine contains human GM-CSF used as an immune adjuvant, multiple tandem epitopes constituting as multi-epitope cancer antigens and hIL-12 used to enhance the immunotherapeutic effects.

Claims

1. The pVec-GM-CSF-hTes, wherein the complete nucleotide sequence of pVec-GM-CSF-hTes is at least 65% identical to SEQ ID NO: 21.

2. The pVec-GM-CSF-hTes of claim 1, wherein the nucleotide sequence of the reverse primer used for PCR amplification and obtaining human GM-CSF (without a stop codon) is at least 69% identical to SEQ ID NO: 3.

3. The pVec-GM-CSF-hTes of claim 1, wherein the nucleotide sequence of the forward primer used for PCR amplification and obtaining hTERT (1540-548)-11 aa-hTERT (572Y-580)-2 aa-hTERT (988Y-997) is at least 69% identical to SEQ ID NO: 16.

4. The pVec-GM-CSF-hTes of claim 1, wherein the nucleotide sequence of the reverse primer used for PCR amplification and obtaining hTERT (1540-548)-11 aa-hTERT (572Y-580)-2 aa-hTERT (988Y-997) is at least 68% identical to SEQ ID NO: 17.

5. The pVec-GM-CSF-hTes of claim 1, wherein the amino acid sequence of hTERT (1540-548)-11 aa-hTERT (572Y-580)-2 aa-hTERT (988Y-997) is at least 39% identical to SEQ ID NO: 8.

6. The pVec-GM-CSF-hTes of claim 1, wherein the nucleotide sequence of hTERT (1540-548)-11 aa-hTERT (572Y-580)-2 aa-hTERT (988Y-997) is at least 32% identical to SEQ ID NO: 18.

7. The pVec-GM-CSF-hTes of claim 1, wherein the amino acid sequence of GM-CSF-hTes is at least 78% identical to SEQ ID NO: 20.

8. The pVec-GM-CSF-hTes of claim 1, wherein the nucleotide sequence of GM-CSF-hTes is at least 76% identical to SEQ ID NO: 19.

9. The pVec-GMKE, wherein the complete nucleotide sequence of pVec-GMKE is at least 64% identical to SEQ ID NO: 36.

10. The pVec-GMKE of claim 9, wherein the amino acid sequence of MUC1 (aa 130-154)-2 aa-Kras 12 Val (aa 5-17)-2 aa-EGFR T790M (aa 788-798) is at least 52% identical to SEQ ID NO: 28.

11. The pVec-GMKE of claim 9, wherein the nucleotide sequence of MUC1 (aa 130-154)-2 aa-Kras 12 Val (aa 5-17)-2 aa-EGFR T790M (aa 788-798) is at least 49% identical to SEQ ID NO: 29.

12. The pVec-GMKE of claim 9, wherein the amino acid sequence of GMKE is at least 72% identical to SEQ ID NO: 34.

13. The pVec-GMKE of claim 9, wherein the nucleotide sequence of GMKE is at least 71% identical to SEQ ID NO: 35.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 pVec-GM-CSF-hTes map

[0013] Human GM-CSF (without a stop codon)-linker-three tandem hTERT epitopes (with a stop codon) is subcloned between NheI and XhoI sites of pVec.

[0014] Full nucleotide sequence of pVec-GM-CSF-hTes: 3,930 bp

[0015] GM-CSF-hTes: bases 801-1,391 bp

[0016] FIG. 2 pVec-GMKE map

[0017] GMKE which stands for human GM-CSF fused to three tandem epitopes respectively from MUC1, Kras and EGFR is subcloned between NheI and XhoI sites of pVec.

[0018] Full nucleotide sequence of pVec-GMKE: 3,966 bp

[0019] GMKE: bases 801-1,427 bp

[0020] FIG. 3 pVec-hIL-12 map

[0021] SalI-hIL-12-NheI is subcloned between XhoI and XbaI sites of pVec.

