Composition containing antigen-presenting cells co-expressing MHC and tumor antigens and cancer treatment using the same

Abstract

The present invention relates to a cancer prevention or treatment vaccine composition comprising antigen-presenting cells in which a complex of MHC (major histocompatibility complex) and tumor antigen is overexpressed on the cell surface, and a cancer treatment using the same.

Description

[Technical field]

[0001] Cross-reference to related applications This application claims the benefit of priority based on Korean Patent Application No. 10-2020-0003910 filed on January 10, 2020, and Korean Patent Application No. 10-2020-0133005 filed on October 14, 2020, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to a vaccine composition for preventing or treating cancer, which comprises an antigen presenting cell in which a complex of major histocompatibility complex (MHC) and a tumor antigen is overexpressed on the cell surface, and a cancer treatment using the same. [Background technology]

[0003] Anticancer therapeutic cell vaccines are expected to act as anticancer vaccines by loading tumor antigens onto specialized antigen-presenting cells, such as dendritic cells, and administering them to cancer patients.

[0004] However, the epitope peptide incubation method commonly used for antigen loading is inevitably limited in the range of epitope sequences that can be presented as antigens and the overall antigen presentation efficacy depending on the specific MHC alleles already expressed in the antigen-presenting cells and their expression levels due to the principle of epitope-MHC restriction between the specific MHC alleles of the antigen-presenting cells and the antigen epitopes. Summary of the Invention [Problem to be solved by the invention]

[0005] In order to use tumor antigens, especially various patient-customized neoantigens derived from individual cancer patients with different cell-specific MHC, in standardized anti-cancer cell vaccines for therapeutic purposes, it is ultimately necessary to develop a new cell vaccine production technology that overcomes the MHC restriction of individual antigen-presenting cells, and this is what we aim to solve.

[0006] One example provides a cancer prevention or treatment vaccine composition comprising an antigen presenting cell in which a complex of a major histocompatibility complex (MHC) and a tumor antigen is overexpressed on the cell surface.

[0007] Another example provides a method for treating cancer, comprising administering to a patient antigen-presenting cells in which a complex of MHC and a tumor antigen is overexpressed on the cell surface.

[0008] Another example provides antigen-presenting cells in which complexes of MHC and tumor antigens are overexpressed on the cell surface for use in cancer therapy.

[0009] Another example provides the use of antigen-presenting cells in which complexes of MHC and tumor antigens are overexpressed on the cell surface for use in the manufacture of drugs for treating cancer.

[0010] Another example provides a method for producing a cancer prevention or treatment vaccine composition, comprising the steps of introducing into an antigen-presenting cell a genetic construct that simultaneously expresses an MHC allele and a tumor antigen gene; an expression vector containing the genetic construct; an RNA transcript transcribed from the genetic construct; or a polypeptide produced from the genetic construct, expression vector, or RNA transcript. [Means for solving the problem]

[0011] The present invention is based on the idea of ​​solving the "constraining factors of antigen-presenting ability due to specific MHC alleles and standard cell expression levels" even in standardized specific antigen-presenting cells by using a bicistronic gene construct or mRNA transcript that simultaneously expresses maximum amounts of patient tumor-derived cell-specific MHC alleles and tumor antigens.

[0012] In particular, in the present invention, the tumor-derived neoantigen sequence obtained from the patient tumor analysis and the tumor-derived MHC alleles of each patient are simultaneously overexpressed in antigen-presenting cells, and the maximum amount of "epitope-MHC immune stimulating complex" is expressed on the cell surface to maximize the tumor antigen presentation efficacy of the antigen-presenting cells. By administering this to the patient, it is ultimately possible to maximize the proliferation and activation of patient-specific anti-cancer T cells and increase and maximize the neoantigen-specific anti-cancer immune effect.

[0013] One aspect of the present invention provides a vaccine composition for preventing or treating cancer, comprising an antigen presenting cell in which a complex of major histocompatibility complex (MHC) and a tumor antigen is overexpressed on the cell surface.

[0014] Also provided is a method for treating cancer, comprising the step of administering to a patient an antigen-presenting cell in which a complex of MHC and a tumor antigen is overexpressed on the cell surface.

[0015] Also provided is an antigen-presenting cell in which a complex of MHC and a tumor antigen is overexpressed on the cell surface for use in cancer treatment.

[0016] Also provided is the use of antigen-presenting cells in which a complex of MHC and a tumor antigen is overexpressed on the cell surface for use in the manufacture of a drug for treating cancer.

[0017] In one embodiment, the antigen-presenting cell may be a dendritic cell.

[0018] In one embodiment, the antigen-presenting cell may contain a genetic construct that simultaneously expresses an MHC allele and a tumor antigen gene; an expression vector containing the genetic construct; or an RNA transcript that is transcribed from the genetic construct and simultaneously expresses an MHC allele and a tumor antigen gene; or a polypeptide produced from the genetic construct, expression vector, or RNA transcript, thereby overexpressing an MHC and tumor antigen complex on the cell surface.

[0019] In one embodiment, the MHC may be MHC class I or MHC class II.

[0020] In one embodiment, the genetic construct may include a nucleic acid sequence of an MHC allele and a nucleic acid sequence encoding a tumor antigen.

[0021] In one embodiment, the nucleic acid sequence of the MHC allele may be fused at its N-terminus to a sequence encoding beta-2-microglobulin (B2M).

[0022] In one embodiment, the MHC alleles and tumor antigens may be derived from tumor cells of a patient with cancer to be treated.

[0023] In one embodiment, the tumor antigen may include a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), or a tumor-derived neoantigen (neoantigen).

[0024] In one embodiment, the tumor-derived neoantigen can comprise a mutation that is specifically expressed in cancer cells.

[0025] In one embodiment, the tumor associated antigen (TAA) is selected from the group consisting of gp100, Melan-A / MART, MAGE-A, MAGE (Melanoma Antigen E), MAGE-3, MAGE-4, MAGE-A3, tyrosinase, TRP2, NY-ESO-1, CEA (Carcinoembryonic Antigen), PSA, p53, Mammaglobin-A, Survivin, Muc1 (Mucin 1) / DF3, metallopanstimulin-1 (MPS-1), cytochrome P450 isoform 1B1, 90K / Mac-2 binding protein, Ep-CAM (MK-1), It may be HSP-70, hTERT (TRT), LEA, LAGE-1 / CAMEL, TAGE-1, GAGE, 5T4, gp70, SCP-1, c-myc, cyclin B1, MDM2, p62, Koc, IMP1, RCAS1, TA90, OA1, CT-7, HOM-MEL-40 / SSX-2, SSX-1, SSX-4, HOM-TES-14 / SCP-1, HOM-TES-85, HDAC5, MBD2, TRIP4, NY-CO-45, KNSL6, HIP1R, Seb4D, KIAA1416, IMP1, 90K / Mac-2 binding protein, MDM2, NY-ESO-1, or LMNA.

[0026] In one embodiment, the gene construct may comprise (a) in the 5' to 3' direction a promoter, a 5'-UTR, a nucleic acid sequence of an MHC allele, a nucleic acid sequence of an IRES (internal ribosome entry site), a nucleic acid sequence encoding a tumor antigen, and a 3'-UTR, or (b) in the 5' to 3' direction a promoter, a 5'-UTR, a nucleic acid sequence encoding a tumor antigen, a nucleic acid sequence of an IRES (internal ribosome entry site), a nucleic acid sequence of an MHC allele, and a 3'-UTR. In a preferred embodiment, the nucleic acid sequence of the MHC allele may be fused to a sequence encoding beta-2-microglobulin (B2M) at its N-terminus.

[0027] In one embodiment, the RNA transcript may be an mRNA transcript.

