Universal dendritic cell and preparation method and application thereof

Abstract

This application discloses a universal DC belonging to the field of immune cell technology, a method for manufacturing the same, and an application. The universal DC is a universal DC that does not express HLA-I class molecules, obtained by knocking out HLA-I class genes from the original DC. When applied, depending on the HLA-I class molecular subtype of the target antigen-specific T cells to be obtained, HLA-I class molecules matching the target antigen-specific T cells can be re-expressed in the universal DC through gene editing, viral, or mRNA technology. This overcomes the cell-specific matching limitations of HLA-I class molecules present in the application of allogeneic DCs and resolves the problem of insufficient coverage of a single DC HLA-I class molecular subtype.

Description

Technology Field

[0001] This application belongs to the field of immune cell technology, and specifically relates to a general-purpose DC and a method for manufacturing the same and its applications. Background Technology

[0002] The immune system can recognize foreign components and eliminate potentially harmful foreign molecules and cells from the body, and it also has the ability to recognize and destroy abnormal cells originating from the host's own tissues. Immune responses include innate immunity and adaptive immunity, where adaptive immunity includes humoral immunity mediated by the B cell response (where B cells develop into plasma cells and secrete soluble antigen-specific antibodies) and cell-mediated immunity mediated by specific subgroups 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 referred to as the Human Leukocyte Antigen (HLA) complex.

[0003] Dendritic cells (DCs) are specialized antigen-presenting cells involved in both cellular and humoral immunity; as a key link in the entire immune system, they significantly influence immune system functions. One of the primary functions of DCs is to recognize captured antigens and present them to naive T lymphocytes, thereby stimulating T cell activation and proliferation to generate large quantities of cytotoxic T lymphocytes (CTLs) that recognize specific antigens and possess strong cytotoxicity. T cell activation requires CD4 or CD8 on the T cell surface to bind to HLA molecules, while simultaneously the alpha and beta peptide chains of the T cell receptor (TCR) on the T cell surface must mutually recognize and interact with the antigen-MHC complex.

[0004] HLA molecules used for antigen presentation mainly include HLA-I class molecules and HLA-II class molecules. Here, HLA-I class molecules include HLA-A, HLA-B, and HLA-C molecules, and the genes encoding these three molecules are, respectively HLA-A gene, HLA-B Genes and HLA-C It is a gene. CD8 T cells primarily react with HLA-I class molecules, then undergo activation and proliferation to recognize specific antigens and generate large quantities of T lymphocytes with strong cytotoxicity.

[0005] HLAGenes are located on human chromosome 6 and constitute the most complex and polymorphic genetic system in the human population to date, possessing numerous alleles. Consequently, HLA molecules exhibit high polymorphism, and there are significant differences in the structure and composition of HLA molecules among different individuals. Furthermore, since HLA molecules on the surface of donor organ cells are foreign antigens to the recipient's immune system, this is also a significant cause of immune responses occurring during the organ transplantation process. The aforementioned problem also exists when T cells are activated by presenting captured antigens using allogeneic 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. Consequently, the TCR and the antigen-MHC complex cannot mutually recognize or interact, making it impossible to achieve the effect of activating target T cells; in other words, there is a limitation in HLA molecule-specific matching.

[0006] In 1996, Hsu FJ et al. of the Department of Oncology at Stanford University Medical Center first reported clinical trials of a dendritic cell vaccine in Nature Medicine, and research on DC tumor vaccines worldwide has continued for decades; currently, several DC tumor vaccines, such as Sipuleucel-T (Provenge), CreaVax RCC, Hybricell, and DCVax Brain, have received market approval. Conventional DC immunotherapy involves first collecting peripheral blood monocytes (PBMCs) from a patient and differentiating them into mature DCs through a series of in vitro induction and cultures; then, loading tumor or viral antigens onto the DCs in vitro; and either directly re-injecting them into the patient's body or stimulating and activating antigen-specific T cells in vitro and then re-injecting the activated antigen-specific T cells.

[0007] In existing treatment methods, the acquisition and differentiation of mature DCs is time-consuming and labor-intensive due to limited sources and restrictions on HLA molecular matching, which limits the use of only autologous peripheral blood monocytes for induced differentiation; this places an additional burden on some individuals with pre-existing poor health.

