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WO2024222886A1 - Vaccins antitumoraux à arnm pour cible mica/b - Google Patents

Vaccins antitumoraux à arnm pour cible mica/b Download PDF

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Publication number
WO2024222886A1
WO2024222886A1 PCT/CN2024/090104 CN2024090104W WO2024222886A1 WO 2024222886 A1 WO2024222886 A1 WO 2024222886A1 CN 2024090104 W CN2024090104 W CN 2024090104W WO 2024222886 A1 WO2024222886 A1 WO 2024222886A1
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seq
mica
mrna
protein
amino acid
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PCT/CN2024/090104
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English (en)
Chinese (zh)
Inventor
马光刚
胡绪鹏
刘小芳
乔亚茹
刘佳雯
田家伦
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北京先声祥瑞生物制品股份有限公司
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Publication of WO2024222886A1 publication Critical patent/WO2024222886A1/fr

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  • the present invention relates to the field of tumor vaccines, and in particular to mRNA tumor vaccines.
  • mice and humans various forms of cellular stress (such as DNA damage) upregulate the ligands of the inducible sex receptor NKG2D.
  • the NKG2D ligands of mice include Rae1, MULT1 and H60
  • 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.
  • NKG2D receptors in NK cells and CD8+ T cells or ⁇ T 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
  • 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 ⁇ 1 and ⁇ 2 domains of MICA/B are responsible for binding to NKG2D and activating NK cell- and T cell-mediated tumor immunity.
  • 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 ⁇ 3 domain near the membrane.
  • Metalloproteinase-mediated cleavage allows part of the ⁇ 3 domain to remain on tumor cells, while the ⁇ 1/2 domains are shed.
  • soluble MICA/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.
  • the Wucherpfennig laboratory used antibodies that bind to the MICA/B ⁇ 3 domain to block MICA/B from falling off the tumor cell membrane.
  • the Fc fragment on the MICA/B ⁇ 3 domain-binding antibody further enhances the immunotherapy effect by binding to the Fc receptor on immune cells.
  • 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 ⁇ 3 domain fragments and identified the ⁇ 3 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 ⁇ , 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.
  • ⁇ 3 domain-specific antibodies increase the stability of MICA/B on tumor cell membranes. They developed a protein vaccine composed of ⁇ 3 domain proteins, fused MICA or MICB ⁇ 3 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.
  • DCs dendritic cells
  • MICB ⁇ 3 domain-ferritin fusion vaccine 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.
  • 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.
  • MICB-vax can play a role in metastatic melanoma models and triple-negative breast cancer disease recurrence models.
  • 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.
  • 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- ⁇ 3 domains, and can also induce antibodies against the MICA and/or MICB- ⁇ 3 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.
  • the present invention includes the following technical solutions:
  • 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.
  • the vaccine is a tumor vaccine.
  • the mRNA encoding the MICA protein domain and the MICB protein domain is expressed separately or in fusion in the host cell.
  • the MICA protein domain and/or the MICB protein domain are MICA ⁇ 3 domain and/or MICB ⁇ 3 domain, respectively;
  • 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 protein encoded by the mRNA further comprises a mutation in a glycosylation site.
  • the protein encoded by the mRNA further comprises a signal peptide
  • the signal peptide is located at the N-terminus of the protein
  • 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).
  • amino acid sequence of the signal peptide is as shown in SEQ ID NO:73-99 respectively.
  • the protein encoded by the mRNA further comprises a molecular adjuvant
  • the molecular adjuvant is located at the N-terminus or C-terminus of the protein
  • the molecular adjuvant includes hXCL1, hCCL19, gD N-terminal sequence, FLT3L, GM-CSF, CD40L, caTLR4, CD70, PADRE, etc.;
  • amino acid sequences of the molecular adjuvants are shown as SEQ ID NO: 107-115 respectively.
  • the MICA/B ⁇ 3 domain protein is combined with a carrier protein to form a fusion protein
  • 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.
