WO2024222886A1 - Mrna tumor vaccines for mica/b target - Google Patents

Abstract

MICA and/or MICB mRNA tumor vaccines and applications thereof, provided are MICA and/or MICB mRNA vaccines, corresponding mRNA constructs, vectors, host cells, fusion proteins, LNP preparations, pharmaceutical uses, and tumor or cancer treatment/prevention methods.

Description

An mRNA tumor vaccine targeting MICA/B Technical Field

The present invention relates to the field of tumor vaccines, and in particular to mRNA tumor vaccines.

Background Art

In mice and humans, various forms of cellular stress (such as DNA damage) upregulate the ligands of the inducible sex receptor NKG2D. Among them, the NKG2D ligands of mice include Rae1, MULT1 and H60, while the NKG2D ligands of humans include ULBP, MICA, MICB and RAE-1G. NKG2D ligands will respond to potentially dangerous cells with genotoxic stress by regulating the immune system. For example, NKG2D receptors in NK cells and CD8+ T cells or γδT cells can directly trigger cell-mediated cytotoxicity through the costimulatory signals provided by NKG2D ligands, killing tumor cells or DNA-damaged cells.

MICA, also known as MHC Class I Polypeptide-Related Sequence A, is a class I MHC molecule that is highly homologous to MICB and both lack the β2M subunit of classical MHC I. Neither MICA nor MICB binds to antigenic peptides, lacks binding sites for CD8, and does not participate in antigen presentation. MICA/B is a class of stress proteins that are rarely expressed in normal cells except for gastrointestinal epithelial cells, endothelial cells, and fibroblasts, but are significantly upregulated when cells are infected or undergo malignant transformation. Many studies have confirmed that MICA/B is highly expressed on the surface of a variety of tumor cells, such as non-small cell lung cancer, colon cancer, breast cancer, etc., and is a tumor-associated antigen.

The α1 and α2 domains of MICA/B are responsible for binding to NKG2D and activating NK cell- and T cell-mediated tumor immunity. However, tumor cells have evolved immune escape pathways - MICA/B is enzymatically cleaved and shed from the cell surface to avoid recognition with NKG2D and thus avoid being killed. The shedding of MICA/B molecules occurs within the α3 domain near the membrane. Metalloproteinase-mediated cleavage allows part of the α3 domain to remain on tumor cells, while the α1/2 domains are shed. When this happens, soluble MICA/B (sMICA/B) can bind to NKG2D on cytotoxic lymphocytes, causing endocytosis and degradation of NKG2D, further reducing NK cell activity and promoting the occurrence of tumor immune escape.

Previously, the Wucherpfennig laboratory used antibodies that bind to the MICA/B α3 domain to block MICA/B from falling off the tumor cell membrane. The Fc fragment on the MICA/B α3 domain-binding antibody further enhances the immunotherapy effect by binding to the Fc receptor on immune cells. For example, the Fc fragment can bind to the low-affinity Fc receptor CD16 on NK cells to trigger the ADCC effect. They immunized mice with recombinant MICA α3 domain fragments and identified the α3 domain-specific antibody 7C6. The antibody 7C6 can better stabilize MICA/B on the surface of tumor cell membranes, activate NK cell-mediated cytotoxicity against human tumor cells, and induce the production of a large amount of interferon γ, that is, by using the 7C6 antibody to increase the level of MICA/B on tumor cells in mice, the growth of tumor cells was significantly reduced. CLN-619 developed by Cullinan Oncology is the first MICA antibody drug to enter the clinic. CLN-619 exerts its anti-tumor activity by preventing MICA/B from falling off tumor cells, antibody ADCC effect and enhancing the binding of MICA/B to NKG2D. It is currently in Phase I clinical trials in combination with Pembrolizumab for the treatment of malignant solid tumors.

Badrinath et al. further demonstrated that α3 domain-specific antibodies increase the stability of MICA/B on tumor cell membranes. They developed a protein vaccine composed of α3 domain proteins, fused MICA or MICB α3 domains to the N-terminus of Helicobacter pylori ferritin for multivalent antigen display. In order to enhance immunogenicity and overcome immune tolerance, they used biodegradable skeleton MSR for vaccine delivery, recruited dendritic cells (DCs), and added granulocyte-macrophage GM-CSF and adjuvant CpG ODN1826. MICB α3 domain-ferritin fusion vaccine (MICB-vax) can induce antibodies with a titer of 10 ⁴ to bind to the MICB stably transferred melanoma cell line B16F10 (MICB), preventing the shedding of MICB protein from the cell line surface. This protein vaccine can inhibit the growth of B16F10 (MICB) subcutaneous tumors and prolong the survival of tumor-bearing mice. In addition, the authors also demonstrated the immune memory of the model by attacking mice that remained tumor-free 4 months after re-tumoring B16F10 (MICB) cells. Furthermore, the authors also found that MICB-vax can play a role in metastatic melanoma models and triple-negative breast cancer disease recurrence models. Although the MICB-vax vaccine showed partial efficacy in animal models, its complex MSR preparation process and low immunogenicity of protein vaccines limit its clinical development.

Therefore, it is necessary to develop tumor vaccines targeting MICA/B targets with simple processes and higher immunogenicity.

Summary of the invention

The first object of the present invention is to provide a tumor vaccine. More specifically, a MICA and/or MICB mRNA vaccine is provided, which can induce an immune response against the MICA and/or MICB-α3 domains, and can also induce antibodies against the MICA and/or MICB-α3 domains.

The second object of the present invention is to provide the mRNA construct contained in the above-mentioned vaccine, and the corresponding vectors and cells.

The third object of the present invention is to provide the pharmaceutical use of the mRNA construct, and the corresponding vectors and cells.

A fourth object of the present invention is to provide a method for inducing an immune response in a subject in need thereof based on the above-mentioned vaccine, or a method for treating cancer in a subject in need thereof.

In order to solve the above technical problems, the present invention includes the following technical solutions:

In one aspect, the present invention provides a vaccine, which is a MICA and/or MICB mRNA vaccine, comprising a MICA/B mRNA construct as an immunogenic component, wherein the mRNA construct comprises an mRNA encoding a MICA protein domain and/or a MICB protein domain. In some specific embodiments, the vaccine is a tumor vaccine.

In some specific embodiments, the mRNA encoding the MICA protein domain and the MICB protein domain is expressed separately or in fusion in the host cell.

In some specific embodiments, the MICA protein domain and/or the MICB protein domain are MICA α3 domain and/or MICB α3 domain, respectively;

Preferably, the MICA protein is MICA*001, MICA*002, MICA*008 or MICA*009, and/or the MICB protein is MICB*004 or MICB*005.

In some specific embodiments, the protein encoded by the mRNA further comprises a mutation in a glycosylation site.

In some specific embodiments, the protein encoded by the mRNA further comprises a signal peptide;

Optionally, the signal peptide is located at the N-terminus of the protein;

Preferably, the signal peptide is derived from mouse H-2Kb, human IgE, HLA-B*46, MICA*008, TfRTM, OSM, VSV-G, mouse Ig Kappa, mouse heavy chain, BM40, human chymosinogen, human chymosinogen-2, human IL-2, human G-CSF, human hemagglutinin IX, human albumin, Gaussia luc, HAS, influenza virus, human Source insulin, silk LC, Erenumab antibody light chain, Pembrolizumab light chain, Ramucirumab light chain, E signal peptide, SP1(LZJ human IgG1, SP2, SP3(ZLQ).

Preferably, the amino acid sequence of the signal peptide is as shown in SEQ ID NO:73-99 respectively.

In some specific embodiments, the protein encoded by the mRNA further comprises a molecular adjuvant;

Optionally, the molecular adjuvant is located at the N-terminus or C-terminus of the protein;

Preferably, the molecular adjuvant includes hXCL1, hCCL19, gD N-terminal sequence, FLT3L, GM-CSF, CD40L, caTLR4, CD70, PADRE, etc.;

Preferably, the amino acid sequences of the molecular adjuvants are shown as SEQ ID NO: 107-115 respectively.

In some specific embodiments, the MICA/B α3 domain protein is combined with a carrier protein to form a fusion protein;

Optionally, the carrier protein includes ferritin, diphtheria toxoid (DT, DT CRM197) and tetanus toxoid (TT), keyhole limpet hemocyanin (KLH), pneumococcal hemolysin (Ply), influenza hemophilin D, pneumococcal PhtA, Pht B, Pht D, Pht DE and artificial protein N19 (Baraldoi et al., 2004, Infect Immun 72:4884-7), T4 Foldon from T4 phage, ESCRT and ALIX-binding region (EABR), BSA and/or OVA, etc.

In some specific embodiments, the carrier protein is T4 Foldon.

In some specific embodiments, the MICA/B α3 domain protein forms a fusion protein with T4 Foldon.

In some specific embodiments, the carrier protein is ferritin.

In some specific embodiments, the MICA/B α3 domain protein forms a fusion protein with ferritin.

The MICA/B α3-ferritin fusion protein of the present invention comprises unit subunits of ferritin, which can be assembled into nanoparticles outside cells, and utilize 24 monomers to form an icosahedron to fully display the immunogenic part of the MICA/B α3 domain.

The ferritin subunit of the present invention is the full length or any part of ferritin, wild type or partial amino acid mutation. In a specific embodiment, the monomer subunit is derived from bacterial ferritin, plant ferritin, algae ferritin, insect ferritin, fungal ferritin and mammalian ferritin.

In a specific embodiment, the ferritin is ferritin from Heliobacter pylori.

In some embodiments, a deglycosylation mutation is introduced into the ferritin;

Optionally, the deglycosylation mutation is a single amino acid deglycosylation mutation or a multiple amino acid deglycosylation mutation.

In some specific embodiments, the protein encoded by the mRNA further comprises a transmembrane domain (TM) and/or an intracellular domain (CTD).

Optionally, the transmembrane domain (TM) and/or intracellular domain (CTD) are from MICA, MICB, MITD, influenza virus HA protein, transferrin, HSV envelope glycoprotein gB, gD, gC, gE, CD8, CD28, SARS-COV-2Spike protein, etc.

Optionally, the transmembrane domain (TM) includes MICATM, MICB TM, MITD, HATM, gD-TMR, TfRTM, CD8α TM, CD28 TM, SARS-COV-2 Spike TM, HSV gB TM, HSV gC TM, HSV gE TM, etc.

Preferably, the amino acid sequences of the transmembrane domain (TM) and/or the intracellular domain (CTD) are as shown in SEQ ID NO: As shown in 100-106.

In some specific embodiments, the protein encoded by the mRNA comprises SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: The amino acid sequence shown in Figure: 37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65 or SEQ ID NO:67 has an amino acid sequence that is at least 90% (91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) identical.

In some specific embodiments, the protein encoded by the mRNA comprises SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 An amino acid sequence having at least 95% identity to the amino acid sequence shown in Q ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65 or SEQ ID NO:67.

In some specific embodiments, the protein encoded by the mRNA comprises SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 An amino acid sequence having at least 99% identity to the amino acid sequence shown in Q ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65 or SEQ ID NO:67.

In some specific embodiments, the protein encoded by the mRNA is relative to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45 at least one of the amino acid sequences set forth in SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65 or SEQ ID NO:67 each independently comprises 1-12 amino acid substitutions;

Optionally, the 1-12 amino acid substitutions are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 amino acid substitutions;

Preferably, the amino acid substitution is a conservative amino acid substitution;

More preferably, the substitution is a highly conservative amino acid substitution.

In a preferred embodiment, the protein encoded by the mRNA includes SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 :33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65 or SEQ ID NO:67.

In a more preferred embodiment, the mRNA is transcribed from DNA, and the DNA comprises SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ The nucleotide sequence shown in SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66 or SEQ ID NO:68.

In some specific embodiments, the mRNA construct further comprises a 5'UTR sequence;

Optionally, the 5'UTR sequence is human alpha globulin 5'UTR, or a non-natural 5'UTR sequence;

Preferably, the human alpha globulin 5'UTR is transcribed from DNA, and the DNA comprises the nucleotide sequence shown in SEQ NO:69;

Preferably, the non-natural 5’UTR is obtained by transcription from DNA, and the DNA sequence contains a nucleotide sequence as shown in SEQ NO:70.

In some specific embodiments, the mRNA construct further comprises a 3'UTR sequence;

Preferably, the 3’UTR is obtained by transcription from DNA, and the DNA sequence contains the nucleotide sequence shown in SEQ NO:71.

