Antigen epitope peptide coded by mRNA and fused in phosphatidylinositol anchoring protein and application of antigen epitope peptide

Abstract

The invention discloses an antigen epitope peptide coded by mRNA and fused in phosphatidylinositol anchoring protein and application of the antigen epitope peptide, and belongs to the field of biological medicine. The antigen epitope peptide comprises a MUC1 (1TR)-CD24 molecule and a MUC1 (1TR, 13aa)-CD24 molecule; the MUC1 (1TR)-CD24 molecule comprises an MUC1 signal peptide, an MUC1 extracellular domain containing one MUC1 VNTR sequence, a CD24 polypeptide and a phosphatidylinositol anchoring signal peptide; the MUC1 (1TR, 13aa)-CD24 molecule contains MUC1 signal peptide, MUC1 (1TR, 13aa) sequence, CD24 polypeptide and phosphatidylinositol anchoring signal peptide, the process is simple, the sequence is controllable, CAR-T cell amplification can be efficiently activated, CAR-T cells can be stimulated to secrete anti-tumor cell factors, and the MUC1 (1TR, 13aa)-CD24 molecule has important medical value in preparation of drugs for treating cancers or preventing cancer relapse.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an mRNA-encoded antigenic epitope peptide fused to a phosphatidylinositol-anchored protein and its applications. Background Technology

[0002] Tumor cells typically express low-glycosylated glycoproteins, such as CA153 (MUC1) and CA125 (MUC16). The genetic mechanism underlying glycosyltransferase dysfunction has been revealed to be due to abnormal function of COSMC, the chaperone protein required for C1GalT1, which catalyzes the transfer of β1,3-galactose to O-chain GalNac residues. In tumor cells, the glycosylation pathway of the core-1 enzyme is blocked, exposing GalNAc on the O-glycan chain as the Tn antigen. Because the Tn antigen is widely expressed in various cancers, several vaccines and antibody therapies targeting the Tn antigen have been developed, targeting either the carbohydrate component or the peptide component alone. In existing technologies, Theratope vaccines induce weak antibody responses targeting Tn antigens (moderate titer 1:320), approximately 200 times weaker than the carrier protein KLH (moderate titer 1:81920). Various chemically synthesized glycopeptide vaccines induce antibody responses targeting cancer cells, but lack specificity for differential recognition between tumor and normal cells. Peptide vaccines targeting the MUC1 VNTR region are undergoing clinical trials, and T-cell responses have been reported. However, because MUC1 is an autoprotein, the immune system's tolerance to its own epitopes makes it difficult to induce T-cell responses targeting autopeptides or glycopeptides.

[0003] Vaccines and antibodies targeting low-glycosylated glycoproteins have been a key focus of cancer immunotherapy research. mRNA vaccines have achieved success in inducing effector T-cell and humoral immune responses targeting viral glycoproteins and tumor antigens. However, the antigenicity of mRNA-encoded cancer glycoproteins (such as MUC1) remains unclear. The efficacy of CAR-T therapy for tumors depends on the in vivo expansion of CAR-T cells; CAR-T cells that cannot persist in vivo are ineffective for cancer patients. For highly glycosylated CAR-T targets like MUC1, there is currently a lack of technologies for effectively expanding CAR-T cells in vivo, and existing vaccine technologies have not shown ideal results in inducing T-cell immune responses.

[0004] Therefore, there is an urgent need to develop an effective mRNA vaccine to stimulate CAR-T cells. This mRNA vaccine, after translation in vivo, produces an antigen that can effectively stimulate CAR-T cells. The MUC1 target in tumors is characterized by specific glycosylation; the antigen translated from the MUC1 mRNA vaccine needs to be identical to the tumor antigen in order to be recognized by CAR-T cells. The stimulatory signal from the MUC1 mRNA vaccine to CAR-T cells needs to be sufficiently strong to effectively stimulate CAR-T cell proliferation. Summary of the Invention

[0005] The first objective of this invention is to provide an mRNA-encoded antigenic epitope peptide fused to a phosphatidylinositol anchoring protein. This involves designing a MUC1 antigenic epitope peptide encoded by mRNA with one or five VTNR sequences, fusing the MUC1 VTNR antigenic epitope peptide sequence with a phosphatidylinositol anchoring protein (CD24) molecule anchored to a cell membrane GPI, and using chimeric antigen receptor T cells to test the intensity of antigen signals from different mRNA-encoded antigenic epitope peptides.