[0008] Therefore, DC immunotherapy capable of overcoming HLA molecular-specific matching limitations, particularly HLA-I class molecular-specific matching limitations, is of great significance. The problem to be solved

[0009] In order to solve the problem of HLA-I class molecular-specific matching limitation when applying the above-mentioned homologous heterologous DC in the prior art, the present application provides a universal DC, a method for manufacturing the same, and an application thereof, and the HLA-I class gene of the original DC ( HLA-A gene, HLA-B Genes and / or HLA-C A universal DC that does not express HLA-I class molecules is obtained by knocking out a gene; upon application, depending on the HLA-I class molecular subtype of the target antigen-specific T cell to be obtained, an HLA-I class molecular subtype matching the target antigen-specific T cell is re-expressed in the universal DC through gene editing, viral, or mRNA technology, thereby overcoming the HLA-I class molecular specific match limitation present in the application of allogeneic DCs and also resolving the problem of insufficient coverage of a single DC HLA-I class molecular subtype. means of solving the problem

[0010] To solve the above problems, the technical solution adopted in this application is as follows.

[0011] In a first aspect, the present application provides a general-purpose DC, and the general-purpose DC HLA-I Class genes are knocked out, HLA-IThe class genes include HLA-A genes, HLA-B genes and / or HLA-C genes, and the universal DC overcomes the HLA-I class molecular-specific match restriction, i.e., HLA-I DCs prior to the knockout of class genes (original DCs) can only stimulate the activation of antigen-specific T cells identical (matching) to their own HLA-A, HLA-B, and / or HLA-C molecular subtypes, but HLA-I Universal DC with knocked-out class genes is new HLA-A gene, HLA-B Genes and / or HLA-C By reintroducing genes, antigen-specific T cells matching new HLA-A, HLA-B, and / or HLA-C molecular subtypes can be stimulated. HLA-I "Class gene" is HLA-A gene, HLA-B gene, HLA-C Although it includes genes, those skilled in the art know that molecular-specific matching restrictions are primarily restrictions of genes (subtypes), so here “ HLA-I "Class gene" is HLA-A gene, HLA-B Genes and HLA-C It may mean any one of the genes or a combination thereof, where “ HLA-I "Class gene" is HLA-A gene, HLA-B gene, HLA-C gene, HLA-A Genes and HLA-B gene, HLA-B Genes and HLA-C gene, HLA-A Genes and HLA-C gene, HLA-A gene, HLA-B Genes and HLA-C It could be a gene.

[0012] In some specific embodiments of the present application, the general-purpose DC HLA-A The gene is knocked out, and the above universal DC releases the HLA-A molecule-specific matching restriction, i.e., HLA-ADCs prior to gene knockout (original DCs) can only stimulate the activation of antigen-specific T cells identical to (matching) their own HLA-A molecular subtype, but HLA-A Gene-knockout universal DC is a new HLA-A By reintroducing the gene, the activation of antigen-specific T cells matching the new HLA-A molecular subtype can be stimulated.

[0013] In some specific embodiments of the present application, the general-purpose DC HLA-B The gene was knocked out, and the above universal DC released the HLA-B molecule-specific matching restriction.

[0014] In some specific embodiments of the present application, the general-purpose DC HLA-C The gene was knocked out, and the above universal DC released the HLA-C molecule-specific matching restriction.

[0015] In some specific embodiments of the present application, the general-purpose DC HLA-A Genes and HLA-B The gene was knocked out, and the above universal DC released the specific matching restriction of HLA-A and HLA-B molecules, i.e. HLA-A Genes and HLA-B DCs prior to gene knockout (original DCs) can only stimulate the activation of antigen-specific T cells identical (matching) to their own HLA-A and / or HLA-B molecular subtypes, but HLA-A Genes and HLA-B Gene-knockout universal DC is a new HLA-A Genes and / or HLA-B By reintroducing the gene, the activation of antigen-specific T cells matching a new HLA-A molecule and / or a second HLA-B molecule subtype can be stimulated.

[0016] In some specific embodiments of the present application, the general-purpose DC HLA-A Genes and HLA-CThe gene was knocked out, and the above universal DC released the specific matching restriction of HLA-A molecules and HLA-C molecules.

[0017] In some specific embodiments of the present application, the general-purpose DC HLA-B Genes and HLA-C The gene was knocked out, and the above universal DC released the specific matching restriction of HLA-B molecules and HLA-C molecules.

