General-purpose DC, its manufacturing method and applications

Abstract

This provides a type of DC immunotherapy that can overcome limitations in the specific matching of HLA molecules, particularly in the specific matching of HLA class I molecules. [Solution] A general-purpose DC, its manufacturing method, and its uses are disclosed, belonging to the field of immunocytology. The general-purpose DC is a general-purpose DC that does not express an HLA class I molecule, obtained by knocking out the HLA class I gene in a primitive DC. When using it, based on the HLA class I molecule subtype of the target antigen-specific T cell to be obtained, an HLA class I molecule that matches the target antigen-specific T cell is reexpressed in the general-purpose DC using genome editing, viral, or mRNA technology, thereby overcoming the limitations of cell-specific matching of HLA class I molecules present when using heterologous DCs and solving the problem of insufficient coverage of HLA class I molecule subtypes in a single DC.

Description

[Technical Field]

[0001] This application belongs to the field of immunotherapy technology and specifically relates to a type of general-purpose DC, its manufacturing method, and its applications. [Background technology]

[0002] The immune system has the ability to recognize non-self components and eliminate potentially harmful non-self molecules and cells from the body, as well as to recognize and destroy abnormal cells originating from the host's own tissues. Immune responses include innate immunity and adaptive immunity. Adaptive immunity includes humoral immunity mediated by B cell responses (B cells developing into plasma cells and secreting soluble antigen-specific antibodies) and cellular immunity mediated by specific subsets of T cells. T cell-dependent (T cell-mediated) adaptive immune responses generally require antigen-presenting cells (APCs) to present antigen peptides bound to major histocompatibility complex (MHC) molecules to T cells. In humans, MHC is also called the human leukocyte antigen (HLA) complex.

[0003] Dendritic cells (DCs) are a type of professional antigen-presenting cell involved in both cellular and humoral immunity, serving as important nodes in the entire immune system and significantly influencing immune system function. One of the main functions of DCs is to recognize captured antigens and present them to naive T lymphocytes, stimulating T cell activation and proliferation, and producing a large number of T lymphocytes that recognize specific antigens and possess potent cytotoxicity, i.e., CTL cells (cytotoxic T lymphocytes). T cell activation requires the binding of CD4 or CD8 on the T cell surface to HLA molecules, as well as mutual recognition and interaction between alpha and beta peptide chains on the T cell receptor (TCR) on the T cell surface and the antigen-MHC complex.

[0004] The HLA molecules used to present antigens mainly consist of two types: HLA class I molecules and HLA class II molecules. Of these, HLA class I molecules mainly consist of three types: HLA-A, HLA-B, and HLA-C molecules, and the genes encoding these three types of molecules are the HLA-A gene, HLA-B gene, and HLA-C gene, respectively. CD8 T cells mainly react with HLA class I molecules, and then undergo activation and proliferation, producing a large number of T lymphocytes that recognize specific antigens and have potent cytotoxicity.

[0005] The HLA gene, located on human chromosome 6, is the most complex and polymorphic genetic system in the population to date, possessing a vast number of alleles. Consequently, the HLA molecule itself exhibits a high degree of polymorphism, resulting in significant structural and compositional differences between HLA molecules of different individuals. This is a crucial cause of immune responses during organ transplantation because the HLA molecules on the cell surface of the donor organ are foreign antigens to the recipient's immune system. The same problem exists when activating T cells by presenting captured antigens using heterologous dendritic cells (DCs) between different individuals. Because the HLA molecules of the recipient T cells do not match those of the donor DCs, CD4 or CD8 on the surface of the recipient T cells cannot bind to the HLA molecules on the surface of the donor DCs. This prevents mutual recognition and interaction between the TCR and the antigen-MHC complex, thus preventing the activation of target T cells. In other words, there is a limitation in the specific matching of HLA molecules.

[0006] In 1996, Hsu FJ et al. from the Department of Oncology at Stanford University Medical Center reported the first clinical trials of a dendritic cell vaccine in Nature Medicine. Research on DC tumor vaccines worldwide has been ongoing for several decades, and currently, several DC tumor vaccines have been approved for market, including Sipuleucel-T (Provenge), CreaVax RCC, Hybricell, and DCVax Brain. Conventional DC immunotherapy involves first collecting peripheral blood mononuclear cells (PBMCs) from the patient, differentiating them into mature DCs through a series of in vitro inductions and cultures, then loading the DCs with tumor or viral antigens in vitro, and then either directly returning them to the patient's body, or stimulating and activating antigen-specific T cells in vitro, and then returning (infusing) the activated antigen-specific T cells.

[0007] Conventional treatment methods for obtaining and differentiating mature DCs are limited by their supply sources, time-consuming and labor-intensive nature, and the limitations of specific HLA molecule matching mean that the only option is to collect peripheral blood mononuclear cells from the individual's own body and induce differentiation. This further increases the burden on some individuals whose bodies are already in an unhealthy state.

[0008] Therefore, a type of DC immunotherapy that can overcome the limitations of specific matching of HLA molecules, particularly HLA class I molecules, is of great importance. [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] To address the limitation in specific matching of HLA class I molecules when using heterologous dendritic cells (DCs) as described above in the prior art, this application provides a general-purpose DC, a method for producing the same, and its uses. By knocking out the HLA class I genes (HLA-A, HLA-B, and / or HLA-C) in primitive DCs, a general-purpose DC that does not express HLA class I molecules is obtained. When using this general-purpose DC, based on the HLA class I molecular subtype of the target antigen-specific T cell to be obtained, the HLA class I molecular subtype that matches the target antigen-specific T cell is reexpressed in the general-purpose DC using genome editing, viral or mRNA technology, thereby overcoming the limitation in specific matching of HLA class I molecules that exists when using heterologous dendritic cells, and also solving the problem of insufficient coverage of HLA class I molecular subtypes in a single DC. [Means for solving the problem]

[0010] To solve the above-mentioned problems, the technical solutions employed in this application are as follows:

[0011] In a first embodiment, the present application provides a general-purpose DC. The HLA class I gene of the general-purpose DC is knocked out, and the HLA class I gene includes the HLA-A gene, the HLA-B gene, and / or the HLA-C gene, and the general-purpose DC overcomes the limitation of specific matching of HLA class I molecules. That is, a DC before the knockout of the HLA class I gene (primitive DC) can only stimulate and activate antigen-specific T cells that are identical (matched) to its own HLA-A molecule, HLA-B molecule, and / or HLA-C molecule subtype. However, a general-purpose DC with the knocked-out HLA class I gene can stimulate antigen-specific T cells that match new HLA-A molecule, HLA-B molecule, and / or HLA-C molecule subtype by reintroducing the HLA-A gene, HLA-B gene, and / or HLA-C gene. The term "HLA class I gene" includes three types: HLA-A, HLA-B, and HLA-C genes. However, as those skilled in the art know, the restriction of specific molecular matching is primarily a restriction of one gene (subtype). Therefore, "HLA class I gene" here can refer to any one or combination of HLA-A, HLA-B, and HLA-C genes. For example, "HLA class I gene" here could be HLA-A, HLA-B, HLA-C, HLA-A and HLA-B, HLA-B and HLA-C, HLA-A and HLA-C, or HLA-A, HLA-B, and HLA-C genes.

[0012] In some specific embodiments of this application, the HLA-A gene of the above-mentioned general-purpose DC is knocked out, and the general-purpose DC is freed from the restriction of specific matching of HLA-A molecules. That is, a DC before the HLA-A gene is knocked out (primitive DC) can only stimulate and activate antigen-specific T cells that are identical (matched) to its own HLA-A molecular subtype, but a general-purpose DC with the HLA-A gene knocked out can stimulate and activate antigen-specific T cells that match a new HLA-A molecular subtype by reintroducing a new HLA-A gene.

[0013] In some specific embodiments of this application, the HLA-B gene of the above-mentioned general-purpose DC is knocked out, thereby removing the restriction on specific matching of the HLA-B molecule in the general-purpose DC.

[0014] In some specific embodiments of this application, the HLA-C gene of the above-mentioned general-purpose DC is knocked out, thereby removing the restriction on specific matching of the HLA-C molecule in the general-purpose DC.

[0015] In some specific embodiments of this application, the HLA-A and HLA-B genes of the above-mentioned general-purpose DC are knocked out, thereby removing the restriction on specific matching of HLA-A and HLA-B molecules in the general-purpose DC. That is, a DC before the knockout of the HLA-A and HLA-B genes (primitive DC) can only stimulate and activate antigen-specific T cells that are identical (match) to its own HLA-A molecule and / or HLA-B molecular subtype. However, a general-purpose DC with knocked-out HLA-A and HLA-B genes can stimulate and activate antigen-specific T cells that match a new HLA-A molecule and / or a second HLA-B molecular subtype by reintroducing a new HLA-A gene and / or HLA-B gene.

[0016] In some specific embodiments of this application, the HLA-A and HLA-C genes of the above-mentioned general-purpose DC are knocked out, thereby removing the restriction on specific matching of HLA-A and HLA-C molecules in the general-purpose DC.

[0017] In some specific embodiments of this application, the HLA-B and HLA-C genes of the above-mentioned general-purpose DC are knocked out, thereby removing the restriction on specific matching of HLA-B and HLA-C molecules in the general-purpose DC.

[0018] In some specific embodiments of the present application, the HLA-A gene, HLA-B gene, and HLA-C gene of the above-mentioned general-purpose DC are knocked out, and the general-purpose DC releases the restrictions of specific matching of HLA-A molecules, HLA-B molecules, and HLA-C molecules. By reintroducing a new HLA-A gene, HLA-B gene, and / or HLA-C gene, antigen-specific T cells matching the new HLA-A molecule, HLA-B molecule, and / or HLA-C molecule subtype can be stimulated and activated.

[0019] In some specific embodiments of the present application, the above-mentioned general-purpose DC of a certain kind is an immortalized general-purpose DC, and the immortalized general-purpose DC can be obtained by knocking out the HLA class I gene of the immortalized DC. The immortalized cells can proliferate infinitely in vitro, meeting the requirements for the quantity and activity of DCs, thereby overcoming the drawback that the quantity of primary DCs is small and it is difficult to culture and amplify them in vitro. At the same time, problems such as the small quantity and high cost based on inducing monocytes to obtain MoDC (monocyte derived DCs, MODC) can be overcome.

[0020] In some specific embodiments of the present application, the above-mentioned immortalized general-purpose DC is UDC981, which is deposited with the China General Microbiological Culture Collection Center (CGMCC). The deposit date is December 25, 2024, the deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 46260.

[0021] In a second aspect, the present application further provides a group of general-purpose DC cells, and the cell group includes any of the above-mentioned general-purpose DCs.

[0022] In a third aspect, the present application provides a general-purpose DC cell line, and the cell line further includes any of the above-mentioned general-purpose DCs.

[0023] In a fourth embodiment, the present application further provides a method for producing a type of general-purpose DC described above, the method comprising the steps of knocking out the HLA class I gene of a primitive DC and selecting DCs that negatively express the HLA class I gene, i.e., general-purpose DCs, wherein the HLA class I gene includes the HLA-A gene, the HLA-B gene, and / or the HLA-C gene.