[0006] Another object of the present invention is to provide the use of the mRNA-encoded antigenic epitope peptide fused to a phosphatidylinositol-anchored protein in the preparation of a drug that efficiently activates CAR-T cells.

[0007] Another object of the present invention is to provide the use of the mRNA-encoded antigenic epitope peptide fused to a phosphatidylinositol-anchored protein in the preparation of a medicament for treating cancer or preventing cancer recurrence.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides an mRNA-encoded antigenic epitope peptide fused to a phosphatidylinositol-anchored protein and its application thereto, comprising MUC1(1TR), MUC1(1TR)-CD24 and MUC1(1TR,13aa)-CD24 molecules.

[0010] Preferably, the MUC1(1TR) molecule comprises a MUC1 signal peptide as shown in SEQ ID NO: 1, an extracellular region of MUC1 containing one MUC1 TR sequence as shown in SEQ ID NO: 2, a transmembrane domain of MUC1 as shown in SEQ ID NO: 3, and an intracellular domain of MUC1 as shown in SEQ ID NO: 4.

[0011] Preferably, the MUC1(1TR)-CD24 molecule comprises the MUC1 signal peptide as shown in SEQ ID NO: 1, the MUC1(1TR) sequence as shown in SEQ ID NO: 6, the CD24 polypeptide as shown in SEQ ID NO: 7, and the CD24 C-terminus (phosphatidylinositol-anchored signal peptide) as shown in SEQ ID NO: 8.

[0012] Preferably, the MUC1(1TR,13aa)-CD24 molecule comprises the MUC1 signal peptide as shown in SEQ ID NO: 1, the MUC1(1TR,13aa) sequence as shown in SEQ ID NO: 9, the CD24 polypeptide as shown in SEQ ID NO: 7, and the CD24 C-terminus (phosphatidylinositol-anchored signal peptide) as shown in SEQ ID NO: 8.

[0013] A second aspect of the invention provides the use of the mRNA-encoded antigenic epitope peptide fused to a phosphatidylinositol-anchored protein in the preparation of a drug that efficiently activates CAR-T cells.

[0014] A third aspect of the invention provides the use of the mRNA-encoded antigenic epitope peptide fused to a phosphatidylinositol-anchored protein in the preparation of a medicament for treating cancer or preventing cancer recurrence.

[0015] In a fourth aspect, the present invention provides an mRNA vaccine in which an antigenic epitope is fused to a phosphatidylinositol anchoring protein and / or a phosphatidylinositol anchoring signal peptide, thereby achieving a faster diffusion rate on the surface of antigen-presenting cells than that of natural antigens, thus achieving strong stimulation of CAR-T cells.

[0016] In a fifth aspect, the present invention provides a CAR molecule for recognizing an antigenic epitope peptide fused to a phosphatidylinositol anchoring protein encoded by any of the mRNAs described in any one of the present invention, the sequence of which is shown in SEQ ID NO: 11.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] I. This invention designs a MUC1 mRNA sequence MUC1(1TR) that encodes a single antigenic epitope, avoiding repetitive sequences of the antigenic epitope. This sequence stimulates CAR-T cells more effectively than the tandem repeat sequences carried by the natural MUC1 protein, resulting in CAR-T cells secreting more than 3 times more IFN-γ.

[0019] II. This invention obtains the MUC1 antigen sequences MUC1(1TR)-CD24 and MUC1(1TR,13aa)-CD24 by fusing the tumor antigen epitope peptide sequence with CD24. The high mobility of the transmembrane region of CD24 promotes the stimulation of CAR-T cells by the MUC1 antigen, resulting in CAR-T cells secreting cytokine IFN-γ at a rate more than 5 times higher than the MUC1 native protein sequence (its own transmembrane region).