[0018] In some specific embodiments of the present application, the general-purpose DC HLA-A gene, HLA-B Genes and HLA-C The gene is knocked out, and the above universal DC has released the specific matching restriction of HLA-A, HLA-B, and HLA-C molecules, and a new HLA-A gene, HLA-B Genes and / or HLA-C By reintroducing genes, the activation of antigen-specific T cells corresponding to new HLA-A, HLA-B, and / or HLA-C molecular subtypes can be stimulated.

[0019] In some specific embodiments of the present application, the general-purpose DC is an immortalized general-purpose DC, and the immortalized general-purpose DC is of the immortalized DC. HLA-I Class genes can be obtained by knocking out immortalized cells, which can proliferate indefinitely in vitro to meet requirements for the number and activity of DCs, thereby overcoming the disadvantages of having a small number of primary DCs and difficulty in culturing and amplifying them in vitro; at the same time, by inducing monocytes, the problem of low numbers and high costs in obtaining MoDCs (monocyte-derived DCs) can be overcome.

[0020] In some specific embodiments of the present application, the immortalized universal DC is UDC981 and is deposited with the General Microbiology Center of the Chinese Commission on Depository and Management of Microbial Species (CGMCC), with the deposit date being December 25, 2024, the deposit address being No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC No. 46260.

[0021] In a second aspect, the present application also provides a universal DC cell population, wherein the cell population comprises any of the universal DCs.

[0022] In a third aspect, the present application also provides a universal DC cell system, wherein the cell system comprises any of the universal DCs.

[0023] In a fourth aspect, the present application also provides a method for manufacturing the general-purpose DC, said manufacturing method,

[0024] Originally DC's HLA-I The step of knocking out class genes - HLA-I Class genes are HLA-A gene, HLA-B Genes and / or HLA-C Contains genes -; and

[0025] HLA-I It includes a step of screening DCs in which the class gene is negatively expressed, i.e., universal DCs.

[0026] In some specific embodiments of the present application, the original DC's HLA-A gene is knocked out; HLA-A Screening for gene-negative DCs, that is, universal DCs with the HLA-A molecule-specific matching restriction lifted.

[0027] In some specific embodiments of the present application, the original DC's HLA-B gene is knocked out; HLA-B Screening for gene-negative DCs, that is, universal DCs with the HLA-B molecule-specific matching restriction lifted.

[0028] In some specific embodiments of the present application, the original DC's HLA-C gene is knocked out; HLA-C Screening for gene-negative DCs, that is, universal DCs with the HLA-C molecule-specific matching restriction lifted.

[0029] In some specific embodiments of the present application, the original DC's HLA-A gene and HLA-B Knock out genes; HLA-A Genes and HLA-B Screening is performed for gene-negative DCs, specifically universal DCs with HLA-A and HLA-B molecule-specific matching restrictions lifted.

[0030] In some specific embodiments of the present application, the original DC's HLA-A gene and HLA-C Knock out genes; HLA-A Genes and HLA-C Screening is performed for gene-negative DCs, specifically universal DCs with HLA-A and HLA-C molecule-specific matching restrictions lifted.

[0031] In some specific embodiments of the present application, the original DC's HLA-B gene and HLA-C Knock out genes; HLA-B Genes and HLA-C Screening is performed for gene-negative DCs, specifically universal DCs with HLA-B and HLA-C molecule-specific matching restrictions lifted.

[0032] In some specific embodiments of the present application, the original DC's HLA-A gene, HLA-B Genes and HLA-C Knock out genes; HLA-A gene, HLA-B Genes and HLA-C Screening is performed for gene-negative DCs, specifically universal DCs with HLA-A, HLA-B, and HLA-C molecule-specific match restrictions removed.

[0033] In some specific embodiments of the present application, the original DC HLA-IMethods for knocking out class genes include homologous recombination methods, CRISPR / Cas gene editing methods, or TALEN (transcriptional activator-like effector nuclease method) methods.

[0034] In some specific embodiments of the present application, the CRISPR / Cas gene editing method is,

[0035] Originally DC's HLA-I Step of designing one or more sgRNAs that bind to class genes - in this application, “original DC’s HLA-I The “step of designing one or more sgRNAs that bind to a class gene” involves binding to the sgRNA according to the universal DC to be manufactured. HLA-I It means determining class genes, and universal DC HLA-A If it is a DC with a knocked-out gene, at least the original DC's HLA-A Designing sgRNA that binds to genes; universal DC HLA-A Genes and HLA-B If it is a DC with a knocked-out gene, at least HLA-A Genes and HLA-B It is possible to design two sgRNAs that bind to each gene, or at least HLA-A Genes and HLA-B It is possible to design sgRNAs that bind to genes simultaneously -; and

[0036] By introducing sgRNA into the original DC using the CRISPR-Cas system, HLA-I It includes a step of knocking out class genes.

