A vaccine for treating glioblastoma multiforme

Abstract

The present application relates to the field of cancer vaccine research and development, in particular to a vaccine for treating glioblastoma multiforme. The present application relates to a nucleic acid vaccine for precisely treating glioblastoma multiforme. The vaccine uses a functionally active variant of the chemokine CX3CL1 after sequence optimization as an aid, and uses a specific in vivo mutation target EGFRvIII of glioblastoma multiforme (GBM) as a targeting antigen. The results show that the new vaccine can induce efficient specific humoral and cellular immune responses in vivo, and ultimately enable the immune system to kill a large number of target tumor cells, and can effectively prevent or treat glioblastoma multiforme after surgery.

Description

Technical Field

[0001] This invention relates to the field of immunotherapy and prevention of tumors, and more specifically to a vaccine for treating glioblastoma multiforme. Background Technology

[0002] Glioblastoma (GBM) is a common malignant neuroepithelial tumor of the central nervous system in adults. Its main treatments include surgery, radiotherapy, and chemotherapy, with a combined average survival of 12–15 months (Cloughesy, Cavenee et al. 2014). This malignant neuroepithelial tumor is characterized by limited treatment options, difficult surgery, and short survival, making it a very difficult-to-cure cancer. The molecular mechanisms that cause glioblastoma are very complex, with mutations in the important human tyrosine kinase receptor EGFR being one of the most significant contributing factors. Overexpression of EGFR protein accounts for up to 57.4% of primary glioblastoma cases (Brennan, Verhaak et al. 2013, Jawhari, Ratinaud et al. 2016).

[0003] EGFR, also known as HER1 or ERBB1, is a tyrosine kinase receptor in the ERBB membrane protein family (Arteaga and Engelman 2014). It is also a cell surface receptor for various human epidermal growth factors such as TGF-α, EGF, amphiregulin, and betacellulin. This transmembrane protein can effectively receive signals from various ligand molecules and transmit these signals intracellularly through multiple intracellular signaling pathways, thereby achieving precise regulation of intracellular protein expression or translation.

[0004] However, EGFR, as the most important receptor protein for human epidermal growth factor, can cause abnormalities in the growth of many human epidermal cells due to its mutations. Among these, five specific deletion mutations have been found in the EGFR gene: vI (N-terminal deletion), vII (exon 14-15 deletion), vIII (exon 2-7 deletion), vIV (exon 25-27 deletion), and vV (exon 25-28 deletion). Both vII and vIII can lead to tumorigenesis (Furnari, Cloughesy et al. 2015). vIII, due to the deletion of too many exons, is the most important deletion mutation leading to glioblastoma. Therefore, the molecular mechanism of EGFR vIII has received widespread attention in the field of glioblastoma treatment.

[0005] EGFRvIII deletion mutations are often co-expressed with wild-type EGFR on the surface of glioblastoma tumor cells. However, EGFRvIII mutations can disrupt signaling pathways in epithelial cells when they receive signals from foreign ligand molecules, leading to overexpression of the EGFR gene and subsequent cancerous transformation and excessive proliferation. Therefore, in the surface of glioblastoma multiforme tumor cells, both wild-type EGFR and EGFRvIII proteins are overexpressed, distinguishing these tumor cells from normal cells. This suggests that EGFRvIII can serve as a specific tumor cell target recognized by the immune system.

[0006] Due to deletions and misplacements in the EGFRvIII gene, its expression product lacks amino acids 6-273 in the wild-type EGFR protein, and a new glycine is introduced at the breakpoint after the deletion as the polypeptide sequence before and after amino acid 6. Therefore, although EGFRvIII is only a deletion mutant of EGFR wild-type, its polypeptide epitopes of amino acids 1-13 are unique to this deletion mutant and do not exist in wild-type EGFR. Thus, this sequence and its subsequent few polypeptide structures will possess unique protein sequences and structural forms. This essential difference can be used as an important marker for targeting this tumor cell.

[0007] CX3CL1 is an immune cell chemokine first discovered in the human genome in 1997 (Bazan, Bacon et al. 1997). This chemokine belongs to a unique family, exhibiting sequence differences in protein structure compared to other conventional CC and CXC family chemokines. CX3CL1 was initially discovered as a transmembrane protein resembling a cell membrane glycoprotein, containing a transmembrane region in its full-length sequence. This protein was later shown to exist in two distinct structural patterns in human tissues. One is a full-length glycoprotein distributed on the cell membrane surface, containing a chemotactic structure at its N-terminal head, a glycosidic structure in the middle, and a cell membrane anchoring structure at its C-terminus. The other form is a secretory protein form, cleaved by enzymes and containing only the chemotactic structure and the glycosidic structure. It has now been shown that the latter secretory protein form is the predominant form of CX3CL1 as a chemokine in tissues, and this form can perform the chemotactic function of immune cells, similar to other common CC or CXC family chemokines.

[0008] Currently, specific vaccines targeting EGFRvIII as a tumor cell target include both active and passive immunization vaccines, with peptide vaccines playing an immunomodulatory role in tumor treatment. Ridopepimut (CDX-110), a synthetically produced 14-peptide with a primary structure similar to EGFRvIII, binds to porphyrin and can induce EGFRvIII-specific humoral and cellular immune responses. Due to its efficacy and safety, ridopepimut has the potential to be a highly promising treatment strategy for GBM; however, its EGFRvIII amino acid sequence specificity as a target needs further improvement, and while current vaccines show significant survival extension, they do not provide a cure. Therefore, there is a need to provide improved EGFRvIII vaccines to enhance survival. Summary of the Invention

[0009] In view of this, the technical problem to be solved by the present invention is a vaccine for treating glioblastoma multiforme.

[0010] The present invention provides a variant of CX3CL1, the amino acid sequence of which is shown in SEQ ID NO:7.

[0011] This invention provides the application of the CX3CL1 variant in improving the cross-presentation effect of antigen molecules.