[0024] In some specific embodiments of this application, the HLA-A gene is knocked out in primitive DCs to select DCs in which the HLA-A gene is negatively expressed, i.e., general-purpose DCs from which the restriction of specific matching of the HLA-A molecule has been removed.

[0025] In some specific embodiments of this application, the HLA-B gene is knocked out in primitive DCs to select DCs in which the HLA-B gene is negatively expressed, i.e., general-purpose DCs from which the restriction of specific matching of the HLA-B molecule has been removed.

[0026] In some specific embodiments of this application, the HLA-C gene of primitive DCs is knocked out, and DCs in which the HLA-C gene is negatively expressed, i.e., general-purpose DCs from which the restriction of specific matching of the HLA-C molecule has been removed, are selected.

[0027] In some specific embodiments of this application, the HLA-A and HLA-B genes of primitive DCs are knocked out, and DCs in which the HLA-A and HLA-B genes are negatively expressed, i.e., general-purpose DCs whose specific matching restrictions for HLA-A and HLA-B molecules are removed, are selected.

[0028] In some specific embodiments of this application, the HLA-A and HLA-C genes of primitive DCs are knocked out, and DCs in which the HLA-A and HLA-C genes are negatively expressed, i.e., general-purpose DCs whose specific matching restrictions for HLA-A and HLA-C molecules are removed, are selected.

[0029] In some specific embodiments of this application, the HLA-B and HLA-C genes of primitive DCs are knocked out, and DCs in which the HLA-B and HLA-C genes are negatively expressed, i.e., general-purpose DCs whose specific matching restrictions for HLA-B and HLA-C molecules are removed, are selected.

[0030] In some specific embodiments of this application, the HLA-A, HLA-B, and HLA-C genes of primitive DCs are knocked out, and DCs in which the HLA-A, HLA-B, and HLA-C genes are negatively expressed are selected, i.e., general-purpose DCs whose specific matching restrictions for HLA-A, HLA-B, and HLA-C molecules are removed.

[0031] In some specific embodiments of this application, the method for knocking out the HLA class I gene in the aforementioned primitive DCs includes homologous recombination and CRISPR / Cas genome editing or TALEN (transmission activator-like effector nuclease) methods.

[0032] In some specific embodiments of this application, the CRISPR / Cas genome editing method described above includes the steps of designing one or more sgRNAs that bind to the HLA class I gene of a primitive DC, and introducing the sgRNAs into the primitive DC using the CRISPR-Cas system to knock out the HLA class I gene. In this application, "designing one or more sgRNAs that bind to the HLA class I gene of a primitive DC" means that the HLA class I gene to which the sgRNAs bind can be determined based on the generic DC to be created. For example, if the generic DC is a DC with the HLA-A gene knocked out, then at least one sgRNA that binds to the HLA-A gene of the primitive DC is designed. Alternatively, if the generic DC is a DC with both the HLA-A and HLA-B genes knocked out, then two sgRNAs that bind to at least the HLA-A and HLA-B genes, respectively, may be designed, or one sgRNA that binds to at least both the HLA-A and HLA-B genes simultaneously may be designed.

[0033] In some specific embodiments of this application, the above-described CRISPR-Cas system includes a CRISPR-Cas9 system or a CRISPR-Cas13 system.

[0034] In some specific embodiments of this application, the above-mentioned sgRNA binds to one or more HLA class I genes.

[0035] In some specific embodiments of this application, the sgRNA described above contains 17 to 23 bases.

[0036] In some specific embodiments of this application, the HLA-A gene subtype of the aforementioned primitive DC is HLA-A0201 and / or HLA-A6801, and / or the HLA-B gene subtype is HLA-B0801 and / or HLA-B1507, and / or the HLA-C gene subtype is HLA-C0303 and / or HLA-C0702.

[0037] In some specific embodiments of this application, the nucleotide sequence of the sgRNA that binds to HLA class I of the aforementioned primitive DC is as shown in SEQ ID NO.1, and this sgRNA can simultaneously target a pair of alleles of the HLA-A gene, HLA-A0201 and HLA-A6801, a pair of alleles of the HLA-B gene, HLA-B0801 and HLA-B1507, and a pair of alleles of the HLA-C gene, HLA-C0303 and HLA-C0702, while simultaneously knocking out all three pairs of alleles, resulting in extremely high genome editing efficiency.

[0038] In some specific embodiments of this application, the primitive DCs described above are immortalized DCs, and immortalized general-purpose DCs can be obtained by knocking out the HLA class I gene of the immortalized DCs. These immortalized general-purpose DCs can be proliferated indefinitely in vitro, meeting the requirements for DC quantity and activity, thereby overcoming the drawbacks of primary DCs, such as their low quantity and difficulty in in vitro culture amplification, as well as the problems of low quantity and high cost of MoDCs.

[0039] In some specific embodiments of this application, the immortalized DC described above is DC0502, deposited with the Center for Ordinary Microorganisms (CGMCC) of the China Microbial Species Preservation and Storage Administration, deposited on December 25, 2024, at the deposit address No. 3, Courthouse 1, Beichen West Road, Chaoyang District, Beijing, with deposit number CGMCC No. 46259, the HLA-A gene subtypes of the immortalized DC are HLA-A0201 and HLA-A6801, the HLA-B gene subtypes are HLA-B0801 and HLA-B1507, and the HLA-C gene subtypes are HLA-C0303 and HLA-C0702.

[0040] In a fifth aspect, the present application further provides an application for target antigen-specific T cell activation of a type of generic DC described above, the application of which involves obtaining a T cell population with an HLA class I molecular subtype (HLA class I molecules including HLA-A molecules, HLA-B molecules and / or HLA-C molecules) that is restrictive to the target antigen, and introducing an HLA class I gene encoding the above-mentioned HLA class I molecular subtype into the generic DC (reintroducing the HLA class I gene so that the HLA class I molecular subtype encoded by the HLA class I gene is different from the original HLA class I molecular subtype of the generic DC). This method includes overcoming the limitations of specific matching of HLA class I molecules in primitive DCs, and also introducing a gene encoding the same HLA class I molecular subtype as the primitive DCs and using it to activate autologous T cells; loading DCs into which the HLA class I gene has been introduced with an antigen peptide that is restrictive to the HLA class I molecular subtype (the antigen epitope of the antigen peptide is an antigen epitope recognized by target antigen-specific T cells); and co-culturing the DCs loaded with the antigen peptide with the aforementioned T cell population to activate specific T cells capable of recognizing the aforementioned antigen epitope.

[0041] In some specific embodiments of this application, the method for introducing the HLA class I gene encoding the HLA class I molecule described above includes genome editing, viral or mRNA methods.

[0042] In some specific embodiments of this application, the antigen peptide described above is derived from a tumor or a virus.

[0043] In some specific embodiments of this application, the tumor described above includes melanoma.

[0044] In some specific embodiments of this application, the above-mentioned virus includes cytomegalovirus.

[0045] In some specific embodiments of this application, the T cell population described above includes PBMCs.

[0046] In a sixth embodiment, the present application further provides applications for the above-described general-purpose DC cell population in target antigen-specific T cell activation, wherein the general-purpose DC cell population includes the above-described general-purpose DC.

[0047] In a seventh embodiment, the present application further provides a use for one of the above-described general-purpose DC cell lines in target antigen-specific T cell activation, wherein the general-purpose DC cell line includes the above-described general-purpose DC.

[0048] In an eighth aspect, the application further provides a use for the prevention or treatment of tumors or viral infections of a type of general-purpose DC described above, the use comprising infusing an effective amount of DC cells loaded with a tumor or viral antigen peptide into the body of a subject, wherein the DC cells are introduced with a gene encoding the same HLA class I molecular subtype as the subject, and the antigen peptide is an antigen peptide that is restricted to the HLA class I molecular subtype.

[0049] In a ninth embodiment, the present application further provides the use of the above-described general-purpose DC cell population in the prevention or treatment of tumors or viral infections.

[0050] In a tenth embodiment, the present application further provides the use of the above-described general-purpose DC cell line in the prevention or treatment of tumors or viral infections.

[0051] In an eleventh embodiment, the present application further provides a method for amplifying a type of target antigen-specific T cell, the method comprising: obtaining a T cell population with an HLA class I molecular subtype (HLA class I molecules including HLA-A molecules, HLA-B molecules and / or HLA-C molecules) that is restrictive to a target antigen; introducing an HLA class I gene encoding the above-mentioned HLA class I molecular subtype into the above-mentioned general-purpose DCs; loading the DCs into which the HLA class I gene has been introduced with an antigen peptide that is restrictive to the HLA class I molecular subtype (the antigen epitope of the antigen peptide is an antigen epitope recognized by target antigen-specific T cells); and co-culturing the DCs loaded with the antigen peptide with the above-mentioned T cell population to activate and amplify specific T cells capable of recognizing the above-mentioned antigen epitope.

[0052] In some specific embodiments of this application, the method for introducing the HLA class I gene encoding the HLA class I molecule described above includes genome editing, viral or mRNA methods.

[0053] In some specific embodiments of this application, the antigen peptide described above is derived from a tumor or a virus.

[0054] In some specific embodiments of this application, the tumor described above includes melanoma.

[0055] In some specific embodiments of this application, the above-mentioned virus includes cytomegalovirus.

[0056] In some specific embodiments of this application, the T cell population described above includes PBMCs.

[0057] In a twelfth aspect, the present application further provides applications for the manufacture of a general-purpose DC target antigen-specific T cell amplification reagent described above.

[0058] In a thirteenth aspect, the present application further provides the use of the above-described general-purpose DC cell population in the production of target antigen-specific T cell amplification reagents.

[0059] In a fourteenth embodiment, the present application further provides the use of one of the above-described general-purpose DC cell lines in the production of a target antigen-specific T cell amplification reagent.

[0060] In some specific embodiments of this application, the reagent further comprises an HLA class I gene encoding an HLA class I molecular subtype of the target antigen-specific T cell described above, wherein the HLA class I molecular subtype comprises an HLA-A molecule, an HLA-B molecule, and / or an HLA-C molecule.

[0061] In a fifteenth embodiment, the present application further provides a modified DC in which the primitive HLA class I gene is knocked out and an exogenous HLA class I gene is introduced, the exogenous HLA class I gene being a single subtype HLA class I gene comprising an HLA-A gene, an HLA-B gene, or an HLA-C gene, and the primitive HLA class I gene comprising at least the same type of HLA class I gene as the exogenous HLA class I gene, the applicant has found through research that DCs into which a single subtype HLA class I gene is introduced have a more potent ability to activate and amplify antigen-specific CD8 T cells compared to primitive DCs, which is presumed to be because the HLA class I gene in primitive DCs contains two subtype alleles, whereas the introduction of an exogenous HLA class I gene has only one subtype, thus having a more potent ability to activate and amplify antigen-specific CD8 T cells.

[0062] In the sixteenth embodiment, the present application further provides a method for manufacturing one type of modified DC as described above, the method being: This includes introducing a single subtype of HLA class I gene into the aforementioned general-purpose DC to obtain modified DCs.