In some specific embodiments, the mRNA construct further comprises a Poly (A) sequence;

Preferably, the Poly (A) sequence comprises a nucleotide sequence as shown in SEQ NO:72.

In a specific embodiment, the vaccine further comprises a delivery formulation;

Preferably, the delivery formulation is a nanoparticle;

Preferably, the delivery preparation includes lipid nanoparticles (LNP), lipid multipolymers (LPP), polymer nanoparticles (PNP), inorganic nanoparticles (INP), cationic nanoemulsion (CNE), exosomes, biological microvesicles, protamine, etc.

More preferably, the nanoparticles are lipid nanoparticles (LNP).

In another aspect, the present invention provides an mRNA construct, which is any one of the mRNA constructs described herein as an immunogenic component in the vaccine, or a combination thereof.

In another aspect, the present invention provides a vector comprising any one of the mRNA constructs described herein, or a combination thereof.

In another aspect, the present invention provides a cell comprising any one of the mRNA constructs described herein, or a combination thereof, or a vector described herein.

In another aspect, the present invention provides a nanoparticle comprising any one of the mRNA constructs described herein, or a combination thereof;

Optionally, the nanoparticles include lipid nanoparticles (LNP), lipid multipolymers (LPP), polymer nanoparticles (PNP), inorganic nanoparticles (INP), cationic nanoemulsion (CNE), exosomes, biological microvesicles, protamine, etc.

Preferably, the nanoparticles are lipid nanoparticles (LNP).

In another aspect, the present invention provides a vaccine comprising any one of the nanoparticles described herein;

In some specific embodiments, the vaccine is a MICA and/or MICB mRNA vaccine.

In another aspect, the present invention provides a fusion protein comprising a protein encoded by any one of the mRNAs described herein and a second protein fused thereto, wherein the protein encoded by the mRNA is an unfused protein.

In some specific embodiments, the second protein is a carrier protein;

Optionally, the carrier protein includes ferritin, diphtheria toxoid (DT, DT CRM197) and tetanus toxoid (TT), keyhole limpet hemocyanin (KLH), pneumolysin (Ply), influenza hemophilin D, pneumococcal PhtA, Pht B, Pht D, Pht DE and artificial protein N19 (Baraldoi et al., 2004, Infect Immun 72:4884-7), T4 Foldon from T4 phage, BSA and/or OVA, etc.

In some specific embodiments, the second protein is T4 Foldon.

In some specific embodiments, the second protein is ferritin.

In some specific embodiments, the ferritin protein comprises a domain that allows the fusion protein to self-assemble into nanoparticles;

Optionally, the ferritin is ferritin from Helicobacter pylori.

In some specific embodiments, the fusion protein comprises an amino acid sequence that is at least 90% identical to an amino acid sequence as shown in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:13, SEQ ID:15, SEQ ID:35 or SEQ ID:37.

In some specific embodiments, the fusion protein independently contains 1-12 amino acid substitutions relative to at least one of the amino acid sequences shown in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:13, SEQ ID:15, SEQ ID:35 or SEQ ID:37.

In some specific embodiments, the fusion protein comprises an amino acid sequence that is at least 95% identical to an amino acid sequence as shown in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:13, SEQ ID:15, SEQ ID:35 or SEQ ID:37.

In some preferred embodiments, the fusion protein comprises an amino acid sequence having at least 99% identity to that shown in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:13, SEQ ID:15, SEQ ID:35 or SEQ ID:37. The amino acid sequence of

In some more preferred embodiments, the fusion protein comprises an amino acid sequence as shown in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:13, SEQ ID:15, SEQ ID:35 or SEQ ID:37.

In another aspect, the present invention provides use of any vaccine, mRNA construct, vector, cell, or nanoparticle described herein in the preparation of a medicament for inducing an immune response in a subject in need thereof.

In another aspect, the present invention provides use of any vaccine, mRNA construct, vector, cell, or nanoparticle described herein in the preparation of a medicament for treating or preventing cancer in a subject in need thereof.

In another aspect, the present invention provides a method of inducing an immune response in a subject in need thereof, comprising administering to the subject any one of the vaccines described herein;

Preferably, the method induces an immune response against MICA/B by using an mRNA vaccine;

More preferably, the mRNA is replicating, non-replicating, trans-self-replicating or circular mRNA.

In another aspect, the present invention provides a method of treating or preventing cancer in a subject in need thereof, comprising administering to the subject any one of the vaccines described herein;

Preferably, the method induces an immune response against MICA/B by using replicating or non-replicating mRNA;

More preferably, the mRNA is replicating, non-replicating, trans-self-replicating or circular mRNA.

In some specific embodiments, the vaccine composition is administered as part of a therapeutic regimen;

Preferably, the treatment regimen is radiation therapy, targeted therapy, immunotherapy or chemotherapy.

In some specific embodiments, the individual is one who tests positive for shed MICA/B in their serum.

In some specific embodiments, the cancer or tumor includes, for example, colon cancer, melanoma, kidney cancer, lymphoma, acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), gastrointestinal tumors, lung cancer, glioma, thyroid tumor, breast cancer, prostate tumor, liver tumor, various virus-induced tumors such as papilloma virus-induced cancer (e.g., cervical cancer), adenocarcinoma, herpes virus-induced tumors (e.g., Burkitt's lymphoma, EBV-induced B cell lymphoma), hepatitis B virus B-induced tumor (hepatoma), HTLV-1 and HTLV-2 induced lymphoma, acoustic neuroma/neurilemmoma, cervical cancer, lung cancer, pharyngeal cancer, rectal cancer, malignant glioma, lymphoma, rectal cancer, astrocytoma, brain tumor, gastric cancer, eye cancer, basal cell carcinoma, brain metastasis, medulloblastoma, vagina cancer, pancreatic cancer, testicular cancer, melanoma, thyroid cancer, bladder cancer, Hodgkin's syndrome, meningioma, Schneeberger's disease, bronchial cancer, pituitary tumors, mycosis fungoides, esophageal cancer, breast cancer, carcinoid tumors, neuromas, spinaliomas, Burkitt's lymphoma, laryngeal cancer, kidney cancer, thymoma, corpus carcinomas, bone cancer, non-Hodgkin's lymphoma, urethral cancer, CUP syndrome, head/neck tumors, oligodendrogliomas, vulvar cancer, intestinal cancer, colon cancer, esophageal cancer, conditions involving warts, small intestine tumors, craniopharyngeal cancer, ovarian cancer, soft tissue tumors/sarcomas, ovarian cancer, liver cancer, pancreatic cancer, cervical cancer, endometrial cancer, liver metastases, penile cancer, tongue cancer, gallbladder cancer, leukemia, plasma cell tumors, uterine cancer, eyelid cancer, prostate cancer, etc.

Beneficial Effects

Compared with the prior art, the technical solution of the present invention has at least one of the following beneficial effects:

1. The MICA/B mRNA vaccine described in the present invention has good immunogenicity, can effectively stimulate the body to produce a high level of immune response, and can not only induce a high level of MICA/B-specific humoral immune response, but also induce MICA/B-targeted cellular immunity.

2. The serum antibodies induced by the MICA/B mRNA vaccine described in the present invention can specifically and effectively bind to cells expressing MICA/B and show a higher antibody titer.

3. Compared with the MICA/B protein vaccine, the MICA/B mRNA vaccine described in the present invention has a simpler preparation process, better efficacy, and is more suitable for clinical development.

Definitions and explanations of terms

Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings understood by those of ordinary skill in the art.

Furthermore, unless otherwise indicated herein, singular terms shall include pluralities and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless expressly indicated otherwise.

The terms "include", "comprising" and "having" are used interchangeably herein and are intended to indicate the inclusiveness of the solution, meaning that the solution may have other elements in addition to the listed elements. At the same time, it should be understood that the use of "include", "comprising" and "having" descriptions herein also provides a "consisting of..." solution.

The term "and/or" as used herein includes the meanings of "and", "or" and "all or any other combination of elements linked by the corresponding term".

The term "and/or" as used herein includes the meanings of "and", "or" and "all or any other combination of elements linked by the corresponding term".

The term "gene" herein refers to a nucleic acid fragment encoding a single protein or RNA (also referred to as a "coding sequence" or "coding region") and associated regulatory regions such as promoters, operators, terminators, etc., which may be located upstream or downstream of the coding sequence.

The term "nucleic acid" herein is used in its broadest sense to include any compound and/or substance comprising a polymer of nucleotides. These polymers are called polynucleotides.

Nucleic acids (also referred to as polynucleotides) may be or may include, for example, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA with a β-D-ribose configuration, α-LNA with an α-L-ribose configuration (diastereomers of LNA), 2′-amino-LNA with 2′-amino functionalization, and 2′-amino-α-LNA with 2′-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), or chimeras or combinations thereof.

The term "mRNA" herein means messenger RNA and refers to any polynucleotide that encodes (at least one) polypeptide (naturally occurring, non-naturally occurring or modified amino acid polymer) and can be translated in vitro, in vivo, in situ or ex vivo to produce the encoded polypeptide.

The basic components of an mRNA molecule generally include at least a coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap, and a poly(A) tail. The polynucleotides of the present disclosure may function as mRNAs, but may differ in their functional and/or structural design characteristics. The mRNAs are distinguished from wild-type mRNA in several aspects that are useful for overcoming existing problems with efficient polypeptide expression using nucleic acid-based therapeutics.

The mRNA described herein refers to mRNA containing a nucleic acid sequence encoding a tumor antigen, which may be 1) mRNA that only encodes and translates a certain tumor antigen, 2) a mixture of mRNAs that encode and translate multiple tumor antigens, or 3) a mixture consisting of 1), 2) and other mRNAs that do not encode tumor antigens.

The term 5'UTR (5'-untranslated region) herein refers to a specific portion of a messenger RNA (mRNA) that is located 5' to the open reading frame of the mRNA. Typically, the 5'UTR starts at the transcription start site and ends at one nucleotide before the start codon of the open reading frame. The 5'UTR may include elements for controlling gene expression, also referred to as regulatory elements. The regulatory element may be, for example, a ribosome binding site or a 5' terminal oligopyrimidine sequence. The 5'UTR may be modified post-transcriptionally, for example by adding a 5'CAP.

The term 3'UTR (3'-untranslated region) herein is the portion of mRNA that is located between the protein coding region (i.e., open reading frame) and the poly (A) sequence of the mRNA. The 3'UTR of the mRNA is not translated into an amino acid sequence. The 3'UTR sequence is usually encoded by a gene that is transcribed into a corresponding mRNA during gene expression. The genomic sequence is first transcribed into a pre-mature mRNA, which includes optional introns. The pre-mature mRNA is then further processed into a mature mRNA during maturation. The maturation process includes the following steps: 5' capping, splicing of the pre-mature mRNA to remove optional introns, and modification of the 3' end, such as polyadenylation of the 3' end of the pre-mature mRNA and optional endo- or exonuclease cleavage.

The term "Poly (A)" herein refers to a (long) sequence of adenosine nucleotides added to the 3' end of RNA, which is up to about 400 adenosine nucleotides, for example, about 25 to about 400, preferably about 50 to about 400, more preferably about 50 to about 300, even more preferably about 50 to about 250, and most preferably about 60 to about 250 adenosine nucleotides.

The term "mutation" herein includes gene mutation and amino acid mutation, wherein gene mutation refers to deletion, insertion, inversion or substitution of heterologous nucleic acid, which may lead to changes in the amino acid sequence of the corresponding protein product; amino acid mutation is also called non-synonymous single nucleotide mutation, which is caused by the change of some single bases, resulting in changes in the amino acid sequence of the protein product. Amino acid changes can affect protein stability, interaction and enzyme activity, thereby leading to the occurrence of diseases.

The terms "protein", "polypeptide" and "peptide" are used interchangeably herein and refer to a peptide-bonded chain of any amino acids, regardless of length or co-translational or post-translational modification. This definition of a protein polypeptide or protein that is not encoded on a nucleic acid construct specifically and additionally includes chains that include one or more unnatural amino acids or amino acid-like building blocks.

The term "amino acid substitution" herein refers to those in which at least one amino acid residue in the native or starting sequence is removed and a different amino acid is inserted in its place at the same position. Substitutions may be single, in which only one amino acid in the molecule has been substituted, or they may be multiple, in which two or more amino acids in the same molecule have been substituted.