[0012] In this invention, CX3CL1 is a chemokine that enables immune cells to exhibit chemotaxis. Following analysis of the CX3CL1 protein structure and sequence, the CX3CL1 chemokine variant was reconstructed. The results showed that CX3CL1-b, among the reconstructed CX3CL1 chemokine variants, exhibited higher chemotactic activity and significantly improved the cross-presentation of cellular antigen molecules. Its amino acid sequence is shown in SEQ ID NO:7.

[0013] Furthermore, the aforementioned cell antigen molecule is a type of antigen located on the cell surface, and each cell type possesses its own unique surface antigens. The cells include: stem cells, blood cells, erythrocytes, leukocytes, platelets, macrophages, and white blood cells; the white blood cells include T lymphocytes, B lymphocytes, nerve cells, muscle cells, and dendritic cells (DCs). In some specific embodiments of the present invention, macrophages, DCs, and T cells are used as test subjects to screen for the cross-presentation effects of different CX3CL1 chemokine variant antigen molecules.

[0014] This invention provides a fusion protein comprising a CX3CL1 variant and an antigen;

[0015] The antigens described in this invention are derived from viruses, pathogens, and / or tumors. In this invention, the antigens may be proteins derived from viruses, pathogens, and / or tumors. In some embodiments, the antigens are derived from viral capsid proteins or non-structural proteins, pathogen membrane proteins, flagellated proteins, or tumor surface antigens. They may be complete fragments or antigenic determinants. They may contain only one antigenic determinant, or they may consist of multiple antigenic determinants tandemly, or a single antigenic determinant may be repeated tandemly two or more times.

[0016] In this invention, the virus includes, but is not limited to, at least one of HPV, EBV, HCV, HIV, HBV, VZV, or coronavirus;

[0017] In this invention, the tumor includes, but is not limited to, at least one of glioblastoma, liver cancer, cervical cancer, ovarian cancer, lung cancer, head and neck cancer, prostate cancer, breast cancer, leukemia, colorectal cancer, gastric cancer, bladder cancer, neurofibrosarcoma, lymphangiosarcoma, or transitional carcinoma.

[0018] In some embodiments, the antigen is the E2, E5, E6, and / or E7 proteins of HPV virus or a mutant epitope thereof. The HPV virus includes various subtypes of HPV virus, such as HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, and / or HPV58.

[0019] In some embodiments, the antigen is LMP1, LMP2, EBNA1 of EB virus, or a mutant epitope thereof.

[0020] In some embodiments, the antigen is the S protein, N protein, E protein, M protein of a coronavirus or an epitope thereof. The coronavirus is SARS virus, MERS virus, and / or COVID-19.

[0021] In some embodiments, the antigen is CD133 and / or EGFRvIII of glioblastoma; the EGFRvIII includes at least one of the following: EGFRvIII extracellular region, EGFRvIII extracellular specific epitope peptide, combination of EGFRvIII extracellular region and EGFRvIII extracellular specific epitope peptide, EGFR extracellular region or full-length EGFR sequence.

[0022] In some embodiments, the antigen is the GPC3 protein and / or AFP protein of liver cancer.

[0023] In some embodiments, the antigen is PSA, PSMA, PSCA, PAP, and / or STEAP1 of prostate cancer.

[0024] In some embodiments, the antigen is a dominant epitope of Her2 / neu and / or BCAR3 in breast cancer.

[0025] In some embodiments, the antigen is melanoma MAGE-A3, ISR2, NY-ESO-1, Melan A, gp100, Tyrosinase, TRP1, and / or TRP2.

[0026] In some embodiments, the antigen is Immunoglobulin idiotype, Immunoglobulin κ-chain, and / or Immunoglobulin λ-chain of leukemia.

[0027] In some embodiments, the antigen is AIM2, HT001, TAF1B, Micoryx and / or TGFβRII of colorectal cancer.

[0028] In some embodiments, the antigen is folate receptor-α of ovarian cancer.

[0029] In some embodiments, the antigen is a variety of proto-oncogenes, tumor suppressor genes and / or tumor-specific antigens such as P53, IDH1 / 2, BAGE, GAGE1, GAGE2, CAG3, RAGE, CEA, CDK4, CASP-8, ras, bcr / abl and / or MUC-1.

[0030] This invention utilizes the chemotactic binding ability of CX3CL1 to receptors on the surface of immune cells such as dendritic cells (DCs) to cross-present the aforementioned antigen proteins to the surface of DCs, thereby improving the efficiency of phagocytosis, processing, and presentation of various antigen proteins by DCs and enhancing their efficacy in preventing and treating related diseases. In the embodiments of this invention, the antigen presentation efficiency of the combination of the EGFRvIII extracellular region of EGFRvIII and the EGFRvIII extracellular specific epitope peptide has been verified to be enhanced by CX3CL1, and the fusion of other proteins with CX3CL1 also exhibits good effects.

[0031] In this invention, the combination of the extracellular region of EGFRvIII and the extracellular specific epitope peptide of EGFRvIII, referred to as vIII antigen or vIII, is designed based on the theories of protein structure and immunology, taking into account the specific stimulation of efficient humoral and cellular immune responses. It selects the T cell epitope of specific EGFRvIII as the cellular immune antigen recognition sequence and the inner layer protein structure sequence not exposed on the surface of the protein structure as the recognition site of new humoral immune antibodies. It has good safety, strong antigen specificity, and can effectively activate cellular immune responses.

[0032] Furthermore, the fusion protein also includes the T2 protein, the T2 sequence of which is derived from a short peptide (T4 phageheadfibritin) at the C-terminus of T4 phageheadfibritin. This exogenous sequence is completely absent in the human body and will not cause damage to other proteins in the body after enhanced immunity. Some reports suggest that this sequence can promote the trimerization of certain proteins under certain circumstances. Experimental results show that the T2 protein at the C-terminus of the antigen protein can effectively enhance the immune strength of the antigen molecule during cellular immunity, significantly increasing the number of specific T cells produced, and playing a decisive role as an immune-enhancing factor.

