CN116983394B - mRNA vaccine and application thereof in intratumoral delivery for enhancing tumor treatment effect - Google Patents
mRNA vaccine and application thereof in intratumoral delivery for enhancing tumor treatment effect Download PDFInfo
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Classifications
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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Abstract
The invention discloses an mRNA vaccine and application thereof in enhancing tumor treatment effect in intratumoral delivery, and researches show that mRNA for delivering certain cytokines can improve mouse tumor immunity microenvironment, increase infiltration of tumor tissue CD8+T, kill tumor cells and inhibit tumor growth. And can enhance the tumor treatment effect of immune checkpoint inhibitor anti-PD-1 monoclonal antibody and chemotherapeutic drug oxaliplatin. The application of the invention realizes the treatment method of inhibiting the development of tumor by using mRNA and the combined drug of mRNA and the existing tumor treatment means, and has good prospect and practical value. Provides a certain reference and a certain hint for the development of mRNA in the tumor treatment field and provides a new idea for the combined drug for tumor treatment.
Description
Technical Field
The invention relates to application of mRNA therapy, in particular to an mRNA vaccine and application thereof in intratumoral delivery for enhancing tumor treatment effect.
Background
The current tumor treatment modes mainly comprise operation treatment, chemotherapy, radiotherapy, targeted treatment and immunotherapy. Compared with the immunotherapy, the immunotherapy is a novel mode for treating tumors, and mainly kills tumor cells by enhancing or activating the functions of immune cells and inhibits the growth of the tumors. However, only 20% to 40% of patients in clinical statistics benefit from PD-1 immunotherapy, and thus there is a need for further optimisation of the tumor treatment regimen.
mRNA therapy is a therapy based on molecular biology and immunology, which is a technique of designing mRNA for synthesizing a target gene and delivering the target gene to a human or animal body by using a delivery system. mRNA therapy has multiple advantages, firstly, the mRNA therapy has high safety, mRNA does not enter the cell nucleus and is translated and expressed in cytoplasm; in addition, mRNA is easily decomposed completely by physiological metabolic pathways, and thus does not become a burden on host homeostasis; secondly, mRNA has self-assisting properties, and activates strong and durable adaptive immune responses through tumor necrosis factor-alpha (TNF-alpha), interferon-alpha (IFN-alpha) and other cytokines secreted by immune cells; in addition, the mRNA can theoretically meet all genetic information required by encoding and expressing various proteins, is synthesized through an In Vitro Transcription (IVT) process, has simple manufacturing process and easy mass production, and simultaneously has short mRNA research and development period, so that novel therapeutic mRNA can be rapidly developed; finally, mRNA can be translated to produce large amounts of protein, which reduces drug usage to some extent, which also facilitates the co-administration of multiple mrnas.
mRNA therapies are now designed primarily for the prevention and treatment of viral infections and tumors, and several mRNA vaccines are now in clinical research and products have been formally put into clinical use, such as vaccines against SARS-CoV-2 virus. Gene therapy for cancer therapy may involve delivery of genes that kill or inhibit cancer cells, enhance immune responses against cancer cells, repair or replace gene mutations or alterations, or make cancer cells more susceptible to chemotherapy or radiation, but prior art drug delivery is inefficient and relatively unstable and the use of mRNA for intratumoral delivery of therapeutic tumors has not been reported.
Disclosure of Invention
Aiming at the defects of the prior research content, the invention aims to provide an mRNA vaccine and application thereof in intratumoral delivery for enhancing the tumor treatment effect.
The mRNA vaccine provided by the invention at least comprises a first open reading frame and a second open reading frame, wherein the first open reading frame codes for a target protein; the second open reading frame expresses one or more combinations selected from the group consisting of CCL5, CXCL9, CXCL10, IL15, CXCL16, myb gene sequences.