The term "conservative amino acid substitution" herein refers to replacing an amino acid normally present in a sequence with a different amino acid of similar size, charge or polarity. Examples of conservative substitutions include replacing a non-polar (hydrophobic) residue such as isoleucine, valine and leucine with another non-polar residue. Likewise, examples of conservative substitutions include replacing one polar (hydrophilic) residue with another residue, such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. In addition, replacing a basic residue such as lysine, arginine or histidine with another residue, Or an acidic residue such as aspartic acid or glutamic acid is substituted with another acidic residue is an additional example of conservative substitution. Examples of non-conservative substitutions include non-polar (hydrophobic) amino acid residues such as isoleucine, valine, leucine, alanine, methionine are substituted with polar (hydrophilic) residues such as cysteine, glutamine, glutamic acid or lysine, and/or polar residues are substituted with non-polar residues.

The term "mutant" herein refers to a "variant" of the protein or peptide that may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity with the amino acid sequence of the protein or peptide.

The term "vector" herein refers to a DNA molecule including single-stranded, double-stranded, circular or supercoiled DNA. Suitable vectors include retroviruses, adenoviruses, adenovirus-associated viruses, poxviruses and bacterial plasmids.

The term "antigen" herein refers to a substance that can be recognized by the immune system and can, for example, trigger an antigen-specific immune response by forming antibodies as part of an adaptive immune response. Antigens described herein include tumor antigens, preferably located on the surface of (tumor) cells. Tumor antigens can also be selected from proteins that are overexpressed in tumor cells compared to normal cells. In addition, tumor antigens also include antigens that are non-self (or initially non-self) degenerate and are associated with hypothetical tumors expressed in cells. Antigens associated with tumors also include antigens from cells or tissues that are usually embedded in tumors. In addition, some substances (such as proteins or peptides) are expressed in patients with (known or unknown) cancer, and their concentration in the body fluids of the patients increases. These substances are also referred to as "tumor antigens", but they are not antigens in the strict sense of immune response inducing substances. In addition, tumor antigens can also be present on the tumor surface in the form of, for example, mutated receptors. In this case, it can be recognized by antibodies.

The term "carrier protein" in this article refers to proteins that are non-toxic to the human body, do not cause allergic reactions and can enhance the immune efficacy of vaccines, including ferritin, diphtheria toxoid (DT, DT CRM197), tetanus toxoid (TT), keyhole limpet hemocyanin (KLH), OMPC from Neisseria meningitidis (N. meningitidis), pneumolysin (Ply), purified protein derivative of tuberculin (PPD), influenza hemophilin D, pneumococcal PhtA, Pht B, Pht D, Pht DE and artificial protein N19, T4 Foldon from T4 bacteriophage, ESCRT and ALIX-binding region (EABR), BSA and/or OVA, etc.

The term "molecular adjuvant" in this article refers to proteins encoded by nucleic acid sequences that act as adjuvants by targeting innate immune receptors or regulating molecular signaling events. Unlike traditional adjuvants or liposomes and nanoparticles, molecular adjuvants are universal because they are directly integrated into plasmids. Molecular adjuvants include pathogen recognition receptor (PRR) agonists, cytokines, chemokines, and immune targeting genes, such as hXCL1, hCCL19, gD N-terminal sequence, FLT3L, GM-CSF, CD40L, caTLR4, CD70, PADRE, C3d, etc.

The term "transmembrane domain (TM)" in this article refers to the structural feature of proteins that span the cell membrane, connecting the inside and outside of the cell, usually composed of alpha helical segments of amino acids, and can be embedded in the hydrophobic interior of the cell membrane. Common transmembrane domains include transmembrane domains (TM) including MICA TM, MICB TM, MITD, HATM, gD-TMR, TfRTM, CD8α TM, CD28 TM, SARS-COV-2 Spike TM, HSV gB TM, HSV gC TM, HSV gE TM, etc.

The term "vaccine" herein refers to a preventive or therapeutic substance that provides at least one antigen or antigenic function that can stimulate an immune response in the body without causing disease. The antigen or antigenic function can stimulate the body's adaptive immune system to provide an adaptive immune response.

The term "delivery formulation" herein refers to a formulation that helps mRNA molecules enter target cells and be successfully expressed. Common delivery formulations include lipid nanoparticles (LNP), lipopolyplex (LPP), Polymer nanoparticles (PNP), inorganic nanoparticles (INP), cationic nanoemulsion (CNE), cationic lipid, exosome, biological microvesicles, protamine polysaccharide particles, etc.

Herein, the terms "nanoparticle" and "nanoparticles" are used interchangeably.

The term "immune system" herein can protect an organism from infection. If a pathogen breaks through the physical barriers of an organism and enters the organism, the innate immune system provides an immediate but non-specific response. If the pathogen avoids the innate response, vertebrates have a second layer of protection, the adaptive immune system. Here, the immune system changes its response during the infection process to improve its recognition of the pathogen. Then, the improved response is retained in the form of immune memory after the pathogen is eliminated, and allows the adaptive immune system to establish a faster and stronger attack each time the pathogen is encountered. Accordingly, the immune system includes an innate and adaptive immune system. Each of these two parts contains so-called body fluids and cellular components.

The term "immune response" herein can typically be a specific response of the adaptive immune system to a specific antigen (so-called specific or adaptive immune response) or a non-specific response of the innate immune system (so-called non-specific or innate immune response). One basis of the present invention relates to the specific response of the adaptive immune system (adaptive immune response); in particular, the adaptive immune response after exposure to an antigen (such as an immunogenic polypeptide). However, this specific response can be supported by additional non-specific responses (innate immune responses). Therefore, one basis of the present invention also relates to compounds for stimulating both the innate and adaptive immune systems to stimulate an effective adaptive immune response. In the context of the present invention, an "antigenic composition" refers to a compound or mixture of compounds (such as in a solution or pharmaceutical preparation) that is capable of, used for, or used for, has the ability or can, in practice, stimulate, increase, produce or cause an immune response (preferably, an effective adaptive immune response) when administered to a subject or otherwise exposed to a subject.

The term "cellular immunity/cellular immune response" herein typically involves the activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T lymphocytes, and the release of various cytokines in response to antigens. In a more general manner, cellular immunity does not involve antibodies but involves the activation of immune system cells. For example, the cellular immune response is characterized by the activation of antigen-specific cytotoxic T lymphocytes, which can induce apoptosis in body cells (such as virus-infected cells, cells with intracellular bacteria) and cancer cells displaying tumor antigens that display antigenic epitopes on their surfaces; Activate macrophages and natural killer cells, allowing them to destroy pathogens; and stimulate cells to secrete a variety of cytokines that affect the functions of other cells involved in adaptive immune responses and innate immune responses.

The term "humoral immunity/humoral immune response" herein typically refers to antibody production and the auxiliary processes that may accompany it. For example, a humoral immune response can typically be characterized by Th2 activation and cytokine production, germinal center formation and allotype switching, affinity maturation and memory cell production. Humoral immunity can also typically refer to the effector functions of antibodies, which include pathogen and toxin neutralization, classical complement activation and phagocytosis and opsonization promotion of pathogen elimination.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the design of mRNA constructs XR-MIC-1 to 4.

FIG. 2 is a schematic diagram of the design of mRNA constructs XR-MIC-5 to 8.

FIG. 3 is a flow cytometric analysis of the expression of XR-MIC-1 to 8 mRNA constructs in BHK21 cells.

FIG4 is a flow cytometry analysis of the expression of LNP formulations of XR-MIC-2/3/5/6/7/8 mRNA constructs in BHK21 cells. Test.

FIG5 shows the ELISA detection of MICA and MICB specific antibody levels in mice after a single immunization with XR-MIC-2/3/5/6/7/8 vaccine.

FIG6 shows the ELISA detection of MICA and MICB specific antibody levels in mice after secondary immunization with XR-MIC-2/3/5/6/7/8 vaccines.

FIG7 shows the ELISPOT detection of IFN-γ secretion levels in spleen cells after secondary immunization of mice with XR-MIC-2/3/5/6/7/8 vaccines.

FIG8 shows the flow cytometry analysis of the proportion of CD4 ⁺ T cells positive for IFN-γ, IL-2, TNF-α, and IL4 after secondary immunization of XR-MIC-2/3/5/6/7/8 vaccines in mice.

FIG9 shows the flow cytometry analysis of the proportion of CD8 ⁺ T cells positive for IFN-γ, IL-2, TNF-α, and IL4 after secondary immunization of XR-MIC-2/3/5/6/7/8 vaccines in mice.

FIG10 shows the expression of MICA/B in MC38(MICA/B)/B16F10(MICA/B)/CT26(MICA/B) stable cell lines detected by flow cytometry.

Figure 11 shows the flow cytometry detection of the binding of MICA/B antibodies in the serum produced by the XR-MIC-2 vaccine to the MICA/B proteins on the surface of the B16F10 (MICA/B) and MC38 (MICA/B) transgenic cell lines.

Figure 12 shows the flow cytometry detection of antibody titers binding to MICA/B on the surface of MC38 (MICA/B) in sera produced by XR-MIC-2/3/5/6/7/8 vaccines; wherein for each concentration, the bar graph represents the data of XR-MIC-2, XR-MIC-3, XR-MIC-5, XR-MIC-6, XR-MIC-7, XR-MIC-8, and normal saline from left to right, respectively.

FIG. 13 is a schematic diagram of the design of mRNA constructs XR-MIC-9 to 15.

FIG. 14 is a flow cytometric analysis of the expression of XR-MIC-9 to 14 mRNA constructs in BHK21 cells.

FIG. 15 shows the ELISA detection of MICB-specific antibody levels in mice after a single immunization with XR-MIC-1 to 14 vaccines.

FIG. 16 shows the ELISA detection of MICB-specific antibody levels in mice after secondary immunization with XR-MIC-1 to 14 vaccines.

Figure 17 shows the preliminary efficacy of XR-MIC-12 vaccine in the mouse MC38 subcutaneous tumor model by different administration routes

FIG. 18 is a schematic diagram of the design of mRNA constructs XR-MIC-12-1 to 17 and XR-MIC-12-13-2 and XR-MIC-12-14-2.

FIG. 19 is a flow cytometric analysis of the expression of XR-MIC-12-2/4/6/7/8/10/11/12/13/14/15/16/17 mRNA constructs in BHK21 cells.

FIG. 20 is a flow cytometric assay of the expression of XR-MIC-12-2/4/6/8/10/11/12/13/14/15/16/17 mRNA construct LNP formulations in BHK21 cells.

FIG21 shows the MICA-specific antibody levels of XR-MIC-12, XR-MIC-15, XR-MIC-12-2/4/6/8/10/11/12/13/14/17 vaccines in mice 20 days after the first immunization detected by ELISA.

Figure 22 shows the ELISA detection of XR-MIC-12, XR-MIC-15, MICB-specific antibody levels in mice 20 days after primary immunization with XR-MIC-12-2/4/6/8/10/11/12/13/14/17 vaccines.

FIG23 shows the MICA-specific antibody levels detected by ELISA after secondary immunization of XR-MIC-12, XR-MIC-15, and XR-MIC-12-2/4/6/8/10/11/12/13/14/17 vaccines in mice.

FIG. 24 shows the ELISA detection of MICB-specific antibody levels in mice after secondary immunization with XR-MIC-12, XR-MIC-15, and XR-MIC-12-2/4/6/8/10/11/12/13/14/17 vaccines.

Figure 25 shows the IFN-γ secretion levels of spleen cells stimulated with MICA peptide library after secondary immunization of mice with XR-MIC-12, XR-MIC-15, and XR-MIC-12-2/4/6/8/10/11/12/13/14/17 vaccines as detected by ELISPOT.

Figure 26 shows the IFN-γ secretion level detected by ELISPOT in spleen cells after secondary immunization of mice with XR-MIC-12, XR-MIC-15, and XR-MIC-12-2/4/6/8/10/11/12/13/14/17 vaccines and stimulation with the MICB peptide library.

DETAILED DESCRIPTION

The present invention will be further described below in conjunction with specific examples, and the advantages and features of the present invention will become clearer as the description proceeds. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

The embodiments of the present invention are only exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

Example 1 XR-MIC-1 to 8 mRNA construct design

In order to obtain an mRNA vaccine against MICA/B with a simple preparation process and higher immunogenicity, multiple MICA/B mRNA vaccine constructs were designed and constructed.