[0028] In one embodiment, the expression vector may be a bicistronic expression vector.

[0029] In one embodiment, the genetic construct, expression vector, RNA transcript, or polypeptide may be introduced into an antigen-presenting cell by electroporation.

[0030] In one embodiment, the antigen presenting cells may be of autologous or allogenic origin from the cancer patient.

[0031] In one embodiment, the vaccine may be a personalized anti-cancer vaccine.

[0032] Another aspect of the present invention provides a method for producing a vaccine composition for preventing or treating cancer, the method comprising the step of introducing into an antigen-presenting cell a gene construct that simultaneously expresses an MHC allele and a tumor antigen gene; an expression vector containing the gene construct; an RNA transcript transcribed from the gene construct; or a polypeptide produced from the gene construct, expression vector or RNA transcript. [Brief description of the drawings]

[0033] [Figure 1] 1 shows the structure of a gene construct that simultaneously expresses an MHC class I allele (ORF1) and a tumor antigen (ORF2) and an in vitro transcribed mRNA transcript therefrom, according to one embodiment of the present application. [Figure 2a] 1 shows the overall sequence structure of the reference template "LGV1007" according to one embodiment of the present application. [Figure 2b] This is a continuation of Figure 2a. [Diagram 3]1 shows the results of electrophoresis confirming that mRNA transcripts were produced by in vitro transcription of the reference template "LGV1007" according to one embodiment of the present application. [Figure 4a] In order to derive the conditions under which the expression level reaches its maximum level after electroporation of LGV1007 mRNA transcript according to one embodiment of the present application into K562 cells, the LGV1007 mRNA transcript was electroporated into K562 cells at an amount of 0, 3, 5, or 10 μg per 1 x 106 cells, and the expression level of HLA-A*0201 molecule was confirmed for 9 days. [Figure 4b] This is a continuation of Figure 4a. [Figure 4c] This is a continuation of Figure 4b. [Diagram 5] 1 shows the results of Western blot analysis confirming that a tumor antigen was expressed as a protein after in vitro translation of LGV1007 mRNA transcript according to one embodiment of the present application. [Figure 6] (a) shows the result of Western blot confirming that the MHC class I allele (ORF1) was expressed as a protein after electroporation of LGV1007 mRNA transcript into K562 cells according to one embodiment of the present application, and (b) shows the result of Western blot confirming that all of the tumor antigens (ORF2) were expressed as a protein. [Figure 7] The figure shows the results of electroporating LGV1007 mRNA transcripts into K562 cells, and confirming the expression level on the K562 cell surface using flow cytometry 24 hours later. [Figure 8] ELISPOT IFNγ secretion assay results measuring immune activation of PBMC T cells by K562 antigen-presenting cells electroporated with LGV1007 mRNA transcripts are shown. [Figure 9]The structure of LGV1032 mRNA transcript and the results of cellular expression in individual-made monocyte-derived dendritic cells (MoDCs) are shown below. LGV1032 has the same bicistronic transcript structure as LGV1007, maintains ORF2 (tumor antigen part), and has a GFP reporter protein located in ORF1 as a cellular expression marker. Like LGV1007, LGV1032 was confirmed to be effectively expressed in individual-derived autologous dendritic cells after being introduced into the cells by electroporation. [Figure 10] ELISPOT IFNγ secretion verification results show that LGV1032 electroporated into individual-derived autologous dendritic cells triggers antigen-specific T cell immune activity through effective antigen presentation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] The present invention will now be described in more detail.

[0035] One example of this application relates to nucleic acid molecules that simultaneously express MHC and tumor antigens.

[0036] Alternatively, it relates to a nucleic acid composition that simultaneously expresses an MHC and a tumor antigen.

[0037] Alternatively, it relates to a genetic construct that simultaneously expresses MHC and tumor antigens.

[0038] Alternatively, it relates to RNA transcripts from genetic constructs that simultaneously express MHC and tumor antigens.

[0039] The present invention also relates to an antigen-presenting cell in which a complex of MHC and a tumor antigen is overexpressed on the cell surface; and a vaccine composition for preventing or treating cancer comprising the same.

[0040] The terms "nucleic acid", "nucleic acid molecule", or "nucleic acid sequence" refer to a DNA or RNA molecule or sequence, and "nucleic acid composition" refers to a composition containing such a nucleic acid, nucleic acid molecule, or nucleic acid sequence.

[0041] The term "genetic construct" refers to a nucleic acid structure in which a gene insert encoding a protein of interest is operably linked for expression. In one embodiment, the genetic construct may be linear or circular, single or double stranded DNA, cDNA, RNA, etc., encoding two or more proteins of interest as a bicistronic transcript. The genetic construct may be, but is not limited to, part of a vector that can be used to transform, transfect or transfect a host, and may itself be transcribed and / or translated in vitro.

[0042] The term "operably linked" refers to a binding between nucleic acid sequences that is functionally linked. For example, a coding sequence (e.g., a sequence encoding a protein of interest) can be operably linked to appropriate regulatory elements to allow for its replication, transcription, and / or translation. For example, a coding sequence is operably linked to a promoter if the promoter is capable of driving the transcription of that coding sequence. Regulatory elements need not be contiguous with the coding sequence so long as they function correctly. For example, intervening sequences that are not translated but are transcribed can be present between the promoter sequence and the coding sequence, and the promoter sequence would still be considered "operably linked" to the coding sequence.

[0043] Each component in the genetic construct must be operably linked to each other, and linking of the component sequences may be accomplished by ligation at convenient restriction enzyme sites or, if no such sites exist, by the use of synthetic oligonucleotide adaptors or linkers in accordance with conventional methods.

[0044] The term "bicistronic" or its interchangeable term "polycistronic" refers to a system in which ribosomes can synthesize polypeptides within mRNA in eukaryotic cells, allowing synthesis of various polypeptides from a single mRNA. In one embodiment of the present application, a gene construct was designed so that a nucleic acid sequence encoding an MHC allele and a nucleic acid sequence encoding a tumor antigen can be simultaneously expressed from a single mRNA.

[0045] Generally, prokaryotic cells have a polycistronic system in which ribosomes bind to various positions on one mRNA to synthesize various proteins at once, whereas eukaryotes have a monocistronic system in which one mRNA is made under one promoter and only that gene is translated to synthesize a polypeptide. However, the gene construct of the present application can express various polypeptides from one mRNA in eukaryotic cells, for example, by placing each gene under its own promoter and expressing it, or by placing an IRES (internal ribosome entry site) between the two genes and simultaneously expressing two genes under one promoter.

[0046] As used herein, "comprising" can mean that it can include ingredients other than the recited ingredients, or that it consists essentially of the recited ingredients.

[0047] The term "MHC (major histocompatibility complex)" refers to proteins that serve to provide antigen fragments to immune cells so that they can distinguish between self and non-self molecules. There are two types of MHC: MHC class I and MHC class II. MHC class I is found in all cells that have a nucleus, whereas MHC class II is found in antigen-presenting cells. The technical concepts provided in this application are applicable to both MHC class I and MHC class II.

[0048] Generally, in adaptive immune responses, when a pathogen or antigen enters the body, antigen-presenting cells ingest it and break it down into short peptide fragments, and the peptides can bind to MHC class I or MHC class II molecules within the cell and be delivered to the cell surface. In this way, when a pathogen or antigen peptide binds to MHC class I or MHC class II and is presented on the cell surface of an antigen-presenting cell, T cells recognize it through the T cell receptor (TCR) and are activated to initiate an immune response. From this perspective, the pathogen or antigen peptide corresponds to the "epitope" of T cells.