[0063] In a seventeenth embodiment, the present application further provides a type of sgRNA whose nucleotide sequence is as shown in SEQ ID NO.1, which can simultaneously target a pair of alleles of the HLA-A gene, HLA-A0201 and HLA-A6801, a pair of alleles of the HLA-B gene, HLA-B0801 and HLA-B1507, and a pair of alleles of the HLA-C gene, HLA-C0303 and HLA-C0702, and can be used to simultaneously knock out the HLA-A, HLA-B, and HLA-C genes in cells containing the above-mentioned gene subtypes.

[0064] In the eighteenth embodiment, the present application further provides the use of the above-described type of sgRNA in cellular HLA class I gene knockout, wherein the HLA class I gene comprises HLA-A, HLA-B and / or HLA-C, and the HLA-A gene subtype in the cell is HLA-A0201 and / or HLA-A6801, and / or the HLA-B gene subtype is HLA-B0801 and / or HLA-B1507, and / or the HLA-C gene subtype is HLA-C0303 and / or HLA-C0702.

[0065] In some specific embodiments of this application, in the use of the above-described type of sgRNA in cellular HLA class I gene knockout, the HLA class I gene comprises HLA-A, and the HLA-A gene subtype in the cell is HLA-A0201 and / or HLA-A6801.

[0066] In some specific embodiments of this application, in the use of the above-described type of sgRNA in cellular HLA class I gene knockout, the HLA class I gene comprises HLA-B, and the HLA-B gene subtype of the cell is HLA-B0801 and / or HLA-B1507.

[0067] In some specific embodiments of this application, in the use of the above-described type of sgRNA in cellular HLA class I gene knockout, the HLA class I gene comprises HLA-C, and the HLA-C gene subtype of the cell is HLA-C0303 and / or HLA-C0702.

[0068] In some specific embodiments of this application, in the use of the above-described type of sgRNA in cellular HLA class I gene knockout, the HLA class I gene comprises HLA-A, HLA-B, and HLA-C, wherein the HLA-A gene subtype in the cell is HLA-A0201 and / or HLA-A6801, the HLA-B gene subtype is HLA-B0801 and / or HLA-B1507, and the HLA-C gene subtype is HLA-C0303 and / or HLA-C0702.

[0069] In some specific embodiments of this application, in the use of the above-described type of sgRNA in cellular HLA class I gene knockout, the cells include DCs and / or T cells.

[0070] In some specific embodiments of this application, in the use of the aforementioned type of sgRNA in cellular HLA class I gene knockout, the cells are DCs.

[0071] In some specific embodiments of this application, the DC described above is an immortalized DC.

[0072] In some specific embodiments of this application, the immortalized DC described above is DC0502, deposited with the Center for Ordinary Microorganisms (CGMCC) of the China Microbial Species Preservation and Storage Administration, deposited on December 25, 2024, at the deposit address No. 3, Courthouse 1, Beichen West Road, Chaoyang District, Beijing, with deposit number CGMCC No. 46259, the HLA-A gene subtypes of the immortalized DC are HLA-A0201 and HLA-A6801, the HLA-B gene subtypes are HLA-B0801 and HLA-B1507, and the HLA-C gene subtypes are HLA-C0303 and HLA-C0702.

[0073] In the nineteenth aspect, the present application further provides immortalized DC DC0502, deposited with the Center for Ordinary Microorganisms (CGMCC), the China Microbial Species Preservation and Storage Administration, on December 25, 2024, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with deposit number CGMCC No. 46259. [Effects of the Invention]

[0074] Compared to the prior art, the beneficial effects of this application are as follows:

[0075] (1) The general-purpose DC provided in this application, its manufacturing method and use involve obtaining a general-purpose DC (UDC) that does not express HLA class I molecules by knocking out the HLA class I genes (HLA-A gene, HLA-B gene and / or HLA-C gene) of primitive DCs, particularly immortalized DCs. When using it, based on the HLA class I molecule subtype of the target antigen-specific T cells to be obtained, i.e., from different individuals, the HLA class I molecule that matches the target antigen-specific T cell can be reexpressed in the general-purpose DC (UDC) by gene transfer technology such as genome editing, virus or mRNA. This removes the limitation of specific matching of HLA class I molecules that exists when using heterologous DCs, solves the problem of long manufacturing cycles and high prices for personalized cell immunotherapy products, and is expected to result in an "off-the-shelf" (off-the-shelf) personalized cell therapy product, drastically reducing costs and having broad application value for allogeneic off-the-shelf cell immunotherapy products.

[0076] (2) The general-purpose DC provided in this application, its manufacturing method and use involve reexpressing an HLA class I molecule that matches a target antigen-specific T cell in a general-purpose DC (UDC), which has only one subtype, whereas HLA class I alleles in primitive DCs, whether self or heterologous, usually have two subtypes, and compared to primitive DCs, this reduces interference from homogeneous HLA class I molecules and improves the ability to activate and amplify antigen-specific CD8 T cells.

[0077] (3) The general-purpose DC provided in this application, its manufacturing method and uses involve reexpressing an HLA class I molecule that matches a target antigen-specific T cell in a general-purpose DC (UDC), thereby avoiding the nonspecific activation and amplification of allogeneic reactive T cells caused by HLA incompatibility, and thereby reducing the risk of graft-versus-host disease (GVHD) or immune rejection.

[0078] (4) The general-purpose DC provided in this application, its manufacturing method and applications, when the protocell is an immortalized DC, can be obtained, possessing the ability to amplify indefinitely, and such as monoclonal UDC981 provided in this application, which, after being cultured for 30 days, has an amplification factor of 18,000 times, can be cultured simply and quickly to meet the requirements for the quantity and activity of DCs, thereby overcoming the disadvantages of primary DCs, which are few in number and difficult to culture and amplify in vitro, as well as the problems of MoDCs, which are few in number and high in cost.

[0079] (5) The general-purpose DC provided in this application, its manufacturing method and use, can be used to simultaneously target a pair of alleles of the HLA-A gene HLA-A0201 and HLA-A6801, a pair of alleles of the HLA-B gene HLA-B0801 and HLA-B1507, and a pair of alleles of the HLA-C gene HLA-C0303 and HLA-C0702 of DC0502, in combination with a genome editing method, and has extremely high genome editing efficiency. [Brief explanation of the drawing]

[0080] [Figure 1]The images show the percentage of HLA-ABC-negative and HLA-A0201-negative cells detected by flow cytometry after genome editing of the original DCs (DC0502) of this application (left arrow), and the percentage of HLA-ABC-negative and HLA-A0201-negative general-purpose DCs (UDCs) after negative selection using magnetic beads (right arrow). "Unstain" represents the unstained control group, and "UDC mix" represents the DCs that underwent genome editing. [Figure 2] These are photographs of the cell morphology of DCs before and after genome editing in this application. The left image shows a primitive DC (DC0502) that has not undergone genome editing, and the right image shows a DC (UDC mix) that has undergone genome editing. [Figure 3] This is a superimposed histogram of flow cytometry analysis of homozygous background DC monoclones in which three genes, HLA-A, HLA-B, and HLA-C (each gene being a pair of alleles), were completely knocked out against the purified genome-edited DCs of this application. [Figure 4] This is a statistical result of the average fluorescence intensity (MFI) values ​​of three types of antibodies used in flow cytometry analysis of homozygous background DC monoclones in which three genes (HLA-A, HLA-B, and HLA-C, each of which is a pair of alleles) were completely knocked out from the purified genome-edited DCs of this application. [Figure 5] These are the sequencing results for the three genes HLA-A, HLA-B, and HLA-C (each gene is a pair of alleles) of the monoclonal UDC981 prepared in this application. [Figure 6] This shows the amplification factor detection results for the original DC (DC0502) and the fabricated single-clone UDC981 in this application. [Figure 7] The flow cytometry results for the phenotype and co-stimulatory molecule identification of monoclonal UDC981 prepared in this application are shown, where isotype refers to the immunoglobulin (Ig) isotype. [Figure 8]The flow cytometry results for the phenotype and co-stimulatory molecule identification of monoclonal UDC981 prepared in this application are shown, where isotype refers to the immunoglobulin (Ig) isotype. [Figure 9] The flow cytometry results for the phenotype and co-stimulatory molecule identification of monoclonal UDC981 prepared in this application are shown, where isotype refers to the immunoglobulin (Ig) isotype. [Figure 10] These are the flow cytometry results for the phenotype and co-stimulatory molecule identification of the primitive DC (DC0502) used in this application. [Figure 11] These are the flow cytometry results for the phenotype and co-stimulatory molecule identification of the primitive DC (DC0502) used in this application. [Figure 12] These are the flow cytometry results for the phenotype and co-stimulatory molecule identification of the primitive DC (DC0502) used in this application. [Figure 13] This document presents the flow cytometry detection results of the HLA-A molecule positivity rates for UDC HLA-A0201, UDC HLA-A1101, and UDC HLA-A2402 after magnetic bead positive screening of UDC981 cells infected with lentiviruses containing HLA-A0201, HLA-A1101, and HLA-A2402. [Figure 14] This application verifies the ability of UDC981 (UDC HLA-A0201), which has HLA-A0201 reintroduced (complemented), to present the CMV epitope (cytomegalovirus epitope). [Figure 15] This application verifies the ability of UDC981 (UDC HLA-A0201), which has HLA-A0201 reintroduced (complemented), to present the Mart-1 epitope (melanoma cell epitope). [Figure 16] This application verifies the ability of UDC981 (UDC HLA-A2402), which has HLA-A2402 reintroduced (complemented), to present the CMV epitope (cytomegalovirus epitope). [Figure 17]This application presents the results of verifying the immunogenicity of UDC981 (UDC HLA-A11), which has HLA-A11 reintroduced (supplemented) in this application, against allogeneic PBMCs. [Modes for carrying out the invention]

[0081] The present application will be further described below in combination with specific examples.

[0082] Unless otherwise defined, all technical and scientific terms used herein are identical to those commonly understood by a person skilled in the art relating to the present application. The term "and / or" used herein includes any and all combinations of one or more related enumerated items.

[0083] Unless otherwise specified in the examples, the procedures should be carried out under normal conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or equipment used are all standard products available commercially.

[0084] As used in this text, the term "about" is used to provide flexibility and impreciseness related to a given term, measure, or value. Those skilled in the art can easily determine the degree of flexibility of a particular variable.

[0085] Concentration, quantity, and other numerical data may be presented in range format in the text. Such range formatting is used solely for convenience and conciseness, and should be interpreted flexibly to include not only the explicitly stated range limits, but also all individual numbers or subranges encompassed within the range, as if each number and subrange were explicitly stated. For example, a numerical range of approximately 1 to approximately 4.5 should be interpreted to include not only the explicitly stated limit of 1 to approximately 4.5, but also individual numbers (e.g., 2, 3, 4, etc.) and subranges (e.g., 1 to 3, 2 to 4, etc.). The same principle applies to ranges containing only a single number, such as "less than approximately 4.5," which should be interpreted to include all the aforementioned values ​​and ranges. Furthermore, this interpretation should apply regardless of the breadth of the stated range or feature.