  • ferritin diphtheria toxoid
  • TT tetanus toxoid
  • KLH keyhole limpet hemocyanin
  • Ply pneumococcal hemolysin
  • influenza hemophilin D pneumococcal PhtA, Pht B, Pht D, Pht DE and artificial protein N19 (Baraldoi et al.
  • the carrier protein is T4 Foldon.
  • the MICA/B ⁇ 3 domain protein forms a fusion protein with T4 Foldon.
  • the carrier protein is ferritin.
  • the MICA/B ⁇ 3 domain protein forms a fusion protein with ferritin.
  • the MICA/B ⁇ 3-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 ⁇ 3 domain.
  • the ferritin subunit of the present invention is the full length or any part of ferritin, wild type or partial amino acid mutation.
  • the monomer subunit is derived from bacterial ferritin, plant ferritin, algae ferritin, insect ferritin, fungal ferritin and mammalian ferritin.
  • the ferritin is ferritin from Heliobacter pylori.
  • a deglycosylation mutation is introduced into the ferritin
  • the deglycosylation mutation is a single amino acid deglycosylation mutation or a multiple amino acid deglycosylation mutation.
  • the protein encoded by the mRNA further comprises a transmembrane domain (TM) and/or an intracellular domain (CTD).
  • TM transmembrane domain
  • CTD intracellular domain
  • 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.
  • the transmembrane domain includes MICATM, MICB TM, MITD, HATM, gD-TMR, TfRTM, CD8 ⁇ TM, CD28 TM, SARS-COV-2 Spike TM, HSV gB TM, HSV gC TM, HSV gE TM, etc.
  • 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.
  • 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%
  • 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
  • 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
  • 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:40, SEQ ID
  • the 1-12 amino acid substitutions are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 amino acid substitutions;
  • the amino acid substitution is a conservative amino acid substitution
  • the substitution is a highly conservative amino acid substitution.
  • 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:40
  • 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:
  • the mRNA construct further comprises a 5'UTR sequence
  • the 5'UTR sequence is human alpha globulin 5'UTR, or a non-natural 5'UTR sequence
  • the human alpha globulin 5'UTR is transcribed from DNA, and the DNA comprises the nucleotide sequence shown in SEQ NO:69;
  • 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.
  • the mRNA construct further comprises a 3'UTR sequence
  • the 3’UTR is obtained by transcription from DNA, and the DNA sequence contains the nucleotide sequence shown in SEQ NO:71.
  • the mRNA construct further comprises a Poly (A) sequence
  • the Poly (A) sequence comprises a nucleotide sequence as shown in SEQ NO:72.
  • the vaccine further comprises a delivery formulation
  • the delivery formulation is a nanoparticle
  • the delivery preparation includes lipid nanoparticles (LNP), lipid multipolymers (LPP), polymer nanoparticles (PNP), inorganic nanoparticles (INP), cationic nanoemulsion (CNE), exosomes, biological microvesicles, protamine, etc.
  • LNP lipid nanoparticles
  • LPP lipid multipolymers
  • PNP polymer nanoparticles
  • INP inorganic nanoparticles
  • CNE cationic nanoemulsion
  • exosomes exosomes
  • biological microvesicles protamine, etc.
  • the nanoparticles are lipid nanoparticles (LNP).
  • LNP lipid nanoparticles
  • 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.
  • the present invention provides a vector comprising any one of the mRNA constructs described herein, or a combination thereof.
  • the present invention provides a cell comprising any one of the mRNA constructs described herein, or a combination thereof, or a vector described herein.
  • the present invention provides a nanoparticle comprising any one of the mRNA constructs described herein, or a combination thereof;
  • the nanoparticles include lipid nanoparticles (LNP), lipid multipolymers (LPP), polymer nanoparticles (PNP), inorganic nanoparticles (INP), cationic nanoemulsion (CNE), exosomes, biological microvesicles, protamine, etc.