[0033] Furthermore, the fusion protein of the present invention also includes an N-terminal IgE signal peptide and / or a C-terminal Flag tag. The amino acid sequence of the IgE signal peptide is MDWTWILFLVAAATRVHS, as shown in SEQ ID NO:15, and its addition at the N-terminus of the recombinant protein promotes the secretion of the fusion protein extracellularly; the amino acid sequence of the Flag tag is DYKDDDDK, as shown in SEQ ID NO:16, and this sequence is only a tag for identifying protein expression and does not affect the immunogenicity of the sequence.

[0034] In this invention, the sequence of the fusion protein is any one or more of the sequences shown in SEQ ID NO:13, SEQ ID NO:3 and / or SEQ ID NO:12.

[0035] In some specific embodiments, the combination of fusion proteins described in this invention may include any of the following:

[0036] CX3CL1 variant and vIII antigen;

[0037] CX3CL1 variant, vIII antigen, signal peptide, and Flag tag;

[0038] CX3CL1 variant, vIII antigen, and T2 protein;

[0039] CX3CL1 variant, vIII antigen, T2 protein, signal peptide, and Flag tag.

[0040] This invention provides a nucleic acid encoding the fusion protein, comprising:

[0041] Nucleic acid encoding the CX3CL1 variant, as shown in SEQ ID NO:4;

[0042] Nucleic acid encoding a combination of the extracellular region of EGFRvIII and an extracellular specific epitope peptide of EGFRvIII, as shown in SEQ ID NO:1;

[0043] The nucleic acid encoding the T2 protein is shown in SEQ ID NO:5.

[0044] Furthermore, the nucleic acid encoding the CX3CL1 variant and the nucleic acid encoding the EGFRvIII extracellular region and the EGFRvIII extracellular specific epitope peptide are linked by linker (G5S)n, where n is 1 to 10; in this embodiment of the invention, the linker sequence is GGGGGSGGGGG, as shown in SEQ ID NO:14.

[0045] Furthermore, in some embodiments, the combination of nucleic acids may be CX3CL1-Linker-vIII.

[0046] In other embodiments, the combination of nucleic acids described in this invention may be CX3CL1-Linker-vIII-T2.

[0047] In other embodiments, the combination of nucleic acids described in this invention may be IgE-CX3CL1-Linker-vIII-Flag.

[0048] In other embodiments, the combination of nucleic acids described in this invention may be IgE-CX3CL1-Linker-vIII-T2-Flag.

[0049] The nucleic acid encoding the fusion protein described in this invention can be DNA, RNA, cDNA, or PNA. In embodiments of this invention, the nucleic acid is in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The nucleic acid can include nucleotide sequences with different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). The nucleic acid can be topologically linear or circular. The nucleic acid can be, for example, a part of a vector (e.g., an expression or cloning vector), or a fragment. The nucleic acid can be obtained directly from a natural source or can be prepared with the assistance of recombinant, enzymatic, or chemical techniques. The RNA form is mRNA obtained by gene transcription, etc.

[0050] In this invention, the DNA sequence for expressing the fusion protein is optimized, including but not limited to: codon usage bias, elimination of secondary structures that are unfavorable to expression (such as hairpin structures), alteration of GC content, CpG dinucleotide content, mRNA secondary structure, hidden splicing sites, early polyadenylation sites, internal ribosome entry and binding sites, negative CpG islands, RNA unstable regions, repetitive sequences (direct repeats, inverted repeats, etc.), and restriction sites that may affect cloning.

[0051] This invention also provides a transcription unit for fusion proteins, wherein the transcription unit refers to a DNA sequence from the start of a promoter to the end of a terminator. Regulatory fragments may also be included on either side of or between the promoter and terminator. These regulatory fragments may include promoters operatively linked to the nucleic acid sequence, enhancers, transcription termination signals, polyadenylation sequences, origins of replication, nucleic acid restriction sites, and homologous recombination sites, such as enhancers of promoters, poly(A) signals, etc.

[0052] The present invention provides a recombinant vector comprising a vector backbone and the nucleic acid described herein.

[0053] The recombinant vector described in this invention refers to a recombinant nucleic acid vector, a recombinant DNA molecule containing the desired coding sequence and suitable nucleic acid sequences or elements essential for the expression of an operatively linked coding gene in a specific host organism. Nucleic acid sequences or elements essential for expression in model animals or mammalian cells include promoters, ribosome binding sites, and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and terminators. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. In this specification, "plasmid" and "vector" are sometimes used interchangeably because plasmids are currently the most commonly used form of vector. However, this invention intends to include other forms of expression vectors that perform equivalent functions and are known or will become known in the art, including but not limited to: plasmids, phage particles, viral vectors, and / or simply potential genomic inserts. In specific embodiments, the nucleic acid encoding the fusion protein provided by this invention can be constructed in various eukaryotic expression vectors. For example, its skeletal carrier can be a pVR series carrier (see Chinese Patent ZL202110624820.8).

[0054] Furthermore, in some specific embodiments, the recombinant vector of the present invention may include any one or more combinations of the following:

[0055] pVR-CX3CL1-vIII-T2;

[0056] pVR-CX3CL1-vIII;

[0057] pVR-vIII.

[0058] This invention provides a host cell for transformation or transfection with the recombinant vector described herein. The host cell is transformed or transfected with a vector constructed using recombinant DNA technology, thereby enabling the transformed host cell to replicate the protein-coding vector or express the desired protein.

[0059] Furthermore, the host includes model animals or mammalian cells. Model animals include commonly used laboratory animals in the biomedical field such as mice, rats, and rabbits; this invention does not limit this to any particular type. Mammalian cells include CHO cells, BHK cells, Sp2 / O, HEK293, and HEK293T.

[0060] Furthermore, this invention uses mice as experimental subjects to conduct in vivo activity and specificity experiments by introducing the aforementioned nucleic acid or vector. This invention uses HEK293T cells as test subjects, transforming or transfecting them with the above-mentioned recombinant vector, and observing protein expression. The results show that plasmids pVR-CX3CL1-vIII-T2, pVR-CX3CL1-vIII, and the control plasmid pVR-vIII can all be successfully and normally expressed in mammalian cells.