[0086] As used in this application, “Dendritic cell (DC)” refers to a professional antigen-presenting cell involved in both cellular and humoral immunity. In this application, unless otherwise specified, “DC” may refer to a cell line or a cell line, where a cell line refers to a population of cells with specific properties or markers obtained from a primary culture or cell line by selection or cloning, for example, UDC981 obtained by selection after genome editing in this application; and a cell line refers to a population of cells that have grown after a primary culture has successfully undergone its first subculturing, for example, DC0502 formed by successful subculturing.

[0087] As used in this application, the term "immune system" refers to the system by which a living organism performs immune responses and immune functions, and is composed of immune organs, immune cells, and immune molecules, which can recognize and eliminate foreign pathogens (e.g., bacteria, viruses, parasites, etc.), as well as monitor and eliminate abnormal cells within the body (e.g., cancer cells) and cells that have aged or been damaged, thereby maintaining the health of the living organism and the stability of its internal environment.

[0088] As used in this application, "T cells," or T lymphocytes, are a type of lymphocyte that originates from hematopoietic stem cells in the bone marrow, undergoes a complex developmental process in the thymus involving positive and negative selection, and ultimately matures to enter the peripheral lymphoid organs and blood circulation, primarily as cytotoxic T cells (CD8). + T cells and helper T cells (CD4 + Cytotoxic T cells (T cells) are primarily responsible for recognizing and killing cells infected with pathogens such as viruses and intracellular parasites, as well as tumor cells. Through the T cell receptor (TCR) on their cell surface, they recognize the antigen-major histocompatibility complex I (MHC-I) molecular complex on the surface of target cells. After recognition, cytotoxic T cells release perforin and granzymes, inducing apoptosis in the target cells. Recognition of the antigen-MHC-I complex by the TCR is necessary for the activation of cytotoxic T cells.

[0089] As used in this application, "stimulating T cells" or "activating T cells" essentially mean the same thing, referring to the process where T cells recognize an antigen, become activated T cells (also called activated T cells), enter the cell cycle, proliferate in large numbers, and provide a sufficient number of effector T cells for the immune response.

[0090] As used in this application, "alleles" refers to a pair of genes located at the same position on a pair of homologous chromosomes that control relative traits. In human chromosomes, different gene forms may exist for a particular locus (a specific location of a gene on a chromosome), and these different forms are alleles. For example, in the cell DC0502 of this application, HLA-A0201 and HLA-A6801 are different gene forms located at the HLA-A locus, so HLA-A0201 and HLA-A6801 are called alleles, and the specific HLA-A0201 is called the HLA-A gene subtype.

[0091] As used in this application, “self” means that which originates from the tissue, cells, or substance of the same solid. “Other” means that which originates from the tissue, cells, or substance of the same species but a different individual.

[0092] As used in this application, "specific matching of HLA molecules" means that a T cell can only be activated when the HLA molecules of the DC and the T cell are identical or similar, or that a DC can only activate T cells whose HLA molecules are identical or similar. It should be noted that "HLA molecules" here does not mean that all HLA molecules are identical or similar, but rather that the HLA molecules that act in a particular T cell activation process are relevant.

[0093] As used in this application, “HLA class I gene” refers to a class of human major histocompatibility complex (MHC) genes that play a crucial role in immune responses and immune recognition processes. The product encoded by “HLA class I gene” is the HLA class I molecule (sometimes called HLA class I protein), which is primarily involved in the presentation of endogenous antigens and helps the immune system recognize self-cells from cells infected with foreign pathogens. Specifically, HLA class I molecules can bind to antigen peptides synthesized within cells, such as viral protein antigens synthesized within cells after a virus infects a cell. After binding to the HLA class I molecule, these antigen peptides are transported to the cell surface and CD8 + CD8 is used for recognition by T lymphocytes. +T cells recognize this antigen peptide-HLA class I molecule complex through their T cell receptor (TCR), thereby initiating a cellular immune response that kills virus-infected cells. The "HLA class I gene" primarily consists of three types: HLA-A, HLA-B, and HLA-C, whose coding products are the HLA-A, HLA-B, and HLA-C molecules, respectively. The HLA-A, HLA-B, and HLA-C molecules differ in their binding specificity to different antigen peptides. For example, the HLA-A molecule may have a high affinity for certain viral antigen peptides, while the HLA-B molecule may have a stronger binding ability to other antigen peptides. This depends on the structural characteristics of their antigen-binding grooves. The HLA-A, HLA-B, and HLA-C genes exhibit a high degree of polymorphism; that is, within a population, there are many different allele forms of the HLA-A, HLA-B, and HLA-C genes, different individuals possess different HLA-A genes, and even two HLA-A alleles possessed by the same individual may be different. The specific alleles of an individual can be detected and classified by specific technical means, and these specific alleles are called "gene subtypes." It should be noted that in this application, "HLA class I gene" includes the three types of HLA-A, HLA-B, and HLA-C genes, but not all of them refer to the HLA-A, HLA-B, and HLA-C genes. Unless otherwise specified, "HLA class I gene" can refer to any one or a combination of the HLA-A, HLA-B, and HLA-C genes, and a person skilled in the art can determine this from the context.

[0094] As used in this application, “Peripheral Blood Mononuclear Cells (PBMCs)” is a general term for mononuclear cells in peripheral blood, which include lymphocytes (T cells, B cells, NK cells) and other monocytes. Monocytes can also be converted into mature DCs through a series of in vitro induction cultures, which are called MoDCs (monocyte-derived DCs, MODCs).

[0095] As used in this application, "knockout," "gene knockout," also known as "gene removal," or "gene knock-out" refers to the removal, loss of activity, or reduction of expression of a specific gene in the cell genome, and such "specific gene" is also called a "target gene," "object gene," etc.

[0096] As used in this application, "primitive DC" refers to initiating cells for producing general-purpose DCs, and may be natural DCs, optimized DCs, such as DCs that have been adapted and made immortalizable, or DCs that have been genome edited and made immortalizable.

[0097] As used in this application, "immortalization" refers to having the ability to proliferate indefinitely. Under appropriate culture conditions, immortalized cells can continuously divide and constantly increase in number, and unlike normal somatic cells, they do not enter the aging and death stage after a certain number of divisions.

[0098] As used in this application, "target antigen-specific T cells" refers to T cells that can recognize a target antigen.

[0099] As used in this application, “negative expression” means that no expected signal or marker appears in a cell, and that the detected target substance (e.g., protein, gene, antigen, etc.) is present in an extremely low amount or is absent in the cell. For example, a DC with negative HLA-A gene expression is a DC in which the HLA-A molecule is present in an extremely low amount or is absent.

[0100] As used in this application, CRISPR / Cas (i.e., Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR-associated proteins) is a type of genome editing technology, and you can refer to the operating manual of the reagent supplier.

[0101] As used in this application, gRNA (guide RNA), or guide RNA, is a key component in the CRISPR / Cas genome editing system that can guide Cas proteins (e.g., Cas9) to position themselves on target DNA sequences, thereby enabling editing of specific gene loci.

[0102] As used in this application, a “restrictive antigen peptide” refers to an antigen peptide that, in the course of an immune response, binds to a specific major histocompatibility complex (MHC) molecule and is presented to and recognized by T lymphocytes through the MHC molecule, and a certain restrictive relationship exists between this antigen peptide and the MHC molecule; that is, only a specific antigen peptide can bind to a specific MHC molecule, and only a specific T cell receptor (TCR) can recognize this antigen peptide-MHC complex. Similarly, the MHC molecule in question is called an antigen-restrictive MHC molecule. For example, an HLA-A0201-restrictive antigen peptide refers to a specific antigen peptide that binds to HLA-A0201, while HLA-A0201 is called an antigen peptide-restrictive HLA-A molecule.

[0103] As used in this application, "antigen epitope" refers to a specific chemical group in an antigen molecule that determines the antigen specificity. For example, in this application, the CMV epitope is a specific region on the cytomegalovirus (CMV) antigen molecule that is recognized by immune cells, and the Mart-1 epitope is a specific region on the melanoma cell-associated antigen (Mart-1 antigen) molecule that is recognized by immune cells.

[0104] As used in this application, "reintroduction (complementary introduction)" refers to the reintroduction of a target gene. For example, reintroducing HLA-A0201 refers to introducing the HLA-A0201 gene into a general-purpose DC through gene transfer technologies such as genome editing, viruses, or mRNA, and reexpressing the HLA-A0201 molecule.

[0105] As used in this application, "HLA-ABC KO DC(s)" refers to DCs in which the HLA-A, HLA-B, and HLA-C genes are negatively expressed; that is, these DCs do not produce HLA-A, HLA-B, and HLA-C molecules, and are also called HLA-ABC negative cells at the protein molecular level. Similarly, "HLA-A0201 KO DC" refers to DCs in which the HLA-A0201 gene is negatively expressed; that is, these DCs do not produce HLA-A0201 molecules.

[0106] As used in this application, "HLA-ABC" + "DCs" refers to DCs in which at least one of the HLA-A, HLA-B, and HLA-C genes is positively expressed; in other words, each DC contains at least one of the HLA-A, HLA-B, and HLA-C molecules.

[0107] As used in this application, flow cytometry (FCM) is a technique for rapid, quantitative analysis and sorting of cells or microparticles (e.g., bacteria, viruses, etc.), and can be referenced from the instructions of the equipment or reagent supplier.

[0108] As used in this application, "genome-edited DC" refers to a DC (UDC mix) that has undergone genome editing.

[0109] As used in this application, "UDC981 HLA-A0201" refers to UDC981 expressing the HLA-A0201 molecular subtype.

[0110] Example 1

[0111] This embodiment provides a method for manufacturing a general-purpose DC and a general-purpose DC manufactured thereby.

[0112] A method for producing general-purpose DCs involves selecting DCs that negatively express the HLA class I gene, i.e., general-purpose DCs, by knocking out the HLA class I gene in primitive DCs. The HLA class I gene is the HLA-A gene, HLA-B gene, and / or HLA-C gene.

[0113] In this embodiment, the original DC was DC0502, the DC was an immortalized DC, deposited with the Center for Ordinary Microorganisms (CGMCC) of the China Microbial Species Preservation and Storage Administration on December 25, 2024, deposited at No. 3, Courthouse 1, Beichen West Road, Chaoyang District, Beijing, deposited at CGMCC No. 46259, the HLA-A gene subtypes of the immortalized DC were HLA-A0201 and HLA-A6801, the HLA-B gene subtypes were HLA-B0801 and HLA-B1507, and the HLA-C gene subtypes were HLA-C0303 and HLA-C0702.

[0114] In this embodiment, the HLA class I genes are the HLA-A gene, HLA-B gene, and HLA-C gene; that is, the HLA-A gene, HLA-B gene, and HLA-C gene of primitive DCs are knocked out.

[0115] In this embodiment, knocking out the HLA class I gene in primitive DCs is performed using CRISPR / Cas genome editing, which includes designing one or more sgRNAs that bind to the HLA class I gene in primitive DCs, and introducing the sgRNAs into the primitive DCs using the CRISPR-Cas system to knock out the HLA class I gene.