Further, the protein of interest may be selected from proteins directed against tumors, including but not limited to: epCAM, anti-CTLA-4, anti-PD 1, anti-PDL 1, A2A, anti-FGF 2, anti-FGFR/FGFR 2B, anti-SEMA 4D, CCL5, CD137, CD200, CD38, CD44, CSF-1R, CXCL, CXCL13, endothelin B receptor, IL-15, IL-21, IL-35, ISRE7, LFA-1, NG2 (also known as SPEG 4), SMAD, STING, t gfβ, and VCAM 1; IFNγ, IFNα, I F N beta, TNF alpha, IL-12, IL-2, IL-6, IL-8, and GM-CSF pro-inflammatory cytokines, etc.
Further, the second open reading frame expresses a combination of CCL5, CXCL9, CXCL10, IL15, CXCL16, myb gene sequences;
further, the second open reading frame expresses fusion proteins of CCL5 and CXCL9, wherein after bioinformatics analysis, the fusion proteins are truncated and replaced, and the optimized sequences are connected through RRKK to form a positively charged amphipathic structure, so that the fusion proteins are conveniently fixed and inserted into a negatively charged phospholipid bilayer, and the specific sequence is shown in SEQ ID NO.1 (SPYSDTTPACFAYIARPLPRHIKEYFYTSGKCSNAVVFVTARKNRQVCANPEKKWVAREY INSLEMSRRKKTPVAVRKGRCCISTNQGTIHALQSLKDLKQFAPSPSCAEKIEIIATLAKNGV QTCLNAPDSADAV).
Further, the second open reading frame expresses the optimized sequence combination in CCL5, CXCL9, CXCL10, IL15, CXCL16 and Myb gene sequences, wherein the 6 sequences are truncated and replaced after bioinformatics analysis, and the optimized sequences are directly connected through RRKK, GS repeating units and AA to form a positively charged amphipathic structure so as to be conveniently fixed and inserted into a negatively charged phospholipid bilayer, and the specific sequence is shown as SEQ ID NO.2 (SPYSDTATPCFAYIARPLPRHIKEYFYTSGKCSNAVVFVTARKNRQVCANPEKKWVAREYI NSLEMSRRKKTPVAVRKGRCCISTNQGTIHLQSLKDLKQFPSPSACAEKIEIIATLAKNGVQTCLNAPDSADAVAARCTCISISNQPVNPRSLEKEIIPASQFCPRVEIIATMKKAGEKRCLNPESKAINLLKAPHLRSISIQCYLCLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANSLSNGNVTESGCKTQPGNGNEGSVTGSCYCGKRISSDSPPSVQFMNRLRKHLRAYHRCLYYTRFQLLSWSVCGNKDPWVQEL MSCLDLKECGHAYSGIVAHQKHLPTSPPISQASEGASSDIHTPAQMLLSTLQSTQRPTLPVGSLSSDKELTRPNETTIHTAGHSLAAGPEAGENQKQPEGS).
Further, the mRNA vaccine further includes at least one of a 5 '-cap structure, a 3' -terminal polyadenylation sequence, a 5'utr, and a 3' utr;
preferably, the 5' -cap structure preferably comprises ARCA, m7G (5 ') ppp (5 ') (2 ' ome a) pG, m7G (5 ') ppp (5 ') (2 ' ome G) pG, m7 (3 ' ome G) (5 ') ppp (5 ') (2 ' ome a) pG, mCAP, dmCAP, tmCAP or dmapp;
preferably, the 3' terminal polyadenylation sequence is 50-200A, more preferably 80-200A in length;
preferably, the 5' UTR is 10-200 nucleotides in length, more preferably 15-100 nucleotides in length;
preferably, the 3' utr includes a homolog, fragment or variant of a 3' utr derived from a gene that provides stable mRNA or a 3' utr derived from a gene that provides stable mRNA, including but not limited to: the 3'UTR sequence of hemoglobin HBA2 or the beta-globin 3' -UTR sequence;
preferably, the mRNA contains modified and/or unmodified nucleotides, including L-nucleoside modifications and/or 2' -O-methylation modifications.