[0020] Third, this invention also designs a CAR molecule that recognizes the MUC1 antigenic epitope peptide, which can be used to accurately measure the immunogenicity of the antigen encoded by mRNA.

[0021] IV. Compared with peptide vaccines, protein subunit vaccines, and adenovirus-encoded vaccines, this invention uses a single antigenic epitope and phosphatidylinositol fusion protein technology to obtain an mRNA-encoded antigenic epitope peptide fused to a phosphatidylinositol anchoring protein. After being fused to both the phosphatidylinositol anchoring protein and the phosphatidylinositol anchoring signal peptide, it achieves a faster diffusion rate on the surface of antigen-presenting cells, provides stronger stimulation to CAR-T cells, and has a simple preparation process with controllable sequence. Attached Figure Description

[0022] Figure 1 The following are examples of MUC1 antigenic epitope peptide sequences encoded by mRNA; A: Amino acid sequences of different MUC1 VNTR sequences; B: Flow cytometry analysis results of 293T cells transfected with MUC1 mRNA vaccine.

[0023] Figure 2 The following are the results of the stimulation of CAR-T cells by the MUC1 antigenic epitope peptide encoded by mRNA in the examples: A: Cytokine secretion of 16ACAR-T cells under MUC1 mRNA vaccine stimulation as measured by enzyme-linked immunosorbent assay; B: Cytokine production of 16ACAR-T cells as measured by intracellular IFN-γ staining; C: Proliferation of 16ACAR-T cells after stimulation by MUC1 mRNA vaccine.

[0024] Figure 3 The following are the results of the cell surface mobility test of CD24-5TR, CD24-1TR and MUC1-1TR molecules in Example 293; A: Observation and recording by total internal reflection fluorescence microscopy (TIRFM) of film; B: cumulative probability; C: single molecule trajectory.

[0025] Figure 4 The following is a schematic diagram of the fusion technology of a single antigenic epitope and phosphatidylinositol anchoring protein in the embodiments: the left figure shows the fusion of a single antigenic epitope and the transmembrane region of natural MUC1; the middle figure shows the fusion of a single antigenic epitope and CD24; and the right figure shows the fusion of multiple tandemly repeated antigenic epitopes and CD24.

[0026] Figure 5 In the examples, the individual antigenic epitope sequences MUC1(1TR), MUC1(4TR), and the CD24 fusion antigen sequences MUC1(1TR)-CD24, MUC1(1TR,13aa)-CD24, and MUC1(5TR)-CD24 are used.

[0027] Figure 6 The amino acid sequence of the CAR molecule used in this example identifies the MUC1 antigenic epitope peptide encoded by mRNA. Detailed Implementation

[0028] To more fully understand and demonstrate the technical solutions, objectives, and advantages of the present invention, the technical effects produced by the present invention will be further described in detail and completely below with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. It should be noted that other embodiments obtained by those skilled in the art without departing from the concept of the present invention are all within the protection scope of the present invention.

[0029] The following examples present an mRNA-encoded antigenic epitope peptide fused to a phosphatidylinositol-anchored protein, comprising MUC1(1TR), MUC1(1TR)-CD24, and MUC1(1TR,13aa)-CD24 molecules.

[0030] In some embodiments, the MUC1(1TR) molecule comprises a MUC1 signal peptide as shown in SEQ ID NO: 1, an extracellular region of MUC1 containing one MUC1 TR sequence as shown in SEQ ID NO: 2, a transmembrane domain of MUC1 as shown in SEQ ID NO: 3, and an intracellular domain of MUC1 as shown in SEQ ID NO: 4.

[0031] In some embodiments, the MUC1(1TR)-CD24 molecule comprises the MUC1 signal peptide as shown in SEQ ID NO: 1, the MUC1(1TR) sequence as shown in SEQ ID NO: 6, the CD24 polypeptide as shown in SEQ ID NO: 7, and the CD24 C-terminus (phosphatidylinositol-anchored signal peptide) as shown in SEQ ID NO: 8.