The MICA/B mRNA vaccine sequence we designed targets the highly conserved α3 domain of the MICA/B protein. This site can be expanded by the disulfide isomerase ERp5 and then cleaved by the matrix metalloenzyme ADAM10/ADAM17/MMP14, causing MICA/B to fall off from tumor cells. Therefore, the vaccine targeting the MICA/B α3 domain is intended to inhibit the shedding of MICA/B on the cell membrane by producing antibodies, thereby inducing tumor immunity mediated by T cells and NK cells.

Just like HLA has polymorphism, MICA/B protein also has polymorphism. So far, 537 MICA and 245 MICB alleles have been identified (IPD/IMGT-HLA database, updated in April 2023). However, whether it is European or Asian population, the most frequently detected MICA or MICB alleles are MICA*008 and MICB*005. Therefore, in the present invention, we selected the α3 domain of MICA*008 and the α3 domain of MICB*005 as target antigens, designed their mRNA sequences, and then expressed them by fusion.

The construct design of the MICA/B mRNA vaccine in the present invention is shown in Figures 1 and 2, wherein XR-MIC-1 to 4 (Figure 1, SEQ NO: 1 to SEQ NO: 8) uses human alpha globin 5'UTR (SEQ NO: 69) (5'UTR-human alpha-globin, hα-globin), and XR-MIC-5 to 8 (Figure 2, SEQ ID NO: 9 to SEQ ID NO: 16) uses a self-designed non-natural 5'UTR sequence, named 5art2 (SEQ ID NO: 70) (refer to See Table 1). Considering that the MICA/B mRNA vaccine we designed will be secreted into the extracellular space in the form of a fusion protein after being administered into the organism and expressed in the host cells, specifically activating B cells to produce antibodies, a signal peptide sequence was added to its N-terminus. In the present invention, the signal peptide sequences used include mouse H-2K ^b signal peptide (SEQ NO:73: MVPCTLLLLLAAALAPTQTRA) and human IgE signal peptide (SEQ NO:74: MDWTWILFLVAAATRVHS), HLA-B*46 signal peptide (SEQ NO:75: MRVTAPRTLILLLSGALALTETWAGS) and MICA*008's own signal peptide (SEQ NO:76: MGLGPVFLLLAGIFPFAPPGAAA); the signal peptides used can also be derived from OSM, VSV-G, mouse Ig Kappa, mouse heavy chain, BM40, human chymosinogen, human chymosinogen-2, human IL-2, human G-CSF, human hemagglutinin IX, human albumin, Gaussia luc, HAS, influenza virus, human insulin, silk LC, Erenumab antibody light chain, Pembrolizumab light chain, Ramucirumab light chain, E signal peptide, SP1(LZJ human IgG1, SP2, SP3(ZLQ), the amino acid sequences of the above signal peptides are shown in SEQ ID NOs: 77-99, respectively.

The seven glycosylation sites or multiple point mutations on the MICA/B protein will change the expression level of MICA/B on the cell surface. In addition, mutations in glycosylation sites may also change some properties of the fusion protein (such as solubility, stability and immunogenicity, etc.). Therefore, when constructing the MICA/B α3 mRNA construct, we constructed two models, wild type and deglycosylation site mutants (mutating the potential N-glycosylation site in the MICA or MICB α3 region from Asn to Gln) to explore the effect of deglycosylation on the immunogenicity of the vaccine of the present invention. Specifically, when designing the deglycosylation mutants, the amino acids at positions 29, 39, 80, and 108 in the MICA α3 region were mutated from Asn to Gln, the amino acids at positions 153, 163, 204, and 232 in the MICB α3 region were mutated from Asn to Gln, and the 179th Thr was mutated to Asn (see Table 1).

The fusion protein bound to ferritin can self-assemble into nanoparticles composed of 24 subunits and be displayed on the surface of the nanoparticles, which can further enhance the immunogenicity of the MICA/B α3-domain peptide. In the present invention, we used ferritin from Heliobacter pylori and introduced single amino acid deglycosylation mutations (SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8) to explore the effect of the introduction of ferritin on the immunogenicity of the vaccine of the present invention.

The main designs and component compositions of the XR-MIC-1 to 8 constructs described in this article are summarized as follows (Figures 1 and 2, the italicized bold parts show the differences between the structures of the constructs, where the deglycosylation site mutants of MICA α3 and MICB α3 are abbreviated as MICA α3m and MICB α3m, respectively):

XR-MIC-1: 5'UTR hα-globin-H-2K ^b signal peptide-MICA α3-GS linker-MICB α3-3'UTR-Poly (A) (its amino acid sequence is SEQ NO: 1; its encoding nucleic acid sequence is SEQ NO: 2);

XR-MIC-2: 5'UTR hα-globin-H-2K ^b signal peptide-MICA α3m-GS linker-MICB α3m-3'UTR-Poly (A) (its amino acid sequence is SEQ NO: 3; its encoding nucleic acid sequence is SEQ NO: 4);

XR-MIC-3: 5'UTR hα-globin-H-2K ^b signal peptide-MICA α3-GS linker-MICB α3-GS linker-ferritin N19Q mutant-3'UTR-Poly (A) (its amino acid sequence is SEQ NO: 5; its encoding nucleic acid sequence is SEQ NO: 6);

XR-MIC-4: 5'UTR hα-globin-H-2K ^b signal peptide-MICA α3m-GS linker-MICB α3m-GS linker-ferritin N19Q mutant-3'UTR-Poly (A) (its amino acid sequence is SEQ NO: 7; its encoding nucleic acid sequence is SEQ NO: 8);

XR-MIC-5: 5'UTR 5art2-H-2K ^b signal peptide-MICA α3-GS linker-MICB α3-3'UTR-Poly (A) (its amino acid sequence is SEQ NO: 9; its encoding nucleic acid sequence is SEQ NO: 10);

XR-MIC-6: 5'UTR 5art2-H-2K ^b signal peptide-MICA α3m-GS linker-MICB α3m-3'UTR-Poly (A) (its amino acid sequence is SEQ NO: 11; its encoding nucleic acid sequence is SEQ NO: 12);

XR-MIC-7: 5'UTR 5art2-H-2K ^b signal peptide-MICA α3-GS linker-MICB α3-GS linker-ferritin N19Q mutant-3'UTR-Poly (A) (its amino acid sequence is SEQ NO: 13; its encoding nucleic acid sequence is SEQ NO: 14);

XR-MIC-8: 5'UTR 5art2-H-2K ^b signal peptide-MICA α3m-GS linker-MICB α3m-GS linker-ferritin N19Q mutant-3'UTR-Poly (A) (its amino acid sequence is SEQ NO: 15; its encoding nucleic acid sequence is SEQ NO: 16).

In the above construct, the sequence of the 3’UTR used is shown in SEQ NO:71, and the sequence of Poly(A) is shown in SEQ NO:72.

Table 1 Each mRNA vaccine construct in this application, its structural composition and corresponding sequence information

Note: For the construction instructions of the second round constructs XR-MIC-9 to 15, and the third round constructs XR-MIC-12-1 to 17, XR-MIC-12-13-2, and XR-MIC-12-14-2, see Examples 9 and 14, respectively.

Example 2 Preparation and detection of mRNA of XR-MIC-1 to 8 constructs

2.1 Synthesis of gene sequences and construction of vectors

According to the SEQ ID.NO 2, 4, 6, 8, 10, 12, 14, 16 sequences of XR-MIC-1 to 8 designed in Example 1, the corresponding nucleotides (Nanjing GenScript) were synthesized, and the corresponding 5'UTR sequence and SpeI restriction endonuclease site sequence were added to the 5' end, and the stop codon and XhoI restriction endonuclease site sequence were added to the 3' end. The DNA fragments after enzyme cutting were cloned into the pUC57 vector backbone containing the T7 promoter sequence, 3'UTR sequence and poly (A) sequence by enzyme cutting and ligation. The sequence between XbaI/NotI in the plasmid was sequenced to confirm that the sequence was correct, and then the plasmid was extracted.

2.2 Plasmid linearization

The eight plasmids XR-MIC-1 to 8 designed in 2.1 were linearized by enzyme digestion according to conventional plasmid linearization and purification methods in the art, and mRNA was prepared by in vitro co-transcription, capping, purification, and so on.

Plasmid linearization was performed according to the restriction digestion volume shown in Table 2, BspQ I (Nearshore Bio, RE07-01-M005)

Table 2 Plasmid restriction enzyme system

After the enzyme digestion is completed, 1% agarose gel electrophoresis and gel imaging are used to observe whether the sample is completely digested.

2.3 In vitro co-transcription and purification

Using the linearized plasmid template, an in vitro transcription system was prepared according to conventional methods in the art, incubated at 37°C for 2 h, and in vitro transcription was performed. Then, DNase I was added to digest at 37°C for 15 min to remove the DNA template, and then the mRNA was purified using ion exchange chromatography media.

2.4 mRNA quality testing

2.4.1. Sample processing: dilute mRNA with 1×TE buffer

2.4.2. Add the RNA dilution standard to the 96-well sample plate, add the diluted test solution and RNALadder solution, seal the wells with a sealing film, centrifuge at 3000rpm/min for 2 minutes, and shake at 2000rpm for 2 minutes to mix. Use 5200FragmentAnalyzer to detect RNA integrity.

2.5 Detection of RNA concentration

Mix Ribogreen dye (ThermoFisher, R11490) with 1×TE buffer and set aside. Dilute RNA standard with 1×TE. Add 100 mL of mRNA sample to be tested into a 96-well plate, then add 100 mL of Ribogreen dye solution and place on a plate shaker for 5 minutes. Detect using SpectraMax iD3 multi-function microplate reader. Calculate total RNA concentration using the standard curve.

The mRNA concentration and integrity of the transcribed and purified XR-MIC-1 to 8 were tested. The mRNA integrity of XR-MIC-1 to 8 was more than 90%, which can meet the subsequent LNP preparation requirements.

Example 3 Preparation and detection of XR-MIC-1 to 8 construct LNP formulations

3.1 Lipid Nanoparticle (LNP) Preparation

When preparing lipid nanoparticles, ALC0315, ALC0159, DSPC and cholesterol are used as lipid components, and the four are prepared in a conventional molar ratio in the art to prepare an organic phase, and RNA stock solution and citric acid buffer are prepared into an aqueous phase, and then a conventional microfluidic method in the art is used to prepare lipid nanoparticles by a microfluidic device, and then the product is used The buffer solution was diluted and concentrated, then diluted again with buffer solution and packaged, and stored frozen at -80°C.

3.2 Lipid nanoparticle size and potential detection

Malvern Zetasizer ultra was used to measure the particle size and potential of lipid nanoparticles. 10 μL of the solution containing LNP particles was diluted to 1 mL with injection water and placed in the detection cell to detect the particle size and potential of the LNP particles.

3.3 Detection of total RNA concentration in lipid nanoparticles

The total RNA concentration of lipid nanoparticles was detected by Ribogreen method. The LNP solution was demulsified with Triton, 100 μL of the demulsified solution was added to a 96-well plate, and then 100 μL of Ribogreen dye solution was added and placed on a plate shaker for 5 minutes at 600 rpm. The SpectraMax iD3 multifunctional microplate reader was used for detection. The total RNA concentration was calculated using the standard curve.

Table 3 Physical and chemical tests of XR-MIC mRNA vaccine LNP preparation

The results in Table 3 show that the particle size, PDI, Zeta potential and total RNA concentration of XR-MIC-LNP all meet the requirements.

Example 4 Expression of XR-MIC-1 to 8 in BHK21 cells

After the LNP preparations of XR-MIC-2/-3/-5/-6/-7/-8 mRNA vaccines were prepared, the expression of XR-MIC-1 to 8 stock solutions and XR-MIC-2/-3/-5/-6/-7/-8 LNP preparations in BHK21 was detected.

On the first day, 1×10 ⁵ BHK21 cells (ATCC#CCL-10) with an activity greater than 95% were plated in a 24-well plate and cultured overnight at 37° C. On the second day, transfection with XR-MIC mRNA stock solution or LNP was performed.