[0049] Human MHC stands for human leukocyte antigen (HLA), and MHC class I (or HLA class I) molecules include HLA-A, HLA-B, and HLA-C. MHC class I molecules are composed of an alpha polypeptide chain and beta-2-microglobulin. MHC class I molecules interact with CD8+ cytotoxic T cells and play an important role in rejecting organ transplants and destroying infected cells.

[0050] MHC class II (or HLA class II) molecules include HLA-DR, HLA-DQ and HLA-DP. MHC class II molecules are composed of alpha and beta polypeptide chains. MHC class II molecules play an important role in recognizing non-self antigens and inducing cell-mediated immunity through interaction with CD4+ accessory T cells.

[0051] Genes that code for MHC are highly polymorphic and have many alleles that can induce immune responses to various pathogens or antigens. For example, as of January 2020, more than 18,000 HLA class I alleles have been reported in humans, and more than 7,000 HLA class II alleles. By individual locus, 5,735 HLA-A, 7,053 HLA-B, 5,653 HLA-C, 2,676 HLA-DRB1, 1,771 HLA-DQB1, and 1,519 HLA-DPB1 have been reported (www.ebi.ac.uk / imgt / hla / ).Another group of patients with HLA-A*2402, HLA-A*3303, HLA-A*0201, 101-101-A* HLA-A*0206、HLA-A*3101、HLA-A*0207、HLA-A*2601、HLA-A*2602、HLA-A*3 001、HLA-A*0101、HLA-A*3004、HLA-A*0203、HLA-A*0301、HLA-A*0205、HLA -A*0215N、HLA-A*2408、HLA-A*2420、HLA-A*2610、HLA-A*2901、HLA-A*260 3、HLA-A*3201、HLA-B*5101、HLA-B*1501、HLA-B*4403、HLA-B*3501、HLA-B *5801、HLA-B*4601、HLA-B*5401、HLA-B*1302、HLA-B*2705、HLA-B*0702、H LA-B*4006、HLA-B*4801、HLA-B*4001、HLA-B*5502、HLA-B*1301、HLA-B*40 02、HLA-B*5201、HLA-B*5901、HLA-B*1401、HLA-B*3901、HLA-B*6701、HLA- B*0801、HLA-B*1507、HLA-B*1518、HLA-B*3701、HLA-B*3802、HLA-B*0705、 HLA-B*1538、HLA-B*3511、HLA-B*4003、HLA-B*4402、HLA-B*5001、HLA-B*5 102、HLA-B*5601、HLA-B*1502、HLA-B*1511、HLA-B*1527、HLA-B*3503、HLA -B*4701、HLA-B*5605、HLA-B*570、HLA-Cw*0102、HLA-Cw*0304、HLA-Cw*14 02、HLA-Cw*0702、HLA-Cw*0801、HLA-Cw*0401、HLA-Cw*0302、HLA-Cw*0303 、HLA-Cw*1403、HLA-Cw*0602、HLA-Cw*0701、HLA-Cw*1202、HLA-Cw*0802、H LA-Cw*0202、HLA-Cw*0704、HLA-Cw*1203、HLA-Cw*0501、HLA-Cw*1505、HLA -Cw*0103、HLA-Cw*1502、HLA-Cw*150 7. You cannot have anything to do with anything else.In addition, since HLA genes are located close to each other across a distance of 4 megabases on the short arm of chromosome 6, they are characterized by being inherited as a single haplotype when passed from parents to children.

[0052] In a preferred embodiment, the MHC allele is an MHC class I allele or an MHC class II allele.

[0053] In a preferred embodiment, the MHC alleles are derived from the subject patient's tumor.

[0054] Also in a preferred embodiment, the genetic construct comprises a nucleic acid sequence for an MHC allele and a nucleic acid sequence encoding a tumor antigen.

[0055] Additionally, the nucleic acid sequence encoding the MHC allele can be fused at its N-terminus to a sequence encoding B2M (beta-2-microglobulin), which can contribute to the structural stability of the MHC allele at the cell surface when expressed in antigen-presenting cells. B2M is a non-glycosylated protein of approximately 12 kDa that acts to stabilize the alpha polypeptide chain of MHC, particularly MHC class I. The human B2M gene encodes a protein of 119 amino acids with 20 N-terminal amino acids encoding a leader sequence. The mature protein contains 99 amino acids. The gene contains 4 exons and 3 introns, the first exon contains the 5' non-coding region, the entire leader sequence and the first 2 amino acids of the mature polypeptide; the second exon encodes the majority of the mature protein; the third exon encodes the last 4 amino acids of the mature protein and a stop codon; and the fourth exon contains the 3' non-coding region. In the present application, fusion with B2M increases the stability of the peptide on the MHC molecule, and can increase the structural stability on the cell surface when expressed on antigen-presenting cells.

[0056] The sequence encoding B2M can include all or part of the nucleic acid residues corresponding to the entire human beta-2-microglobulin gene.

[0057] The term "tumor antigen" or "cancer antigen" refers to a molecule expressed in a tumor that elicits an immune response. Such an immune response can involve the production of antibodies, or the activation of specific immunologically-competent cells, or both.

[0058] Tumor antigens can be derived from the tumor-bearing organism, from dead or inactivated whole tumor cells, or from lysates, and include any antigen derived from a tumor. Lysates refer to materials resulting from the application of processes that cause the division of normal structures of cells. Tumor antigens also include any protein or other material with antigenic properties that is contained in tumor cells and is expressed differently from normal cells.

[0059] For example, tumor antigens include tumor-associated antigens (TAA) and tumor-specific antigens (TSA).

[0060] Tumor-associated antigens (TAA) are antigens that are more abundant in cancer cells than in normal cells or that appear at a different differentiation stage than normal cells, and are tumor shared antigens that are present in trace amounts in normal cells as well. Therefore, immune responses using these antigens are likely to fail due to self-tolerance, an immunosuppressive mechanism that prevents damage to self-cells, or conversely, unwanted organs may be attacked by autoimmunity.

[0061] Examples of tumor-associated antigens (TAA) include gp100, Melan-A / MART, MAGE-A, MAGE (melanoma antigen E), MAGE-3, MAGE-4, MAGE-A3, tyrosinase, TRP2, NY-ESO-1, CEA (carcinoembryonic antigen), PSA, p53, mammaglobin-A, survivin, Muc1 (mucin 1) / DF3, metallopanstimulin-1 (MPS-1), cytochrome P450 isoform 1B1, 90K / Mac-2 binding protein, Ep-CAM (MK-1), and HSP-70. , hTERT (TRT), LEA, LAGE-1 / CAMEL, TAGE-1, GAGE, 5T4, gp70, SCP-1, c-myc, cyclin B1, MDM2, p62, Koc, IMP1, RCAS1, TA90, OA1, CT-7, HOM-MEL-40 / SSX-2, SSX-1, SSX-4, HOM-TES-14 / SCP-1, HOM-TES-85, HDAC5, MBD2, TRIP4, NY-CO-45, KNSL6, HIP1R, Seb4D, KIAA1416, IMP1, 90K / Mac-2 binding protein, MDM2, NY-ESO-1, or LMNA.

[0062] Tumor-specific antigens (TSA) are antigens that are present specifically only in cancer cells. In particular, when tumors grow in cancer patients, cancer cell-specific genetic mutations occur, generating new antigen epitopes that can stimulate T cells, which are called neoantigens. In other words, neoantigens contain cancer cell-specific genetic mutations, and unlike tumor-shared antigens that are expressed in trace amounts in normal cells, they are selectively expressed only in cancer cells. They are recognized by the immune system as non-self foreign epitopes, inducing strong anti-cancer immune activity.