[0037] In some specific embodiments of the present application, the CRISPR-Cas system includes a CRISPR-Cas9 system or a CRISPR-Cas13 system.

[0038] In some specific embodiments of the present application, the sgRNA is one or more HLA-I It binds to the class gene.

[0039] In some specific embodiments of the present application, the sgRNA comprises 17 to 23 bases.

[0040] In some specific embodiments of the present application, the original DC HLA-A Gene subtypes are HLA-A0201 and / or HLA-A6801 and; and / or HLA-B Gene subtypes are HLA-B0801 and / or HLA-B1507 is; and / or HLA-C Gene subtypes are HLA-C0303 and / or HLA-C0702 am.

[0041] In some specific embodiments of the present application, coupled to the original DC HLA-I The nucleotide sequence of the sgRNA bound to the class is denoted by SEQ ID NO.1, and the sgRNA is of the original DC HLA-A A pair of alleles of a gene HLA-A0201 and HLA-A6801 ; HLA-B A pair of alleles of a gene HLA-B0801 and HLA-B1507 ; and HLA-C A pair of alleles of a gene HLA-C0303 and HLA-C0702 It can target all three pairs of alleles simultaneously and has very high gene editing efficiency by knocking out three pairs of alleles at once.

[0042] In some specific embodiments of the present application, the original DC is an immortalized DC, and the immortalized DC HLA-I By knocking out class genes, immortalized universal DCs can be obtained, and immortalized universal DCs can be infinitely multiplied in vitro to meet the requirements for the number and activity of DCs, thereby overcoming the disadvantages of primary DCs, which are small in number and difficult to culture and amplify in vitro; at the same time, the problem of low number and high cost of MoDCs can be overcome.

[0043] In some specific embodiments of the present application, the immortalized DC is DC0502, is deposited with the General Microbiology Center of the Chinese Commission on Depository and Management of Microbial Species (CGMCC), the deposit date is December 25, 2024, the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 46259; the immortalized DC HLA-A Gene subtypes are HLA-A0201 and HLA-A6801 is; HLA-B Gene subtypes are HLA-B0801 and HLA-B1507 And; HLA-C Gene subtypes are HLA-C0303 and HLA-C0702 am.

[0044] In a fifth aspect, the present application also provides an application of the universal DC in the activation of target antigen-specific T cells, said application,

[0045] Acquire HLA-I class molecular subtypes of a T cell population restricted to the target antigen, wherein the HLA-I class molecules include HLA-A molecules, HLA-B molecules and / or HLA-C molecules;

[0046] coding the HLA-I class molecular subtype in the above universal DC HLA-I Introducing class genes; HLA-I reintroduce class genes, and the above HLA-I If the HLA-I class molecular subtype coded by the class gene differs from the original HLA-I class molecular subtype of the universal DC, it overcomes the HLA-I class molecular-specific match restriction of the original DC; it may also be used to activate autologous T cells by introducing the same gene coding for the original HLA-I class molecular subtype;

[0047] HLA-I The DC into which the class gene has been introduced loads the HLA-I class molecular subtype restriction antigen peptide, wherein the antigen epitope of the antigen peptide is an antigen epitope recognized by target antigen-specific T cells;

[0048] DCs loaded with antigen peptides are co-cultured with the T cell population to activate specific T cells capable of recognizing the antigen epitope.

[0049] In some specific embodiments of the present application, encoding the HLA-I class molecule HLA-I The above method of introducing class genes includes gene editing, virus, or mRNA methods.

[0050] In some specific embodiments of the present application, the antigen peptide is derived from a tumor or a virus.

[0051] In some specific embodiments of the present application, the tumor includes melanoma.

[0052] In some specific embodiments of the present application, the virus includes a cytomegalovirus.

[0053] In some specific embodiments of the present application, the T cell population includes PBMCs.

[0054] In a sixth aspect, the present application also provides the application of the universal DC cell population in the activation of target antigen-specific T cells, wherein the universal DC cell population comprises the universal DC.