[0032] In some embodiments, the MUC1(1TR,13aa)-CD24 molecule comprises the MUC1 signal peptide as shown in SEQ ID NO: 1, the MUC1(1TR,13aa) sequence as shown in SEQ ID NO: 9, the CD24 polypeptide as shown in SEQ ID NO: 7, and the CD24 C-terminus (phosphatidylinositol-anchored signal peptide) as shown in SEQ ID NO: 8.

[0033] The following examples also propose the use of the above-mentioned mRNA-encoded antigenic epitope peptide fused to phosphatidylinositol anchoring protein for the preparation of drugs that can effectively activate CAR-T cells, or for the preparation of drugs that treat cancer or prevent cancer recurrence.

[0034] The following examples also present a CAR molecule for recognizing the antigenic epitope peptide fused to the phosphatidylinositol anchoring protein encoded by the above-mentioned mRNA, the sequence of which is shown in SEQ ID NO: 11.

[0035] Example 1

[0036] I. Design of mRNA sequences with different MUC1 VNTR sequences

[0037] To further optimize the mRNA sequence, untranslated regions (UTRs) of human β-globulin were added to the 5' and 3' ends, respectively. Additionally, a Kozak sequence was added to the open reading frame (ORF) at the 5' end, and a polyadenylated nucleotide (PolyA) sequence was added to the 3' end. These gene fragments were synthesized and cloned into the pVAX1 vector to obtain template plasmids pVAX1-PcrV and pVAX1-OprF-I. The mRNA was then transcribed and encapsulated in lipid nanoparticles.

[0038] like Figure 1 and 5 As shown, five antigen sequences were designed to test their stimulatory effects on CAR-T cells. The single antigenic epitope sequences MUC1(1TR) and MUC1(4TR) are antigenic epitope peptide antigens containing the MUC1 transmembrane domain and having different numbers of VNTR sequences. The CD24-fused antigen sequences MUC1(1TR)-CD24, MUC1(1TR,13aa)-CD24, and MUC1(5TR)-CD24 are antigenic epitope peptide antigens fused to the CD24 molecule and anchored to glycosylphosphatidylinositol. Figure 1 A), the specific sequence is as follows: MUC1(1TR):

[0039] MUC1 signal peptide: MTPGTQSPFFLLLLLTVLTVVTG (SEQ ID NO: 1)

[0040] MUC1 extracellular domain:

[0041] SGHASSTPGGEKETSATQRSSVPSSTEKNAVSMTSSVLSSSPGSGSSTTQGQ

[0042] DVTLAPATEPASGSAATWGQDVTSVPVTRPALGSTTPPAHDVTSAPDNK

[0043] RPAPGSTAPPAHGVTSAPDT

[0044] PALGSTAPPVHNVTSASGSASGSASTLVHNGTSARATTTPASKSTPFSIPSHHS

[0045] DTPTTLASHSTKTDASSTHHSTVPPLTSSNHSTSPQLSTGVSFFFLSFHISNLQF

[0046] NSSLEDPSTDYYQELQRDISEMFLQIYKQGGFLGLSNIKFRPGSVVVQLTLAF

[0047] REGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAGVPG (SEQ ID NO: 2)

[0048] MUC1 transmembrane domain:

[0049] WGIALLVLVCVLVALAIVYLIAL(SEQ ID NO: 3)

[0050] MUC1 intracellular domain:

[0051] AVCQCRRKNYGQLDIFPARDTYHPMSEYPTYHTHGRYVPPSSTDRSPYEKVSAGNGGSSLSYTNPAVAATSANL (SEQ ID NO: 4)

[0052] MUC1(4TR):

[0053] MUC1 signal peptide: MTPGTQSPFFLLLLLTVLTVVTG (SEQ ID NO: 1) MUC1 extracellular domain:

[0054] SGHASSTPGGEKETSATQRSSVPSSTEKNAVSMTSSVLSSHSPGSGSSTTQGQ

[0055] DVTLAPATEPASGSAATWGQDVTSVPVTRPALGSTTPPAHDVTSAPDNK

[0056] PAPGSTAPPAHGVTSAPDTR

[0057] PPPGSTAPPAHGVTSAPDTR

[0058] PPPGSTAPAAHGVTSAPDTR

[0059] PAPGSTAPPAHGVTSAPDNR

[0060] PALGSTAPPVHNVTSASGSASGSASTLVHNGTSARATTTPASKSTPFSIPSHHS

[0061] DTPTTLASHSTKTDASSTHHSTVPPLTSSNHSTSPQLSTGVSFFFLSFHISNLQF

[0062] NSSLEDPSTDYYQELQRDISEMFLQIYKQGGFLGLSNIKFRPGSVVVQLTLAF

[0063] REGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAGVPG(SEQ ID NO: 5)

[0064] MUC1 transmembrane domain: WGIALLVLVCVLVALAIVYLIAL(SEQ ID NO: 3) MUC1 intracellular domain:

[0065] AVCQCRRKNYGQLDIFPARDTYHPMSEYPTYHTHGRYVPPSSTDRSPYEKVSAGNGGSSLSYTNPAVAATSANL(SEQ ID NO: 4)

[0066] MUC1(1TR)-CD24:

[0067] MUC1 signal peptide: MTPGTQSPFFLLLLLTVLTVVTG(SEQ ID NO: 1) MUC1(1TR): RPAPGSTAPPAHGVTSAPDT(SEQ ID NO: 6) CD24 peptide: SETTTGTSSNSSQSTSNSGLAPNPTNATTKAAG (SEQ ID NO: 7)

[0068] CD24 C-terminal hydrophobic stretch:

[0069] GALQSTASLFVVSLSLLHLYS (SEQ ID NO: 8)

[0070] MUC1(1TR,13aa)-CD24:

[0071] MUC1 signal peptide: MTPGTQSPFFLLLLLTVLTVVTG (SEQ ID NO: 1)MUC1(1TR, 13aa): RPAPGSTAPPAHG (SEQ ID NO: 9)

[0072] CD24 peptide: SETTTGTSSNSSQSTSNSGLAPNPTNATTKAAG (SEQ ID NO: 7) CD24 C-terminus: GALQSTASLFVVSLSLLHLYS (SEQ ID NO: 8) MUC1(5TR)-CD24:

[0073] MUC1 signal peptide: MTPGTQSPFFLLLLLTVLTVVTG (SEQ ID NO: 1)MUC1(5TR):

[0074] GVTSAPDT

[0075] RPAPGSTAPPAHGVTSAPDT

[0076] RPAPGSTAPPAHGVTSAPDT

[0077] RPAPGSTAPPAHGVTSAPDT

[0078] RPAPGSTAPPAHGVTSAPDT RPAPGSTAPPAHGVTSA (SEQ ID NO: 10)

[0079] CD24 polypeptide: SETTTGTSSNSSQSTSNSGLAPNPTNATTKAAG (SEQ ID NO: 7) CD24 C-terminus: GALQSTASLFVVSLSLLHLYS (SEQ ID NO: 8).

[0080] The cell surface antigen translated from mRNA vaccine in 293T cells was stained using monoclonal antibody 16A, such as... Figure 1 As shown in Figure B, the translated VNTR epitope peptides were stained with a 16A monoclonal antibody, which preferentially binds to GalNAc-modified VNTRs rather than unglycosylated VNTRs. Two cell populations were observed after staining: one with moderate 16A staining and the other with high 16A staining. The 16A antibody preferentially binds to the GalNAc-modified VNTR peptide ST(GalNAc)APPAHG, exhibiting a 30-fold greater affinity for ST(GalNAc)APPAHG than for STAPPAHG. Therefore, these two populations of 293T cells reflect unglycosylated and low-glycosylated epitope peptides.