4.1 Transfection with mRNA stock solution or LNP preparation

4.1.1 Transfection of mRNA.

Before use, equilibrate TransIT-mRNA and mRNA Boost Reagent (Miurs, MIR-2250) to room temperature and vortex gently. Aspirate 50 μL of Opti-MEM I medium into a sterile tube. Add 0.5 μg (0.5 μL of 1 μg/μL stock solution) of mRNA. Aspirate gently to mix thoroughly. Add 1 μL of mRNA Boost Reagent to the diluted mRNA mixture. Aspirate gently. Add 1 μL of TransIT-mRNA Reagent to the diluted mRNA mixture and pipette gently to mix thoroughly. Incubate at room temperature for 2-5 minutes to allow enough time for complex formation, then gently drop the complex into the cell culture wells and gently shake the cell plate to mix.

4.1.2 Transfection of LNPs

When transfecting LNP, a certain amount of LNP (1 μg/single well of 24-well plate) was thoroughly mixed with OptiMEM and then added dropwise to different areas of the 24-well plate.

4.2 Processing steps of cell samples after transfection with XR-MIC-1 to 8 mRNA stock solution/XR-MIC-2/-3/-5/-6/-7/-8 LNP (fixation + penetration)

4.2.1 Preparation of fixation and permeabilization solution: Take 1 ml of Fixation/Permeabilization Concentrate (Thermo Fisher, 00-5123) and add 3 ml of Fixation/Permeabilization Diluent (Thermo Fisher, 00-5223).

4.2.2 After the cells were transfected with XR-MIC-1 to 8 mRNA construct stock solution or corresponding LNP for 24 hours, 200 μL of the prepared fixation and permeabilization solution was added and incubated for 40 minutes.

4.2.3 Prepare permeabilization buffer (PB): add 9 ml ddH ₂ O to 1 ml 10×Permeabilization Buffer (Thermo Fisher, 00-8333).

4.2.4 Centrifuge and discard the supernatant, add 200 μL permeabilization buffer (PB), and after 5 minutes, add fetal bovine serum with a final concentration of 2% and block at room temperature for 15 minutes.

4.2.5 Add 200 μL of MICA mouse monoclonal 6G6 antibody (Santa Cruz, sc-56995) diluted 1:200 with permeabilization buffer and incubate at room temperature for 1 hour

4.2.6 Centrifuge and discard the supernatant, add 200 μL of permeabilization buffer (PB), and wash twice

4.2.7 Add goat anti-mouse FITC secondary antibody (Thermo Fisher, A16079) diluted 1:800 with permeabilization buffer and incubate at room temperature for 30 min

4.2.8 Centrifuge and discard the supernatant, add 200 μL of permeabilization buffer (PB), wash twice, add 200 μL of PBS for detection by Attune NxT acoustic focusing flow cytometer (Thermo Fisher, 2AFC236901121)

As shown in Figure 3 or Figure 4 (Control is PBS), the expression of transfected mRNA or LNP in the BHK21 cell line can be detected by FACS, indicating that XR-MIC-1 to 8 mRNA stock solutions and XR-MIC-2/3/5/6/7/8 LNP preparations can be normally expressed in cells.

Example 5 XR-MIC-2/-3/-5/-6/-7/-8 molecule immunogenicity test in mice

After the LNP preparation of XR-MIC-2/-3/-5/-6/-7/-8 mRNA vaccine was prepared, its immunogenicity was tested.

SPF female C57BL/6 mice aged 6-8 weeks were randomly divided into 7 groups, 5 mice in each group. The grouping and dosing details are shown in Table 4:

Table 4 Immunity test of mice with XR-MIC-2/-3/-5/-6/-7/-8 mRNA vaccine

Each group was vaccinated with 5 μg (100 μL) of the corresponding XR-MIC vaccine LNP preparation, and immunized twice, with an interval of 3 weeks between the two immunizations, recorded as D0 and D21 respectively. Blood was collected on D14 and D35 days, and ELISA was performed to detect the levels of MICA-specific antibodies and MICB-specific antibodies. After the spleen of the mice was collected on D35 day, they were stimulated with the MICA peptide library or the MICB peptide library, and then IFN-γ ELISPOT detection and cytokine detection were performed.

5.1 ELISA to detect MICA-specific antibody and MICB-specific antibody levels in mouse serum

96-well ELISA plates were coated with 2 μg/ml MICA protein (MICA ECD, Sino Biological, 12302-H08H-1mg) or 10 μg/ml MICB protein (MICB ECD, Sino Biological, Sino#10759-H08H-1-1mg) at 4°C overnight. The plates were washed 5 times with 260 μL/well PBST and blocked with PBST containing 3% BSA for 2 hours at 37°C. The immune mouse serum was diluted two-fold and added to each well, incubated at 37°C for 1 hour, washed 5 times with PBST, patted dry, and HRP-labeled goat anti-mouse IgG (Sino Biological, #SSA007) was added. TMB was added for 10 minutes, and the ELISA stop solution was added to terminate the reaction. The absorbance value of each well at 450nm was read with an ELISA reader. The maximum dilution corresponding to the sample OD450 ≥ the average value of all negative control samples × 2.1.

The results 14 days after a single administration are shown in FIG5 . The XR-MIC vaccine groups were able to produce high titers of IgG binding antibodies, including MICA and MICB specific antibodies. Among them, XR-MIC-2, XR-MIC-3, and XR-MIC-12 showed higher antibody titers: the endpoint dilution of XR-MIC-2 exceeded 200,000, and the antibody titer of XR-MIC-3 also exceeded 70,000. On the 14th day (D35) after the booster immunization on D21, the titers of antibodies against MICA or MICB in the six administration groups of the XR-MIC vaccine all exceeded 10 ⁶ , indicating that the XR-MIC vaccines of different molecules in the present invention have good immunogenicity in C57BL/6 mice ( FIG6 ).

5.2 ELISpot detection of IFN-γ levels secreted by mouse spleen cells

The spleen cell suspension (2×10 ⁵ cells) was incubated with PRIM 1640 medium (negative control), MICA peptide library or MICB peptide library (ordered by GenScript, 6.67 μg/ml), or ConA (6.67 μg/ml, positive control) in a 37°C incubator for 16-20 h, and the mouse IFN-γ ELISPOT kit (Abcam, ab64029) was used according to the manufacturer's instructions. The number of spots was determined using an enzyme-linked immunospot analyzer (Cellular Technology Limited, S6Entry).

The six groups immunized with XR-MIC vaccine were able to produce high levels of IFN-γ secretion after stimulation with the MICA peptide library or the MICB peptide library (Figure 7), indicating that the XR-MIC vaccine can not only induce a high level of humoral immune response, but also induce cellular immunity targeting MICA/B.

5.3 Detection of intracellular factors in mouse spleen cells

1.5×10 ⁶ spleen cells were incubated with 0.2 μg of each peptide from the MICA or MICB peptide library or 1×eBioscience ^TM cell stimulation mixture (Thermo Fisher, 00-4970-03) at 37°C for 1 h, followed by the addition of 1×eBioscience ^TM protein Transport inhibitor cocktail (Thermo, 00-4980-93) was added and cultured in an incubator at 37°C with 5% carbon dioxide for 5 h to block the release of cytokines.

Cell surface marker staining: Block the cells with 50 μL of PBS containing 4% TruStain FcX ^TM anti-mouse CD16/32 (Biolegend, #101320), incubate at 4°C for 8-10 min, add 50 μL of PBS containing 1:500 LIVE/DEAD ^TM fixable light green dead cell stain/L34957 (Thermo Fisher, #L34957), 2% CD45 (Biolegend, #103116), CD8 (Biolegend, #100734), CD4 (Biolegend, #100422) directly labeled fluorescent antibodies and incubate at 4°C for 20 min.

Cytokine staining: Fix with IC Fixation Buffer (Thermo Fisher, 00-8222-49) at 4°C for 20 min. Transmembrane staining was performed with 100 μL (1% IL-2 (Biolegend, 503808), IFN-γ (Biolegend, 505830), TNF-α (Biolegend, 506304) and IL-4 (APC anti-mouse IL4 Antibody, Biolegend, 504106) directly labeled with fluorescent antibodies 1× Permeabilization Buffer (Thermo Fisher, 00-8333-56) and incubated at 4°C in the dark for 20 min. After staining, cells were gated (forward and side scatter, FSC/SSC) and samples were analyzed using Attune NxT acoustic focusing flow cytometer (Thermo Fisher, 2AFC236901121).

The test results are shown in Figures 8 and 9. After stimulation with the MICB peptide library, the CD4 ⁺ T cells from the vaccine group mice can specifically secrete Th1 cytokines IFN-γ, IL-2 and TNF-α, while the Th2 cytokine IL4 is produced less; while the activated CD8 ⁺ T cells from the vaccine group mice can only detect a small amount of Th1 cytokine IFN-γ, while IL-2 and TNF-α and Th2 cytokine IL-4 are almost undetectable.

Through the IFN-γ detection results in CD4 ⁺ T cells and combined with the ELISpot (Figure 7) and D14 ELISA detection results (Figure 5), it can be found that 14 days after the first immunization, XR-MIC-6 and XR-MIC-8 showed higher antibody titers than XR-MIC-5 and XR-MIC-7, that is, the secretion levels of IgG binding antibodies and IFN-γ induced by XR-MIC-6 and XR-MIC-8 were higher than those of XR-MIC-5 and XR-MIC-7, respectively, indicating that the introduction of glycosylation site mutations helps to improve the immunogenicity of the vaccine.

Example 6 Construction of MC38 (MICA/B)/B16F10 (MICA/B)/CT26 (MICA/B) stable cell line

In order to evaluate the in vitro and in vivo efficacy of the present invention, it is necessary to construct a cell line that can stably express MICA/B. In this example, three commonly used mouse tumor cell lines were selected, and cell lines that can stably express the MICA/B target protein were screened by recombinant lentiviral transfection.

6.1 Virus packaging

The target genes MICA and MICB were constructed into the plasmid pLVX-XR-MIC-P2A-EGFP-IRES-Puro through P2A concatenation. The auxiliary plasmids pGP and pVSVG were used with a plasmid mass ratio of 15μg:9μg:6μg to transfect 293T cells. The virus was collected after 24 hours of culture. Lenti-X concentrate was used to concentrate the supernatant of 293T culture virus. Resuspended in DMEM and stored at -80℃.

6.2 Virus transfection

18-24 hours before lentiviral transfection, MC38/B16F10/CT26 cells were plated into T25 flasks at 5×10 ⁵ /well. The number of cells during lentiviral transfection was about 5×10 ⁵ /flask; 40ul of melted lentivirus was added dropwise into the T25 cell culture flask, and cultured for 24 hours, and the virus-containing culture medium was replaced with fresh culture medium. After 48-72 hours of culture, GFP fluorescence was observed.

6.3 Puro resistance screening

Different concentrations of Puro antibiotics were added to the MC38/B16F10/CT26 untransfected cell lines to explore the concentration suitable for Puro screening. Among them, B16F10 used 1μg/ml, CT26 used 10μg/ml, and MC38 used 4μg/ml. The corresponding concentration of Puro was added to the stable cell line pool, and the stable cell line positive pool was obtained after multiple rounds of screening.

6.4 Obtaining Monoclonal Stable Cell Lines

According to the common methods in the field, the CytKick Max (auto sampler) of Thermo Fisher Company was used to sort the monoclonal cells based on the GFP signal of the constructed stable cell line pool co-expressing MICA/B and GFP. Single cell clones with high and medium protein expression were selected.

6.5 Detection of monoclonal stable transfected cells

The monoclonal stable cells obtained by screening were detected using Attune NxT acoustic focusing flow cytometer (Thermo Fisher, 2AFC236901121). The antibody was MICA/B antibody 6D4 (PE anti-human MICA/MICB antibody, BioLegend, #320906), which was diluted at 1:40.

The test results are shown in Figure 10. We obtained monoclonal clones of MC38 (MICA/B), B16F10 (MICA/B) and CT26 (MICA/B) stable cell lines. These monoclonal cell lines will be used as target cells for subsequent serum antibody binding to cell surface MICA/B or NK cell killing efficiency evaluation or tumor-bearing cells in animal pharmacodynamic analysis.

Example 7 Binding of MICA/B antibodies in serum of D35 of XR-MIC-2 vaccine group to MICA/B on cell surface

In order to verify whether the antibody can bind to MICA/B on various cells, we took XR-MIC-2 as an example to characterize its cell binding ability.