[0055] In a seventh aspect, the present application also provides an application of the universal DC cell system in the activation of target antigen-specific T cells, wherein the universal DC cell system comprises the universal DC.

[0056] In an eighth aspect, the present application also provides an application of the universal DC for preventing or treating a tumor or viral infection, said application comprising injecting an effective amount of DC cells loaded with a tumor or viral antigen peptide into the body of a subject; said DC cells having an HLA-I class molecular subtype coding gene identical to that of the subject introduced therein, said antigen peptide being the HLA-I class molecular subtype restriction antigen peptide.

[0057] In a ninth aspect, the present application also provides an application of the universal DC cell population for preventing or treating tumors or viral infections.

[0058] In a tenth aspect, the present application also provides an application of the universal DC cell system for preventing or treating tumors or viral infections.

[0059] In the eleventh aspect, the present application also provides a method for amplifying target antigen-specific T cells, said method,

[0060] A step of obtaining HLA-I class molecular subtypes of a T cell population restricted to a target antigen, wherein the HLA-I class molecules include HLA-A molecules, HLA-B molecules and / or HLA-C molecules;

[0061] coding the HLA-I class molecular subtype in the above universal DC HLA-I Step of introducing class genes;

[0062] HLA-I The DC into which the class gene has been introduced loads the above HLA-I class molecular subtype restriction antigen peptide - the antigen epitope of the antigen peptide is an antigen epitope recognized by target antigen-specific T cells -;

[0063] The method includes the step of co-culturing DCs loaded with antigen peptides with the T cell population and activating and amplifying specific T cells capable of recognizing the antigen epitope.

[0064] In some specific embodiments of the present application, encoding the HLA-I class molecule HLA-I Methods for introducing class genes include gene editing, viral, or mRNA methods.

[0065] In some specific embodiments of the present application, the antigen peptide is derived from a tumor or a virus.

[0066] In some specific embodiments of the present application, the tumor includes melanoma.

[0067] In some specific embodiments of the present application, the virus includes a cytomegalovirus.

[0068] In some specific embodiments of the present application, the T cell population includes PBMCs.

[0069] In a 12th aspect, the present application also provides an application of the general-purpose DC in the preparation of a reagent for amplifying target antigen-specific T cells.

[0070] In a 13th aspect, the present application also provides the application of the universal DC cell population in the preparation of a reagent for amplifying target antigen-specific T cells.

[0071] In a 14th aspect, the present application also provides an application of the universal DC cell system in the preparation of a reagent for amplifying target antigen-specific T cells.

[0072] In some specific embodiments of the present application, the reagent codes for the HLA-I class molecular subtype of the target antigen-specific T cell. HLA-I It includes class genes, and the HLA-I class molecular subtype includes HLA-A molecules, HLA-B molecules, and / or HLA-C molecules.

[0073] In a 15th aspect, the present application also provides a modified DC, and the original of said modified DC HLA-I Class genes are knocked out, and at the same time, exogenous HLA-I Class genes are introduced, but the above exogenous HLA-I Class genes are a single subtype HLA-I It is a class gene and exogenous HLA-I Class genes are HLA-A gene, HLA-B Gene or HLA-C It contains genes, and originally HLA-I Class genes are at least exogenous HLA-I Class genes and homologous HLA-I Includes class genes, and the applicant, through research, a single subtype HLA-I It was discovered that DCs with introduced class genes have a stronger ability to activate and amplify antigen-specific CD8 T cells compared to the original DCs, and this is the original DCs' HLA-I Class genes are mostly alleles containing two subtypes, and exogenous HLA-I It is speculated that the introduction of the class gene is due to its stronger ability to activate and amplify antigen-specific CD8 T cells because there is only one subtype.

[0074] In a 16th aspect, the present application also provides a method for manufacturing the modified DC, said method,

[0075] Single subtype in the above general-purpose DC HLA-I It includes the step of obtaining a modified DC by introducing a class gene.

[0076] In the 17th aspect, the present application also provides an sgRNA, the nucleotide sequence of said sgRNA is denoted by SEQ ID NO.1, and said sgRNA is HLA-A A pair of alleles of a gene HLA-A0201 and HLA-A6801 ; HLA-B A pair of alleles of a gene HLA-B0801 and HLA-B1507 ; HLA-C A pair of alleles of a gene HLA-C0303 and HLA-C0702 It can simultaneously target and cells including the said gene subtype HLA-A , HLA-B and HLA-C It can be used to simultaneously knock out genes.