[0022] Full nucleotide sequence of pVec-hIL-12: 5,145 bp

DETAILED DESCRIPTION OF THE INVENTION

[0023] The object of the present invention is to provide an mRNA cancer vaccine encoding human granulocyte macrophage colony-stimulating factor (GM-CSF) fused to multiple tandem epitopes, which is obtained using conventional molecular biotechnologies through the following steps.

[0024] Taking pCMV-SPORT6-GM-CSF (purchased from Open Biosystems, GM-CSF GenBank accession number: BC108724) as a template, and using the forward primer designed according to Kozak sequence as SEQ ID NO: 1 and the reverse primer designed by deleting human GM-CSF stop codon (tga) and adding a linker (SEQ ID NO: 2) to the 3 end as SEQ ID NO: 3, the product obtained by polymerase chain reaction (PCR) amplification is subcloned into NheI and HindIII sites of our proprietary pVec, and transformed into top10 chemically competent E. coli cells or DH5 alpha competent cells, obtaining pVec-NheI-GM-CSF (without a stop codon)-linker-HindIII.

[0025] pYEX-BX encoding KAP123-flu (purchased from Addgene, plasmid number: 24048) is digested with restriction endonuclease San. Subsequently, the fragment containing vector backbone is isolated by 1% agarose gel electrophoresis, self-ligated with T4 DNA ligase by head to tail connection and transformed into top10 chemically competent E. coli cells or DH5 alpha competent cells, obtaining pYEX-BX vector.

[0026] Three epitopes including 1540-548 (SEQ ID NO: 4), 572Y-580 (SEQ ID NO: 5) and 988Y-997 (SEQ ID NO: 6) are selected from hTERT (GenBank accession number: AF015950). Two linkers including an 11 amino acid (aa) linker (SEQ ID NO: 7) and a 2 amino acid linker (GlyGly) are designed and used to tandem connect the above three hTERT epitopes.

[0027] The designed amino acid sequence of hTERT (1540-548)-11 aa-hTERT (572Y-580)-2 aa-hTERT (988Y-997) is as SEQ ID NO: 8 and the corresponding nucleotide sequence (with a start codon, atg) is as SEQ ID NO: 9.

[0028] To obtain the fragment containing BamHI-hTERT (1540-548)-11 aa-hTERT (572Y-580)-2 aa-hTERT (988Y-997)-SalI, the following designed oligonucleotides are synthesized and ligated.

[0029] hTERT F1 nucleotide sequence is as SEQ ID NO: 10.

[0030] hTERT F2 nucleotide sequence is as SEQ ID NO: 11.

[0031] hTERT F3 nucleotide sequence is as SEQ ID NO: 12.

[0032] hTERT R1 nucleotide sequence is as SEQ ID NO: 13.

[0033] hTERT R2 nucleotide sequence is as SEQ ID NO: 14.

[0034] hTERT R3 nucleotide sequence is as SEQ ID NO: 15.

[0035] Two g of pYEX-BX is digested with BamHI and SalI, dephosphorylated with alkaline phosphatase (calf intestinal, CIP, New England Biolabs, Cat #: M0290S) and purified.

[0036] All the above indicated oligonucleotides (0.25 g/each oligonucleotide), 2.5 l of 10 reaction buffer, 2 l T4 polynucleotide kinase (New England Biolabs, Cat #: M0201S) and the appropriate amount of water to a total volume of 25 l are put into a PCR reaction tube. After mixing, the above reaction tube is incubated for phosphorylation at 37 C. for 1 hour, subsequently denatured at 94 C. for 10 minutes, annealed at room temperature for 30 minutes and then put on ice for 10 minutes.

[0037] Four l of the above annealed reaction products, an equal amount of pYEX-BX vector digested with BamHI and SalI and dephosphorylated, 2 l of 10T4 ligation buffer, 1 l T4 DNA ligase, and the appropriate water to a total volume of 20 l are put into a PCR reaction tube, incubated at 16 C. overnight and then transformed into top10 chemically competent E. coli cells or DH5 alpha competent cells, obtaining pYEX-BX-BamHI-hTERT (1540-548)-11 aa-hTERT (572Y-580)-2 aa-hTERT (988Y-997)-SalI.