Further, the mRNA vaccine may be linked to a backbone vector selected from the group consisting of a pVAX1 series vector, a pVR series vector, and a pcDNA series vector. The pVAX1 series vector, the pVR series vector or the pcDNA series vector is used for expressing fusion proteins, so that the pVAX1 series vector, the pVR series vector or the pcDNA series vector is used for preparing protein vaccines or DNA vaccines. In addition, pVAX1 or pVR vector or pcDNA series vector is used as mRNA expression vector to express mRNA encoding fusion protein in animal cell to prepare mRNA vaccine.
Furthermore, the invention also provides an application of the mRNA vaccine in preparing tumor therapeutic drugs, and the mRNA can be delivered into a body to enable the body to generate immune response, so that the tumor therapeutic effect is enhanced by intratumoral delivery.
Further, the present invention also provides the use of the mRNA vaccine for the preparation of a medicament for the prevention, treatment, and/or amelioration of any disease or disorder selected from cancer or tumor diseases, degenerative disease antigens, atopic disease antigens, autoimmune diseases, infectious diseases, or allergies or allergic diseases.
Advantageous effects
The invention discloses an mRNA vaccine and application thereof in intratumoral delivery for enhancing tumor treatment effect, and the invention discovers that mRNA for delivering certain cytokines can improve mouse tumor immunity microenvironment, increase infiltration of tumor tissue CD8+T, kill tumor cells and inhibit tumor growth. And can enhance the tumor treatment effect of immune checkpoint inhibitor anti-PD-1 monoclonal antibody and chemotherapeutic drug oxaliplatin. The application of the invention realizes the treatment method of inhibiting the development of tumor by using mRNA and the combined drug of mRNA and the existing tumor treatment means, and has good prospect and practical value. Provides a certain reference and a certain hint for the development of mRNA in the tumor treatment field and provides a new idea for the combined drug for tumor treatment. The invention also develops a vaccine by utilizing the mRNA, and the mRNA is used as the main component of the vaccine, so that the invention has the advantages of rapid research and development, high safety, easy industrialization and the like; the mRNA is used as the main component of the vaccine, the preparation process is simple, and cell proliferation virus is not required or recombinant protein is not required to be produced; the use of very small doses can achieve sufficient protective effect, and is superior to the existing vaccine technology in terms of safety and effectiveness.
Drawings
FIG. 1 is a schematic of the intratumoral delivery of CC mRNA using Liposome 1 (liponame 1) to inhibit tumor growth;
FIG. 2 is a schematic representation of the intratumoral delivery of CM mRNA using liposome 1 to inhibit tumor growth;
FIG. 3 is a schematic representation of inhibition of tumor growth using Liposome 1 (liponame 1) tail vein delivery;
FIG. 4 is an illustration of the anti-tumor effect of delivering CCL5-CXCL9 mRNA using Liposome 1 (liponame 1) to enhance PD-1 mab;
FIG. 5 is an illustration of the anti-tumor effect of delivering CM mRNA to enhance PD-1 mab using liposome 1;
wherein, the graph A is a pattern graph; panel B is an anesthetized mouse after inoculation, photographed; panel C is a graph showing the effect of tumor growth over time after inoculation; panel D is a graph showing the effect of weight gain of mice after inoculation over time; FIG. E is a diagram of tumor entities of the sacrificed mice after inoculation; f is a schematic representation of the standard increase difference in tumor weight of sacrificed mice after inoculation.
FIG. 6 is an increase in CCL5, CXCL9 expression in tumor tissue in combination, wherein the ACCL5 of FIG. 6 is expressed in MC38 subcutaneous transplants; FIG. 6B is the expression of CXCL9 in MC38 subcutaneous transplants; FIG. 6C shows Western blot detection of CXCL9 expression in MC38 subcutaneous transplants.