[0061] The present invention provides a method for preparing the fusion protein, which involves culturing the recombinant host described in the present invention to obtain a culture containing the fusion protein.

[0062] This invention provides the use of at least one of the following (I) to (V) in the preparation of a drug for preventing and treating tumors:

[0063] I) The fusion protein described in this invention;

[0064] II) The nucleic acid described in this invention;

[0065] III) The recombinant vector described in this invention;

[0066] IV) The host described in this invention;

[0067] V) The fusion protein prepared by the method described in this invention.

[0068] Furthermore, the tumor described in this invention is a malignant tumor.

[0069] In some embodiments, the malignant tumor is a tumor that highly expresses EGFR. The tumor that highly expresses EGFR includes brain cancer, non-small cell lung cancer, breast cancer, colorectal cancer, esophageal cancer, prostate cancer, ovarian cancer, and head and neck squamous cell carcinoma.

[0070] In some specific embodiments, the tumor is lung cancer. The lung cancer includes non-small cell lung cancer or small cell lung cancer.

[0071] Furthermore, the prevention and treatment methods described in this invention include any one of inhibiting tumor growth, reducing tumor volume, or slowing down the growth rate of tumors, and this invention does not limit these methods.

[0072] In this invention, the application also includes its use in the preparation of drugs for mouse xenografts. Experimental results show that the pVR-CX3CL1-vIII-T2 plasmid, CX3CL1-vIII-T2-mRNA, and CX3CL1-vIII-T2 protein completely disappeared in mouse xenografts around day 20 after tumor inoculation, achieving a complete tumor treatment effect.

[0073] This invention provides a drug for preventing and treating tumors, comprising any one or more of the following: i) to v)

[0074] i) The fusion protein described in this invention;

[0075] ii) The nucleic acid described in this invention;

[0076] iii) The recombinant vector described in this invention;

[0077] iv) The host described in this invention;

[0078] v) The fusion protein prepared by the method described in this invention.

[0079] Furthermore, the antitumor drugs provided by the present invention also include nucleotide sequences that are reverse complementary to the nucleic acid sequence, transcription mRNA or translation initiation products of the nucleic acid, etc., which are not limited by the present invention.

[0080] Furthermore, the drug also includes lipid nanoparticles containing the nucleic acid, recombinant nucleic acid, or mRNA. The preparation process of the lipid nanoparticles involves encapsulating the nucleic acid, recombinant nucleic acid, or mRNA using an in vivo transfection reagent to form a vaccine in the form of lipid nanoparticles containing the nucleic acid or mRNA for tumor prevention and treatment.

[0081] Furthermore, the medicament described in this invention also includes pharmaceutically acceptable excipients or carriers.

[0082] This invention provides a method for preventing and treating tumors, which involves administering the drug described herein. The administration method may include injection, oral administration, or a gene gun.

[0083] This invention relates to a nucleic acid vaccine for the precise treatment of glioblastoma multiforme. This vaccine utilizes a functionally active variant of the chemokine CX3CL1, optimized for sequence adaptation, as an adjuvant, and employs EGFRvIII, a specific in vivo mutant target of glioblastoma multiforme (GBM), as a targeting antigen. Results show that this novel vaccine can induce highly efficient specific humoral and cellular immune responses in vivo, ultimately leading to a massive killing of target tumor cells by the immune system, effectively preventing or treating glioblastoma multiforme postoperatively. Attached Figure Description

[0084] Figure 1 Comparative analysis of the ability of CX3CL1 and its various functional active variants to chemotact with various professional antigen-presenting cells;

[0085] Figure 2 The complete structure of the N-terminal extracellular domain of EGFR and EGFRvIII proteins (blue) and the EGFRvIII antigen recognition protein (red) are shown.

[0086] Figure 3 The diagrams of three constructed plasmids, pVR-CX3CL1-vIII-T2, pVR-CX3CL1-vIII, and pVR-vIII, are shown.

[0087] Figure 4 The expression of the target gene in three plasmids, pVR-CX3CL1-vIII-T2, pVR-CX3CL1-vIII, and pVR-vIII, was detected. The expression of the fusion protein with the C-terminus Flag tag was detected by Western blot.

[0088] Figure 5 The timeline of mice subjected to prophylactic immunization with different fusion genes for subsequent testing of specific T cell responses;

[0089] Figure 6 Elispot assay results of specific T cell responses in mice after immunization with different fusion genes;

[0090] Figure 7 Timelines of prophylactic immunization and tumor inoculation in mice using different fusion gene, mRNA, and protein vaccines;

[0091] Figure 8 The above-mentioned preventive immunosuppression and tumor detection results are shown in the figure.

[0092] Figure 9 The timeline of therapeutic immunization and tumor inoculation in mice using different fusion gene, mRNA and protein vaccines;

[0093] Figure 10 The above-mentioned therapeutic immunotherapy and tumor detection results are shown in the figure. Detailed Implementation

[0094] This invention provides a vaccine for treating glioblastoma multiforme. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the vaccine. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0095] Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art. For definitions and terminology in this field, those skilled in the art may refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in this field to refer to one of the 20 commonly used L-amino acids.

[0096] The vIII antigen gene sequence is:

[0097]

[0098] The CX3CL1-vIII-T2 fusion gene sequence is as follows:

[0099]

[0100] The amino acid sequence of the CX3CL1-vIII-T2 fusion protein is as follows:

[0101]

[0102] The CX3CL1 nucleotide sequence is as follows:

[0103]

[0104] The T2 nucleotide sequence is:

[0105]

[0106] The amino acid sequence of the vIII antigen protein is:

[0107]

[0108] The amino acid sequence of CX3CL1 is as follows:

[0109]

[0110] The sequence of the plasmid pVR-CX3CL1-vIII-T2 fusion gene is as follows:

[0111]

[0112] The pVR-vIII fusion gene sequence is as follows:

[0113]

[0114] The sequence of the plasmid pVR-CX3CL1-vIII fusion gene is as follows:

[0115]

[0116] The mRNA sequence encoding the fusion protein by CX3CL1-vIII-T2-mRNA is as follows:

[0117]

[0118] The amino acid sequence of the CX3CL1-vIII-T2 fusion protein is as follows:

[0119]

[0120] The amino acid sequence of the CX3CL1-Linker-vIII fusion protein is as follows:

[0121]

[0122] The linker amino acid sequence is: GGGGGSGGGGG (as shown in SEQ ID NO:14).