[0063] When peptides generated through mutated DNA are presented on the cell surface by MHCI, they are recognized by the T cell receptor (TCR), and because mutations do not occur in normal cells or tissues, neoantigen-specific T cells are free from self-tolerance and autoimmune problems. Because of these advantages, neoantigens are considered ideal targets for T cell-based cancer immunotherapy.

[0064] The causes of neoantigens include, but are not limited to, frame-shift deletion or insertion, which causes a shift in the genetic code due to the deletion or insertion of one or more nucleotides in DNA, point mutation, which occurs when one nucleotide is replaced by another, splice-site mutation, read-through mutation, or gene-fusion mutation.

[0065] Neoantigens are predicted through the analysis of specific cancer cell genomes of individual cancer patients. For example, after obtaining cancer cells from the patient's tumor and extracting DNA, the base sequence is analyzed, and this is compared with the base sequence of normal cells to select the part where mutations have occurred, and neoantigens that stimulate T cells can be identified from the base sequence of various parts where mutations have occurred. This can be done using big data processing such as next-generation base sequencing (NGS), whole-exome sequencing (WES) or RNA sequencing, MHC binding prediction computer programs, or artificial intelligence (AI) for neoantigen prediction, but is not limited to these. Since mutation forms are not shared between patients, neoantigens can be made into personalized anti-cancer vaccines.

[0066] The term "loading" or "pulsing" refers to the process by which antigen-presenting cells (APCs) such as dendritic cells capture and break down antigens, then display them on their surface on MHC molecules. These antigen-loaded cells can then induce potent antigen-specific T lymphocyte activity.

[0067] A genetic construct may include transcriptional and translational expression control sequences that allow the gene to be expressed in a selected host. Expression control sequences may include a promoter to effect transcription, an optional operator sequence to regulate such transcription, and / or sequences that regulate the termination of transcription and translation. Initiation and termination codons are generally considered to be part of the nucleic acid sequence that encodes the protein of interest, and must be functional in the individual when the genetic construct is administered, and must be in frame with the coding sequence.

[0068] For example, the genetic construct can include (a) from 5' to 3' direction, a promoter, a 5'-UTR, a nucleic acid sequence encoding an MHC allele, a nucleic acid sequence for an internal ribosome entry site (IRES), a nucleic acid sequence encoding a tumor antigen, and a 3'-UTR, or (b) from 5' to 3' direction, a promoter, a 5'-UTR, a nucleic acid sequence encoding a tumor antigen, a nucleic acid sequence for an internal ribosome entry site (IRES), a nucleic acid sequence encoding an MHC allele, and a 3'-UTR.

[0069] The term "promoter" refers to a DNA base sequence site to which a transcriptional regulatory factor binds, and for the purposes of the present invention, a promoter capable of inducing strong and stable gene expression can be used to increase the gene expression rate.

[0070] The promoter may be constitutive or inducible. Examples of promoters include, but are not limited to, early and late promoters of adenovirus, simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, HIV long terminal repeat (LTR) promoter, Moloney virus, cytomegalovirus (CMV) promoter, Epstein-Barr virus (EBV) promoter, Rous sarcoma virus (RSV) promoter, RNA polymerase ± promoter, T3 and T7 promoters, major operator and promoter regions of phage lambda, and the like.

[0071] The term "5'-UTR (untranslated region)" or "5'-non-translated region" refers to a region that is present at the 5'-end of an mRNA transcript but is not translated into amino acids. In a genomic sequence, the 5'-UTR is generally defined as the region between the transcription initiation site and the start codon. The 5'-UTR of a vertebrate mRNA can be from a few dozen bases to a few hundred bases long. The process of mRNA translation into protein begins with the binding of the 30S subunit of the ribosome to the 5'-UTR. Specifically, translation into protein begins when the 16S rRNA (16S ribosomal RNA) in the 30S subunit of the ribosome binds to the ribosome binding site (RBS) of the 5'-UTR and tRNA recognizes and binds to the start codon (AUG) of the mRNA. The ribosome binding site and the initiation codon of the 5'-UTR are located approximately 6 to 8 nucleotides apart, and within the ribosome binding site there is a sequence that is complementary to the 3' end sequence of 16S rRNA; this sequence is called the Shine-Dalgarno sequence.

[0072] The term "IRES (internal ribosome entry site)" refers to a base sequence that ribosomes recognize as the start of translation during the translation of a transcript. That is, IRES is a specific region present inside an mRNA to which ribosomes directly bind so that various polypeptides can be synthesized from one mRNA in eukaryotic cells, and it plays a role in making the gene construct of the present application bicistronic. That is, during the process of translation, the polypeptide coding sequence located on the 5' side of the IRES is translated from the 5' cap structure, and the polypeptide coding sequence located on the other side of the IRES is translated by binding a ribosomal subunit to the IRES, so that various polypeptides can be obtained separately.

[0073] IRES can be naturally occurring or synthetically derived and may be virally derived, such as poliovirus, EMC virus, or cellular derived, such as, but not limited to, immunoglobulin heavy chain binding protein (BiP), Drosophila antenna gene (Antp).

[0074] The term "3'-untranslated region (3'-UTR)" or "3'-untranslated region" refers to a region that is present at the 3'-end of an mRNA transcript but is not translated into amino acids.

[0075] Additionally, the genetic construct may include adaptors or linkers, enhancers, selection markers (e.g., antibiotic resistance markers), replicable units, polyA sequences, purification tags (e.g., GST, poly-Arg, FLAG, His-tags or c-my, etc.) or other constitutive and inducible sequences known to regulate expression of genes in prokaryotic or eukaryotic cells or viruses thereof, as appropriate, and various combinations thereof.

[0076] The present application also relates to RNA transcripts transcribed from the aforementioned genetic constructs, which simultaneously express MHC alleles and tumor antigens. The RNA transcripts are bicistronic in that the MHC alleles and tumor antigens can be simultaneously translated and synthesized from the RNA transcript.

[0077] The RNA transcript may be an mRNA transcript and may be isolated and purified after the genetic construct is transcribed in a cell, but is preferably transcribed in a test tube by in vitro transcription methods.

[0078] The present application also relates to a bicistronic expression vector comprising the gene construct. The term "vector" refers to a genetic construct that contains essential regulatory elements operably linked to a gene insert encoding a target protein so that the gene insert is expressed in the cells of an individual, and various types of vectors can be used, such as plasmids, viral vectors, bacteriophage vectors, cosmid vectors, etc.

[0079] The explanation for each component of the expression vector and the RNA transcript is substituted for the explanation for each component of the gene construct described above.

[0080] Other examples of the present application relate to polypeptides produced from said genetic constructs, expression vectors comprising said genetic constructs, or RNA transcripts transcribed from said genetic constructs.

[0081] Other examples of the present application relate to compositions for simultaneously expressing MHC alleles and tumor antigens, comprising the aforementioned genetic constructs; expression vectors comprising said genetic constructs; RNA transcripts transcribed from said genetic constructs; or polypeptides produced from said genetic constructs, expression vectors or RNA transcripts.

[0082] The present application also relates to an antigen-presenting cell comprising the genetic construct; an expression vector comprising the genetic construct; an RNA transcript transcribed from the genetic construct; or a polypeptide produced from the genetic construct, expression vector or RNA transcript.

[0083] The present application also relates to a method for producing a vaccine composition for preventing or treating cancer, comprising the step of introducing the genetic construct, an expression vector containing the genetic construct, an RNA transcript transcribed from the genetic construct, or a polypeptide produced from the genetic construct, expression vector or RNA transcript into an antigen-presenting cell.