  • LNP lipid nanoparticles
  • LPP lipid multipolymers
  • PNP polymer nanoparticles
  • INP inorganic nanoparticles
  • CNE cationic nanoemulsion
  • exosomes exosomes
  • biological microvesicles protamine, etc.
  • the nanoparticles are lipid nanoparticles (LNP).
  • LNP lipid nanoparticles
  • the present invention provides a vaccine comprising any one of the nanoparticles described herein;
  • the vaccine is a MICA and/or MICB mRNA vaccine.
  • 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.
  • the second protein is a carrier protein
  • 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.
  • ferritin diphtheria toxoid
  • TT tetanus toxoid
  • KLH keyhole limpet hemocyanin
  • Ply pneumolysin
  • 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/
  • the second protein is T4 Foldon.
  • the second protein is ferritin.
  • the ferritin protein comprises a domain that allows the fusion protein to self-assemble into nanoparticles
  • the ferritin is ferritin from Helicobacter pylori.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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;
  • the method induces an immune response against MICA/B by using an mRNA vaccine
  • the mRNA is replicating, non-replicating, trans-self-replicating or circular mRNA.
  • 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;
  • the method induces an immune response against MICA/B by using replicating or non-replicating mRNA
  • the mRNA is replicating, non-replicating, trans-self-replicating or circular mRNA.
  • the vaccine composition is administered as part of a therapeutic regimen
  • the treatment regimen is radiation therapy, targeted therapy, immunotherapy or chemotherapy.
  • the individual is one who tests positive for shed MICA/B in their serum.
  • 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, lymphom
  • AML acute
  • the technical solution of the present invention has at least one of the following beneficial effects:
  • 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.
  • 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.
  • the MICA/B mRNA vaccine described in the present invention has a simpler preparation process, better efficacy, and is more suitable for clinical development.
  • 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.
  • 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 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 ⁇ -D-ribose configuration, ⁇ -LNA with an ⁇ -L-ribose configuration (diastereomers of LNA), 2′-amino-LNA with 2′-amino functionalization, and 2′-amino- ⁇ -LNA with 2′-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), or chimeras or combinations thereof.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • TAA threose nucleic acid
  • GNA glycol nucleic acid
  • PNA peptide nu
  • 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.
  • 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.
  • 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.
  • 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.
  • mutant 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.
  • protein 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.
  • amino acid substitution 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.
  • conservative amino acid substitution refers to replacing an amino acid normally present in a sequence with a different amino acid of similar size, charge or polarity.
  • conservative substitutions include replacing a non-polar (hydrophobic) residue such as isoleucine, valine and leucine with another non-polar residue.
  • 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.
  • 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.
  • 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.
  • vector 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.
  • antigen 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.
  • tumor antigens are expressed in patients with (known or unknown) cancer, and their concentration in the body fluids of the patients increases.
  • tumor antigens are also referred to as “tumor antigens”, but they are not antigens in the strict sense of immune response inducing substances.
  • 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.
  • 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.
  • Ply pneumolysin
  • PPD purified protein derivative of tuberculin
  • 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.
  • 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.
  • PRR pathogen recognition receptor
  • transmembrane domain 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, CD8 ⁇ TM, CD28 TM, SARS-COV-2 Spike TM, HSV gB TM, HSV gC TM, HSV gE TM, etc.
  • 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.
  • delivery formulation 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.
  • 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.
  • immune response 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).
  • specific or adaptive immune response a specific antigen
  • non-specific or innate immune response a non-specific response of the innate immune system
  • 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).
  • an antigen such as an immunogenic polypeptide
  • innate immune responses innate immune responses
  • 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.
  • 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.
  • cellular immunity/cellular immune response 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.
  • 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.
  • 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.
  • humoral immunity/humoral immune response typically refers to antibody production and the auxiliary processes that may accompany it.
  • 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.
  • 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- ⁇ 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- ⁇ , IL-2, TNF- ⁇ , 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- ⁇ , IL-2, TNF- ⁇ , 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- ⁇ 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- ⁇ 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.
  • the MICA/B mRNA vaccine sequence we designed targets the highly conserved ⁇ 3 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 ⁇ 3 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.