[0081] II. Stable expression of different MUC1 molecules in 293T-COSMC- / - cells

[0082] Complementary DNA sequences encoding different MUC1 molecules were cloned into the pcDNA3.1 plasmid (Thermo Fisher, Waltham, MA) and stably transfected into 293T-COSMC- / - cells using Lipofectamine (Thermo Fischer). Stably transfected cells were selected using hygromycin-containing medium. MUC1-positive cells were obtained by flow cytometry-based cell sorting, and the mean fluorescence intensity was measured using 16A antibody staining (BioLegend, San Diego, CA). The mean fluorescence intensities of MUC1 (1TR), CD24-MUC1 (1TR), and CD24-MUC1 (5TR) were 1.08E, respectively. 6 ,4.98E 5 and 7.74E 5 .

[0083] III. Measurement of the Mobility of GPI-Anchored Antigenic Epitope Peptides

[0084] TIRF imaging was performed using a Nikon ECLIPSETi microscope equipped with a TIRF port, combined with a Digital Camera C11440, a TIRF100×SRApo, a 1.49NA eyepiece, and five lasers (405nm, 445nm, 488nm, 561nm, and 647nm) (Nikon). Image acquisition was controlled using NIS-Elements AR software (Nikon). The imaging buffer contained: 138mM NaCl, 5mM KCl, 1.3mM CaCl2, 0.5mM MgCl2, 0.4mM MgSO4, 0.4mM KH2PO4, 0.3mM Na2HPO4, and 5mM Mglucose. For live-cell single-molecule imaging, a live-cell incubator (37°C, 5% CO2) was used. 293 cells labeled with Cy516A-scFv (5 nM) (expressing TR) were placed on the glass bottom of a PLL plate and incubated for 10 minutes to allow cell attachment. The TIRF mode and focal plane were then adjusted, and imaging was performed using a 647 nm laser (output power approximately 5 mW). A 256 × 256 pixel region was recorded at a frame rate of 50 frames per second. The mean square displacement (MSD) and diffusion coefficient of single-molecule trajectories were analyzed using the preceding Matlab code.

[0085] like Figure 3 As shown, cells were labeled with Cy5-linked 16A-scFv, placed on polylysine (PLL)-coated glass slides, and imaged using total internal reflection fluorescence microscopy (TIRFM). Single-molecule tracking quantification results showed that the average diffusion velocities of MUC1(1TR)-CD24 and MUC1(5TR)-CD24 were 0.26 μm. 2 / s and 0.12μm 2 The diffusion rate of MUC1 (1TR) is higher than that of antigens fused to the transmembrane region of the native MUC1 protein; however, the average diffusion rate of MUC1 fused to both the original transmembrane domain and the extracellular region is only 0.03 μm² / s. Figure 3 The AC indicates that the diffusion rate of the MUC1 epitope on the glycosyl phosphatidylinositol anchoring structure is much faster.

[0086] IV. Preparation of recombinant lentiviruses encoding chimeric antigen receptors

[0087] By assembling the heavy chain variable region (VH), hinge region (GGGGSGGGGSGGGGS), light chain variable region (VL), transmembrane region of CD8α, intracellular domain of 41BB, and complementary DNA sequence of CD3, the pHAGE-16A-chimeric antigen receptor-IRES-red fluorescent protein (RFP) plasmid was constructed. The sequence of the plasmid is as follows: Figure 6 As shown, specifically:

[0088] MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGSLRLSCVVSGIDFSRY

[0089] WMSWVRQAPGKGLEWVGEITPDSNTINYVPSVKGRFGISRDNAKNSLYLOM

[0090] NSLRAEDTAVYFCASYYEGFAYWGQGTLVTVSSGGGGSGGGGSGGGGSQAV

[0091] VTQEPSLTVSPGGTVTLTCGSSTGAVITSNYANWVQQKPGQAPTGLIGRTYNK

[0092] VPWTPARFSGSLLGDKAALTLSGAQPEDEAEYFCALWYSNHFVFGGGTKLTV

[0093] LSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF

[0094] ACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCS

[0095] CRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR

[0096] GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 11).