[0116] In the example, the nucleotide sequence of the sgRNA is as shown in SEQ ID NO.1 (CGGCTACTACAACCAGAGCG). This sgRNA can simultaneously target a pair of alleles of the HLA-A gene HLA-A0201 and HLA-A6801, a pair of alleles of the HLA-B gene HLA-B0801 and HLA-B1507, and a pair of alleles of the HLA-C gene HLA-C0303 and HLA-C0702 in primitive DCs, while simultaneously knocking out all three pairs of alleles, exhibiting extremely high genome editing efficiency. This sgRNA was synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0117] In the examples, specifically, introducing sgRNA into primitive DCs using the CRISPR-Cas system is: The logarithmic growth phase DC0502 is collected in a 15 mL centrifuge tube, centrifuged at 400 g for 5 minutes, the supernatant is removed, 10 ml of phosphate-buffered saline (PBS) is added, the centrifuge tube is inverted and gently washed, and then centrifuged again at 400 g for 5 minutes. The supernatant is aspirated and discarded, the cell precipitate is resuspended using PBS, and then counted. Prepare the cell count according to the standard electroporation procedure: 1 × 10^6 cells / test, centrifuge at 400 g for 5 min, remove the supernatant, resuspend the cell precipitate in 50 μL / test of Gene Pulser electroporation buffer (purchased from BIO-RAD, USA, product number 1652676) equilibrated at room temperature, gently mix, aspirate the cell suspension and add it to a sterile 1.5 ml EP tube. GenCRISPR TM According to the instructions for using Cas9V1.2 (purchased from Nanjing Jinsirui Biotechnology Co., Ltd., product number Z03702), GenCRISPR TM The steps include adding Cas9V1.2 and sgRNA in a molar ratio of 1:3 together with electroporation buffer to a sterile EP tube, The steps include incubating the mixture at room temperature for 30 minutes to form the RNP complex, The steps include: aspirating the cell suspension from the EP tube, adding it to the RNP complex, mixing thoroughly, aspirating the mixture, and adding it to a sterile electroporation cuvette; The procedure involves performing electroporation of an RNP complex using the BIO-RAD electroporation system, the Gene Pulser Xcell eukaryotic electroporation system (catalog no. 165-2661) (electroporation conditions: pulse type, square wave, voltage, 200V, pulse, 2ms), and The procedure includes the steps of: immediately aspirating the cells after electroporation and transferring them to a preheated cell culture medium (the cell culture medium being XVIVO15 (LONZA, 04-418Q) + 400 IU / mL IL-2 (Tetracyclic Biotechnology, National Pharmaceutical Code S10970015); and continuing the culture by placing the culture plate in a 37°C constant temperature incubator to obtain a UDC mix.

[0118] In this embodiment, DCs that negatively express the HLA class I gene are selected using a magnetic bead negative sorting method, and purified DCs that negatively express the HLA class I gene are obtained and referred to as HLA-ABC KO DCs.

[0119] In this embodiment, cell separation is performed using the Miltenyi MACS® Separation System. The procedure is as follows, referring to the instructions for Anti-PE MicroBeads (miltenyi biotec, Order no. 130-048-801): After culturing the UDC mix obtained after genome editing, the cells are amplified and then collected. Primary antibodies are then labeled. 5 μL of PE anti-human HLA-A, B, C Antibody (Biolegend, 311406) is added to each 100 μL cell suspension (1 × 10^8 cells / mL), mixed thoroughly, and incubated in a light-shielded refrigerator at 4°C for 30 minutes. Wash the labeled cells, resuspend the cell suspension using the buffer prepared according to the instructions, centrifuge at 300 g for 6 minutes, and repeat the washing process. Thoroughly aspirate and discard the supernatant, add 20 μL of Anti-PE MicroBeads to each 1 × 10^7 cells, mix thoroughly, and incubate in a 4°C refrigerator for 15 minutes. Wash the cells and centrifuge at 300 g for 6 minutes. Thoroughly aspirate and discard the supernatant, and resuspend the cell precipitate in 500 μL of buffer. Place the magnetic stand inside the biosafety cabinet, attach the preparative LD column to the magnetic stand, and place the collection tube below the preparative column. Before cell sorting, add 2 ml of buffer to the sorting column to thoroughly infiltrate it. Allow the buffer to pass through the sorting column and flow into the collection tube. When the MACS buffer has run out, replace it with a new collection tube. The cell suspension labeled with Anti-PE MicroBeads is filtered through a 40 μm mesh to obtain a single-cell suspension. The filtered cell suspension is added to the preparative column. When the droplets dripping from the preparative column begin to stagnate, another 2 ml of buffer is added to the preparative column for washing, and the cell suspension that flows into the collection tube, i.e., HLA-ABC KO Dcs, is collected. When the droplets dripping from the preparative column begin to stagnate, remove the preparative column from the magnetic stand and insert it into a new collection tube. Add another 3 ml of buffer to the preparative column, attach a plunger to the column opening, and push the liquid from the preparative column into the new collection tube to create a cell suspension, i.e., HLA-ABC. + Obtain DCs. After sorting, HLA-ABC molecule-negative UDCs are seeded onto cell culture plates. The cell culture medium consists of XVIVO15 (LONZA, 04-418Q) + 400 IU / ml IL-2 (Tetracyclic Biomedical Laboratory, National Pharmaceutical Code S10970015). The culture plates are placed in a 37°C incubator and culture is continued.

[0120] This embodiment further provides identification of HLA-ABC KO DCs after magnetic bead sorting.

[0121] In this embodiment, the ratio of HLA-ABC KO DC and HLA-A0201 KO DC in the DC after separation of magnetic beads is detected using flow cytometry technology, and the purity of HLA-ABC KO DC and HLA-A0201 KO DC is confirmed. The detection results are shown in Figure 1.

[0122] As can be seen from the right side of the arrow in Figure 1, genome-edited DCs that underwent two rounds of Anti-PE MicroBeads negative screening consisted of 100% HLA-ABC KO DCs and 100% HLA-A0201 KO DCs, and the purified genome-edited DCs were named UDCs.

[0123] Figure 2 shows the cell morphology of DCs before and after genome editing, and it can be seen that the genome-edited DCs (UDC mix) maintain morphological similarity to wild-type DCs (DC0502).

[0124] Example 2

[0125] This example provides a method for detecting the genome editing efficiency of the CRISPR / Cas genome editing method in Example 1.

[0126] Specifically, we will detect the efficiency of simultaneously performing genome editing on three types of genes (HLA-A, HLA-B, and HLA-C genes, each containing two alleles, i.e., a total of six genes) in DC0502.

[0127] In this embodiment, genome editing efficiency detection is performed by first detecting the overall HLA-ABC KO efficiency in a UDC mix obtained by culturing after electroporation using an HLA-ABC antibody (Biolegend, 311406), and then detecting the efficiency of HLA-A0201 single gene editing in a UDC mix obtained by culturing after electroporation using an HLA-A0201 antibody (BD bioscience, 567739). Specifically, this includes the following: After electroporation of DC0502 cells, they were cultured for 48 hours. The UDC mix after genome editing electroporation and the DC0502 cells without genome editing were collected in EP tubes, and the cell surface HLA-ABC molecules and HLA-A0201 molecules were stained according to the standard staining procedure of flow cytometry.

[0128] The flow cytometry detection results are shown in Figure 1. As can be seen from the left of the arrow in Figure 1, 15% of the UDC mix obtained after culture following electroporation was negative for HLA-ABC molecules, and 55.2% was negative for HLA-A0201 molecules. This explains that the CRISPR / Cas genome editing method in Example 1, particularly the designed target sgRNA, can simultaneously knock out HLA-A, HLA-B, and HLA-C genes, and that it has high genome editing efficiency.

[0129] Example 3

[0130] This example provides further validation and selection of purified genome-edited DC (UDC) monoclones.

[0131] The selection target for this embodiment is a homozygous background UDC monoclonal in which all three pairs of alleles, HLA-A, HLA-B, and HLA-C, are completely knocked out.

[0132] After purification, genome-edited DCs are collected and seeded into 96-well plates at a density of 0.5-1 cell / well for subclonal culture. The culture medium consists of XVIVO15 (LONZA, 04-418Q) + 400 IU / ml IL-2 (Tetracyclic Biomedical Laboratory, National Drug Code S10970015). After 2-3 weeks of culture amplification, the expression of HLA-A, HLA-B, and HLA-C molecules in the DC monoclonals is performed, and the HLA-A, HLA-B, and HLA-C genes are identified.

[0133] From several dozen UDC monoclones, seven monoclones numbered 1-2, 1-3, 1-9, 1-10, 1-11, 1-19, and 1-20 were preferentially selected. HLA-A, HLA-B, and HLA-C molecule expression was verified, and flow cytometry samples were prepared according to the standard flow cytometry staining procedure using the flow cytometry antibodies PE anti-human HLA-A (BD bioscience, 567739), Alexa Fluor® 647 anti-human HLA-B (BD bioscience, 569662), and FITC anti-human HLA-ABC (Biolegend, 311404). Analysis was performed using a flow cytometer (Beckman), and the data was visualized using FlowJo software (BD).

[0134] Simultaneously, cell genomic DNA is extracted, and its HLA-A, HLA-B, and HLA-C gene sequences are detected through sequencing.

[0135] The results are shown in Figures 3 to 5.

[0136] Figure 3 shows superimposed histograms of flow cytometry analysis of homozygous background UDC monoclones in which all three pairs of alleles (HLA-A, HLA-B, and HLA-C) were completely knocked out, compared to purified genome-edited DCs (UDCs). The first row of each histogram represents the DC0502-negative control, the second row represents the UDC mix-positive control, and the third row represents the HLA class I molecule expression intensity of each monoclone. The third row of the first group corresponds to the HLA-A molecule expression intensity, the third row of the second group corresponds to the HLA-B molecule expression intensity, and the third row of the third group corresponds to the HLA-ABC molecule expression intensity. The results are as follows. Among these seven monoclones, 1-2, 1-3, 1-9, 1-10, 1-11, 1-19, and 1-20, the HLA-A, HLA-B, and HLA-ABC molecules in monoclones 1-11, 1-19, and 1-20 are at background levels.

[0137] As shown in Figure 4, analysis of the average fluorescence intensity of HLA-A, HLA-B, and HLA-ABC molecules revealed that monoclonal HLA-A, HLA-B, and HLA-ABC molecules in 1-11, 1-19, and 1-20 exhibit negative expression.

[0138] Sequencer analysis was performed on UDC single clones 1-19, and the results are shown in Figure 5.

[0139] The nucleotide sequence of the partial fragment of the HLA-A0201 gene is ACCCTGCGCGGCTACTACAACCAGAGCGAGGCCGG (SEQ ID NO. 8), and the nucleotide sequence of the same fragment after genome editing is ACCCTGCGCGGCTACTACAACCAGAGGCCGG (SEQ ID NO. 9).

[0140] The partial nucleotide sequence of the HLA-A6801 gene is ACCCTGCGCGGCTACTACAACCAGAGCGAGG (SEQ ID NO. 10), and the nucleotide sequence of the same fragment after genome editing is ACCCTGCGCGGCTACTACAACCAGAAGCGAGG (SEQ ID NO. 11).

[0141] The partial nucleotide sequence of the HLA-B0801 gene is AACCTGCGCGGCTACTACAACCAGAGCGAGG (SEQ ID NO. 12), and the nucleotide sequence of the same fragment after genome editing is AACCTGCGCGGCTACTACAACCAGAAGCGAGG (SEQ ID NO. 13).