[0084] The term "antigen-presenting cells" refers to any cell that achieves the purpose of the present application by promoting an immune response to an antigen (e.g., a tumor antigen). Antigen-presenting cells include dendritic cells (DCs), macrophages, or B cells. Antigen-presenting cells may be of autologous or allogenic origin to the cancer patient. The term "autologous" refers to cells obtained from an individual and used to treat the same individual, and "allogenic" refers to cells obtained from another individual of the same species.

[0085] Dendritic cells (DCs) are the body's most efficient antigen-presenting cell type, capable of phagocytosing foreign antigens and presenting them to naive (CD4+ or CD8+T) and memory T cells. Dendritic cells generally absorb antigens by cellular micropinocytosis and / or phagocytosis, process such antigens intracellularly, and then present the antigens to T cells of the immune system. While such processing generally occurs within the body, it is possible to isolate dendritic cells from the body, culture them in vitro, provide antigen to the in vitro cultured dendritic cells, and then return the cells to the body where they can interact with T cells to elicit an increased immune response to the antigen of interest. The process of providing the antigen to dendritic cells is generally referred to as loading or pulsing or co-cultivation.

[0086] In one embodiment, dendritic cell precursor cells can be obtained from a patient or an allogeneic or xenogeneic individual, differentiated into dendritic cells, and then the gene construct, RNA transcript, expression vector, or polypeptide can be introduced. For example, dendritic cells can be obtained by differentiation of mononuclear cells isolated from the blood of a patient to be vaccinated. Techniques for differentiation of dendritic cells from mononuclear cells are well established in the art. As a non-limiting example, in a typical protocol, peripheral blood mononuclear cells (PBMCs) can be isolated from whole blood by density gradient centrifugation, and the separation of mononuclear cells from PBMCs can be performed using the adherence of mononuclear cells or using immuno-autologous beads. The isolated mononuclear cells can be cultured in the presence of GM-CSF and IL-4 to differentiate into immature dendritic cells. The immature dendritic cells can be cultured in the presence of maturation factors, typically TLR-4 agonists, such as LPS, or cultured and matured using a mononuclear cell maturation cocktail containing TNFα, IL-6, IL-1β, and PGE2. Dendritic cell maturation and vector transduction may be carried out simultaneously.

[0087] Gene constructs, RNA transcripts, or expression vectors can be introduced into cells using standard techniques known in the art, including, but not limited to, electroporation, electroinjection, microinjection, calcium phosphate co-precipitation, calcium chloride / rubidium chloride, retroviral infection, DEAE-dextran, cationic liposome, polyethylene glycol-mediated uptake, gene gun, etc. In this case, circular constructs can be cut with appropriate restriction enzymes to introduce them in a linear form.

[0088] In a preferred embodiment, a gene construct, an RNA transcript, an expression vector, or a polypeptide can be introduced by electroporation. Electroporation refers to the application of an electric current or electric field to cells to promote the entry of a non-permeable substance into the cells. For example, when cells are suspended in a solution containing the non-permeable substance to be introduced (e.g., nucleic acid, vector, etc.) and a high-voltage DC pulse is passed through the cells, holes are formed in the cell membrane by electricity, and the non-permeable substance is introduced into the cells by the action of electrophoresis. The electroporation device can be a stationary electroporation device or a flow-type electroporation device. A stationary electroporation device includes a specialized cuvette containing electrodes submerged in a fluid that is in contact with a fixed amount of target cells. The molecule of interest is placed between the two electrodes and pulsed with high voltage.

[0089] Other examples relate to cancer prevention or treatment vaccine compositions and methods for their preparation, which comprise the above-mentioned genetic constructs, RNA transcripts, expression vectors, or polypeptides.

[0090] Other examples relate to cancer prevention or treatment vaccine compositions and methods for producing the same, which comprise antigen-presenting cells into which the above-mentioned gene constructs, RNA transcripts, expression vectors, or polypeptides have been introduced.

[0091] Another example relates to a cancer prevention or treatment vaccine composition comprising an antigen-presenting cell on whose cell surface a complex of an MHC allele and a tumor antigen is overexpressed, and a method for producing the same.

[0092] The term "vaccine" refers to a dosage form containing the antigen-presenting cells of the present application that can be administered to a subject. Thus, the vaccine composition of the present invention can be conveniently used to prevent, ameliorate, or treat a disease. After being introduced into a subject or host, the vaccine can induce an immune response, including, but not limited to, the production of antibodies, cytokines, and / or other cellular responses.

[0093] In a preferred embodiment, the MHC alleles and tumor antigens may be derived from tumor cells of the cancer patient to whom the composition is to be administered.

[0094] The cancer vaccine composition of the present application can simultaneously overexpress tumor antigen sequences, particularly neoantigen sequences, obtained from patient tumor analysis and tumor-derived MHC alleles of each patient in antigen-presenting cells (APCs), thereby expressing the maximum amount of antigen (or epitope)-MHC immune stimulating complexes on the cell surface to maximize the tumor antigen presentation efficacy of APCs, ultimately maximizing the proliferation and activation of patient-specific anti-cancer T cells and enhancing the anti-cancer immune effect.

[0095] Specifically, the antigen-presenting cells (e.g., dendritic cells) contained in the cancer vaccine composition of the present application can simultaneously overexpress MHC alleles and tumor antigens, and thus MHC alleles and tumor antigens are bound and expressed on the surface of the antigen-presenting cells, thereby presenting antigens to T lymphocytes, and when T lymphocytes are activated through this, strong cancer-specific cytotoxic T lymphocytes (CTLs) are induced to destroy cancer cells, thereby immunologically treating cancer. In addition, costimulatory substances abundantly expressed in antigen-presenting cells (e.g., dendritic cells) play an important role in the reaction between T lymphocyte receptors and antigen-MHC complexes, and can secrete various cytokines related to the differentiation, growth or influx of T lymphocytes to regulate the activity of immune responses, making them useful for anti-cancer immunotherapy.

[0096] The vaccine compositions of the present application may also include additional adjuvants that enhance the effectiveness of the vaccine. Suitable adjuvants include: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, and the like; (2) oil-in-water emulsion formulations (with or without the same specific immunostimulants as muramyl peptides or bacterial cell wall components), such as (a) 5% squalene, 0.5% Tween 80, and 0.5% Span 85 formulated into submicron particles (optionally, but not necessarily, containing various amounts of N-acetylglucosamine-L-alanyl-D-isoglutaminyl-L-alanine-2-(1',2'-diphenylphosphine)-2-phosphate dehydrogenase (PDE) and / or N-acetylglucosamine-2-phosphate dehydrogenase (PDE)). (b) SAF containing 10% squalene, 0.4% Tween 80, 5% Pluronic blocked polymer and N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP) that have been microfluidized to submicron particles or shaken to produce a large particle size emulsion; and (c) monophosphoryl lipid A (MPL), trehalose, and trehalose. (3) saponin immunoadjuvant; (4) Freund's complete immunoadjuvant (CFA) and incomplete immunoadjuvant (IFA); (5) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., gamma interferons), and inflammatory cytokines (e.g., inflammatory bowel disease, inflammation, and inflammatory diseases); (6) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., gamma interferons), and inflammatory cytokines (e.g., inflammatory bowel disease, inflammation, and inflammatory diseases); (7) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., gamma interferons), and inflammatory cytokines (e.g., inflammatory bowel disease, inflammation, and inflammatory diseases); (8) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., gamma interferons), and inflammatory cytokines (e.g., inflammatory bowel disease, inflammation, and inflammatory diseases); (9) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., gamma interferons), and inflammatory cytokines (e.g., inflammatory (6) bacterial ADP-ribosylating toxins, such as cholera toxin (CT), pertussis toxin (PT), or the heat-labile toxins (LT) of Escherichia coli (E. coli), particularly detoxified mutants of LT-R72, CT-S109, PT-K9 / G129 (WO93 / 13302 and WO92 / 19265); and (7) other substances that act as immune stimulants to enhance the effectiveness of vaccines.