[0038] Taking the above constructed vector pYEX-BX-BamHI-hTERT (1540-548)-11 aa-hTERT (572Y-580)-2 aa-hTERT (988Y-997)-SalI as a template, and using the forward primer deleting a start codon (atg) as SEQ ID NO: 16 and the reverse primer adding a stop codon (tga) as SEQ ID NO: 17, the fragment containing hTERT (1540-548)-11 aa-hTERT (572Y-580)-2 aa-hTERT (988Y-997)-stop codon (tga) is obtained by PCR amplification, and then subcloned into HindIII and XhoI sites of pVec-NheI-GM-CSF (without a stop codon)-linker-HindIII, and transformed into top10 chemically competent E. coli cells or DH5 alpha competent cells, obtaining pVec-NheI-GM-CSF (without a stop codon)-linker-HindIII-hTERT (1540-548)-11 aa-hTERT (572Y-580)-2 aa-hTERT (988Y-997)-stop codon (tga)-XhoI, referred to as pVec-GM-CSF-hTes. pVec-GM-CSF-hTes is deposited as PTA-122795 at the American Type Culture Collection (ATCC).

[0039] The nucleotide sequence of hTERT (1540-548)-11 aa-hTERT (572Y-580)-2 aa-hTERT (988Y-997) of pVec-GM-CSF-hTes is as SEQ ID NO: 18. The nucleotide sequence of GM-CSF (without a stop codon)-linker-HindIII-hTERT (1540-548)-11 aa-hTERT (572Y-580)-2 aa-hTERT (988Y-997) or GM-CSF-hTes of pVec-GM-CSF-hTes is as SEQ ID NO: 19, the corresponding amino acid sequence is as SEQ ID NO: 20. The full nucleotide sequence of pVec-GM-CSF-hTes has been sequenced by Genewiz Company and is as SEQ ID NO: 21.

[0040] The amino acid sequence of MUC1 (aa 130-154) selected from MUC1 (GenBank accession number: J05582) is as SEQ ID NO: 22, and the corresponding nucleotide sequence is as SEQ ID NO: 23.

[0041] The amino acid sequence of Kras 12 Val (aa 5-17) selected from Kras (GenBank accession number: M54968) is as SEQ ID NO: 24, and the corresponding nucleotide sequence is as SEQ ID NO: 25.

[0042] The amino acid sequence of EGFR T790M (aa 788-798) selected from EGFR (GenBank accession number: GU255993) is as SEQ ID NO: 26, and the corresponding nucleotide sequence is as SEQ ID NO: 27.

[0043] The amino acid sequence of the linker used to tandem connect the above mentioned epitopes is as Gly Gly, and the corresponding nucleotide sequence is as gga ggt.

[0044] The amino acid sequence of the designed MUC1 (aa 130-154)-2 aa-Kras 12 Val (aa 5-17)-2 aa-EGFR T790M (aa 788-798) is as SEQ ID NO: 28, and the corresponding nucleotide sequence is as SEQ ID NO: 29. In addition, a stop codon (tga) is added to the 3 end.

[0045] The inserter containing MUC1 (aa 130-154)-2 aa-Kras 12 Val (aa 5-17)-2 aa-EGFR T790M (aa 788-798)-stop codon (tga) is gradually subcloned into HindIII and XhoI sites of pVec-NheI-GM-CSF (without a stop codon)-linker-HindIII, transformed into top10 chemically competent E. coli cells or DH5 alpha competent cells.

[0046] Taking pVec-NheI-GM-CSF (without a stop codon)-linker-HindIII as a template and using the above mentioned forward primer as SEQ ID NO: 1 as well as the reverse primer as SEQ ID NO: 30, the product obtained by PCR amplification is subcloned into NheI and XhoI sites of pVec-NheI-GM-CSF (without a stop codon)-linker-HindIII, and transformed into top10 chemically competent cells or DH5 alpha competent cells, obtaining pVec-NheI-GM-CSF (without a stop codon)-linker-HindIII-MUC1a-XhoI.