[0142] The partial nucleotide sequence of the HLA-B1507 gene is AACCTGCGCGGCTACTACAACCAGAGCGAGGCC (SEQ ID NO. 14), and the nucleotide sequence of the same fragment after genome editing is AACCTGCGCGGCTACTACAACCAGCGAGGCC (SEQ ID NO. 15).

[0143] The partial nucleotide sequence of the HLA-C0303 gene is AACCTGCGCGGCTACTACAACCAGAGCGAGGCCA (SEQ ID NO. 16), and the nucleotide sequence of the same fragment after genome editing is AACCTGCGCGGCTACTACAACCAGAAGCGAGGCCA (SEQ ID NO. 17).

[0144] The partial nucleotide sequence of the HLA-C0702 gene is AACCTGCGCGGCTACTACAACCAGAGCGAGGAC (SEQ ID NO. 18), and the nucleotide sequence of the same fragment after genome editing is AACCTGCGCGGCTACTACAACCAGAAGCGAGGAC (SEQ ID NO. 19).

[0145] This gene declares that all three pairs of alleles—HLA-A, HLA-B, and HLA-C—are completely knocked out, and is therefore classified as an HLA-ABC KO DC.

[0146] The above flow cytometry and sequencing results prove that UDC monoclonal 1-19 is homozygous, with all three pairs of alleles (HLA-A, HLA-B, and HLA-C) completely knocked out. UDC monoclonal 1-19 was preferentially selected and named UDC981. It was deposited with the Center for Ordinary Microorganisms (CGMCC), China Microbial Species Preservation and Management Commission, on December 25, 2024, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with deposit number CGMCC No. 46260.

[0147] Example 4

[0148] This embodiment provides a method for detecting the proliferation ability of primitive DC (DC0502) and UDC monoclonal 1-19 (UDC981).

[0149] DC0502 and UDC981 cells were collected and counted, and inoculated into T25 culture flasks according to a cell density of 5 × 10^5 cells / mL. The starting total number of cells was 5 × 10^6 cells. 10 ml of culture medium was added, along with XVIVO15 (LONZA, 04-418Q) + 400 IU / ml IL-2 (Tetracyclic Biologics, National Pharmaceutical Code S10970015). The color of the culture medium and the cell status were observed daily. After the culture medium turned yellow, a half-volume change and sampling count were performed, and fresh culture medium was added to adjust the cell density to 5-8 × 10^5 cells / mL. Subculturing counting was continued for 30 days.

[0150] The results of DC0502 and UDC981 cell amplification culture are shown in Figure 6. After 30 days of culture, the amplification factor reached 18,000 times, demonstrating that DC0502 and UDC981 can be continuously subcultured and possess immortalization properties.

[0151] Example 5

[0152] This embodiment provides detection and identification of the UDC monoclonal 1-19 (UDC981) cell phenotype.

[0153] Phenotypic identification of UDC981 cells is performed comprehensively through several methods, including the expression of lineage markers on the DC surface, DC maturation markers, and function-related molecules such as antigen-presenting molecules, costimulatory molecules, and T cell activation-related molecules.

[0154] This study investigates the effects of genome editing on the phenotype and expression of related functional molecules in DCs by performing comprehensive expression detection of UDC981 phenotypes and co-stimulatory molecules. The specific procedure is as follows:

[0155] UDC monoclonal cells UDC981 and DC0502 were collected and placed in 1.5 ml EP tubes. Flow cytometry samples were prepared according to the standard flow cytometry staining procedure, and analysis was performed using a flow cytometer (Beckman). The data was then visualized using FlowJo software (BD). The antibody reagents used were PE anti-human CD4 (Biolegend, 300441), FITC anti-human CD19 (Biolegend, 363008), PE anti-human CD56 (Biolegend, 362508), BV421 anti-human CD14 (Biolegend, 325628), and FITC anti-human. CD4 (Biolegend, 300506), FITC anti-human DC-SIGN (Biolegend, 330103), PE / Cyanine7 anti-human CCR7 (Biolegend, 353226), Alexa Fluor700 anti-human HLA-ABC (Biolegend, 311438), BV510 anti-human HLA-DR (Biolegend, 307646), PE anti-human CD205 (Biolegend, 342204), PE / Cyanine7 The results for anti-human CD83 (Biolegend, 305330), BV510 anti-human CD80 (Biolegend, 305234), PE anti-human CD86 (Biolegend, 305406), BV650 anti-human CD40 (Biolegend, 334338), PE anti-human CD70 (Biolegend, 355104), PE / Cyanine7 anti-human CD137L (Biolegend, 311512), FITC anti-human CD11C (Biolegend, 301604), PE anti-human CD274 (Biolegend, 153611), and their corresponding isotype controls (Biolegend) are shown in Figures 7 to 12.

[0156] As can be seen from FIGS. 7 to 12, the expression intensities of the UDC981 and DC0502 phenotypes and costimulatory molecules were compared. After the DC genome was edited to knockout the HLA-A gene, HLA-B gene, and HLA-C gene encoding HLA class I molecules, the antigen-presenting HLA class I classical molecules HLA-A molecule, HLA-B molecule, and HLA-C molecule did not express, and it was verified that other DC surface molecules still retained the phenotype and costimulatory molecule expression ability consistent with DC WT (DC0502).

[0157] Example 6

[0158] This example provides the expression of HLA class I molecules after reintroducing (complementing) the gene encoding HLA class I molecules into UDC single clone 1-19 (UDC981).

[0159] The antigen-presenting ability of DC is one of its core abilities as a professional antigen-presenting cell. DC presents endogenous antigens to the TCR on the surface of CD8 + T cells for recognition and binding, and further stimulates the activation of CD-eight + T cells to initiate a specific immune cell response. UDC981 has knocked out the genes expressing HLA-A molecule, HLA-B molecule, and HLA-C molecule, does not express HLA-A molecule, HLA-B molecule, and HLA-C molecule, and itself does not have antigen-presenting ability. Therefore, genes encoding different types of HLA class I molecules are reintroduced through different gene transfer pathways to verify whether the stimulation activation and proliferation of different HLA class I antigen-specific cells can be achieved.

[0160] In this example, the reintroduction of different HLA class I molecules into UDC981 includes the following. Based on UDC981, the lentivirus method is used to integrate the genes encoding different HLA class I molecules into the UDC981 genome.

[0161] In this embodiment, we selected the construction of UDCs by reintroducing the HLA-A0201, HLA-A1101, and HLA-A2402 molecules, based on some of the most common types of human HLA-A molecular subtypes. The amino acid sequences of the HLA-A0201, HLA-A1101, and HLA-A2402 molecules are shown in SEQ ID NO.2 to SEQ ID NO.4, and their coding genes were synthesized by Nanjing Jinsirui Biotechnology Co., Ltd.

[0162] HLA-A0201 molecule: MAVMAPRTLVLLLSGALALTQTWAGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAE QLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEPSSQPTIPIVGIIAGLVLFGAVITGAVVAAVMWRRKSSDRKGGSYSQAASSDSAQGSDVSLTACKV(SEQ ID NO.2).

[0163] HLA-A1101 molecule: MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRGYYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAE QQRAYLEGRCVEWLRRYLENGKETLQRTDPPKTHMTHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWELSSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSSDRKGGSYTQAASSDSAQGSDVSLTACKV(SEQ ID NO.3).

[0164] HLA-A2402 molecule: MAVMAPRTLVLLLSGALALTQTWAGSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIALRYYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAE QQRAYLEGTCVDGLRRYLENGKETLQRTDPPKTHMTHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEPSSQPTVPIVGIIAGLVLLGAVITGAVVAAVMWRRNSSDRKGGSYSQAASSDSAQGSDVSLTACKV(SEQ ID NO.4).

[0165] As an example, incorporating genes encoding different HLA class I molecules into the UDC981 genome using a lentiviral approach involves viral vector construction and lentiviral packaging, specifically including the following: The genes encoding the HLA-A0201, HLA-A1101, and HLA-A2402 molecules were inserted into the pCDH-CMV-MCS-EF1 commercial lentiviral vector, transformed into Stbl3 competent cells, and after confirming the correct sequencing, the plasmids were extracted and purified using an endotoxin-free plasmid extraction kit. 293T cells are cultured in a 10 cm cell culture dish, and plasmid transfection is initiated before exceeding 80% confluence. The target plasmid, packaging plasmids psPAX2, pMD2.G, and transfection reagent PEI (polyethylenimine) are mixed in a serum-free DMEM culture medium in a mass ratio of 3:2:1. Subsequently, a mixture of lentiviral plasmids containing genes encoding HLA-A0201, HLA-A1101, or HLA-A2402 molecules is added to the 293T cells, and the cells are cultured at 37°C. The cell culture supernatants are ultracentrifugally concentrated after 48 h and 72 h to collect viral particles carrying the target gene, and viral titers are measured. Collect and count UDC981 cells, seed them into four wells of a 6-well plate according to 5 × 10^5 cells / mL, and add 2 ml / well of the quantified cell suspension. Infect each experimental well of UDC981 with a lentivirus containing genes encoding HLA-A0201, HLA-A1101, or HLA-A2402 molecules according to MOI=50. After infection, the UDC981 cells are cultured and amplified for 3 weeks, and then HLA-ABC positive cell separation is performed using the Miltenyi MACS® Separation System. Refer to the instructions for Anti-PE MicroBeads (miltenyi biotec, Order no. 130-048-801) for the procedure.

[0166] The results are shown in Figure 13. As can be seen from Figure 13, UDC981 HLA-A0201, UDC981 HLA-A1101, and UDC981 HLA-A2402 were obtained after purification following positive fractionation, with a 95% HLA-A positivity rate.

[0167] The results described above indicate that after reintroducing the gene encoding an HLA class I molecule into UDC981, it can reexpress the HLA class I molecule.

[0168] Example 7

[0169] This example verifies the antigen-presenting ability of UDC981 HLA-A0201, that is, whether it can stimulate the proliferation of antigen-specific T cells that match the HLA-A0201 molecule.

[0170] Stimulating the proliferation of antigen-specific T cells of the HLA-A0201 molecular subtype includes the following: UDC981 HLA-A0201 is loaded with an antigen peptide that is restricted to the HLA-A0201 molecular subtype. The antigen epitope of this antigen peptide is an antigen epitope recognized by target antigen-specific T cells. DCs loaded with antigen peptides are co-cultured with T cell populations to activate and amplify specific T cells capable of recognizing the aforementioned antigen epitopes.

[0171] In this embodiment, the T cell population is PBMC (Bokan Biotechnology (Shandong) Co., Ltd.), and its HLA-A subtype is HLA-A0201. Resuscitation is performed before use. In this embodiment, resuscitation is performed as follows: PBMCs that have been left standing for 2 hours are removed from the incubator, centrifuged at 600g for 5 minutes, the supernatant is aspirated and discarded, Optivitro (EXCEL, TE000-N022) + 2% Clin-SFM (Beijing Kelinen Biotechnology Co., Ltd., 1007) culture medium is added to the centrifuge tube to resuspend the cells, the cell suspension density is adjusted to 2 × 10⁶ cells / mL, and inoculated into a 24-well plate at 1 ml / well.