  • 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 ⁇ 3 domain of MICA*008 and the ⁇ 3 domain of MICB*005 as target antigens, designed their mRNA sequences, and then expressed them by fusion.
  • FIG. 1 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, h ⁇ -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).
  • 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.
  • 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
  • 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.
  • 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 ⁇ 3 mRNA construct, we constructed two models, wild type and deglycosylation site mutants (mutating the potential N-glycosylation site in the MICA or MICB ⁇ 3 region from Asn to Gln) to explore the effect of deglycosylation on the immunogenicity of the vaccine of the present invention.
  • the amino acids at positions 29, 39, 80, and 108 in the MICA ⁇ 3 region were mutated from Asn to Gln
  • the amino acids at positions 153, 163, 204, and 232 in the MICB ⁇ 3 region were mutated from Asn to Gln
  • 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 ⁇ 3-domain peptide.
  • ferritin from Heliobacter pylori 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.
  • XR-MIC-1 5'UTR h ⁇ -globin-H-2K b signal peptide-MICA ⁇ 3-GS linker-MICB ⁇ 3-3'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 h ⁇ -globin-H-2K b signal peptide-MICA ⁇ 3m-GS linker-MICB ⁇ 3m-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 h ⁇ -globin-H-2K b signal peptide-MICA ⁇ 3-GS linker-MICB ⁇ 3-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 h ⁇ -globin-H-2K b signal peptide-MICA ⁇ 3m-GS linker-MICB ⁇ 3m-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 ⁇ 3-GS linker-MICB ⁇ 3-3'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 ⁇ 3m-GS linker-MICB ⁇ 3m-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 ⁇ 3-GS linker-MICB ⁇ 3-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 ⁇ 3m-GS linker-MICB ⁇ 3m-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).
  • sequence of the 3’UTR used is shown in SEQ NO:71
  • sequence of Poly(A) is shown in SEQ NO:72.
  • 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)
  • 1% agarose gel electrophoresis and gel imaging are used to observe whether the sample is completely digested.
  • 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.
  • 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.
  • 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.
  • 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.
  • Malvern Zetasizer ultra was used to measure the particle size and potential of lipid nanoparticles. 10 ⁇ L 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.
  • the total RNA concentration of lipid nanoparticles was detected by Ribogreen method.
  • the LNP solution was demulsified with Triton, 100 ⁇ L of the demulsified solution was added to a 96-well plate, and then 100 ⁇ L 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.
  • TransIT-mRNA and mRNA Boost Reagent (Miurs, MIR-2250) to room temperature and vortex gently.
  • 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.
  • 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.
  • Each group was vaccinated with 5 ⁇ g (100 ⁇ L) 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- ⁇ ELISPOT detection and cytokine detection were performed.
  • 96-well ELISA plates were coated with 2 ⁇ g/ml MICA protein (MICA ECD, Sino Biological, 12302-H08H-1mg) or 10 ⁇ g/ml MICB protein (MICB ECD, Sino Biological, Sino#10759-H08H-1-1mg) at 4°C overnight.
  • MICA ECD MICA ECD
  • MICB ECD MICB ECD
  • the plates were washed 5 times with 260 ⁇ L/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.
  • 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.
  • 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 ⁇ g/ml), or ConA (6.67 ⁇ g/ml, positive control) in a 37°C incubator for 16-20 h, and the mouse IFN- ⁇ 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- ⁇ 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.
  • spleen cells 1.5 ⁇ 10 6 spleen cells were incubated with 0.2 ⁇ g 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.
  • 1 ⁇ eBioscience TM protein Transport inhibitor cocktail Thermo, 00-4980-93
  • Cell surface marker staining Block the cells with 50 ⁇ L of PBS containing 4% TruStain FcX TM anti-mouse CD16/32 (Biolegend, #101320), incubate at 4°C for 8-10 min, add 50 ⁇ L 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 ⁇ L (1% IL-2 (Biolegend, 503808), IFN- ⁇ (Biolegend, 505830), TNF- ⁇ (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.