[0123] The IgE amino acid sequence is: MDWTWILFLVAAATRVHS (as shown in SEQ ID NO:15).

[0124] The amino acid sequence of Flag is: DYKDDDDK (as shown in SEQ ID NO:16).

[0125] In this embodiment of the invention, the amino acid sequences of the involved fragments and the encoded nucleic acid fragments are shown in Table 1:

[0126] Table 1 shows the amino acid sequence and encoded nucleic acid sequence of the fragment.

[0127] Serial Number amino acids SEQ ID NO:1 vIII antigen gene sequence SEQ ID NO:2 CX3CL1-vIII-T2 fusion gene sequence SEQ ID NO:3 Amino acid sequence of CX3CL1-vIII-T2 fusion protein SEQ ID NO:4 CX3CL1 nucleotide sequence SEQ ID NO:5 T2 nucleotide sequence SEQ ID NO:6 EGFRvIII antigen protein amino acid sequence SEQ ID NO:7 CX3CL1 amino acid sequence SEQ ID NO:8 plasmid pVR-CX3CL1-vIII-T2 fusion gene sequence SEQ ID NO:9 plasmid pVR-vIII fusion gene sequence SEQ ID NO:10 plasmid pVR-CX3CL1-vIII fusion gene sequence SEQ ID NO:11 The mRNA sequence of the CX3CL1-vIII-T2-mRNA encoding the fusion protein SEQ ID NO:12 Amino acid sequence of CX3CL1-vIII-T2 fusion protein SEQ ID NO:13 CX3CL1-Linker-EGFRvIII amino acid sequence SEQ ID NO:14 linker SEQ ID NO:15 IgE SEQ ID NO:16 Flag

[0128] In this invention, the combination of the extracellular region of EGFRvIII and the extracellular specific epitope peptide of EGFRvIII is also referred to as EGFRvIII antigen, vIII antigen, vIII or EGFRvIII.

[0129] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0130] The present invention will be further illustrated below with reference to the embodiments:

[0131] Example 1: Construction of CX3CL1 chemokine variants

[0132] The protein structure and sequence of the CX3CL1 chemokine were analyzed. Analysis revealed that the molecule contains three domains: an N-terminal chemokine domain, a glycosidic domain, and a C-terminal transmembrane domain. To determine the most efficient chemokine sequence for various immune cells, multiple sequences of the CX3CL1 protein were constructed, and the chemotactic ability of the protein molecule was simultaneously tested. Since the C-terminal domain is a transmembrane domain and cannot secrete antigen proteins outside the cell, the entire C-terminal transmembrane region was deleted in different variant tests, defining amino acid sequences 25–338 of the CX3CL1 protein as the full-length CX3CL1 molecule. Based on this, and combined with protein structural analysis, the following molecular variants were designed and constructed (amino acid numbers are shown in parentheses): CX3CL1-a (25–100); CX3CL1-b (25–128); CX3CL1-c (25–185); CX3CL1-d (25–265); CX3CL1-e (25–309); CX3CL1-f (25–338, full length) and CX3CL1-g (37–388). Sequences of all the above molecular variants were synthesized and constructed into the *E. coli* pET28a vector for protein expression and purification. Crude purified samples of all seven protein molecules were obtained for subsequent experiments.

[0133] Monocytes and T cell subsets were isolated from mouse bone marrow and peripheral blood, respectively. Bone marrow monocytes were induced to differentiate into macrophages and dendritic cells (DCs) by adding M-CSF and GM-CSF, respectively, and then subjected to chemotaxis experiments. The isolated or induced cells were placed in the upper chamber of a chemotaxis chamber (carbonate membrane Transwell chamber: 5 μm; Costa, Cat: 3422), with a cell count of 1 × 10⁻⁶ cells based on previous laboratory work. 6 / 100μl / well. A spontaneous migration control group and a group containing all functional variants of the CX3CL1 cytokine were also set up, with the same number of cells added. Based on previous work in the laboratory, 100ng / ml was determined to be the optimal dose for chemotactic efficiency. Cells in the lower chemotactic chamber were collected after 4 hours, and flow cytometry analysis was performed to analyze the chemotactic ability of the CX3CL1 functional variants on various immune cells. The results showed that different molecular variants of CX3CL1 could effectively recruit various immune cells from the upper chamber to the lower chamber (P < 0.001), and the chemotactic ability of different protein molecules varied. The most chemotactically active variant was not the chemokine domain protein as understood through traditional sequence analysis, but rather the molecular variant CX3CL1-b (with a relatively longer sequence). Figure 1Its amino acid sequence is shown in SEQ ID NO:7.

[0134] Example 2: Structural prediction and sequence selection of EGFRvIII antigen sequence and CX3CL1 chemokine protein sequence

[0135] EGFRvIII is a deletion misplacement mutation of the human EGFR gene, with exons 2-7 deleted and a new glycine residue introduced as a linker. Therefore, to ensure the uniqueness and specificity of the antigen recognition sequence and prevent immunosuppression, the full-length EGFRvIII antigen sequence cannot be used. Otherwise, the antigen protein may trigger the body to recognize its own wild-type EGFR, leading to an allergic reaction, or it may trigger immunosuppression, preventing the immune system from recognizing the antigen sequence as a foreign substance for attack. Therefore, guided by theories of protein structure and immunology, and considering the specific stimulation of efficient humoral and cellular immune responses, it is necessary to select specific EGFRvIII T-cell epitopes as cellular immune antigen recognition sequences; and inner layer protein structural sequences not exposed on the protein surface as novel humoral immune antibody recognition sites.