[0077] In the 18th aspect, the present application also includes cells HLA-I Providing the application of the above sgRNA in the knockout of class genes, HLA-I Class genes are HLA-A , HLA-B and / or HLA-C Includes, and the cell HLA-A Gene subtypes are HLA-A0201 and / or HLA-A6801 is; and / or HLA-B Gene subtypes are HLA-B0801 and / or HLA-B1507 and; and / or HLA-C Gene subtypes are HLA-C0303 and / or HLA-C0702 am.

[0078] In some specific embodiments of the present application, cells SONGS-I Providing the application of the above sgRNA in the knockout of class genes, SONGS-I Class genes are HLA-A Includes, and the cell HLA-A Gene subtypes are HLA-A0201 and / or HLA-A6801 am.

[0079] In some specific embodiments of the present application, cells SONGS-I Providing a knockout application of the above sgRNA in the knockout of class genes, SONGS-I Class genes are HLA-B Includes, and the cell HLA-B Gene subtypes are HLA-B0801 and / or HLA-B1507 am.

[0080] In some specific embodiments of the present application, cells SONGS-I Providing a knockout application of the above sgRNA in the knockout of class genes, SONGS-I Class genes are HLA-C Includes, and the cell HLA-C Gene subtypes are HLA-C0303 and / or HLA-C0702 am.

[0081] In some specific embodiments of the present application, cells SONGS-I Providing the application of the above sgRNA in the knockout of class genes, SONGS-I Class genes are HLA-A , HLA-B and HLA-C Includes, and the cell HLA-A Gene subtypes are HLA-A0201 and / or HLA-A6801 is; HLA-B Gene subtypes are HLA-B0801 and / or HLA-B1507 And; HLA-C Gene subtypes are HLA-C0303 and / or HLA-C0702 am.

[0082] In some specific embodiments of the present application, cells SONGS-I The application of the sgRNA is provided in the knockout of class genes, and the cells include DCs and / or T cells.

[0083] In some specific embodiments of the present application, cells SONGS-I The application of the above sgRNA is provided in the knockout of class genes, and the cell is a DC.

[0084] In some specific embodiments of the present application, the DC is an immortalized DC.

[0085] In some specific embodiments of the present application, the immortalized DC is DC0502, is deposited with the General Microbiology Center of the Chinese Commission on Depository and Management of Microbial Species (CGMCC), the deposit date is December 25, 2024, the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 46259; the immortalized DC HLA-A Gene subtypes are HLA-A0201 and HLA-A6801 is; HLA-B Gene subtypes are HLA-B0801 and HLA-B1507 And; HLA-C Gene subtypes are HLA-C0303 and HLA-C0702 am.

[0086] In the 19th aspect, the present application further provides immortalized DC DC0502 and is deposited with the General Microbiology Center of the Chinese Commission on Microbial Species Depositary Control (CGMCC), with a deposit date of December 25, 2024, a deposit address of No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and deposit number CGMCC No. 46259. Effects of the invention

[0087] The advantageous effects of the present application compared with the prior art are as follows.

[0088] (1) In the general-purpose DC and the method of manufacturing and application thereof provided in the present application, the original DC, in particular the immortalized DC SONGS-I Class gene ( HLA-A gene, HLA-B Genes and / or HLA-C By knocking out the gene, universal DCs (UDCs) that do not express HLA-I class molecules are obtained. When applied, depending on the HLA-I class molecular subtype of the target antigen-specific T cells to be acquired—that is, different individuals—HLA-I class molecules matching the target antigen-specific T cells can be re-expressed in the universal DCs (UDCs) through gene introduction technologies such as gene editing, viruses, or mRNA. This removes the HLA-I class molecular specific matching limitations present in the application of allogeneic DCs, resolves the problems of long manufacturing cycles and high costs associated with personalized cellular immunotherapy products, and enables "off-the-shelf" personalized cell therapy products. This significantly reduces costs and holds broad application value for allogeneic cellular immunotherapy products.

[0089] (2) In the universal DC and the method of manufacturing and application thereof provided in the present application, an HLA-I class molecule matching a target antigen-specific T cell is re-expressed in the universal DC (UDC), and said HLA-I class molecule has only one subtype, and regardless of whether it is self or homologous, the original DC SONGS-IClass alleles often have two subtypes and, compared to the original DC, reduce interference from allogeneic HLA-I class molecules and enhance the ability to activate and amplify antigen-specific CD8 T cells.