Transgenic MC38 cell lines expressing full-length MICA/MICB (MC38(MICA/B) or B16F10(MICA/B)) were added to 96-well U-bottom plates, centrifuged at 400 g for 5 min, washed twice with PBS, incubated with PBS containing 2% FBS at 4°C for 10 min, incubated with 100 μL of serum samples serially diluted 10-fold in FACS buffer for 1 h at room temperature, washed twice with FACS buffer, stained with 100 μL Alexa Fluor 647-conjugated goat anti-mouse IgG secondary antibody (1 μg ml–1 FACS buffer; BioLegend) for 30 min at room temperature, washed twice with FACS buffer, and analyzed using a flow cytometer (BD Biosciences) and FlowJo software. Sera from control immunized mice, parental cells without MICA or MICB expression, and the corresponding secondary antibodies were used as negative controls for flow cytometry.

The results are shown in Figure 11. Both MC38 (MICA/B) and B16F10 (MICA/B) can bind to the antibodies in the serum of the XR-MIC-2 vaccine group. The positive signal could not be detected after the MC38 (MICA/B) cells were diluted to 10 ⁵ times, while the positive signal could not be detected after the B16F10 (MICA/B) cells were diluted to 10 ⁶ times, which may be related to the higher level of MICA/B expression in B16F10 (MICA/B) cells.

Example 8 Binding of MICA/B antibodies in XR-MIC-2/-3/-5/-6/-7/-8 vaccine serum to cell surface MICA/B

In order to verify the maximum effective antibody dilution titer that the antibodies produced by the vaccine described in Example 5 can show to cells, we took the transgenic MC38 cell line (MC38 (MICA/B) as an example to detect the effective antibody dilution titer of the XR-MIC-2/-3/-5/-6/-7/-8 vaccine.

Transgenic MC38 cells expressing full-length MICA/MICB (MC38(MICA/B)) were harvested into 96-well U-bottom plates at 2 × 10 ⁵ cells per well; centrifuged at 400 g for 5 min, washed twice with PBS; incubated with PBS containing 2% FBS at 4°C for 10 min; incubated with 100 μL of XR006 vaccine secondary immunization D35 serum samples serially diluted in FACS buffer for 1 h at room temperature; washed twice with FACS buffer; stained with 100 μL Alexa Fluor 647-conjugated goat anti-mouse IgG secondary antibody (1 μg/ml FACS buffer; BioLegend) at room temperature for 30 min; washed twice with FACS buffer; and analyzed using a flow cytometer (BD Biosciences) and FlowJo software. Sera from control immunized mice, parental cells without MICA or MICB expression, and the corresponding secondary antibodies were used as negative controls for flow cytometry.

The results are shown in Figure 12. The highest antibody titers detected in each vaccine group were between 10 ⁵ and 10 ^6. In addition, consistent with the ELISA antibody titer test results of the six XR-MIC vaccine groups in Figure 6, XR-MIC-3 showed a higher antibody titer after the second immunization, indicating better immunogenicity.

Example 9 Second round of mRNA construct design

Comparison of the immunogenicity of XR-MIC-2/3/5/6/7/8 construct vaccines in mice showed that the introduction of glycosylation site mutations helps to improve the immunogenicity of MICA/B mRNA vaccines. Therefore, glycosylation site mutations were introduced into the MICA and MICB protein sequences in subsequent construct designs.

In order to compare different signal peptides and whether the addition of transmembrane regions can help further improve the immunogenicity of MICA/B mRNA vaccines, a second round of mRNA constructs XR-MIC-9 to 15 were designed, and their schematic diagrams are shown in Figure 13. For ease of comparison, the above constructs all use the same 5'UTR (SEQ NO: 69), 3'UTR (SEQ NO: 71) and Poly (A) (SEQ NO: 72) sequences.

The main designs and component compositions of the XR-MIC-9 to 15 constructs are summarized as follows (the italicized bold parts show the differences between the structures of the constructs, where the deglycosylation site mutants of MICA α3 and MICB α3 are abbreviated as MICA α3m and MICB α3m, respectively):

XR-MIC-9: 5’UTR-human IgE signal peptide-MICA α3m-GS linker-MICB α3m-3’UTR-Poly(A) (its amino acid sequence is SEQ NO:17; its encoding nucleic acid sequence is SEQ NO:18);

XR-MIC-10: 5’UTR-human IgE signal peptide-MICA α3m-GS linker-MICB α3m-MICB TM+CTD-3’UTR-Poly(A) (its amino acid sequence is SEQ NO:19; its encoding nucleic acid sequence is SEQ NO:20);

XR-MIC-11: 5’UTR-MICA*008 signal peptide-MICA α3m-GS linker-MICB α3m-3’UTR-Poly (A) (its amino acid sequence is SEQ NO: 21; its encoding nucleic acid sequence is SEQ NO: 22);

XR-MIC-12: 5’UTR-MICA*008 signal peptide-MICA α3m-GS linker-MICB α3m-MICB TM+CTD-3’UTR-Poly(A) (its amino acid sequence is SEQ NO:23; its encoding nucleic acid sequence is SEQ NO:24);

XR-MIC-13: 5’UTR-HLA-B*46 signal peptide-MICA α3m-GS linker-MICB α3m-3’UTR-Poly (A) (its amino acid sequence is SEQ NO: 25; its encoding nucleic acid sequence is SEQ NO: 26);

XR-MIC-14: 5’UTR-HLA-B*46 signal peptide-MICA α3m-GS linker-MICB α3m-MICB TM+CTD-3’UTR-Poly(A) (its amino acid sequence is SEQ NO:27; its encoding nucleic acid sequence is SEQ NO:28);

XR-MIC-15: 5’UTR-Tranferrin Receptor TM-GS linker-MICA α3m-GS linker-MICB α3m-3’UTR-Poly(A) (its amino acid sequence is SEQ NO:29; its encoding nucleic acid sequence is SEQ NO:30);

The human IgE signal peptide sequence used in the above construct is shown in SEQ NO: 74, the HLA-B*46 signal peptide sequence is shown in SEQ NO: 75, and the MICA*008 signal peptide is shown in SEQ NO: 76; the MICB TM+CTD sequence used is shown in SEQ NO: The sequence is shown in SEQ NO:101, and the sequence of Tranferrin receptor TM is shown in SEQ NO:100.

Example 10 Preparation and detection of mRNA of XR-MIC-9 to 15 constructs

According to the SEQ ID.NO: 18, 20, 22, 24, 26, 28 and 30 sequences of XR-MIC-9 to 15 designed in Example 9, the corresponding nucleotides (Nanjing GenScript) were synthesized, and the corresponding 5'UTR sequence and SpeI restriction endonuclease site sequence were added to the 5' end, and the stop codon and XhoI restriction endonuclease site sequence were added to the 3' end. The DNA fragments after enzyme cutting were cloned into the pUC57 vector backbone containing the T7 promoter sequence, 3'UTR sequence and poly (A) sequence by enzyme cutting and ligation. The sequence between XbaI/NotI in the plasmid was sequenced to confirm that the sequence was correct, and then the plasmid was extracted.

The linearized plasmid was prepared according to the method in Example 2.2, and the mRNA molecules were prepared and purified by in vitro transcription according to the method in Example 2.3. The quality and concentration of mRNA were tested according to the methods in Examples 2.4 and 2.5. The test results showed that the mRNA integrity of XR-MIC-9 to 15 was more than 90%, and the concentration was between 1.4 mg/ml and 2.5 mg/ml, which could meet the subsequent LNP preparation requirements.

Example 11 Expression of XR-MIC-9 to 14 in BHK21 cells

In order to verify whether the above construct can express the target protein, the XR-MIC-9 to 14 mRNA stock solution was first transfected into BHK21 cells, and the specific method is shown in Example 4.1. The transfected cells were stained according to the method of Example 4.2 and detected using a flow cytometer. The results are shown in Figure 14. The BHK21 cell population transfected with XR-MIC-9 to 14 mRNA showed obvious translation relative to the negative control, indicating that the above mRNA molecules can normally express the target protein in the cells.

Example 12 Comparison of immunogenicity of XR-MIC-1 to 14 constructs in mice

After verifying that the mRNA stock solutions of the XR-MIC-9 to 14 constructs can normally express the target protein, LNP preparations were prepared and tested according to the method of Example 3. The results showed that the particle size, PDI, Zeta potential and total RNA concentration of the LNP preparations of XR-MIC-9 to 14 met the requirements.

Subsequently, immunogenicity comparisons were performed in mice for all constructs of XR-MIC-1 to 14. SPF female C57BL/6 mice aged 6-8 weeks were randomly divided into 15 groups, 5 mice in each group, and the grouping and dosing details are shown in Table 5.

Table 5 Comparison of immunogenicity of XR-MIC-1 to 14 mRNA vaccines in mice

Each group was vaccinated with 1 μg (50 μL) of the corresponding XR-MIC vaccine LNP preparation, and immunized twice, with an interval of 3 weeks between the two immunizations, D0 and D21, respectively. Blood was collected on D14 and D35, and the MICB-specific antibody level was detected by ELISA. For specific methods, see Example 5.1.

The results 14 days after a single dose are shown in Figure 15. XR-MIC-1 to 14 vaccine groups were able to produce high titers of MICB IgG binding antibodies. By comparing XR-MIC-1 with XR-MIC-2, XR-MIC-3 with XR-MIC-4, and XR-MIC-5 with XR-MIC-6, it can be found that the antibody titers of constructs with MICA/B glycosylation modifications are higher than those of unmodified constructs. Among the newly constructed XR-MIC-9 to 14 constructs, XR-MIC-12 induced the highest antibody level, which was higher than that of XR-MIC-1 to 8 constructs.

On the 14th day (D35) after the booster immunization on D21, the antibody levels of all vaccine groups increased significantly, with the antibody titers of XR-MIC-2/4/9/10/12 exceeding 2×10 ⁶ and the antibody titer of XR-MIC-3 exceeding 3.5×10 ⁶ ( FIG. 16 ). The above results indicate that the XR-MIC vaccines of different molecules in the present invention have good immunogenicity in C57BL/6 mice.

Example 13 Preliminary efficacy verification of the second round of XR-MIC mRNA vaccine in MC38-TgMICA/B subcutaneous tumor-bearing mice

In order to verify the therapeutic efficacy of the second round of XR-MIC mRNA vaccine, we selected the LNP preparation of the XR-MIC-12 construct with higher immunogenicity and verified the tumor inhibitory activity of different doses and different administration routes in MC38-TgMICA/B subcutaneous tumor-bearing C57BL/6 mice. At the same time, the recombinant MICB protein vaccine (with CpG and GM-CSF as adjuvants) was selected as a control. The specific experimental group design is shown in Table 6.

Table 6 Preliminary efficacy verification of the second round of XR-MIC mRNA vaccine in MC38-TgMICA/B subcutaneous tumor-bearing mice

First, MC38-TgMICA/B tumor cells were inoculated subcutaneously on the back of C57BL/6 mice (6-8 weeks old) with an inoculation volume of 6×10 ⁵ live cells. Four days after tumor loading, the mice were grouped according to tumor size and given different vaccines or saline. The day of administration was counted as D0, and the drug was administered once a week thereafter for a total of 3 times. The results are shown in Figure 17. The tumor in the control group given saline showed continuous growth, while the administration of 1 μg of XR-MIC-12 mRNA vaccine and recombinant protein MICB vaccine could inhibit tumor growth, with the highest tumor inhibition rates of 34.18% and 36.09%, respectively, and the overall trends of the two were consistent. When the dose of XR-MIC-12 mRNA vaccine was increased to 10 μg, its tumor inhibition efficiency was further improved, reaching 58% at the highest (2 days after two doses, D9), indicating that the efficacy of XR-MIC-12 mRNA vaccine has a dose-dependent effect. When the XR-MIC-12 mRNA vaccine was administered intradermally, its therapeutic effect was significantly improved compared with the same dose administered intramuscularly. The tumor inhibition rate reached 90% at the highest point (D9) and still reached 53.37% at the end point of the trial (D23), which was significantly better than the recombinant protein vaccine and saline control groups.

The above results preliminarily verified the therapeutic efficacy of XR-MIC mRNA vaccine against MC38-TgMICA/B tumors, showing a significant dose-dependent effect, and the most significant efficacy was achieved through intradermal administration.