[0136] After theoretical structural analysis and attempts combining various length sequences, the final antigen structural sequence obtained through screening was as follows: Figure 2 As shown, the antigen sequence ultimately selected was the red structure region of the N-terminal extracellular domain of EGFR. Structurally, this region is located inside the wild-type EGFR extracellular domain and cannot be recognized and anchored by antibody proteins in normal human cells. However, in the EGFRvIII mutant lacking exons 2–7, this region is exposed on the outermost side of the entire protein, allowing for effective antibody recognition and binding. This fragment also contains the N-terminal sequence of the missing EGFRvIII and added glycine-containing epitopes. Human HLA epitope prediction revealed multiple effective MHC-binding epitope peptides in this fragment, which can effectively activate cellular immune responses.

[0137] Based on the analysis of the chemokine structural sequence of CX3CL1 in Example 1, the optimized CX3CL1-b was used as the sequence in subsequent examples of this invention (CX3CL1 was used instead of CX3CL1-b as the chemokine name in subsequent experiments).

[0138] Example 3: Antigen design scheme for fusion gene or protein vaccines and construction and preparation of mammalian expression plasmids

[0139] The pVR-CX3CL1-vIII-T2 plasmid was constructed as follows: the EGFRvIII specific antigen sequence was as shown in SEQ ID NO:6, and the human CX3CL1 protein sequence was as shown in SEQ ID NO:7. After adding the T2 polypeptide sequence (as shown in SEQ ID NO:5), the fusion protein CX3CL1-linker-EGFRvIII-T2 was constructed in 5' to 3' order, with the nucleotide sequence shown in SEQ ID NO:2 and the amino acid sequence shown in SEQ ID NO:3. An IgE signal peptide with the amino acid sequence MDWTWILFLVAAATRVHS was attached to the N-terminus of the fusion protein CX3CL1-vIII-T2; an 8-amino acid flag tag consisting of DYKDDDDK was attached to the C-terminus of the fusion protein CX3CL1-vIII-T2.

[0140] The resulting fusion protein, from N-terminus to C-terminus, includes: IgE signal peptide, human CX3CL1 protein sequence, linker sequence (GGGGGSGGGGG), EGFRvIII protein sequence, T2 protein sequence, and Flag tag sequence.

[0141] The amino acid sequence of the fusion protein was optimized using codons preferred for mammalian cell expression, and its fusion gene sequence was determined to be SEQ ID NO:8. This fusion gene sequence was then synthesized (its amino acid sequence is shown in SEQ ID NO:12), and subsequently constructed into the corresponding multiple cloning site region of the pVR plasmid vector (sequence and map shown in patent: 202110624820.8), enabling it to express the fusion protein with the correct codon translation sequence. The final constructed plasmid was named pVR-CX3CL1-vIII-T2 plasmid. Figure 3 As shown in Figure A.

[0142] Constructing the pVR-vIII plasmid: An IgE signal peptide with the amino acid sequence MDWTWILFLVAAATRVHS is linked before the EGFRvIII-specific antigen sequence, followed by an 8-amino acid flag tag consisting of DYKDDDDK. This results in a fusion protein that, from N-terminus to C-terminus, comprises: the IgE signal peptide, the EGFRvIII-specific protein sequence, and the flag tag sequence. Figure 3 As shown in C.

[0143] The amino acid sequence of the fusion protein was optimized using codons preferred for mammalian cell expression, and its fusion gene sequence (SEQ ID NO:9) was determined. This fusion gene sequence was then synthesized and constructed into the corresponding multiple cloning site region of the pVR plasmid vector, enabling it to express the fusion protein with the correct codon translation sequence. The final constructed plasmid was named pVR-vIII plasmid.

[0144] Constructing plasmid pVR-CX3CL1-vIII: Similarly, the final constructed fusion gene includes, from N-terminus to C-terminus, the following sequence: IgE signal peptide, human CX3CL1 protein sequence, linker sequence (GGGGGSGGGGG), EGFRvIII specific protein sequence, and Flag tag sequence.

[0145] The amino acid sequence of the fusion protein was optimized using codons preferred for mammalian cell expression, and its fusion gene sequence was determined to be SEQ ID NO:10. This fusion gene sequence was synthesized and then constructed into the corresponding multiple cloning site region of the pVR plasmid vector, enabling it to express the fusion protein with the correct codon translation sequence. The final constructed plasmid was named pVR-CX3CL1-vIII plasmid. Figure 3 As shown in B.

[0146] Example 4: In vitro cell transfection experiment for constructing plasmids

[0147] 24 hours before transfection, seed 2.5 × 10⁶ cells into 6-well cell culture plates. 5 HEK293T cells were transfected when the cell density reached 60%–70%. The cell culture medium and serum-free Opti-MEM medium were preheated in a 37°C water bath before transfection. For transfection, 5 μg of empty vector, pVR-CX3CL1-vIII-T2 expression vector, pVR-CX3CL1-vIII expression vector, pVR-vIII expression vector, and 20 μL of PEI transfection reagent were added sequentially to 200 μL of serum-free Opti-MEM, mixed thoroughly, and incubated at room temperature for 20 minutes. The cells to be transfected were then replaced with fresh medium, and the mixture was gently added to the transfection system and gently shaken. The cells were returned to the cell culture incubator and cultured for 6 hours, after which the medium was changed. 48 hours after transfection, the cells were harvested and Western blot was used to detect the expression of the EGFRvIII fusion gene plasmid in HEK293T cells.