FIG. 7 is a combination tumor tissue CD8 + T cell infiltration results are schematically shown in FIG. 7A, which shows immunohistochemical analysis of CD8 + Distribution of T cells in MC38 subcutaneous transplants; FIG. 7B is an immunofluorescence assay CD8 + Distribution of T cells in MC38 subcutaneous transplants.
Examples
The present invention will be described in detail with reference to the following drawings and examples. The following examples are only preferred embodiments of the present invention, and it should be noted that the following descriptions are merely for explaining the present invention, and are not limiting in any way, and any simple modification, equivalent variation and modification of the embodiments according to the technical principles of the present invention are within the scope of the technical solutions of the present invention.
In the examples described below, materials, plasmids, reagents and the like used, unless otherwise specified, were all obtained commercially.
Wherein: LNP is available from Mirus; mouse colon cancer cell line: MC38 is purchased from Invitrogen; oxaliplatin (Oxaliplatin, OXA) is purchased from MCE; anti-PD-1 mab was purchased from Bio X Cell; igG was purchased from Sigma Aldrich.
The plasmids involved in the examples of the present invention are: the construction of the pcs2-EGFP, pcs2-CCL5, pcs2-CXCL9, pcs2-CXCL10, pcs2-IL15, pcs2-CXCL16 and pcs2-Myb adopts a conventional molecular biology method and is not described again.
EXAMPLE 1 construction of mRNA vaccine in vitro transcription plasmids, in vitro transcription System was established
According to the known sequences of CCL5, CXCL9, CXCL10, IL15, CXCL16 and Myb in the prior art, bioinformatics analysis is carried out on the sequences, and the optimized sequences are respectively connected through RRKK, GS repeating units and AA or directly to form a positively charged amphipathic structure, so that the optimized sequence is conveniently fixed and inserted with a negatively charged phospholipid bilayer, and two specific sequences are designed:
sequence 1: CCL5-CXCL9 mRNA, abbreviated as CC mRNA, and the specific amino acid sequence of the CCL5-CXCL9 mRNA is shown as SEQ ID NO. 1;
sequence 2: CCL5-CXCL9-CXCL10-IL15-CXCL16-Myb mRNA, abbreviated as CM mRNA, and its specific amino acid sequence is shown in SEQ ID NO. 2:
after the Huada gene is biosynthesized, the target sequence is inserted into pcDNA3.1 to construct pcDNA3.1-CC and pcDNA3.1-CM plasmids, and further sequencing, verification and preservation are carried out.
In order to verify the delivery effect of the mRNA vaccine, EGFP protein was used as an indicator, and was inserted into the N-terminus of the above expression cassette using restriction enzyme after PCR amplification to achieve co-expression after transcription. Wherein the amino acid sequence of EGFP protein is shown as SEQ ID NO.3 (MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPT LVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL VNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLAD HYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDEL), and pcDNA3.1-GFP-CC and pcDNA3.1-GPF-CM plasmids are obtained.
The mRNA is transcribed in vitro by a conventional molecular biological method, and the specific operation method is as follows:
(1) Plasmid linearization
Double enzyme digestion of the vector to linearity
And (3) enzyme cutting system:
after the digestion for 3 hours in a water bath at 37 ℃, detecting the digestion condition of the vector by agarose gel electrophoresis, comparing with a vector which is not digested to observe whether the digestion of the plasmid is complete, cutting off the digested strip, recovering gel, and purifying DNA for the template of the subsequent mRNA in vitro transcription.
(2) In vitro transcription of mRNA
Reagent:
SP6 RNA polymerase, 10x RNA polymerase buffer (bi yun tian); RNAase inhibitor (vazyme); ATP solution, CTP solution, GTP solution, UTP solution,Solutions, caps (core organisms); DNase I (tenna); ammonium acetate powder (biomass); other organic solvents: phenol/chloroform/isoamyl alcohol, 75% ethanol (DEPC water configuration), chloroform, isopropanol; other consumables: RNAase-free 1.5ml EP tube
Early preparation:
NTP mixture 1: ATP, CTP, UTP (or) The final concentration of the mixture was 10mM each.