[0097] Vaccine compositions can include a conventional saline or buffered aqueous medium in which the vaccine is suspended or dissolved. For example, diluents such as water, saline, glycerol, ethanol, etc., can be included, and auxiliary substances such as wetting agents, emulsifying agents, pH buffering agents, etc., can be present in the composition.

[0098] Suitable forms for injection include sterile aqueous solutions (water-soluble) or dispersions for extemporaneous preparation of sterile injectable water or dispersions, and sterile powders. They must be stable under the conditions of manufacture and must be preserved against the contamination of microorganisms such as bacteria or fungi. Microbial contamination can be prevented by using various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of the injection composition can be achieved by using an agent that delays absorption, for example, aluminum monostearate or gelatin, in the composition.

[0099] Sterile water for injection is produced by combining the required amount of antigen-presenting cells and various other ingredients listed above in the above-mentioned solvent, and sterilizing the remaining ingredients, for example, a buffer solution such as PBS, excluding antigen-presenting cells and / or heat-sensitive immunopotentiator cytokines, if necessary. In general, dispersions are produced by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and other necessary ingredients from those mentioned above. In the case of sterile powders for the production of sterile water for injection, the preferred production method is vacuum drying and freeze-drying techniques. By such techniques, powders of active ingredients and any desired ingredients are obtained from the above-mentioned sterile-filtered solutions.

[0100] Without limiting the action of the present invention in any manner, the delivery of dendritic cells according to the present invention is particularly useful for inducing an immune response, in particular a cytotoxic T-lymphocyte response against an antigen, said immune response may be a specific (T cell and / or B cell) and / or a non-specific immune response.

[0101] Thus, another aspect of the present invention relates to a method for generating, inducing or enhancing an immune response to an antigen in a human, particularly a cancer patient, which method comprises administering to the cancer patient an effective amount of a vaccine composition or antigen-presenting cells as described above.

[0102] The immune response preferably comprises a cytotoxic T-lymphocyte response, which in accordance with the present invention may occur in conjunction with or independently of a helper T-cell response, a humoral response or other specific or non-specific immune response.

[0103] Yet another aspect of the present invention relates to the use of the dendritic cells of the present invention in connection with the treatment and / or prevention of disease states. Examples of diseases that can be treated by the methods of the present invention include various cancer diseases and intractable cancer diseases.

[0104] For example, the cancer may be a solid cancer or a blood cancer, including, but not limited to, breast cancer, lung cancer, prostate cancer, ovarian cancer, brain cancer, liver cancer, cervical cancer, endometrial cancer, uterine cancer, colon cancer, large intestine cancer, colorectal cancer, rectal cancer, kidney cancer, nephroblastoma, skin cancer, oral squamous cell carcinoma, epidermal cancer, nasopharyngeal cancer, head and neck cancer, bone cancer, esophageal cancer, bladder cancer, lymphatic cancer (e.g., Hodgkin's lymphoma or non-Hodgkin's lymphoma), stomach cancer, pancreatic cancer, The cancer may be testicular cancer, thyroid cancer, follicular thyroid cancer, melanoma, myeloma, multiple myeloma, mesothelioma, osteosarcoma, myelodysplastic syndrome, tumor of mesenchymal origin, soft tissue sarcoma, liposarcoma, gastrointestinal stromal tumor, malignant peripheral nerve sheath tumor (MPNST), Ewing's sarcoma, leiomyosarcoma, mesenchymal chondrosarcoma, lymphosarcoma, fibrosarcoma, rhabdomyosarcoma, teratocarcinoma, neuroblastoma, medulloblastoma, glioma, benign tumor of the skin, or leukemia. The lung cancer may be, for example, small cell lung carcinoma (SCLC) or non-small cell lung carcinoma (NSCLC). The leukemia may be, for example, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL) or chronic lymphocytic leukemia (CLL). The subject to be treated may be a subject undergoing a second line antihyperproliferative therapy. For example, the secondary anti-hyperproliferative therapy may be chemotherapy, radiation therapy, immunotherapy, phototherapy, cryotherapy, toxin therapy, hormone therapy, or surgery.

[0105] Thus, another aspect of the invention provides a method of treating cancer by enhancing a tumor-specific immune response using the cancer vaccine composition, where administration of the composition generates, induces or promotes an immune response that inhibits, halts, delays or prevents the onset or progression of a disease state.

[0106] Direct delivery of the composition can generally be systemic, subcutaneous, intradermal, intraperitoneal, intravascular (intravenous), intramuscular or local, or delivered to the interstitial space of tissue. The composition can also be administered to a lesion. The administration regimen can be a single dose or a multiple dose schedule.

[0107] The term "effective amount" means an amount sufficient to achieve a desired result when administered to an individual, including humans, e.g., an amount effective to treat or prevent cancer. The effective amount will vary depending on factors such as the disease state, age, sex, and weight of the individual. Dosage or treatment regimen can be adjusted to provide an optimal therapeutic response, as will be appreciated by those skilled in the art.

[0108] A treatment regimen for an individual using a therapeutically effective amount may consist of a single administration, or, as another example, may include a series of applications. The duration of treatment is determined by various factors such as the severity of the disease, the age of the individual, the concentration of the cancer vaccine, the patient's responsiveness to the cancer vaccine, or a combination thereof. It should also be understood that the effective dosage of the cancer vaccine used for treatment may be increased or decreased during the course of an individual treatment regimen. Dosage variations may occur and may be evident through standard diagnostic assays known in the art. In some aspects, the cancer vaccine of the present invention may be administered before, during, or after treatment with conventional anticancer drugs, radiation therapy, hormone therapy, biotherapy, and / or surgical tumor resection.

[0109] The present invention will now be described in more detail with reference to the following examples, which are merely for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. EXAMPLES

[0110] Example 1. Construction of a bicistronic expression vector The bicistronic vector for simultaneously expressing MHC class I alleles and tumor antigens is constructed as shown in FIG. 1, specifically, it contains the following gene construct from 5' to 3' direction: 5'UTR-ORF1 (open reading frame containing a fusion of recombinant B2M and MHC class I alleles; specifically, a coding sequence for a fusion protein containing B2M at the N-terminus and a tumor-derived MHC class I allele at the C-terminus)-IRES-ORF2 (coding sequence for a recombinant protein composed of multiple antigenic epitopes in a beads-on-a-string structure)-3'UTR. Specifically, the reference template was named "LGV1007" and had a sequence coding for a fusion protein (SEQ ID NO: 1) including a signal sequence, B2M, and HLA-A*0201 in the N-terminal to C-terminal direction as ORF1, and a coding sequence for a shared antigen recombinant protein (SEQ ID NO: 2) including NY-ESO1, MAGE-A3, SURVIVIN, MULTI-MAGE-A, and MELAN-A as ORF2. The entire sequence structure of the LGV1007 is shown in FIG. 2, and the entire DNA sequence (3,523 bp) including the BamHI site at the 5' end to the XbaI site at the 3' end of the LGV1007 is shown in SEQ ID NO: 3, and was obtained by requesting gene synthesis (GeneArt, Thermo Fisher). However, LGV1007 is merely a representative example according to one embodiment of the present application, and as described above, the ORF1 and ORF2 base sequences can be altered to include autologous sequences in a custom-made manner for individual cancer patients, and the base sequence excluding ORF1 and ORF2 can be optimized and standardized for antigen presentation using sequences commonly known in the art.