[0047] Taking the above obtained pVec-NheI-GM-CSF (without a stop codon)-linker-HindIII-MUC1a-XhoI as a template and using the mentioned forward primer as SEQ ID NO: 1 as well as the reverse primer as SEQ ID NO: 31, the product obtained by PCR amplification is subcloned into NheI and XhoI sites of pVec-NheI-GM-CSF (without a stop codon)-linker-HindIII, and transformed into top10 chemically competent cells or DH5 alpha competent cells, obtaining pVec-NheI-GM-CSF (without a stop codon)-linker-HindIII-MUC1-XhoI.

[0048] Taking the above obtained pVec-NheI-GM-CSF (without a stop codon)-linker-HindIII-MUC1-XhoI as a template and using the mentioned forward primer as SEQ ID NO: 1 as well as the reverse primer as SEQ ID NO: 32, the product obtained by PCR amplification is subcloned into NheI and XhoI sites of pVec-NheI-GM-CSF (without a stop codon)-linker-HindIII, and transformed into top10 chemically competent cells or DH5 alpha competent cells, obtaining pVec-NheI-GM-CSF (without a stop codon)-linker-HindIII-MUC1-2 aa-Kras 12 Val-2 aa-XhoI.

[0049] Taking the above obtained pVec-NheI-GM-CSF (without a stop codon)-linker-HindIII-MUC1-2 aa-Kras 12 Val-2 aa-XhoI as a template and using the mentioned forward primer as SEQ ID NO: 1 as well as the reverse primer as SEQ ID NO: 33, the product obtained by PCR amplification is subcloned into NheI and XhoI sites of pVec-NheI-GM-CSF (without a stop codon)-linker-HindIII, and transformed into top10 chemically competent cells or DH5 alpha competent cells, obtaining pVec-NheI-GM-CSF (without a stop codon)-linker-HindIII-MUC1 (aa 130-154)-2 aa-Kras 12 Val (aa 5-17)-2 aa-EGFR T790M (aa 788-798)-stop codon (tga)-XhoI, referred to as pVec-GMKE. pVec-GMKE is deposited as PTA-122796 at the American Type Cell Collection (ATCC).

[0050] The amino acid sequence of GM-CSF (without a stop codon)-linker-HindIII-MUC1 (aa 130-154)-2 aa-Kras 12 Val (aa 5-17)-2 aa-EGFR T790M (aa 788-798) or GMKE of pVec-GMKE is as SEQ ID NO: 34, and the corresponding nucleotide sequence is as SEQ ID NO: 35. The full nucleotide sequence of pVec-GMKE has been sequenced by Genewiz Company and is as SEQ ID NO: 36.

[0051] Human interleukin-12 (hIL-12) gene is obtained by digesting pORF-hIL-12 G2 (InvivoGen) with SalI and NheI, and subcloned into XhoI and XbaI sites of pVec, obtaining pVec-hIL-12. The complete nucleotide sequence of pVec-hIL-12 is as SEQ ID NO: 37.

[0052] The above obtained pVec-GM-CSF-hTes, pVec-GMKE and pVec-hIL-12 are amplified, purified with Qiaprep spin miniprep kit (Qiagen, Cat #: 27106), digested with restriction endonuclease SpeI respectively, obtaining the corresponding linearized plasmid DNAs. A small amount of each of the above SpeI cut plasmid DNAs is used to detect whether each of the plasmid DNAs is completely linearized by 1% agarose gel electrophoresis. Each of the obtained linearized plasmid DNAs is purified through the following protocol. The mixture of 100 l SpeI cut plasmid DNA reaction solution with 500 l Buffer PB is transferred into a spin column, centrifuging for 30 seconds and discarding the effluent (flow-through). 750 l Buffer PE is added to the above spin column, centrifuging for 30 seconds, draining the effluent and then centrifuging again for 1 minute. The above spin column is put into a clean micro-centrifuge tube, adding 30 l of water to the spin column, standing for 1 minute and centrifuging for 1 minute. The purified linearized plasmid DNA is used to determine the DNA concentration and adjust to the concentration of 0.5 to 1 g/1.