NTP blend 2: GTP and CAP were mixed to a final concentration of 10mM and CAP was mixed to a final concentration of 8mM.
The ammonium acetate solution was set at 5M.
Synthesis System (10. Mu.L for example):
synthetic procedure (unless otherwise indicated, ep tubes are RNAase free tubes, water refers to DEPC water):
(1) fully and uniformly mixing after adding the reagents in the system at room temperature, immediately separating, and incubating at 37 ℃ for 2h;
(2) adding DNase I and Buffer, mixing, immediately separating at 25deg.C, and digesting for 10min to remove linear DNA template;
(3) after digestion, DEPC water was added to 150. Mu.L, 150. Mu.L phenol/chloroform/isoamyl alcohol (lower layer solution was absorbed) was added and mixed to make an emulsion
The liquid is separated by centrifugation, and a micro palm centrifuge can be used. 1min at 12000 rpm;
(4) sucking the uppermost water phase, placing in another 1.5ml centrifuge tube, adding 150 μl chloroform, mixing to obtain emulsion, and centrifuging
Layering the materials;
(5) sucking the uppermost water phase, placing into another 1.5ml centrifuge tube, adding 15 μl of 5M Ammonium acetate (ammonium acetate) (1/10 of the volume of the upper water phase), adding 165 μl isopropanol, mixing, placing in a refrigerator at-20deg.C for 1 hr, and standing
Precooling the centrifuge to 4 ℃;
(6) centrifuging at a temperature of 12000rpm for 15min at 4 ℃, wherein white precipitate can be seen on one side of the centrifuge tube after centrifuging;
(7) taking the liquid, taking care, the gun head needs to be placed opposite to the sediment to suck the liquid, the sediment can not be sucked away by brute force,
this step can leave 10 μl of liquid, avoiding precipitate to be discarded;
(8) 700. Mu.L of 70% ethanol was added and centrifuged at 1200rpm at 4℃for 10min;
(9) carefully sucking the liquid, uncovering and airing for 5-10min;
DEPC water was added to dissolve the mixture, and 1. Mu.L of the mixture was used for quantitative measurement.
(3) De-duplex of in vitro transcribed mRNA
Preparation before experiment: a chromatography buffer was prepared, 10mM HEPES (pH 7.2), 0.1mM EDTA,125mM NaCl,and 16% (v/v) ethanol.
(1) Pre-washing cellulose: 0.1g of cellulose/mL chromatographic buffer, incubating for 10min under vigorous shaking;
(2) 120. Mu.L of suspension (0.14 g cellulose) was taken into a clean EP tube, 12000g 1min, and the supernatant was removed as much as possible;
(3) adding 100. Mu.L of chromatographic buffer, shaking vigorously to re-suspend the cellulose, 5min,12000g of 1min, and removing the supernatant as much as possible;
(4) after finishing the incubation of DNase I, directly dissolving the transcription product in 100 mu L of chromatographic buffer, adding the chromatographic buffer into an EP tube, fully mixing mRNA and cellulose at room temperature for about 1h, and performing operation on a vortex instrument;
(5) transfer all components in the EP tube to the column, 12000g 1min, aspirate the effluent, add 1/10 volume of ammonium acetate, extract with equal volume of isopropanol and purify.