[0090] (3) In the universal DC and the method of manufacturing and application provided in this application, an HLA-I class molecule that matches a target antigen-specific T cell is re-expressed in the universal DC (UDC), and the risk of graft-versus-host disease or immune rejection is reduced by preventing the non-specific activation and amplification of allogeneic reactive T cells due to HLA mismatch.

[0091] (4) In the general-purpose DC and the method of manufacturing and application thereof provided in this application, if the original cell is an immortalized DC, an immortalized general-purpose DC with infinite amplification ability can be obtained. For example, the single clone UDC981 provided in this application has an amplification multiplier of 18,000 times after being cultured for 30 days, so the requirements for the number and activity of DCs after convenient and rapid culture can be met, thereby overcoming the disadvantages of primary DCs which are small in number and difficult to culture and amplify in vitro; at the same time, the problem of the number of MoDCs being small and the cost being high can be overcome.

[0092] (5) In the general-purpose DC and the method of manufacturing and application thereof provided in this application, the applicant designs the nucleotide sequence of sgRNA represented by SEQ ID NO.1 for the immortalized DC DC0502 previously screened by the applicant, and DC0502's HLA-A A pair of alleles of a gene HLA-A0201 and HLA-A6801 , HLA-B A pair of alleles of a gene HLA-B0801 and HLA-B1507 and HLA-C A pair of alleles of a gene HLA-C0303 and HLA-C0702 It can simultaneously target and, in conjunction with genome editing methods, DC0502's HLA-Agene, HLA-B Genes and HLA-C It can be used to simultaneously knock out genes, resulting in very high gene editing efficiency. Brief explanation of the drawing

[0093] FIG. 1 shows the ratio of HLA-ABC negative and HLA-A0201 negative cells detected in flow cytometry after gene editing of the original DC (DC0502) in the present application (left arrow), and the ratio of HLA-ABC negative and HLA-A0201 negative universal DC (UDC) after magnetic bead negative selection (right arrow), where unstain is an unstained control; and UDC mix is ​​a population of gene-edited DCs. Figure 2 is a cell morphology image of a DC before and after gene editing in the present application, where the left image is the original DC (DC0502) that has not undergone gene editing, and the right image is the gene-edited DC (UDC mix). Figure 3 is from the purified gene-edited DC in the present application HLA-A , HLA-B , HLA-C This is an overlapping histogram obtained by flow cytometry analysis of selected homozygous background DC monoclones in which three genes (each gene having a pair of alleles) were completely knocked out. Figure 4 is from the purified gene-edited DC in the present application HLA-A , HLA-B , HLA-C This is the statistical result of the mean fluorescence intensity (MFI) values ​​of three antibodies analyzed via flow cytometry on selected homozygous background DC monoclones in which three genes (each gene having a pair of alleles) were completely knocked out. FIG. 5 is a single clone of UDC981 manufactured according to the present application. HLA-A , HLA-B , HLA-C This is the sequencing result of three genes (each gene consists of a pair of alleles). Figure 6 shows the amplification multiple detection results of the original DC (DC0502) and the manufactured single clone UDC981 in the present application. FIGS. 7 to 9 are flow cytometry results for the phenotype and co-stimulatory molecule identification of a single clone UDC981 produced according to the present application, where isotype refers to an isotype of immunoglobulin (Ig). Figures 10 to 12 are flow cytometry results for the phenotype and co-stimulatory molecule identification of the original DC (DC0502) used in the present application. FIG. 13 is from the present application HLA-A0201 , HLA-A1101 , HLA-A2402 This is the result of flow cytometry detection on the HLA-A molecular positivity rates of UDC HLA-A0201, UDC HLA-A1101, and UDC HLA-A2402 after self-bead positive selection of UDC981 infected with a lentivirus containing [the component]. FIG. 14 is from the present application HLA-A0201 This is the result of verifying the ability of this reintroduced UDC981 (UDC HLA-A0201) to present CMV epitopes (cytomegalovirus epitopes). FIG. 15 is from the present application HLA-A0201 This is the result of verifying the ability of this reintroduced UDC981 (UDC HLA-A0201) to present the Mart-1 epitope (melanoma cell epitope). FIG. 16 is from the present application HLA-A2402 This is the result of verifying the ability of reintroduced UDC981 (UDC HLA-A2402) to present CMV epitopes (cytomegalovirus epitopes). Figure 17 shows the results of verifying the immunogenicity response test of UDC981 (UDC HLA-A11) with reintroduced HLA-A11 in the present application against homologous and heterologous PBMCs. Specific details for implementing the invention

[0094] The present application is further explained in conjunction with the specific embodiments below.