[0111] Example 2. Generation of mRNA transcripts using in vitro transcription (IVT) To generate the bicistronic mRNA transcript, the bicistronic vector (DNA plasmid vector) obtained in Example 1 above was linearized by cleavage with the restriction enzyme Xbal, and the linearized DNA was purified by ExpinTM The purified linearized plasmid template was purified using the combo GP kit (GeneAll, 112-102). TM The T7 Ultra transcription kit (ThermoFisher) was used for IVT (in vitro transcription) mRNA production according to the manufacturer's instructions. The generated mRNA transcripts were finally purified using the LiCl precipitation method. Specifically, the mRNA products were gently mixed with LiCl precipitation solution (50 μL per 20 μL IVT reaction) and incubated at -20°C for at least 30 min. The samples were then spun down by centrifugation at 20,000xg for 14 min at 4°C. The pellet was then washed with 70% ethanol. The final mRNA products were resuspended in nuclease-free deionized water. The amount of mRNA product was measured using a Nanodrop2000.

[0112] FIG. 3 shows the results of in vitro transcription of the reference template LGV1007, followed by PAGE electrophoresis to confirm that mRNA transcripts were produced, and the expected size of the product was confirmed (~3.5 Kb, lane 1).

[0113] Example 3. Cell electroporation for intracellular mRNA delivery The mRNA transcripts obtained in item 2 above were transferred into human cells using cell electroporation. K562 human myeloid-derived lymphoblastic leukemia cells (ATCC CCL-243 TM ) by Amaxa TM 4D-Nucleofector TM X Unit Instrument and SF Cell Line Nucleofector TM K562 cells were electroporated using 3×10 PBS Solution (Lonza) according to the manufacturer's protocol. Two days before electroporation, 3×10 PBS was added to the 5×10 5 On the day of electroporation, count the cells and prepare the required number of cells (1–2 × 10 6Before electroporation, cells were washed twice with serum-free RPMI or Opti-MEM (Gibco) and then immersed in 100 μl of SF Cell Line 4D Nucleofector ... TM The cells were finally resuspended in 1× Solution (82 μl Nucleofection Solution, 18 μl Supplement). Then, 0.1 ml of the cell suspension was mixed with 0–20 μg of in vitro transcribed (IVT) LGV1007 mRNA using program FF-120. After electroporation, fresh culture medium was added to the cell suspension, and the electroporated cells were incubated at 37°C and CO 2 Immediate incubation was performed in a cell culture incubator.

[0114] Figure 4 shows the results of electroporation of LGV1007 mRNA transcripts into K562 cells. 6 The expression level of HLA-A*0201 molecules was confirmed for 9 days after electroporation into K562 cells at 0, 3, 5, or 10 μg per cell, and the maximum expression level (maximum 65 times the standard expression level) was observed 24 hours after electroporation of 10 μg of the transcript, and after 48 hours, it rapidly decreased and was continuously maintained at an expression level of about 5 to 2 times the standard value. Such maximum overexpression of MHC-I alleles of non-antigen presenting cells (i.e., individual patient tumor custom-made type) can be expected to have the effect of substantially unifying the MHC-I phenotype of specific antigen presenting cells with the patient tumor custom-made type, thereby maximizing the activation of anti-cancer immune T cells of individual patients.

[0115] Example 4. In vitro translation of mRNA products For in vitro translation of Flag-tagged polyantigen recombinant proteins, LGV1007 template DNA plasmid was used in a single-tube in vitro transcription / in vitro translation reaction using the TnT Quick Coupled Transcription / Translation kit (Promega) or the 1-Step Human High-Yield Mini IVT kit (Thermo Scientific), following the manufacturer's manual. The reaction volume was scaled down from 100 μl to 25 μl. For the TnT Quick Coupled Transcription / Translation kit (Promega), the reaction mixture was incubated at 30°C for 60–90 min, and for the 1-Step Human High-Yield Mini IVT kit (Thermo Scientific), the reaction time was 6–16 h. FIG. 5 shows the results of in vitro translation of LGV1007 mRNA transcripts to produce Flag-tagged polyantigen recombinant proteins, confirmed by Western blot analysis.

[0116] Example 5. Protein Expression Analysis After mRNA electroporation into K562 cells, Western blot analysis was performed for the cellular expression of recombinant MHC-I proteins fused with B2M and HLA-A*0201. Western blot of in vitro translated flag-tagged polyantigen recombinant proteins was also performed to confirm the protein expression of polyantigen recombinant proteins. Specifically, K562 cells electroporated with LGV1007 mRNA were collected, washed twice with PBS, and lysed in RIPA lysis buffer containing protease inhibitor cocktail (ATTO) at 4°C. Following standard Western blot protocols, 20-30 μg of protein was mixed with SDS sample buffer and analyzed by running SDS-PAGE on Tris-Glycine precast 12% or 4-20% gels (KOMABIOTECH). Immunoblot antibodies were obtained from the following sources: beta-2 microglobulin (Cell Signaling) and FLAG (Sigma-Aldrich). Anti-beta-2 microglobulin or anti-FLAG M2 antibodies were applied overnight at 4°C. Horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology) were applied for 1 h at room temperature. Enhanced chemiluminescence detection kits (ECL, Amersham or Pierce) were used to detect immunoreactive proteins. Western blotting signals were detected using an Amersham Imager680.

[0117] Figure 6 shows the results of Western blot analysis confirming that the MHC class I allele (ORF1) and tumor antigen (ORF2) were all expressed as proteins after electroporation of LGV1007 mRNA transcripts into K562 cells. The ORF1 (B2M-HLA-A*0201) protein was confirmed to be ~51 kDa in size (Figure 6a), and ORF2 (tumor antigen fusion protein) was confirmed to be expressed at the expected size of ~30 kDa (Figure 6b). However, the tumor antigen fusion protein was confirmed to be clearly expressed only by the in vitro translation method, as it was rapidly degraded by the proteasome when expressed in cells.

[0118] Example 6. Flow cytometry analysis Intellicyt iQue TM Flow cytometry was performed with a Screener PLUS (Sartorius) flow cytometer. Specifically, 24 to 72 hours or up to 2 weeks after nucleofection, K562 cells electroporated with LGV series mRNA were collected and centrifuged at 300xg for 5 minutes. The cell pellet was resuspended in flow cytometry buffer (DPBS containing 1% FBS and 2mM EDTA). For cell staining, APC-conjugated-mouse anti-human HLA-A2 (BD Pharmigen) was added at the recommended concentration, and the cells were then stained by incubation for 30 minutes to 1 hour in the dark and at room temperature. After staining incubation, the cells were pelleted and resuspended in PBS. Flow cytometry results were analyzed with Intellicyt ForeCyt software (Sartorius).

[0119] Figure 7 shows that LGV1007 mRNA transcripts were 1 × 10 6 K562 cells were electroporated with 1 ug per cell, and the expression level on the K562 cell surface was examined 24 hours later using flow cytometry, confirming overexpression of ~10-fold compared to baseline.

[0120] Example 7. Immune Cell Activation Using ELISPOT IFNγ Cytokine Secretion Assay A comparative study was conducted on the efficacy of immune activation between peptide pulse and RNA antigen loading, which are traditional antigen loading methods, using K562apc antigen-presenting cells. Specifically, on the first day, an ELISPOT IFNγ immune activity comparison experiment was conducted between K562apc cells electroporated with LGV1007 mRNA transcripts (containing five antigen sequences: SURVIVIN, MART1, NYESO1, MAGEA3, MULTIMAGE) prepared in Example 3, K562apc cells loaded with a reference antigen (SURVIVIN 9 amino acids peptide), or K562apc control cells not treated with peptide. Purified PBMC (peripheral blood mononuclear cell) CD8+ T cells in a cell suspension of 2.5 x 10e5 cells / ml were stimulated with APC. 100 μL / well T cells and 100 μL APCs / well were added (mixed at 2:1 to 10:1 ratios of T cells vs. APCs) and the cell mixture was incubated at 37°C for 24 h. On day 2, the culture medium was replaced with 10 U / mL IL-2 and 5 ng / mL IL-15, and IL-2 was replenished 7 days later. 11 days after APC stimulation, IFNγ-secreting T cells were analyzed by ELISPOT IFNγ protocol (BD Bioscience), following the manufacturer's instructions. The positive spot development was carefully monitored, and the assay plate was dried in air at room temperature for 2 h to overnight until completely dry. PBMC T cell activation analysis was performed by directly counting the positive spots under a dissecting microscope or automatically counting the positive spots using an ELISPOT plate reader.