[0097] To generate viral particles, the pHAGE16A-chimeric antigen receptor-RFP plasmid, or the control empty vector pHAGE-IRES-RFP plasmid, was transfected into Lenti-293X cells (Shanghai Sangon Biotech, China) along with the packaging plasmids psPAX2 and pMD2.G. After collection, the viral particles were mixed with polyethylene glycol 8000 (Sigma-Aldrich, St. Louis, MO), incubated at 4°C for 12 hours, and then concentrated 100-fold by centrifugation at 1600×g for 30 minutes at 4°C.

[0098] V. Generation of chimeric antigen receptor T cells

[0099] Peripheral blood mononuclear cells (PBMCs) obtained from healthy donors were activated in cell culture plates pre-coated with antibodies against CD3 (5 μg / mL, BioLegend, San Diego, CA) and CD28 (1 μg / mL, BD Biosciences, San Jose, CA). The culture medium consisted of intact human lymphocyte culture medium (Gibco RPMI Medium 1640), 10% Gibco heat-inactivated bovine serum, 100 units / mL penicillin, 100 μg / mL streptomycin, and 200 IU / mL recombinant human interleukin-2 (Tetracycline Biotech, Beijing, China). After 48 hours of culture, PBMCs were infected with viral supernatant at a multiplicity of infection (MOI) of 10 to 40. Simultaneously, an empty pHAGE vector was transduced along with T cells to generate control T cells. After 12 hours, fresh intact culture medium was added to the cells, followed by expansion over 10 days, and then functional experiments were performed.

[0100] VI. Stimulation of chimeric antigen receptor T cells

[0101] Use 4 293T cells were transfected with a g mRNA vaccine and allowed to remain submerged for 24 hours before being mixed with chimeric antigen receptor T cells. Chimeric antigen receptor T cells were then mixed with 100,000 target cells expressing MUC1 at a 1:2 effector cell / target cell ratio. Chimeric antigen receptor T cells and MUC1-expressing target cells were co-cultured in 96-well plates for 24 hours, and the release of cytokines in the culture medium was measured by enzyme-linked immunosorbent assay (ELISA). As a positive control, chimeric antigen receptor T cells were stimulated with phorbol myristate acetate (500 ng / mL) and iomycin (10 μg / mL, both from Dakowei Biotechnology Co., Ltd., China).

[0102] A sequence was designed to fuse MUC1(1TR) with CD24, and the fusion molecule was anchored to glycosylphosphatidylinositol. This molecule was then compared with a MUC1(1TR) molecule possessing the original transmembrane domain to assess its stimulatory activity on chimeric antigen receptor T cells. The stimulatory effect of the mRNA-encoded MUC1 antigenic epitope peptide on CAR-T cells was evaluated. Figure 2 As shown in the figure. The cytokine secretion of 16A CAR-T cells stimulated by MUC1 mRNA vaccine, as measured by enzyme-linked immunosorbent assay (ELISA), is as follows. Figure 2 As shown in Figure A, the ability of chimeric antigen receptor T cells to produce cytokines using glycosylphosphatidylinositol-anchored MUC1(1TR) antigenic epitope peptides is more than 5 times higher than that of the MUC1(1TR) molecule. Figure 2A). MUC1(1TR,13aa)-CD24 encodes a shorter version of MUC11TR and also exhibits a similar strong stimulatory effect; intracellular IFN-γ staining measures cytokine production in 16ACAR-T cells as follows: Figure 2 As shown in B; the proliferation of 16ACAR-T cells stimulated by the MUC1 mRNA vaccine is as follows. Figure 2 As shown in C.

[0103] Enzyme-linked immunosorbent assay (ELISA) revealed that the stimulatory activity of MUC1(5TR)-CD24 was more than three-fold lower than that of MUC1(1TR)-CD24, indicating that parallel epitope columns are superior to contiguous tandem repeat epitopes in activating chimeric antigen receptor T cells. Target cells expressing the MUC1 repeat sequence showed lower stimulatory activity towards chimeric antigen receptor T cells, suggesting that this contiguous epitope arrangement may lead to lower binding efficiency of chimeric antigen receptors to the cell surface.