  • the CD4 + T cells from the vaccine group mice can specifically secrete Th1 cytokines IFN- ⁇ , IL-2 and TNF- ⁇ , 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- ⁇ , while IL-2 and TNF- ⁇ and Th2 cytokine IL-4 are almost undetectable.
  • a cell line that can stably express MICA/B 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.
  • 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.
  • 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 15 ⁇ g:9 ⁇ g:6 ⁇ g 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°C.
  • 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.
  • 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.
  • 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.
  • Transgenic MC38 cell lines expressing full-length MICA/MICB 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 ⁇ L of serum samples serially diluted 10-fold in FACS buffer for 1 h at room temperature, washed twice with FACS buffer, stained with 100 ⁇ L Alexa Fluor 647-conjugated goat anti-mouse IgG secondary antibody (1 ⁇ g 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.
  • Example 5 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.
  • MC38 transgenic MC38 cell line
  • Transgenic MC38 cells expressing full-length MICA/MICB 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 ⁇ L 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 ⁇ L Alexa Fluor 647-conjugated goat anti-mouse IgG secondary antibody (1 ⁇ g/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 cytometer (BD Biosciences) and
  • XR-MIC-9 5’UTR-human IgE signal peptide-MICA ⁇ 3m-GS linker-MICB ⁇ 3m-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 ⁇ 3m-GS linker-MICB ⁇ 3m-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 ⁇ 3m-GS linker-MICB ⁇ 3m-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 ⁇ 3m-GS linker-MICB ⁇ 3m-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 ⁇ 3m-GS linker-MICB ⁇ 3m-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 ⁇ 3m-GS linker-MICB ⁇ 3m-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 ⁇ 3m-GS linker-MICB ⁇ 3m-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.
  • 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.
  • 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.
  • 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.
  • mice for all constructs of XR-MIC-1 to 14 were randomly divided into 15 groups, 5 mice in each group, and the grouping and dosing details are shown in Table 5.
  • Each group was vaccinated with 1 ⁇ g (50 ⁇ L) 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.
  • Example 13 Preliminary efficacy verification of the second round of XR-MIC mRNA vaccine in MC38-TgMICA/B subcutaneous tumor-bearing mice
  • 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 ⁇ g 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.
  • XR-MIC-12-1 5’UTR-MICA*008 signal peptide-MICA ⁇ 3m-(GGGGS)2 linker-MICB ⁇ 3m-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 ⁇ 3m-(EAAAK)2 linker-MICB ⁇ 3m-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 ⁇ 3m-(GGGGS)2 linker-MICB ⁇ 3m-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 ⁇ 3m-(EAAAK)2 linker-MICB ⁇ 3m-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 ⁇ 3m-(GGGGS)2 linker-MICA ⁇ 3m-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 ⁇ 3m-(GGGGS)2 linker-MICA ⁇ 3m-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 ⁇ 3m-(GGGGS)2 linker-MICB ⁇ 3m-(GGGGS)2 linker-MICA ⁇ 3m-(GGGGS)2 linker-MICB ⁇ 3m-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 ⁇ 3m-(EAAAK)2 linker-MICB ⁇ 3m-(GGGGS)2 linker-MICA ⁇ 3m-(EAAAK)2 linker-MICB ⁇ 3m-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 ⁇ 3m-(GGGGS)2 linker-MICB ⁇ 