Example 14 Third round XR-MIC mRNA construct design

By comparing the immunogenicity of XR-MIC-1 to 14 construct vaccines in mice, it can be found that the modification of the target protein structure, including signal peptides, transmembrane regions, glycosylation sites, etc., can affect the immunogenicity of the vaccine, and the strength of the immunogenicity is crucial to the efficacy of the vaccine. In order to further improve the immunogenicity of the vaccine, we conducted a new round of construct design based on the XR-MIC-12 construct, constructed the third round of XR-MIC mRNA constructs XR-MIC-12-1 to 17, as well as XR-MIC-12-13-2 and XR-MIC-12-14-2, and compared the effects of different linker sequences, transmembrane regions, vector sequences, the order and number of repetitions of MICA and MICB, and the addition of chemokine sequences on immunogenicity. The schematic diagram of the elements of the target protein coding sequence is shown in Figure 18. The above constructs all use the same 5’UTR (SEQ NO:69), 3’UTR (SEQ NO:71) and Poly (A) (SEQ NO:72) sequences, and the encoded MICA and MICB proteins both contain glycosylation site mutations.

The main designs and component compositions of the XR-MIC-12-1 to 17 constructs are summarized as follows (the italicized bold parts show the differences between the structures of the constructs, among which the deglycosylation site mutants of MICA α3 and MICB α3 are abbreviated as MICA α3m and MICB α3m, respectively)

XR-MIC-12-1: 5’UTR-MICA*008 signal peptide-MICA α3m-(GGGGS)2 linker-MICB α3m-MICB TM+CTD-3’UTR-Poly(A) (its amino acid sequence is SEQ NO:31; its encoding nucleic acid sequence is SEQ NO:32);

XR-MIC-12-2: 5'UTR-MICA*008 signal peptide-MICA α3m-(EAAAK)2 linker-MICB α3m-MICB TM+CTD-3'UTR-Poly(A) (its amino acid sequence is SEQ NO: 33; its encoding nucleic acid sequence is SEQ NO: 34);

XR-MIC-12-3: 5’UTR-MICA*008 signal peptide-MICA α3m-(GGGGS)2 linker-MICB α3m-Foldon-3’UTR-Poly(A) (its amino acid sequence is SEQ NO:35; its encoding nucleic acid sequence is SEQ NO:36);

XR-MIC-12-4: 5’UTR-MICA*008 signal peptide-MICA α3m-(EAAAK)2 linker-MICB α3m-Foldon-3’UTR-Poly(A) (its amino acid sequence is SEQ NO:37; its encoding nucleic acid sequence is SEQ NO:38);

XR-MIC-12-5: 5’UTR-MICA*008 signal peptide-MICB α3m-(GGGGS)2 linker-MICA α3m-MICB TM+CTD-3’UTR-Poly(A) (its amino acid sequence is SEQ NO:39; its encoding nucleic acid sequence is SEQ NO:40);

XR-MIC-12-6: 5’UTR-MICA*008 signal peptide-MICB α3m-(GGGGS)2 linker-MICA α3m-MICA TM+CTD-3’UTR-Poly(A) (its amino acid sequence is SEQ NO:41; its encoding nucleic acid sequence is SEQ NO:42);

XR-MIC-12-7: 5’UTR-MICA*008 signal peptide-MICA α3m-(GGGGS)2 linker-MICB α3m-(GGGGS)2 linker-MICA α3m-(GGGGS)2 linker-MICB α3m-MICB TM+CTD-3’UTR-Poly(A) (its amino acid sequence is SEQ NO:43; its encoding nucleic acid sequence is SEQ NO:44);

XR-MIC-12-8: 5’UTR-MICA*008 signal peptide-MICA α3m-(EAAAK)2 linker-MICB α3m-(GGGGS)2 linker-MICA α3m-(EAAAK)2 linker-MICB α3m-MICB TM+CTD-3’UTR-Poly(A) (its amino acid sequence is SEQ NO:45; its encoding nucleic acid sequence is SEQ NO:46);

XR-MIC-12-9: 5’UTR-MICA*008 signal peptide-MICA α3m-(GGGGS)2 linker-MICB α3m-(GGGGS)2 linker-MICA α3m-(GGGGS)2 linker-MICB α3m-(GGGGS)2 linker-MICA α3m-(GGGGS)2 linker-MICB α3m-(GGGGS)2 linker-MICA α3m-(GGGGS)2 linker-MICB α3m-MICB TM+CTD-3’UTR-Poly(A) (its amino acid sequence is SEQ NO:47; its encoding nucleic acid sequence is SEQ NO:48);

XR-MIC-12-10: 5’UTR-MICA*008 signal peptide-MICA α3m-(EAAAK)2 linker-MICB α3m-(GGGGS)2 linker-MICA α3m-(EAAAK)2 linker-MICB α3m-(GGGGS)2 linker-MICA α3m-(EAAAK)2 linker-MICB α3m-(GGGGS)2 linker-MICA α3m-(EAAAK)2 linker-MICB α3m-MICB TM+CTD-3’UTR-Poly(A) (its amino acid sequence is SEQ NO:49; its encoding nucleic acid sequence is SEQ NO:50);

XR-MIC-12-11: 5’UTR-hXCL1-(GGGGS)2 linker-MICA α3m-(GGGGS)2 linker-MICB α3m-3’UTR-Poly(A) (its amino acid sequence is SEQ NO:51; its encoding nucleic acid sequence is SEQ NO:52);

XR-MIC-12-12: 5’UTR-hCCL19-(GGGGS)2 linker-MICA α3m-(GGGGS)2 linker-MICB α3m-3’UTR-Poly(A) (its amino acid sequence is SEQ NO:53; its encoding nucleic acid sequence is SEQ NO:54);

XR-MIC-12-13: 5’UTR-MICA*008 signal peptide-MICA α3m-(GGGGS)2 linker-MICB α3m-MITD-3’UTR-Poly(A) (its amino acid sequence is SEQ NO:55; its encoding nucleic acid sequence is SEQ NO:56);

XR-MIC-12-14: 5’UTR-MICA*008 signal peptide-MICA α3m-(GGGGS)2 linker-MICB α3m-HA TM+CTD-3’UTR-Poly(A) (its amino acid sequence is SEQ NO:57; its encoding nucleic acid sequence is SEQ NO:58);

XR-MIC-12-15: 5’UTR-gDN-terminal sequence-MICA α3m-(GGGGS)2 linker-MICB α3m-gD TMR-3’UTR-Poly(A) (its amino acid sequence is SEQ NO:59; its encoding nucleic acid sequence is SEQ NO:60);

XR-MIC-12-16: 5’UTR-MICA*008 signal peptide-MICB α3m-(GGGGS)2 linker-MICB α3m-MITD-3’UTR-Poly(A) (its amino acid sequence is SEQ NO:61; its encoding nucleic acid sequence is SEQ NO:62);

XR-MIC-12-17: 5'UTR-MICA*008 signal peptide-MICA α3m-(GGGGS)2 linker-MICA α3m-(GGGGS)2 linker-MICB α3m-(GGGGS)2 linker-MICB α3m-(GGGGS)2 linker-MICB α3m-MICB TM+CTD-3'UTR-Poly(A) (its amino acid sequence is SEQ NO: 63; its encoding nucleic acid sequence is SEQ NO: 64);

XR-MIC-12-13-2: 5’UTR-MICA*008 signal peptide-MICA α3m-(EAAAK)2 linker-MICB α3m-MITD-3’UTR-Poly(A) (its amino acid sequence is SEQ NO: 65; its encoding nucleic acid sequence is SEQ NO: 66);

XR-MIC-12-14-2: 5’UTR-MICA*008 signal peptide-MICA α3m-(EAAAK)2 linker-MICB α3m-HA TM+CTD-3’UTR-Poly(A) (its amino acid sequence is SEQ NO:67; its encoding nucleic acid sequence is SEQ NO:68);

The MICB TM+CTD sequence used in the above construct is shown in SEQ NO:101, the MICATM+CTD sequence is shown in SEQ NO:102, the T4 foldon sequence is shown in SEQ NO:103, the MITD sequence is shown in SEQ NO:104, and the HATM+CTD sequence is shown in SEQ NO:105.

Example 15 Preparation and detection of mRNA of XR-MIC-12-1 to 17 constructs

The corresponding nucleotides were synthesized according to the sequences of XR-MIC-12-1 to 17 designed in Example 14 (Nanjing GenScript), and the synthesized DNA fragments were cloned into the pUC57 vector backbone containing the T7 promoter sequence, 5'UTR sequence, 3'UTR sequence and poly(A) sequence. The sequence between XbaI/NotI in the plasmid was sequenced to confirm that the sequence was correct, and then the plasmid was extracted.

The linearized plasmid was prepared according to the method in Example 2.2, and the mRNA molecules were prepared and purified by in vitro transcription according to the method in Example 2.3. The quality and concentration of mRNA were tested according to the methods in Examples 2.4 and 2.5. The test results showed that the mRNA integrity of XR-MIC-12-1/2/4/6/7/8/10/11/12/13/14/15/16/17 was more than 90%, and the concentration was between 0.78 mg/ml-2.5 mg/ml, which could meet the subsequent LNP preparation requirements.

Example 16 Expression of XR-MIC-12-2/4/6/7/8/10/11/12/13/14/15/16/17 mRNA stock solution in BHK21 cells

In order to verify whether the third round construct can express the target protein, the XR-MIC-12-2/4/6/7/8/10/11/12/13/14/15/16/17 mRNA stock solution was first transfected into BHK21 cells. The specific method is shown in Example 4.1. The transfected cells were stained according to the method of Example 4.2 and detected by flow cytometry. The results are shown in Figure 19. All molecules can normally express the target protein in cells, and the positive cell rate is between 40% and 50%.

Example 17 Expression of XR-MIC-12-2/4/6/8/10/11/12/13/14/15/16/17 LNP formulations in BHK21 cells

According to the protein expression after mRNA stock solution transfection of cells and the design of different antigen structures, XR-MIC-12-2/4/6/8/10/11/12/13/14/15/16/17 were selected to prepare LNP preparations according to the method of Example 3 and tested. The results showed that the particle size, PDI, Zeta potential and total RNA concentration of all prepared LNP preparations met the requirements.

The prepared LNP preparation was transfected into BHK21 cells again according to the method in Example 4.1, and the expression of the target protein was detected according to the method in Example 4.2. The results are shown in Figure 20. In the above LNP preparations, the expression levels of XR-MIC-12-15 and XR-MIC-16 were low, so they were discarded in the subsequent mouse test. The remaining constructs can express the target protein normally.

Example 18 XR-MIC-12/12-2/12-4/12-6/12-8/12-10/12-11/12-12/12-13/12-14/12-17/15 Comparison of immunogenicity of constructs in mice

The immunogenicity of XR-MIC-12/12-2/12-4/12-6/12-8/12-10/12-11/12-12/12-13/12-14/12-17/15 constructs was compared again in mice, including humoral and cellular immunity level detection, to further screen the preferred molecules. SPF female C57BL/6 mice aged 6-8 weeks were randomly divided into 13 groups, 5 mice in each group, and each group was vaccinated with 5 μg (100 μL) of the corresponding XR-MIC vaccine LNP preparation for a total of 2 immunizations, with an interval of 3 weeks between the two immunizations, D0 and D21, respectively. The grouping and dosing details are shown in Table 7.

Table 7 Comparison of immunogenicity of XR-MIC mRNA vaccines in mice

18.1 MICA and MICB ELISA Antibody Detection

Blood was collected on D20 and D43, and the MICA and MICB specific antibody levels were detected by ELISA. For specific methods, see Example 5.1.

The MICA ELISA antibody levels detected at D20 are shown in Figure 21. All constructs induced high levels of specific antibodies against MICA. Compared with XR-MIC-12, the new constructs XR-MIC-12-2, 12-8, 12-10, and 12-11 induced higher levels of MICA antibodies. The MICB ELISA antibody levels detected at D20 are shown in Figure 22. Similarly, all constructs induced high levels of specific antibodies against MICB. Compared with XR-MIC-12, the new constructs XR-MIC-12-2, 12-8, 12-10, 12-11, 12-13, 12-14, and 12-17 induced higher levels of antibodies.

The test results of D43 are shown in Figures 23 and 24, respectively. After booster immunization, the antibody titers of all construct vaccines increased significantly. Compared with XR-MIC-12, there was no significant difference in the MICA antibody level among the constructs, while the antibody levels of XR-MIC-12-6, 12-10 and 12-12 were higher in MICB antibody.

18.2 ELISpot detection of IFN-γ levels secreted by mouse spleen cells

On D43, all mice were euthanized and spleens were collected. Splenocytes were isolated after grinding according to the method of Example 5.2. After stimulation with the peptide libraries of MICA and MICB, ELISpot was used to detect the number of cells secreting IFN-γ in order to compare the differences in cellular immunogenicity of different constructs.