[0172] In this embodiment, the UDC981 HLA-A0201 is a general-purpose DC incorporating the HLA-A0201, specifically as described in Example 6.

[0173] In this embodiment, the antigen peptides are the CMV epitope and the Mart-1 epitope, and are HLA-A molecule-restricted antigen peptides.

[0174] In this embodiment, the loading of the antigen peptide onto UDC981 HLA-A0201 includes the following: Cell suspensions of DC0502 (positive control), UDC981 (negative control), and UDC981 HLA-A0201 were added to centrifuge tubes, centrifuged at 600 g for 5 minutes, the supernatant was aspirated and discarded, and each tube was resuspended in culture medium. The cell culture medium was prepared as XVIVO15 (LONZA, 04-418Q) + 400 IU / ml IL-2 (Tetracyclic Biotechnology, National Pharmaceutical Code S10970015), and the cell suspension density was adjusted to 1.0 × 10⁶ cells / mL. This was inoculated into 48-well plates, and each of the DC0502, UDC981, and UDC981 HLA-A0201 cells was seeded into four culture wells. In each culture well, CMV-A0201, Mart-1-A0201 antigen peptide (synthesized by Nanjing Jinsirui Biotechnology Co., Ltd., with amino acid sequences as shown in SEQ ID NO. 5 and SEQ ID NO. 6) (10 μg / μL), and a blank control were added according to a 1:1000 ratio. The cell culture plates were placed in a 37°C incubator and incubated for 4 hours to obtain three types of dendritic cells (DCs) loaded with the antigen peptides.

[0175] In the example, the stimulation of antigen-specific T cell proliferation by co-culturing DCs loaded with antigen peptides and T cell populations includes the following: Different DCs loaded with antigen peptides and corresponding blank control DCs are taken and counted, and the number of DCs is added to PBMC culture wells according to DC:PBMC=1:100, gently mixed, and the DC / PBMC co-culture plate is cultured in a 37°C, 5% CO2 incubator for approximately 14 days, with fluid replenishment (feeding) or amplification operations performed every two days during the process.

[0176] The detection method, which involves detecting the percentage of antigen-specific T cell proliferation stimulated by UDC981 HLA-A0201 loaded with an antigen peptide during the culture process, includes the following: Cells from different sample groups were collected on day 7 and day 9 of DC / PBMC co-cultures loaded with antigen peptides. Each sample was placed in a 1.5 ml EP tube and centrifuged at 400 g for 5 minutes to collect the cells. Discard the supernatant, resuspend the cell precipitate in 50 μL of FACS buffer (purchased from Beijing Tianjingsha Genetics Co., Ltd.), add 1 μL of Human TruStain FcX / EP tube to the sample tube, and incubate at room temperature in the dark for 15 minutes. After incubation is complete, add the corresponding Tetramer-CMV-A0201 and Tetramer-Mart-1 A0201 to each tube based on the loading of different antigen peptides, add 0.5 μL of Tetramer / EP tube, and incubate at 37°C for 30 minutes in the dark. After incubation is complete, wash the cells with 200 μL of FACS buffer, centrifuge at 400 g for 5 minutes to collect the cells, aspirate and discard the supernatant, and prepare the flow cytometry sample according to the standard flow cytometry staining procedure. The PBMC surface marker antibody molecules used were Alexa Fluor 700 anti-human CD3, APC anti-human CD4, and BrilliantViolet 510. TM anti-human CD8, PE / Cyanine7 anti-human CD56 (NCAM). Analysis was performed using a flow cytometer (Beckman), and the flow cytometry data was analyzed with Beckman CytExpert software. The results are shown in Figures 14 and 15, where Figure 14 shows the results with CMV-A0201 antigen addition and Figure 15 shows the results with Mart-1-A0201 antigen addition. The statistical results are shown in Tables 1 and 2.

[0177] [Table 1]

[0178] [Table 2]

[0179] Looking at Figures 14 and 15 and Tables 2 and 3 together, Co-culture time points 7 and 9 of UDC981 (negative control) and PBMCs showed specific CD8 positive for antigen peptide CMV-tetramer and antigen peptide Mart-1-tetramer, respectively. + The T cells were not stimulated, which is consistent with the expected result because UDC981 lacks the HLA-A molecule and therefore does not possess antigen-presenting ability. DC0502 (HLA-A0201 positive) and PBMC co-culture at day 7 and day 9 stimulated CMV-tetramer-positive specific CD8 + The percentages of T cells were 1.06%, 1.7%, and Mart-1-tetramer-positive specific CD8. + The percentage of T cells was 0.36% and 0.49%. UDC981 HLA-A0201 and PBMC co-culture at day 7 and day 9 stimulated CMV-tetramer-positive specific CD8 + The percentages of T cells were 1.27%, 2.98%, and Mart-1-tetramer-positive specific CD8. + The percentages of T cells were 0.53% and 1.09% (calculated as: percentage of tetramer-positive cells in the antigen peptide-added group - percentage of tetramer-positive cells in the mock group).

[0180] The above example illustrates that UDC981 HLA-A0201 is a different type of antigen-specific CD8, similar to wild-type DC0502 (HLA-A0201 positive). + The study demonstrated that T cells could be stimulated, and the positive rate gradually increased over time from day 7 to day 9. It also showed that after reintroducing the HLA-A0201 molecule onto the UDC981 basal body, UDC981 HLA-A0201 possessed antigen-presenting ability consistent with wild-type DC0502 (HLA-A0201), and that its effect was superior to that of wild-type DC0502.

[0181] Example 8

[0182] This example verifies the antigen-presenting ability of UDC981 HLA-A2402, that is, whether it can stimulate the activation and proliferation of antigen-specific T cells that match the HLA-A2402 molecule.

[0183] Referring to Example 7, in this example the T cell population is PBMC (Bokan Biotechnology (Shandong) Co., Ltd.) with HLA-A subtype HLA-A2402, and is resuscitated before use. In this example, resuscitation includes the following: Remove the PBMCs, which have been left standing for 2 hours, from the incubator, centrifuge at 600 g for 5 minutes, aspirate and discard the supernatant, add Optivitro (EXCEL, TE000-N022) + 2% Clin-SFM (Beijing Kelinen Biotechnology Co., Ltd., 1007) culture medium to the centrifuge tube to resuspend the cells, adjust the cell suspension density to 2 × 10⁶ cells / mL, and inoculate into a 24-well plate at 1 ml / well.

[0184] In this embodiment, UDC981 HLA-A2402 is a general-purpose DC into which HLA-A2402 has been introduced, specifically as described in Example 6. The HLA-A gene subtypes of the original DC (DC0502) of UDC981 HLA-A2402 are HLA-A0201 and HLA-A6801, meaning that a different HLA-A gene subtype from the original DC has been introduced into the general-purpose DC.

[0185] In this embodiment, the antigen peptide is a CMV epitope and is an HLA-A2402 molecule-restricted antigen peptide.

[0186] In this example, the loading of antigen peptides onto UDC981 HLA-A2402 includes the following: Cell suspensions of DC-0801 and DC-0802 (both containing HLA-A2402 and HLA-A1101 molecular phenotypes, positive controls) and UDC981 HLA-A2402, respectively, were added to centrifuge tubes, centrifuged at 600g for 5 minutes, the supernatant was aspirated and discarded, and each tube was resuspended in culture medium. The cell culture medium was prepared as XVIVO15 (LONZA, 04-418Q) + 400 IU / ml IL-2 (Tetracyclic Biotechnology, National Pharmaceutical Code S10970015), the cell suspension density was adjusted to 1.0 × 10⁶ cells / mL, and the cells were inoculated into 48-well plates. Each HLA-A2402 cell was seeded into three culture wells. In each culture well, CMV-A2402 antigen peptide (synthesized by Nanjing Jinsirui Biotechnology Co., Ltd., with its amino acid sequence shown in SEQ ID NO. 7) (10 μg / μL) and a blank control were added according to a 1:1000 ratio. The cell culture plate was placed in a 37°C incubator and incubated for 4 hours to obtain four types of dendritic cells (DCs) loaded with the antigen peptide.

[0187] In the examples, the stimulation of antigen-specific T cell proliferation by co-culturing DCs loaded with antigen peptides and T cell populations includes the following: Take different DCs loaded with antigen peptides and corresponding blank control DCs, count them, add the number of DCs to the PBMC culture wells according to a DC:PBMC ratio of 1:100, gently mix, and incubate the DC / PBMC co-culture plate in a 37°C, 5% CO2 incubator for approximately 14 days, performing fluid replenishment (feeding) or amplification every two days during the incubation period.

[0188] The detection method, which involves detecting the percentage of antigen-specific T cell proliferation stimulated by UDC981 HLA-A0201 loaded with an antigen peptide during the culture process, includes the following: Cells from different sample groups were collected on day 9 and day 11 of DC / PBMC co-cultures loaded with antigen peptides. Each sample was placed in a 1.5 ml EP tube and centrifuged at 400 g for 5 minutes to collect the cells. Discard the supernatant, resuspend the cell precipitate in 50 μL FACS buffer, add 1 μL Human TruStain FcX / EP tube to the sample tube, and incubate at room temperature in the dark for 15 minutes. After incubation is complete, add the corresponding Tetramer-CMV-A2402 based on the antigen peptide load to each tube, add 0.5 μL Tetramer / EP tube, and incubate at 37°C in the dark for 30 minutes. After incubation is complete, wash the cells with 200 μL of FACS buffer, centrifuge at 400 g for 5 minutes to collect the cells, aspirate and discard the supernatant, and prepare the flow cytometry sample according to the standard flow cytometry staining procedure. The PBMC surface marker molecule antibodies used were Alexa Fluor 700 anti-human CD3, APC anti-human CD4, and BrilliantViolet 510. TM The drugs tested were anti-human CD8 and PE / Cyanine7 anti-human CD56 (NCAM). Analysis was performed using a flow cytometer (Beckman), and the flow cytometry data was analyzed using Beckman CytExpert software. The results are shown in Figure 16, and the statistical results are shown in Table 3.

[0189] [Table 3]

[0190] As can be seen from Figure 16 and Table 3, Two wild-type DC strains (including the HLA-A2402 subtype), DC-0801 and DC-0802, were co-cultured with PBMCs at day 9 and day 11, and specific CD8 cells were stimulated with the antigen peptide CMV-tetramer. + The percentages of T cells were 0.63%, 0.15%, and 2.47% and 2.15%, respectively. UDC981 HLA-A2402 and PBMC co-culture at day 9 and day 11 stimulated specific CD8 antigen peptide positive for CMV-tetramer. +The proportion of T cells was 4.9% and 9.78%, respectively, and specific CD8 cells were positive for the antigen peptide CMV-tetramer. + The proportion of T cells increased over time, becoming approximately twice as high on day 11 compared to day 9, indicating that UDC981 possesses extremely strong antigen-presenting capabilities after HLA-A2402 reintroduction.