[0121] Figure 8 shows the results of ELISPOT IFNγ secretion analysis to measure the immune activity of PBMC T cells from K562apc cells electroporated with LGV1007 mRNA transcripts, K562apc cells loaded with a reference antigen (SURVIVIN9 amino acids peptide), and K562apc control cells not treated with peptide. K562 antigen-presenting cells activated through electroporation with the reference RNA LGV1007 primed and activated PBMC CD8+ T cells with the same MHC class I allele type (HLA-A*0201), and positive spots secreting IFNγ were confirmed. In contrast, CD8 T cells derived from a heterologous MHC class I allele type (non-HLA-A*0201), used as an MHC genotype-restricted control group, were not primed by APC. Through this experiment, we confirmed that K562apc cells electroporated with LGV1007 mRNA transcripts induced superior ex vivo human PBMC immune cell activation compared to traditional peptide antigen loading.

[0122] Example 8. Preparation of autologous dendritic cells transfected with LGV1032 and activation of immune cells using the same We conducted experiments to verify whether the LGV RNA antigen loading method can effectively trigger antigen-specific immune cell activation through antigen loading and antigen presentation not only in laboratory-optimized K562apc but also in personalized autologous dendritic cells with diverse MHC class I and class II extracted from each patient and cultured and matured. The results are shown in Figures 9 and 10.

[0123] FIG. 9 shows the results of FACS analysis confirming the structure of LGV1032 mRNA transcript and its cellular expression in autologous dendritic cells derived from individuals. LGV1032 has the same bicistronic transcript structure as LGV1007 (Example 1), with ORF2 (tumor antigen part) maintained and GFP reporter protein located in ORF1 as a cellular expression marker. Therefore, the method for producing LGV1032 is the same as that for producing LGV1007. As a result of flow cytometry as described above, it was confirmed that LGV1032, like LGV1007, is effectively expressed in autologous dendritic cells derived from individuals after being introduced into the cells by electroporation. Autologous dendritic cells derived from individuals or made to order for individuals are produced by collecting blood, differentiating monocytes into dendritic cells in an immature state, and then undergoing a maturation process to obtain mature dendritic cells. This in vitro cell differentiation and maturation process takes about a week, and it is known that the final matured dendritic cells have the highest antigen presentation ability. As can be seen from the FACS analysis results of the GFP reporter protein in Figure 9, it was confirmed that LGV1032 was effectively electroporated into cells at all stages (monocyte, immature DC, mature DC) and expressed after cell inflow.

[0124] Figure 10 shows the ELISPOT IFNγ secretion spot increase results, proving that LGV1032 electroporated into individual-derived autologous dendritic cells (MoDCs) effectively stimulates antigen-specific T cell immune activity. It was clearly verified through the ELISPOT IFNγ secretion spot increase that LGV1032 electroporated autologous dendritic cells (Figure 10, columns 3 & 4) stimulated MART1 antigen-specific TCR T cell activation very effectively compared to autologous dendritic cells pulsed with peptide antigens (Figure 10, column 2), which is the traditional method.

[0125] From the above description, it should be understood by those skilled in the art that the present invention can be embodied in other specific forms without changing its technical ideas or essential features. Therefore, the above-described embodiments should be understood as illustrative and not limiting in all respects. The scope of the present invention should be interpreted as including all modifications and variations derived from the meaning and scope of the claims below, rather than the above detailed description, and the equivalent concepts thereof.

Claims

1. The present invention relates to an antigen-presenting cell in which a complex of MHC (major histocompatibility complex) and a tumor antigen is recombinantly expressed on the cell surface. A cancer prevention or treatment vaccine composition, comprising: The antigen-presenting cell is Bicistronic gene constructs that simultaneously express MHC alleles and tumor antigen genes; an expression vector comprising the bicistronic gene construct; or an RNA transcript transcribed from said bicistronic gene construct, which simultaneously expresses an MHC allele and a tumor antigen gene; A composition, wherein the MHC alleles and tumor antigens are derived from tumor cells of a patient with cancer to be treated.

2. The composition of claim 1 , wherein the antigen-presenting cell is a dendritic cell.

3. The composition of claim 1 , wherein the MHC is MHC class I or MHC class II.

4. The composition of claim 1 , wherein the bicistronic gene construct comprises a nucleic acid sequence of an MHC allele and a nucleic acid sequence encoding a tumor antigen.

5. The composition of claim 4, wherein the nucleic acid sequence of the MHC allele is fused at its N-terminus to a sequence encoding beta-2-microglobulin (B2M).

6. 2. The composition of claim 1, wherein the tumor antigen comprises a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), or a tumor-derived neoantigen (neoantigen).

7. The composition of claim 6, wherein the tumor-derived neoantigen contains a mutation that is specifically expressed in cancer cells.

8. Tumor-associated antigens (TAAs) include gp100, Melan-A / MART, MAGE-A, MAGE (melanoma antigen E), MAGE-3, MAGE-4, MAGE-A3, tyrosinase, TRP2, NY-ESO-1, CEA (carcinoembryonic antigen), PSA, p53, Mammaglobin-A, Survivin, Mucl (mucin 1) / DF3, metallopanstimulin-1 (MPS-1), cytochrome P450 isoform 1B1, 90K / Mac-2 binding protein, Ep-CAM (MK-1), HSP-70, hT 7. The composition of claim 6, wherein the protein is ERT (TRT), LEA, LAGE-1 / CAMEL, TAGE-1, GAGE, 5T4, gp70, SCP-1, c-myc, cyclin B1, MDM2, p62, Koc, IMP1, RCAS1, TA90, OA1, CT-7, HOM-MEL-40 / SSX-2, SSX-1, SSX-4, HOM-TES-14 / SCP-1, HOM-TES-85, HDAC5, MBD2, TRIP4, NY-CO-45, KNSL6, HIP1R, Seb4D, KIAA1416, IMP1, 90K / Mac-2 binding protein, MDM2, NY-ESO-1, or LMNA.

9. The bicistronic gene construct comprises: (a) comprising, in the 5' to 3' direction, a promoter, a 5'-UTR, a nucleic acid sequence for an MHC allele, a nucleic acid sequence for an IRES (internal ribosome entry site), a nucleic acid sequence encoding a tumor antigen, and a 3'-UTR; (b) the composition of claim 1, comprising, from 5' to 3', a promoter, a 5'-UTR, a nucleic acid sequence encoding a tumor antigen, a nucleic acid sequence of an IRES (internal ribosome entry site), a nucleic acid sequence of an MHC allele, and a 3'-UTR.

10. 10. The composition of claim 9, wherein the nucleic acid sequence of the MHC allele is fused at its N-terminus to a sequence encoding beta-2-microglobulin (B2M).

11. The composition of claim 1 , wherein the RNA transcript is an mRNA transcript.

12. The composition of claim 1 , wherein the antigen-presenting cells are autologous or allogenic to the cancer patient.

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