The test results are shown in Figures 25 and 26. All constructs can induce high levels of cellular immune responses against MICA and MICB. When stimulated with the MICA peptide library, the number of cells secreting IFN-γ of all constructs except XR-MIC-12-4, 12-6 and 12-8 was higher than that of XR-MIC-12, among which the construct XR-MIC-12-13 was the highest. When stimulated with the MICB peptide library, except for XR-MIC-4, the number of cells secreting IFN-γ of all other constructs was higher than that of XR-MIC-12, among which XR-MIC-12-6 was the highest, followed by XR-MIC-12-14, 12-13, 12-17 and 12-2.

The above results indicate that in the construct design of MICA and MICB, the humoral immunity and/or cellular immunogenicity of MIC mRNA vaccines can be significantly improved by changing the linker sequence, adjusting the connection order of the two proteins, and selecting appropriate transmembrane and intracellular region sequences.

18.3 Preliminary efficacy verification of the third round of XR-MIC mRNA vaccine in MC38-TgMICA/B subcutaneous tumor-bearing mice

In this study, the immunogenicity of the XR-MIC mRNA vaccine was continuously improved through three rounds of construct design, in vitro screening and in vivo immunogenicity comparison. Given that immunogenicity and therapeutic efficacy are highly correlated, construct vaccines with better immunogenicity are expected to also have better efficacy. Therefore, in order to verify the therapeutic efficacy of the third round of XR-MIC mRNA vaccines, we intend to verify the tumor inhibitory activity of the LNP formulation of the third round of XR-MIC mRNA constructs in MC38-TgMICA/B subcutaneous tumor-bearing C57BL/6 mice at different doses and different administration routes. For specific verification methods, see Example 13. As mentioned above, the present invention has proved that XR-MIC mRNA has good tumor inhibition efficiency by verifying the tumor inhibition ability of the XR-MIC-12 construct. It is expected that the third round of further improved mRNA constructs will have better tumor inhibition efficiency and more potential therapeutic effects.

The teachings of all patents, published applications, and references cited herein are incorporated by reference in their entirety.

While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments as covered by the appended claims.

Claims (32)

A vaccine, characterized in that the vaccine is a MICA and/or MICB mRNA vaccine, which comprises a MICA/B mRNA construct as an immunogenic component, wherein the mRNA construct comprises mRNA encoding a MICA protein domain and/or a MICB protein domain. The vaccine according to claim 1 is characterized in that the vaccine is a tumor vaccine. The vaccine according to claim 1 or 2, characterized in that the mRNA encoding the MICA protein domain and the MICB protein domain are expressed alone or in fusion in the host cell. The vaccine according to any one of claims 1 to 3, characterized in that the MICA protein domain and/or the MICB protein domain are respectively the MICA α3 domain and/or the MICB α3 domain; preferably, the MICA protein is MICA*001, MICA*002, MICA*008 or MICA*009, and/or the MICB protein is MICB*004 or MICB*005. The vaccine according to any one of claims 1-4 is characterized in that the protein encoded by the mRNA also contains a mutation in a glycosylation site. The vaccine according to any one of claims 1 to 5, characterized in that the protein encoded by the mRNA further comprises a signal peptide sequence, and optionally, the signal peptide is located at the N-terminus of the protein; preferably, the signal peptide is derived from mouse H-2K ^b signal peptide, human IgE signal peptide, HLA-B*46 signal peptide, MICA*008 self-signal peptide, OSM, VSV-G, mouse Ig Kappa, mouse heavy chain, BM40, human chymosinogen, human chymosinogen-2, human IL-2, human G-CSF, human hemagglutinin IX, human albumin, Gaussia luc, HAS, influenza virus, human insulin, silk LC, Erenumab antibody light chain, Pembrolizumab light chain, Ramucirumab light chain, E signal peptide, SP1 (LZJ human IgG1), SP2, SP3 (ZLQ). The vaccine according to claim 6 is characterized in that the amino acid sequences of the signal peptides are shown in SEQ ID NO: 73-99 respectively. The vaccine according to any one of claims 1-5 is characterized in that the protein encoded by the mRNA also contains a molecular adjuvant, and optionally, the molecular adjuvant is located at the N-terminus or C-terminus of the protein; preferably, the molecular adjuvant includes hXCL1, hCCL19, gD N-terminal sequence, FLT3L, GM-CSF, CD40L, caTLR4, CD70, PADRE, etc. The vaccine according to claim 8 is characterized in that the amino acid sequences of the molecular adjuvants are shown in SEQ ID NO: 107-115 respectively. The vaccine according to any one of claims 1 to 9, characterized in that the mRNA construct further comprises a portion encoding a carrier protein, and the mRNA construct encodes a MICA protein domain that binds to a carrier protein to form a fusion protein and/or a MICB protein domain that binds to a carrier protein to form a fusion protein; Optionally, the carrier protein includes ferritin, diphtheria toxoid (DT, DT CRM197), tetanus toxoid (TT), keyhole limpet hemocyanin (KLH), pneumolysin (Ply), influenza hemophilin D, pneumococcal PhtA, Pht B, Pht D, Pht DE and artificial protein N19, T4 Foldon from T4 bacteriophage, ESCRT and ALIX-binding region (EABR), BSA and/or OVA, etc.; Preferably, the carrier protein is T4 Foldon; Preferably, the carrier protein is ferritin; Preferably, the ferritin protein comprises a domain that allows the fusion protein to self-assemble into nanoparticles; Preferably, the ferritin is ferritin from Helicobacter pylori. The vaccine according to claim 10, characterized in that a deglycosylation mutation is introduced into the ferritin, and optionally, the deglycosylation mutation is a single amino acid deglycosylation mutation or a multiple amino acid deglycosylation mutation. The vaccine according to any one of claims 1-11, characterized in that the protein encoded by the mRNA contains SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO: an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65 or SEQ ID NO:67; Preferably, the protein encoded by the mRNA comprises SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65 or SEQ ID NO:67; More preferably, the protein encoded by the mRNA comprises SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65 or SEQ ID NO:67. The vaccine according to any one of claims 1 to 11, characterized in that the protein encoded by the mRNA is relative to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 At least one of the amino acid sequences shown in SEQ ID NO:65 or SEQ ID NO:67 Independently contain 1-12 amino acid substitutions; Optionally, the 1-12 amino acid substitutions are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 amino acid substitutions; Preferably, the amino acid substitution is a conservative amino acid substitution; more preferably, the substitution is a highly conservative amino acid substitution. The vaccine according to claim 12 or 13, characterized in that the protein encoded by the mRNA comprises SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 The amino acid sequence shown in Q ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65 or SEQ ID NO:67. The vaccine according to any one of claims 1-14, characterized in that the mRNA is transcribed from DNA, and the DNA contains SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 The nucleotide sequence shown in SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66 or SEQ ID NO:68. The vaccine according to any one of claims 1 to 15, characterized in that the mRNA construct further comprises a 5'UTR sequence, a 3'UTR sequence and a Poly (A) sequence; optionally, the 5'UTR sequence is a human alpha globulin 5'UTR, or a non-natural 5'UTR sequence. The vaccine according to claim 16, characterized in that the human alpha globulin 5'UTR is obtained by transcription from DNA, and the DNA comprises the nucleotide sequence shown in SEQ NO:69; or the non-natural 5'UTR is obtained by transcription from DNA, and the DNA sequence comprises the nucleotide sequence shown in SEQ NO:70; The 3’UTR is obtained by transcription from DNA, and the DNA sequence comprises the nucleotide sequence shown in SEQ NO:71; The Poly(A) sequence contains the nucleotide sequence shown in SEQ NO:72. The vaccine according to any one of claims 1-17 is characterized in that the protein encoded by the mRNA further comprises a transmembrane domain (TM) and/or an intracellular domain (CTD). The vaccine according to any one of claims 1 to 18, characterized in that the vaccine further comprises a delivery preparation; preferably, the delivery preparation is a nanoparticle; preferably, the delivery preparation comprises lipid nanoparticles (Lipid nanoparticle, LNP), lipid multipolymers (lipopolyplex, LPP), polymer nanoparticles (Polymer nanoparticles, PNP), inorganic nanoparticles (Inorganic nanoparticles, INP), cationic nanoemulsion (Cationic nanoemulsion, CNE), exosomes, biological microvesicles, protamine, etc.; more preferably, the nanoparticles are lipid nanoparticles (Lipid nanoparticle, LNP). An mRNA construct, characterized in that it is the mRNA construct according to any one of claims 1-18, or a combination thereof, wherein the mRNA construct is used as an immunogenic component in the vaccine according to any one of claims 1-19. A vector, characterized in that it comprises the mRNA construct according to any one of claims 1-18, 20, or a combination thereof. A cell, characterized in that it comprises the mRNA construct according to any one of claims 1-18, 20, or a combination thereof, or the vector according to claim 19. A nanoparticle, characterized in that it comprises the mRNA construct according to any one of claims 1 to 18 and 20, or a combination thereof; Optionally, the nanoparticles include lipid nanoparticles (LNP), lipid multipolymers (LPP), polymer nanoparticles (PNP), inorganic nanoparticles (INP), cationic nanoemulsion (CNE), exosomes, biological microvesicles, protamine, etc. Preferably, the nanoparticles are lipid nanoparticles (LNP). A vaccine, characterized in that it comprises the nanoparticles according to claim 23, and the vaccine is a MICA and/or MICB mRNA vaccine. A fusion protein, characterized in that it comprises a protein encoded by the mRNA according to any one of claims 1 to 18 and a second protein fused thereto, wherein the protein encoded by the mRNA is an unfused protein; Optionally, the second protein is a carrier protein; Optionally, the carrier protein includes ferritin, diphtheria toxoid (DT, DT CRM197) and tetanus toxoid (TT), keyhole limpet hemocyanin (KLH), pneumolysin (Ply), influenza hemophilin D, pneumococcal Pht A, Pht B, Pht D, Pht DE and artificial protein N19, T4 Foldon from T4 bacteriophage, BSA and/or OVA, etc.; Preferably, the second protein is T4 Foldon or ferritin; Preferably, the ferritin protein comprises a domain that allows the fusion protein to self-assemble into nanoparticles; Preferably, the ferritin is ferritin of Helicobacter pylori; Preferably, the fusion protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID: 15, SEQ ID: 35 or SEQ ID: 37; or The fusion protein independently comprises 1-12 amino acid substitutions relative to at least one of the amino acid sequences shown in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID: 15, SEQ ID: 35 or SEQ ID: 37; Preferably, the fusion protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID: 15, SEQ ID: 35 or SEQ ID: 37; Preferably, the fusion protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID: 15, SEQ ID: 35 or SEQ ID: 37; More preferably, the fusion protein comprises an amino acid sequence as shown in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:13, SEQ ID:15, SEQ ID:35 or SEQ ID:37. Use of the vaccine according to any one of claims 1-19 and 24, the mRNA construct according to claim 18, the vector according to claim 19, the cell according to claim 20, or the nanoparticle according to claim 21 in the preparation of a medicament for inducing an immune response in a subject in need thereof. Use of the vaccine according to any one of claims 1-19 and 24, the mRNA construct according to claim 18, the vector according to claim 19, the cell according to claim 20, or the nanoparticle according to claim 21 in the preparation of a medicament for treating or preventing cancer in a subject in need thereof; optionally, the cancer is primary cancer or metastatic cancer. A method for inducing an immune response in a subject in need thereof, characterized in that the vaccine according to any one of claims 1-19 and 24 is administered to the subject; preferably, the method induces an immune response against MICA/B by using an mRNA vaccine. A method for treating or preventing cancer in a subject in need thereof, characterized in that the vaccine according to any one of claims 1-19 and 24 is administered to the subject; optionally, the cancer is a primary cancer or a metastatic cancer. The method according to claim 28 or 29 is characterized in that the method induces an immune response against MICA/B by using an mRNA vaccine, preferably, the mRNA is a replicating, non-replicating, trans-self-replicating or circular mRNA. The method according to claim 29 or 30 is characterized in that the vaccine composition is administered as part of a treatment regimen; preferably, the treatment regimen is radiation therapy, targeted therapy, immunotherapy or chemotherapy. The method according to any one of claims 29-31, characterized in that the individual is one whose serum is tested positive for MICA/B shedding.