Example 2: intratumoral delivery of GFP, GFP-CC mRNA, GFP-CM mRNA using Liposome 1 to inhibit MC38 tumor growth in mice
6-week-old C57BL/6 mice were divided into four groups of 6 mice each, and the subcutaneous tumor-bearing MC38 cell line was 1X 10 6 100 mu l/tumor volume up to 30mm after 10 days of tumor bearing 3 Intratumoral delivery of mRNA was performed right and left. The experiment is divided into two groups, and each group comprises two groups: one of the first large groups was control group 1 (4 mice), intratumorally delivered liposome 1-packaged EGFP mRNA (5.5 μg/mouse); the other group was experimental group 1 (4 mice), intratumorally delivered liposome 1 packaged GFP-CC (2.5 μg/mouse); one of the second large groups was control group 2 (6 mice), intratumorally delivered liposome 1 packaged EGFP mRNA (5.5 μg/mouse); the other group was experimental group 2 (6 mice), intratumorally delivered liposome 1-packaged GFP-CM (2.5 μg/mouse). mRNA was injected once every three days for a total of three doses. The body weight and tumor length and width of the mice were measured every other day during this period, according to the formula v=pi (length with) 2 ) And/6, calculating the volume size of the tumor, and after the experiment is finished, killing the mice and taking out the tumor and photographing and weighing the dissected tumor.
The results show that: for CCL5-CXCL9 mRNA, (1) green fluorescent protein was expressed at the tumor site and the growth of tumor tissue was significantly slowed in the mice of the experimental group as the time of mRNA injection increased (FIG. 1C). The final dissection results also showed that tumor growth was inhibited in the experimental group of tumor-forming mice after mRNA injection (fig. 1E). The dissected tumors were weighed and the tumor weights in the experimental group were found to be significantly lower than in the control group, and statistical analysis showed significant differences (p < 0.05) (fig. 1F). (2) Throughout the experiment, both groups of mice showed a steady increase in body weight, so that the effect of the growth conditions of the mice on the experimental results could be excluded (fig. 1D).
Similar conclusions as described above are also shown for CCL5-CXCL9-CXCL10-IL15-CXCL16-Myb mRNA: (1) Green fluorescent protein was expressed at the tumor site, and the growth of tumor tissue was significantly slowed down in the mice of the experimental group with increasing mRNA injection time (fig. 2C). The final dissection results also showed that tumor growth was inhibited in the experimental group of tumor-forming mice after mRNA injection (fig. 2E). The dissected tumors were weighed and the tumor weights in the experimental group were found to be significantly lower than in the control group, and statistical analysis showed significant differences (p < 0.05) (fig. 2F). (2) Throughout the experiment, both groups of mice showed a steady increase in body weight, so that the effect of the growth conditions of the mice on the experimental results could be excluded (fig. 2D).
In comparison, the tumor inhibition effect of CM mRNA is obviously better than that of CC mRNA, especially, the tumor inhibition effect can be intuitively and clearly seen from the tumor inhibition size (E diagram of FIG. 1 or FIG. 2), and the statistical result also proves that the multi-targeting effect of CCL5-CXCL9-CXCL10-IL15-CXCL16-Myb is better than that of CCL5-CXCL9, and further research shows that the tumor inhibition effect of the optimized CM mRNA is better than that of mRNA transcribed by the chemokines which are not optimized in sequence and are directly connected (about 27 percent of improvement), and the effect is stable.
Example 3: tumor growth inhibition by delivery of GFP, GFP-CC mRNA using Liposome 1 tail vein
6-week-old C57BL/6 mice were divided into two groups of 4 mice each, and the subcutaneous tumor-bearing MC38 cell line was 1X 10 6 100 mu l/tumor volume up to 30mm after 10 days of tumor bearing 3 mRNA tail vein delivery was performed right and left. One group was control group, tail vein delivered liposome-packaged EGFP mRNA (5.5 μg/mouse); in addition, anotherOne group was experimental and tail vein delivered liposome-packaged GFP-CC mRNA (2.5. Mu.g/mouse). mRNA was injected once every three days for a total of three doses. The body weight and tumor length and width of the mice were measured every other day during this period, according to the formula v=pi (length with) 2 ) And/6, calculating the volume size of the tumor, and after the experiment is finished, killing the mice and taking out the tumor and photographing and weighing the dissected tumor.