3m-(GGGGS)2 linker-MICA ⁇ 3m-(GGGGS)2 linker-MICB ⁇ 3m-(GGGGS)2 linker-MICA ⁇ 3m-(GGGGS)2 linker-MICB ⁇ 3m-(GGGGS)2 linker-MICA ⁇ 3m-(GGGGS)2 linker-MICB ⁇ 3m-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 ⁇ 3m-(EAAAK)2 linker-MICB ⁇ 3m-(GGGGS)2 linker-MICA ⁇ 3m-(EAAAK)2 linker-MICB ⁇ 3m-(GGGGS)2 linker-MICA ⁇ 3m-(EAAAK)2 linker-MICB ⁇ 3m-(GGGGS)2 linker-MICA ⁇ 3m-(EAAAK)2 linker-MICB ⁇ 3m-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 ⁇ 3m-(GGGGS)2 linker-MICB ⁇ 3m-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 ⁇ 3m-(GGGGS)2 linker-MICB ⁇ 3m-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 ⁇ 3m-(GGGGS)2 linker-MICB ⁇ 3m-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 ⁇ 3m-(GGGGS)2 linker-MICB ⁇ 3m-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 ⁇ 3m-(GGGGS)2 linker-MICB ⁇ 3m-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 ⁇ 3m-(GGGGS)2 linker-MICB ⁇ 3m-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 ⁇ 3m-(GGGGS)2 linker-MICA ⁇ 3m-(GGGGS)2 linker-MICB ⁇ 3m-(GGGGS)2 linker-MICB ⁇ 3m-(GGGGS)2 linker-MICB ⁇ 3m-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 ⁇ 3m-(EAAAK)2 linker-MICB ⁇ 3m-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 ⁇ 3m-(EAAAK)2 linker-MICB ⁇ 3m-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
  • the HATM+CTD sequence is shown in SEQ NO:105.
  • 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.
  • 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%.
  • 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.
  • 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.
  • 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 ⁇ g (100 ⁇ L) 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.
  • 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.
  • mice 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- ⁇ in order to compare the differences in cellular immunogenicity of different constructs.
  • 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.
  • 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.

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Abstract

L'invention concerne des vaccins antitumoraux à ARNm pour MICA et/ou MICB et des applications de ceux-ci, des vaccins à ARNm pour MICA et/ou MICB, ainsi que des constructions d'ARNm, des vecteurs, des cellules hôtes, des protéines de fusion, des préparations de LNP, des utilisations pharmaceutiques, et des méthodes pour traiter/prévenir les tumeurs et les cancers correspondants.
PCT/CN2024/090104 2023-04-28 2024-04-26 Vaccins antitumoraux à arnm pour cible mica/b WO2024222886A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150165065A1 (en) * 2009-12-31 2015-06-18 Avidbiotics Corp. Non-natural mic proteins
CN106456728A (zh) * 2014-03-14 2017-02-22 达纳－法伯癌症研究所公司 恢复对抗癌症的nkg2d通路功能的疫苗组合物和方法
CN108770343A (zh) * 2015-12-04 2018-11-06 达纳－法伯癌症研究所公司 用于治疗癌症的使用MICA/Bα3结构域的疫苗接种
US20220401538A1 (en) * 2021-06-09 2022-12-22 Oncocine Llc Therapeutic mrna vaccine for malignancies
CN115611982A (zh) * 2021-07-14 2023-01-17 浙江大学 一种抗人MICA/Bα3区的单克隆抗体及其应用
CN115944722A (zh) * 2023-01-09 2023-04-11 浙江大学 一种抗肿瘤mRNA疫苗及其制备方法和应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150165065A1 (en) * 2009-12-31 2015-06-18 Avidbiotics Corp. Non-natural mic proteins
CN106456728A (zh) * 2014-03-14 2017-02-22 达纳－法伯癌症研究所公司 恢复对抗癌症的nkg2d通路功能的疫苗组合物和方法
CN108770343A (zh) * 2015-12-04 2018-11-06 达纳－法伯癌症研究所公司 用于治疗癌症的使用MICA/Bα3结构域的疫苗接种
US20220401538A1 (en) * 2021-06-09 2022-12-22 Oncocine Llc Therapeutic mrna vaccine for malignancies
CN115611982A (zh) * 2021-07-14 2023-01-17 浙江大学 一种抗人MICA/Bα3区的单克隆抗体及其应用
CN115944722A (zh) * 2023-01-09 2023-04-11 浙江大学 一种抗肿瘤mRNA疫苗及其制备方法和应用

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