[0104] like Figure 4 As shown, the stimulation mechanism of CAR-T cells by single antigenic epitopes and CD24 fusion antigenic epitope peptides is demonstrated; when tumor antigenic epitopes are carried by lipids located on lipid rafts containing cholesterol and glycosphingolipids, the stimulation effect on CAR-T cells is strong. Figure 4 (in Chinese); compared to the same antigenic epitope carried by lipids, the stimulatory effect is weaker when the antigenic epitope is carried by a glycoprotein. Figure 4 (Left); When tumor antigen epitopes are arranged in a tandem repeat pattern, the stimulatory effect is significantly reduced. Figure 4 (Right). First, aggregation of chimeric antigen receptors around repetitive sequence regions may lead to inefficiency because repetitive sequence regions of mucins are generally considered disordered and lack secondary structure (α-helices or β-sheets). Second, mechanosensitivity of parallel epitope columns may produce stronger signal intensity than that of sequentially arranged epitopes because lipid-carried parallel epitopes may be more elastic than epitopes with repetitive sequences. Third, protein dissociation in immune synapses around parallel epitope columns may be faster than that of sequentially arranged epitopes.

[0105] In summary, this invention uses chimeric antigen receptor T cells as a benchmark to measure the signal intensity of the MUC1 antigenic epitope peptide encoded by mRNA vaccines. The results show that antigenic epitope peptides with a single tandem repeat sequence have a higher stimulation intensity than those with multiple tandem repeat sequences. Furthermore, when the MUC1 antigenic epitope peptide is fused with a GPI anchor, the stimulation intensity increases by more than 5-fold. Molecular migration of the antigenic epitope peptide was measured using total internal reflection fluorescence microscopy, revealing that the moderate diffusion rate of the GPI-anchored MUC1 antigen is 5 times faster than that of the MUC1 antigenic epitope peptide with its own transmembrane domain. The study demonstrates that the intrinsic disordered regions of glycoproteins interact with CAR-T cells, and the combined use of mRNA vaccines targeting CAR-T cells and mRNA vaccines targeting B cells can exert a synergistic effect of cellular and humoral immunity.

[0106] The above are merely preferred embodiments of the present invention and are not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An mRNA-encoded fusion of an epitope peptide of an antigen fused to a phosphatidylinositol-anchored protein, characterized in that, It comprises MUC1(1TR)-CD24 and MUC1(1TR, 13aa)-CD24 molecules.

2. The antigenic epitope peptide of the fusion encoded by the mRNA according to claim 1, characterized in that, The MUC1(1TR)-CD24 molecule comprises a MUC1 signal peptide with sequence as shown in SEQ ID NO: 1, a MUC1(1TR) sequence as shown in SEQ ID NO: 6, a CD24 polypeptide as shown in SEQ ID NO: 7, and a phosphatidylinositol anchor signal peptide as shown in SEQ ID NO:

8.

3. The antigenic epitope peptide of the fusion protein encoded by the mRNA according to claim 1, wherein, The MUC1(1TR, 13aa)-CD24 molecule comprises a MUC1 signal peptide with sequence as shown in SEQ ID NO: 1, a MUC1(1TR, 13aa) sequence as shown in SEQ ID NO: 9, a CD24 polypeptide as shown in SEQ ID NO: 7, and a phosphatidylinositol anchor signal peptide as shown in SEQ ID NO:

8.

4. Use of the mRNA-encoded fusion antigen epitope peptide of the phosphatidylinositol-anchored protein in the preparation of a medicament for efficiently activating CAR-T cells.

5. Use of the mRNA-encoded fusion antigen epitope peptide of the phosphatidylinositol-anchored protein in the preparation of a medicament for treating or preventing recurrence of cancer.

6. An mRNA vaccine, characterized in that, After the antigen epitope is fused to the phosphatidylinositol-anchored protein and / or the phosphatidylinositol anchor signal peptide, a faster diffusion speed than the natural antigen is obtained on the surface of antigen-presenting cells, thereby achieving strong stimulation of CAR-T cells.

7. A CAR molecule for recognizing the mRNA-encoded fusion antigen epitope peptide of the phosphatidylinositol-anchored protein of any one of claims 1 to 3, with sequence as shown in SEQ ID NO: 11.

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