[0148] Collect cells and add 60 μL of 0.5% NP40 lysis buffer containing PMSF or a cocktail protease inhibitor. Resuspend cells thoroughly and lyse by rotating at 4°C for 30 minutes. Centrifuge the lysis buffer at 12000 rpm at 4°C for 10 minutes, collect the supernatant into a new 1.5 mL EP tube, and discard the precipitate. Add 5×SDS-PAGE protein loading buffer according to the actual sample volume, mix well, and heat the sample in a 100°C air bath for 10 minutes. Immediately perform Western blot analysis using a flag-tagged antibody (Sigma, F3165). Results are as follows: Figure 4 The results showed that the negative control in the empty vector had no protein expression, while the size and location of the expressed proteins in pVR-CX3CL1-vIII-T2 and pVR-CX3CL1-vIII were significantly higher than those in pVR-vIII. This indicates that the experimental plasmids pVR-CX3CL1-vIII-T2 and pVR-CX3CL1-vIII, as well as the control plasmid pVR-vIII, can be successfully and normally expressed in mammalian cells.

[0149] Example 5: Construction of a TC-1 mouse cell line stably transfected with full-length EGFRvIII protein

[0150] The full-length EGFRvIII protein sequence, deleting exons 2-7, was codon-optimized for mammalian cells. The synthesized gene was then constructed into the corresponding cloning site of the pEZ-lv201 plasmid vector via enzyme digestion and ligation. This allowed the stable transfected plasmid vector to simultaneously express the target gene and the EGFP indicator gene. Following a similar method to Example 4, 12 μg of the pEZ-lv201 plasmid containing the EGFRvIII target gene, 10 μg of the packaging plasmid pAX2, and 3 μg of pMD2.G were simultaneously transfected into HEK293T cells. After 3 days of cell culture, observation under a fluorescence microscope revealed that some cells had successfully transfected the target plasmid and exhibited green fluorescence. The cell culture supernatant was aspirated and added to prepared, high-quality TC-1 cell line culture dishes. After 2 days of viral infection, the cell supernatant was changed, and after another 2 days of culture, puromycin was added to the infected cell culture supernatant at a ratio of 1000:1 for selection. Three days later, the culture medium was changed to remove dead cells, and the remaining live cells were cultured. During culture, the cells were observed every other day, and the medium was changed and the cells were passaged as needed. This continued until all cells were infected with a stable lentivirus and showed green fluorescence under a fluorescence microscope. The expanded cells at this point were then processed and lysed according to the method described in Example 4, and finally, a Western blot experiment was performed to detect whether the target protein was expressed. The results confirmed that the cell line expressing the target protein was the TC-1 stable cell line stably transfected with the full-length EGFRvIII protein.

[0151] Example 6 explores the induction of cell-specific T cell responses by fusion gene vaccines.

[0152] Given that fusion genes can be normally expressed in mammalian cells, we extracted individual pVR-CX3CL1-vIII-T2, pVR-CX3CL1-vIII, and pVR-vIII plasmids and performed immunoplasmid electroporation on mice using a TERESA in vivo gene delivery system. The plasmid dose was 25 μg, and there were four groups (n=5 mice in each group) including a negative control PBS group. Figure 5 Mice were immunized using an immunization strategy marked on a timeline. On day 14, the spleens of mice in each group were dissected and added to heparinized PBS solution. The spleens were ground into cells and filtered to remove connective tissue and other impurities. The cell solution was centrifuged at 1500 rpm for 3 min. The supernatant was discarded, and the remaining precipitate was dispersed by shaking. 2 mL of lysing buffer was added, and the mixture was incubated at room temperature for 1 min. Immediately after lysing, 10 mL of PBS was added to stop the lysing process. All samples were then centrifuged at 1500 rpm for 3 min. The supernatant was discarded, and the cells were washed once with 5 mL of PBS solution. A second centrifugation at 1500 rpm for 3 min was performed. The supernatant was discarded, and 5 mL of inactivated 10% FBS 1640 medium was added to resuspend the precipitate. Cell counting was then performed.

[0153] Remove the pre-coated strips from the Elispot kit (catalog number: 3321-4APW-2) and wash four times with sterile PBS (200 μL / well / wash). Discard the last wash and pat dry on absorbent paper. Add 200 μL of working medium to each well and incubate at room temperature for 30 min. Discard the medium from the wells and pat dry on absorbent paper. Add 2.5 × 10⁻⁶ ppm of working medium to each well according to the kit's recommended method. 5 Single-nuclear cells were used, and 2 μg / well of a polypeptide library (a mixture of full-length polypeptide libraries containing the EGFRvIII antigen sequence) was added to each well in the experimental group. Cell Activation Cocktail (catalog number: 423301) was added to the positive control group, while no substance was added to the negative control group. Cells were cultured at 37°C for 18 hours. The next day, samples were stained and washed with antibodies according to the kit's instructions, with a final staining time of 5 minutes. The number of Elispot spots was counted and the data compared. Results are as follows: Figure 6As shown in the results, compared with the three plasmids pVR-CX3CL1-vIII-T2, pVR-CX3CL1-vIII, and pVR-vIII, pVR-CX3CL1-vIII-T2 exhibited the strongest specific T response, while pVR-vIII plasmid showed the weakest. This indicates that the CX3CL1 chemokine at the N-terminus of the antigen protein can effectively induce the binding of antigen molecules to specific immune cells, thereby greatly enhancing the cross-presentation effect of antigen molecules, ultimately enabling CX3CL1 to induce a stronger specific immune response to antigen molecules. In addition, the T2 peptide at the C-terminus of the antigen protein can effectively enhance the immune intensity of antigen molecules in the cellular immunity process, greatly increasing the number of specific T cells produced, playing a decisive role as an immune-enhancing factor. Therefore, the pVR-CX3CL1-vIII-T2 fusion gene sequence designed in this project is currently the most effective immunizing combination.

[0154] Example 7: Effect of a vaccine combining gene DNA, mRNA, and protein on the development of stable TC-1 allograft tumors in mice.