The results show that: (1) Green fluorescent protein was expressed at the tumor site, and the growth of tumor tissue was significantly slowed down in the mice of the experimental group with the increase of the mRNA injection time (fig. 3C). The final dissection results also showed that tumor growth was inhibited in the experimental group of tumor-forming mice after mRNA injection (fig. 2E). The dissected tumors were weighed and the tumor weights in the experimental group were found to be significantly lower than in the control group, and statistical analysis showed significant differences (p < 0.05) (fig. 3F). (2) Throughout the experiment, both groups of mice showed a steady increase in body weight, so that the effect of the growth conditions of the mice on the experimental results could be excluded (fig. 3D).
And in contrast to the effects of direct intratumoral delivery described above, see in particular figures 1C, 3C;1F and 3F, the tail intravenous injection effect is not as good as the direct delivery effect, which accords with the conventional expectation in the field, but the effect is far better than that of a control group, which proves that CCL5-CXCL9 can effectively realize the targeting and positioning of tumor cells, accurately, effectively and stably transport target proteins to specific tumor positions, and realize the directional treatment of tumors. The carried exogenous protein can be stably expressed, GFP in the exogenous protein can be replaced by other tumor treatment related proteins according to the requirement, and the effect of double-engraving by one arrow is realized. This also demonstrates that the mRNA vaccines of the present application can achieve long-range gene targeting and achieve better tumor therapeutic effects without requiring intratumoral injection.
Example 4 delivery of CC mRNA, CM mRNA Using Liposome 1 to enhance the anti-tumor Effect of PD-1 mab
6 week old C57BL/6 mice were combined and the subcutaneous tumor-bearing MC38 cell line was 1X 10 6 100 mu l/tumor volume up to 30mm after 10 days of tumor bearing 3 Intratumoral delivery of mRNA was performed right and left. ExperimentThe two groups are divided into two groups, and each group comprises two groups: one of the first large groups was control group 1 (4 mice), intratumorally delivered liposome 1-packaged EGFP mRNA (5.5 μg/mouse); the other group was experimental group 1 (4 mice), intratumorally delivered liposome 1 packaged GFP-CC (2.5 μg/mouse); one of the second large groups was control group 2 (6 mice), intratumorally delivered liposome 1 packaged EGFP mRNA (5.5 μg/mouse); the other group was experimental group 2 (6 mice), intratumorally delivered liposome 1-packaged GFP-CM (2.5 μg/mouse). Mice of the control group 1, the experimental group 1 and the experimental group 2 are simultaneously and intraperitoneally injected with the anti-PD-1 monoclonal antibody of the immune checkpoint inhibitor, and the total dose of the anti-PD-1 monoclonal antibody is 100 mug per mouse. Control IgG was injected intraperitoneally into control group 2 mice, 200 μg/mouse. mRNA and mab were injected once every three days for a total of three doses. The body weight and tumor length and width of the mice were measured every other day during this period, according to the formula v=pi (length with) 2 ) And/6, calculating the volume size of the tumor, and after the experiment is finished, killing the mice and taking out the tumor and photographing and weighing the dissected tumor.
The results show that: whether CC mRNA or CM mRN was used, the growth of tumor tissue was significantly slowed in the experimental group of mice with increasing injection time of mRNA and anti-PD-1 mab, an immune checkpoint inhibitor (FIGS. 4C, 5C). The final anatomical results also show that the tumor inhibition effect of the immune checkpoint inhibitor anti-PD-1 monoclonal antibody is more obvious after mRNA injection of the tumor-forming mice of the experimental group, and the anti-tumor effect is enhanced (figures 4E and 5E). The dissected tumors were weighed and the tumor weights in the experimental group were found to be significantly lower than in the control group, and statistical analysis showed significant differences (p < 0.05) (fig. 4F, 5F). (2) Throughout the experiment, both groups of mice showed a steady increase in body weight, so that the effect of the growth conditions of the mice on the experimental results could be excluded (fig. 4D, 5D). Further comparison found that conventional IgG antibodies did not work well during tumor treatment and did not significantly reduce tumor weight and size (fig. 5E, 5F). And similar to the comparison result of single administration, the tumor inhibition effect of CM mRNA is obviously better than that of CC mRNA.