[0053] Using HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs, Cat #: E2040S) and 3-0-Me-m.sup.7G(5)ppp(5)G RNA Cap Structure Analog (ARCA, New England Biolabs, Cat #: S1411S), the IVT GM-CSF-hTes mRNA, GMKE mRNA and hIL-12 mRNA are respectively generated through the following steps.

[0054] At room temperature, the following reagents are respectively added to a 1.5 ml micro-centrifuge tube.

TABLE-US-00001 Nuclease-free water x l 10 X reaction buffer 2 l ATP (100 mM) 2 ul 10 mM final UTP (100 mM) 2 l 10 mM final CTP (100 mM) 2 l 10 mM final GTP (20 mM) 2 l 2 mM final ARCA (40 mM) 4 l 8 mM final Template DNA (linearized) x l 1 g T7 RNA polymerase mix 2 l Total reaction volume 20 l

[0055] After mixing well and pulse-spinning, the above reaction tube is incubated at 37 C. for 2 hours. To remove the template DNA, 70 l nuclease-free water, 10 l of 10DNase I buffer and 2 l DNase I (New England Biolabs, Cat #: M0303S) are added to the above reaction tube, incubating at 37 C. for 15 minutes.

[0056] Using RNeasy mini kit (Qiagen, Cat #: 74104), the IVT GM-CSF-hTes mRNA, GMKE mRNA and hIL-12 mRNA are respectively purified through the following steps. 20 to 30 l of the IVT mRNA diluted with nuclease-free water is taken and transferred into a micro-centrifuge tube (nuclease-free) each time, 350 l Buffer RLT with 1% -mercaptoethanol (-ME) is added to the above tube. After thoroughly mixing with pipette, an equal volume of 70% ethanol is added to the above tube. After mixing, the above mixture is transferred into a spin column for centrifuging and draining the effluent (flow-through). 700 l Buffer RW1 is added to the above spin column, centrifuging and draining the effluent. 500 l Buffer RPE is added to the above spin column, centrifuging and draining the effluent, repeating twice. After centrifuging again for 1 minute, the above spin column is transferred into a clean micro-centrifuge tube (nuclease-free) and 30 l of nuclease-free water is added to the above spin column, standing for 1 minute and then centrifuging for 1 minute. The purified IVT mRNA is used to determine the mRNA concentration using a Nanodrop spectrophotometer and its quality is detected by 1% formaldehyde agarose gel electrophoresis.

[0057] Each of the purified IVT GM-CSF-hTes mRNA (5 g), GMKE mRNA (5 g) and hIL-12 mRNA (5 g) is respectively electroporated into 110.sup.6 cells (e.g., mouse cell lines) in a 0.2 cm cuvette at the condition of 350 V, 500 s. Subsequently the cells electroporated with the IVT mRNA are cultured in a cell growth medium at 5% CO.sub.2, 37 C. for 36 hours and then the above cell supernatants are collected.

[0058] The collected supernatants of the cells electroporated with GM-CSF-hTes mRNA and the cells with GMKE mRNA are respectively used to detect human GM-CSF expression with human GM-CSF enzyme-linked immunosorbent assay (ELISA) kit (eBioscience, Cat #: 88-8337-22) through the following steps.

[0059] The ELISA plate is coated with 100 l capture antibody diluted with 1 coating buffer at the ratio of 1:250 each well, sealed and put at 4 C. overnight.

[0060] Next day, discarding the excess capture antibody solution and washing the above ELISA plate with wash buffer [1 phosphate-buffered saline (PBS) containing 0.05% Tween-20] 3 times, at least 1 minute each time and patting dry, 200 l of 1ELISA/ELISPOT Diluent is added to each well of the above plate, then incubating at room temperature for 1 hour.