[0155] Given that fusion genes can be normally expressed in mammalian cells, we extracted individual pVR-CX3CL1-vIII-T2 and pVR-vIII plasmids, performed electroporation of the plasmids in mice using the TERESA in vivo gene delivery system, and prepared an mRNA vaccine capable of transcribing pVR-CX3CL1-vIII-T2 in vitro. This mRNA vaccine was then encapsulated with the in vivo transfection reagent in vivo-jetPEI into lipid nanoparticles as the mRNA vaccine (denoted as CX3CL1-vIII-T2-mRNA, sequence shown in SEQ ID NO:11).

[0156] The fusion protein was purified and used as a protein vaccine. The fusion protein corresponding to the pVR-CX3CL1-vIII-T2 plasmid was designated as CX3CL1-vIII-T2 (SEQ ID NO:12).

[0157] The inhibitory effect of fusion gene immunization on the growth of TC-1 xenograft tumor cells was observed after allogeneic transplantation of TC-1 cells that were stably transgenic to EGFRvIII.

[0158] After confirming TC-1 cell tumorigenesis, we followed... Figure 7The timeline-annotated immunization strategy involved plasmid electroporation, intramuscular injection of mRNA, and subcutaneous injection of protein in mice. Female and male C57B6 mice (purchased from Wetonlife) were divided into groups A and B. Each group received five injections of PBS, pVR-vIII plasmid, pVR-CX3CL1-vIII-T2 plasmid, CX3CL1-vIII-T2 mRNA, and CX3CL1-vIII-T2 protein, respectively. Hair was removed from the right side of the mice near the inguinal lymph nodes using depilatory cream. Following this, mRNA and protein were injected directly, or plasmids were injected at the hair removal site using an electroporator, at a dose of 25 μg per mouse, every two weeks for a total of two injections. One week after the last injection, before inoculation, TC-1 tumor cells were selected to establish tumorigenesis. Tumor formation time was observed, and the long axis (a) and short axis (b) of the tumor were measured every two days. Tumor volume was calculated as a × b × b / 2, and a tumor growth curve was plotted. Results are as follows: Figure 8 As shown, immunization with pVR-CX3CL1-vIII-T2 plasmid, pVR-vIII plasmid, CX3CL1-vIII-T2 mRNA, and CX3CL1-vIII-T2 protein all prevented tumor formation in mice, demonstrating significant vaccine efficacy. Furthermore, a second tumor challenge on day 30 showed that the transplanted tumors still failed to form. This indicates that all four vaccine groups have a very significant preventative effect against tumors.

[0159] Example 8: Therapeutic effect of fusion gene vaccine on mouse xenograft tumor cells TC-1 allograft tumors

[0160] Given the excellent efficacy of vaccines such as CX3CL1-vIII-T2 in prophylactic immunization experiments, we further investigated their therapeutic effects on TC-1 allograft tumors treated with fusion gene immunization. We employed methods such as... Figure 9 The timeline in the figure shows the timeline for tumor inoculation and therapeutic plasmid immunization in mice. Each immunization dose was 25 μg. Mice were immunized twice after tumor inoculation, on days 4 and 11. Tumor volume was then measured and recorded using the same method. Tumor growth curves were plotted as shown in the figure. Figure 10 As shown in the figure. The results showed that the three groups of pVR-CX3CL1-vIII-T2 plasmid, CX3CL1-vIII-T2-mRNA, and CX3CL1-vIII-T2 protein had a more significant tumor growth inhibition effect than the pVR-vIII plasmid group. Moreover, the tumors in all three groups completely disappeared around day 20 after tumor grafting, achieving a complete tumor treatment effect.

[0161] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The CX3CL1 variant, whose amino acid sequence is shown in SEQ ID NO:

7.

2. The use of the CX3CL1 variant of claim 1 in the preparation of products that improve the cross-presentation effect of antigen molecules.

3. Fusion proteins, From N-terminus to C-terminus, it consists of: IgE signal peptide, the CX3CL1 variant as described in claim 1, linker sequence, EGFRvIII protein sequence, T2 protein sequence, and Flag tag sequence; Alternatively, from the N-terminus to the C-terminus, the sequence is as follows: IgE signal peptide, the CX3CL1 variant as described in claim 1, linker sequence, EGFRvIII protein sequence, and Flag tag sequence; The amino acid sequence of the EGFRvIII protein is shown in SEQ ID NO:

6.

4. The fusion protein according to claim 3, characterized in that, Its amino acid sequence is at least one of SEQ ID NO:13, SEQ ID NO:3 or SEQ ID NO:

12.

5. The nucleic acid encoding the fusion protein of claim 4.

6. The nucleic acid according to claim 5, characterized in that, in: Nucleic acid encoding the CX3CL1 variant, as shown in SEQ ID NO:4; Nucleic acid encoding a combination of the extracellular region of EGFRvIII and an extracellular specific epitope peptide of EGFRvIII, as shown in SEQ ID NO:1; The nucleic acid encoding the T2 protein is shown in SEQ ID NO:

5.

7. Recombinant vectors, including: The vector backbone and the nucleic acid as described in claim 5 or 6.

8. The recombinant vector according to claim 7, characterized in that, The carrier skeleton includes pVR.

9. Transforming or transfecting a host of the recombinant vector of claim 7 or 8, wherein the host is a mammalian cell.

10. The method for preparing the fusion protein according to claim 3 or 4, characterized in that, Cultivate the host according to claim 9 to obtain a culture containing the fusion protein.

11. The use of at least one of the following (I) to (V) in the preparation of drugs for the prevention and treatment of glioblastoma or lung cancer: I) The fusion protein according to claim 3 or 4; II) The nucleic acid as described in claim 5 or 6; III) The recombinant vector as described in claim 7 or 8; IV) The host as described in claim 9; V) The fusion protein prepared by the preparation method according to claim 10.

12. Drugs for the prevention and treatment of glioblastoma or lung cancer, wherein the raw materials include any one or more of the following i) to v: i) The fusion protein according to claim 3 or 4; ii) The nucleic acid as described in claim 5 or 6; iii) The recombinant vector as described in claim 7 or 8; iv) The host as described in claim 9; v) The fusion protein prepared by the preparation method according to claim 10.

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