[0191] In this experiment, antigen-specific CD8 of UDC981 HLA-A2402 + The ability to stimulate T cells was significantly stronger than that of wild-type DCs (HLA-A2402 subtypes) DC-0801 and DC-0802. This is likely because UDC981 HLA-A2402 is a single subtype of HLA-A2402, while DC-0801 and DC-0802 are heterozygotes of HLA-A2402 and HLA-A1101, resulting in greater specificity in the amplification of UDC981 HLA-A2402 against antigen-specific T cells.

[0192] Example 9

[0193] This example verifies the immunogenicity of UDC981 HLA-A11 against allogeneic PBMCs.

[0194] T cells are stimulated to proliferate when they come into contact with allogeneic somatic cells that do not match their HLA class I. To investigate whether matching a single allele of HLA class I can reduce immune stimulation to allogeneic T cells, we will evaluate the immunogenicity of UDC981 cells reintroduced (supplemented) with HLA class I molecules through T cell proliferation experiments. Specifically, the following will be conducted. Allogeneic PBMCs (Bokan Biotechnology (Shandong) Co., Ltd.) possessing a single allele HLA-A*11:01 but not matching in other alleles are labeled with CFSE. The staining procedure is as follows: PBMCs (Bokan Biotechnology (Shandong) Co., Ltd.) with HLA-A subtype HLA-A02:HLA-A11 are resuscitated before use. Resuscitation includes the following: PBMCs left standing for 2 hours are removed from the incubator, centrifuged at 600 g for 5 minutes, the supernatant is aspirated and discarded, and Optivitro (EXCEL, TE000-N022) + 2% Clin-SFM (Beijing Kelinen Biotechnology Co., Ltd., 1007) culture medium is added to the centrifuge tube to resuspend the cells, and the cell suspension density is adjusted to 1 × 10^7 cells / mL. Add CFSE Cell Division Tracker Kit (Biolegend, GYS0004) to the cell suspension to a final concentration of 5 μM. Also, remove some cells and set aside unstained cells as a control. Place the stained cell suspension in a 37°C incubator, shielded from light, and incubate for 15 minutes. Add five times the volume of pre-warmed culture medium (1640 + 10% FBS) to the stained cell suspension, incubate for 5 minutes, and remove any excess free dye. Centrifuge the cell suspension at 600 g for 10 min, resuspend the cell precipitate using pre-warmed culture medium (1640 + 10% FBS + 20 IU IL-2) to adjust the cell density to 1 × 10^6 cells / mL. Inoculate stained and unstained PBMCs into a 6-well plate and add 2 mL / well of cell suspension. Add wild-type DC0502 with HLA-A subtype HLA-A02:HLA-A68 in a 200:1 ratio to the CFSE-labeled PBMC culture wells, and add UDC981 HLA-A11 (specifically HLA-A1101, whose amino acid sequence is as shown in SEQ ID NO.3, and whose coding gene was synthesized by Nanjing Jinsirui Biotechnology Co., Ltd., see Example 6 for the construction of UDC981 HLA-A11) to another well. Set up individual PBMC culture wells (CFSE labeled, unlabeled) and a positive control well: 2 μL T Cell TransAct TM Add (miltenyi, 130-128-758). Co-culture was performed for 6 days, and CD3 was analyzed via flow cytometry (FACS). + The fluorescence intensity of CFSE in a T cell subset was detected. Analysis was performed using a flow cytometer (Beckman), and the flow cytometry data was analyzed with Beckman CytExpert software. The results are shown in Figure 17. The results are as follows: CD3 proliferated in the wild-type DC0502 group (DC0502 WT) where the HLA class I subtype did not match. + The percentage of T cells reached 27.57%. In the UDC981 HLA-A11 group, this percentage decreased to 4.86%, and the background level (unstimulated CD3) + It is relatively similar to the cells (2.62%). UDC981, which matches a single allele of HLA class I, has been shown to reduce non-antigen-specific immune stimulation against allogeneic T cells.

[0195] As can be seen from the above, the general-purpose DC, its manufacturing method, and applications provided in this application can prevent the expression of HLA-A, HLA-B, and HLA-C molecules in genome-edited modified DCs, and sequencing results indicate that the corresponding sites of all six alleles of HLA-A, HLA-B, and HLA-C were mutated in the resulting UDC monoclonal. Modified UDCs can reexpress any subtype of HLA class I molecule through genome editing, viral vectors, and mRNA technology, overcoming the limitations of the DC's own HLA class I molecule and enabling the amplification of any HLA class I-restricted antigen-specific T cells as APCs. At the same time, allogeneic immunogenicity tests performed after reintroducing (supplementing) HLA class I molecules into UDC981 demonstrate that UDC981 possesses antigen presentation and specific immune activation functions in actual applications while simultaneously avoiding the core risk of allogeneic immune rejection. The low nonspecific T cell activation characteristics of UDC981 cells provide important justification for realizing safe allogeneic general-purpose applications. By combining this with antigen peptide loading or mRNA antigen expression technology, DC vaccine products can be developed for any HLA class I type.

Claims

1. A general-purpose DC characterized in that the HLA class I gene of the general-purpose DC is knocked out, and the HLA class I gene includes an HLA-A gene, an HLA-B gene and / or an HLA-C gene.

2. The general-purpose DC according to claim 1, characterized in that the HLA class I gene includes an HLA-A gene, an HLA-B gene, and an HLA-C gene.

3. The general-purpose DC according to claim 1, characterized in that the general-purpose DC is an immortalized general-purpose DC.

4. The general-purpose DC according to claim 2, characterized in that the general-purpose DC is an immortalized general-purpose DC.

5. The general-purpose DC according to claim 3, characterized in that the immortalized general-purpose DC is UDC981, deposited with the Center for Ordinary Microorganisms (CGMCC) of the China Microbial Species Preservation and Management Commission, deposited on December 25, 2024, deposited at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and deposited at CGMCC No. 46260.

6. The general-purpose DC according to claim 4, characterized in that the immortalized general-purpose DC is UDC981, deposited with the Center for Ordinary Microorganisms (CGMCC) of the China Microbial Species Preservation and Management Commission, deposited on December 25, 2024, deposited at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and deposited at CGMCC No. 46260.

7. A general-purpose DC cell population, characterized in that the cell population includes the general-purpose DC described in any one of claims 1 to 6.

8. A method for manufacturing a general-purpose DC, wherein the manufacturing method is: A step of knocking out the HLA class I gene of a primordial DC, wherein the HLA class I gene includes the HLA-A gene, the HLA-B gene and / or the HLA-C gene, and the knockout of the HLA class I gene of the primordial DC comprises knocking out the HLA class I gene of the primordial DC using homologous recombination, CRISPR / Cas genome editing, or a transcription activator-like effector nuclease method, A method for producing general-purpose DCs, characterized by comprising the step of selecting DCs that negatively express the HLA class I gene, i.e., general-purpose DCs.

9. The aforementioned CRISPR / Cas genome editing method is A step of designing one or more sgRNAs that bind to the HLA class I gene of a primitive DC, A method for producing a general-purpose DC according to claim 8, characterized by comprising the step of introducing sgRNA into a primitive DC using the CRISPR-Cas system to knock out the HLA class I gene.

10. A method for producing a general-purpose DC according to claim 9, characterized in that the HLA-A gene subtype of the primitive DC is HLA-A0201 and / or HLA-A6801, and / or the HLA-B gene subtype is HLA-B0801 and / or HLA-B1507, and / or the HLA-C gene subtype is HLA-C0303 and / or HLA-C0702, and the nucleotide sequence of the sgRNA is as shown in SEQIDNO.

1.

11. A method for producing a general-purpose DC according to any one of claims 8 to 10, characterized in that the original DC is an immortalized DC.

12. The method for producing a general-purpose DC according to claim 11, characterized in that the immortalized DC is DC0502, deposited with the Center for Ordinary Microorganisms (CGMCC) of the China Microbial Species Preservation and Storage Administration, deposited on December 25, 2024, deposited at No. 3, Courthouse 1, Beichen West Road, Chaoyang District, Beijing, and deposited at CGMCC No. 46259.

13. A method for amplifying target antigen-specific T cells, We obtained HLA class I molecular subtypes that are restrictive to target antigens in T cell populations, and the HLA class I molecules include HLA-A molecules, HLA-B molecules and / or HLA-C molecules. The HLA class I gene encoding the HLA class I molecular subtype is introduced into the aforementioned general-purpose DC. The dendritic cells into which the HLA class I gene has been introduced are loaded with an antigen peptide that is restrictive to the HLA class I molecular subtype, and the antigen epitope of the antigen peptide is an antigen epitope recognized by target antigen-specific T cells. A method for amplifying target antigen-specific T cells, characterized by co-culturing DCs loaded with the antigen peptide with the T cell population to activate and amplify specific T cells capable of recognizing the antigen epitope.

14. The method for amplifying target antigen-specific T cells according to claim 13, characterized in that the antigen peptide is derived from a tumor or a virus.

15. A method for amplifying target antigen-specific T cells according to claim 13 or 14, characterized in that the method for introducing the HLA class I gene includes genome editing, a virus, or an mRNA method.

16. The method for amplifying target antigen-specific T cells according to claim 15, characterized in that the T cell group includes PBMCs.

17. A target antigen-specific T cell amplification reagent, wherein the reagent comprises a general-purpose DC as described in any one of claims 1 to 6.

18. A target antigen-specific T cell amplification reagent, characterized in that the reagent includes the general-purpose DC cell population described in claim 7.

19. The reagent according to claim 18, further comprising an HLA class I gene encoding an HLA class I molecular subtype of the target antigen-specific T cell, wherein the HLA class I molecule comprises an HLA-A molecule, an HLA-B molecule and / or an HLA-C molecule.

20. Modified DC characterized in that the primitive HLA class I gene is knocked out and an exogenous HLA class I gene is introduced, the exogenous HLA class I gene is a single subtype of HLA class I gene, the exogenous HLA class I gene includes an HLA-A gene, an HLA-B gene, or an HLA-C gene, and the primitive HLA class I gene includes at least an HLA class I gene of the same type as the exogenous HLA class I gene.

21. sgRNA characterized by having a nucleotide sequence as shown in SEQIDNO.

1.

22. The use of sgRNA according to claim 21 in knocking out an HLA class I gene in a cell, characterized in that the HLA class I gene comprises an HLA-A gene, an HLA-B gene and / or an HLA-C gene, the HLA-A gene subtype of the cell is HLA-A0201 and / or HLA-A6801, and / or the HLA-B gene subtype is HLA-B0801 and / or HLA-B1507, and / or the HLA-C gene subtype is HLA-C0303 and / or HLA-C0702.

23. The use according to claim 22, characterized in that the cells include DCs or T cells.

24. The use according to claim 23, characterized in that the DC is an immortalized DC.

25. The use according to claim 24, characterized in that the immortalized DC is DC0502, deposited with the Center for Ordinary Microorganisms (CGMCC) of the China Microbial Species Preservation and Storage Administration, deposited on December 25, 2024, deposited at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and deposited at CGMCC No. 46259.

26. An immortalized DC, named DC0502, deposited with the Center for Ordinary Microorganisms (CGMCC) of the China Microbial Species Preservation and Storage Administration, deposited on December 25, 2024, deposited at No. 3, Courthouse 1, Beichen West Road, Chaoyang District, Beijing, with deposit number CGMCC No. 46259.

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