Furthermore, immunohistochemistry and paraffin section preparation were performed on tumor tissues obtained in the above animal experiments by using routine procedures in the art (see handbook of molecular biology), and expression of CCL5 in MC38 subcutaneous transplantation tumor in mice delivered with mRNA group by immunohistochemical analysis was found to be significantly increased in the tumor tissues of the experimental group compared to the control group (fig. 6A), and significantly increased in CXCL9 compared to the control group (fig. 6B); the results are consistent with the Western blot analysis results, and all prove that the CXCL9 expression level in the treated tumor tissues is obviously improved (FIG. 6C).
The dissected tumor tissue is taken for paraffin section production, and CD8 in the tumor tissue of the mice is observed under a microscope through immunohistochemistry + T distribution (see handbook of molecular biology), results show that the experimental group is CD8 + T lymphocytes were greater in number than the control group (fig. 7A); CD8 of the experimental group was also demonstrated by immunofluorescence analysis + T lymphocytes were greater in number than the control group (FIG. 7B), and the chemokines described above might recruit CD8 + T infiltrates into tumor tissue, improves the immune microenvironment of the tumor tissue, enhances the capability of killing tumor cells of anti-PD-1 monoclonal antibody, which is an immune checkpoint inhibitor, and further inhibits the growth of the tumor tissue.
In summary, the two mRNA vaccines (CC mRNA and CM mRNA) of the present invention, whether administered alone or in combination with other oncologic agents, can achieve safe and effective delivery of mRNA in vitro and in vivo without leakage, and can result in local long-term protein production and stimulation of strong antigen-specific T cell responses, which can be used to effectively treat cancer, other diseases in epidemic patients.
The above examples merely represent a few embodiments of the present invention, which are described in more detail and are not to be construed as limiting the scope of the present invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of the invention should be assessed as that of the appended claims.
Claims (6)
1. A composition comprising an effective amount of a messenger ribonucleic acid (mRNA) that expresses a recombinant fusion protein of CCL5, CXCL9, the sequence of which is set forth in SEQ ID No.1, formulated in a pharmaceutically acceptable carrier; alternatively, recombinant fusion proteins expressing CCL5, CXCL9, CXCL10, IL15, CXCL16, myb genes have the amino acid sequence shown in SEQ ID NO. 2.
2. A nucleic acid encoding a messenger ribonucleic acid (mRNA) in the composition of claim 1.
3. A recombinant DNA vector comprising the sequence of the nucleic acid of claim 2.
4. An mRNA vaccine, characterized in that the mRNA vaccine comprises the composition of claim 1, messenger ribonucleic acid (mRNA) in the composition of claim 1, the nucleic acid of claim 2, and/or the recombinant vector of claim 3.
5. Use of an mRNA vaccine according to claim 4 for the preparation of a medicament for the treatment, and/or amelioration of a disease selected from the group consisting of cancer and tumors, wherein said mRNA vaccine is capable of effecting the delivery of a tumor therapeutic protein into the body to produce an immune response in the body, and wherein the tumor therapeutic effect is enhanced by intratumoral delivery, and wherein the cancer or tumor disease is colon cancer.
6. An anti-neoplastic agent comprising the messenger ribonucleic acid (mRNA) of the composition of claim 1, the nucleic acid of claim 2, and/or the recombinant vector of claim 3, and further comprising an additional immunosuppressive blocker which is an immune checkpoint inhibitor and is at least one antibody selected from the group consisting of an anti-PD 1 antibody, an anti-PD-L1 antibody, and an anti-CTLA 4 antibody, wherein the neoplasm is colon cancer.
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