[0061] The above ELISA plate is washed following the previous indicated method once. 100 l of 1ELISA/ELISPOT Diluent diluted standard human GM-CSF or 100 l of the collected cell supernatant is added to each well, then sealing and incubating at room temperature for 2 hours.

[0062] The above plate is washed according to the above indicated method 3 to 5 times. 100 l of 1ELISA/ELISPOT Diluent diluted detection antibody is added to each well, then sealing and incubating at room temperature for 1 hour.

[0063] The above plate is washed according to the previous mentioned method 3 to 5 times. 100 l of 1ELISA/ELISPOT Diluent diluted Avidin-horseradish peroxidase (HRP) is added to each well, then sealing and incubating at room temperature for 30 minutes.

[0064] The plate is washed according to the above indicated method 5 to 7 times. 100 l of 1 tetramethylbenzidine (TMB) solution is added to each well, incubating at room temperature for 15 minutes.

[0065] Then 50 l of 2 M H.sub.2SO.sub.4 stop solution is added to each well of the above ELISA plate. Further, the concentration of human GM-CSF expressed in the cell supernatant is determined by measuring optical density (OD) value at 450 nm using a micro-plate reader.

[0066] The experimental results show that both the cells electroporated with GM-CSF-hTes mRNA and the cells with GMKE mRNA can express human GM-CSF.

[0067] The collected supernatants from the cells electroporated with the IVT hIL-12 mRNA are used to detect human IL-12 expression with human IL-12 ELISA kit (eBioscience, Cat #: 88-7126-88) by the previous mentioned protocol.

[0068] The ELISA plate is coated with 100 l capture antibody diluted with 1 coating buffer at the ratio of 1:250 for each well, sealed and incubated at 4 C. overnight.

[0069] After discarding the coating solution containing capture antibody, rinsing with wash buffer (1PBS containing 0.05% Tween-20) 3 times, at least 1 minute each time, and patting dry, 200 l of 1ELISA/ELISPOT Diluent is added to each well of the above plate, then incubating at room temperature for 1 hour.

[0070] According to the previous mentioned method, the above plate is washed. 100 l of 1ELISA/ELISPOT Diluent diluted standard human IL-12 or 100 l of the collected supernatant is added to each well, then sealing and incubating at room temperature for 2 hours.

[0071] The plate is washed according to the previous method 3 to 5 times. 100 l of 1ELISA/ELISPOT Diluent diluted detection antibody is added to each well, then sealing and incubating at room temperature for 1 hour.

[0072] The plate is washed according to the above method 3 to 5 times. 100 l of 1ELISA/ELISPOT Diluent diluted Avidin-HRP is added to each well of the above plate, sealing and incubating at room temperature for 30 minutes.

[0073] The plate is washed according to the above method 5 to 7 times, 100 l of 1TMB solution is added to each well, then incubating at room temperature for 15 minutes.

[0074] Then 50 l of 2 M H.sub.2SO.sub.4 stop solution is added to each well of the above plate. Further, the concentration of human IL-12 expressed in the cell supernatant is determined by measuring OD value at 450 nm using a micro-plate reader.

[0075] The experimental results show that the cells electroporated with the IVT hIL-12 mRNA can express human IL-12.

[0076] The percentage identity between a query sequence and a subject is obtained using basic local alignment search tool (BLAST).

[0077] The mRNA components of an mRNA cancer vaccine encoding human GM-CSF fused to multiple tandem epitopes include GM-CSF-hTes mRNA, GMKE mRNA and hIL-12 mRNA. This mRNA cancer vaccine contains human GM-CSF used as an immune adjuvant, multiple tandem epitopes constituting as multi-epitope cancer antigens and hIL-12 used to enhance the immunotherapeutic effects. Therefore, this mRNA cancer vaccine would be a very effective vaccine for cancer immunotherapy, especially targeting non-small cell lung cancer (NSCLC) patients.