[0095] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by a person skilled in the art to which this application pertains; and the term “and / or” used herein includes any and all combinations of one or more related listed items.

[0096] Unless specific conditions are specified in the examples, the procedure is performed under ordinary conditions or conditions provided by the manufacturer. If the manufacturer is not specified for any reagents or equipment used, they are all generic products available on the market.

[0097] As used herein, the term “about” is used to provide flexibility and inaccuracy regarding a given term, measurement, or value. A person skilled in the art can easily determine the degree of flexibility of a specific variable.

[0098] Concentration, quantity, and other value data may be presented in the form of ranges in this specification. It should be understood that such range formats are used merely for convenience and brevity and should be interpreted flexibly to include not only the values ​​explicitly mentioned as the limits of the range, but also all individual values ​​or sub-ranges included within said range, as each value and sub-range is explicitly mentioned. For example, a value range of about 1 to about 4.5 should be interpreted to include not only the explicitly mentioned limits of 1 to about 4.5, but also individual numbers (e.g., 2, 3, 4) and sub-ranges (e.g., 1 to 3, 2 to 4, etc.). The same principle applies to ranges mentioning only one value, such as “less than about 4.5,” and should be interpreted to include all described values ​​and ranges. Furthermore, this interpretation should apply regardless of the width of the described range or feature.

[0099] As used in this application, “dendritic cell (DC)” is a specialized antigen-presenting cell involved in both cellular and humoral immunity. In this application, unless otherwise specified, “DC” may represent a cell strain or a cell line. A cell strain refers to a population of cells with specific properties or markers obtained from a primary culture or cell line through selection or cloning methods, such as UDC981 obtained by screening after gene editing in this application; a cell line refers to a population of cells propagated from a primary culture after successful first passage, such as DC0502 formed by successful passage.

[0100] As used in this application, the “immune system” refers to a system in which an organism performs immune responses and immune functions, and is composed of immune organs, immune cells, and immune molecules, capable of recognizing and eliminating external pathogens (e.g., bacteria, viruses, parasites, etc.), and simultaneously monitoring and eliminating abnormal cells (e.g., cancer cells), auto-aging, and damaged cells within the body, thereby maintaining the health of the organism and the stability of the internal environment.

[0101] As used in this application, “T cells,” or T lymphocytes, are a type of lymphocyte derived from hematopoietic stem cells in the bone marrow. They undergo a complex developmental process in the thymus, including positive and negative selection, to finally mature and enter peripheral lymphoid organs and blood circulation; they primarily consist of cytotoxic T cells (CD8 T cells) and helper T cells (CD4 T cells). Cytotoxic T cells primarily play a role in recognizing and killing cells infected with pathogens, such as viruses and intracellular parasites, as well as tumor cells. They recognize antigen-major histocompatibility complex I (MHC-I) molecules on the surface of target cells via T cell receptors (TCRs) on their cell surfaces. Upon recognition, cytotoxic T cells release perforin and granzyme to induce the death of the target cells. The activation of cytotoxic T cells requires the TCR to recognize the antigen-MHC-I complex.

[0102] As used in this application, “stimulation of T cells” or “activation of T cells” have substantially the same meaning and refer to a process in which T cells, after recognizing an antigen, are activated into activated T cells (also referred to as activated T cells), enter the cell cycle, proliferate in large numbers, and provide a sufficient number of effector T cells for an immune response.

[0103] As used in this application, “allele” refers to a pair of genes located at the same position on a pair of homologous chromosomes that control relative traits, and in human chromosomes, different gene forms may exist at a specific locus (a specific position of a gene on a chromosome), and these different forms are precisely alleles. For example, in cell DC0502 of this application, HLA-A0201 and HLA-A6801 this HLA-A If different gene forms are located at the locus, HLA-A0201 and HLA-A6801 It is referred to as an allele, and specific HLA-A0201 second HLA-AIt is referred to as a genetic subtype.

[0104] As used in this application, “autologous” means tissue, cell, or material derived from the same individual itself; and “homologous heterologous” means tissue, cell, or material derived from different individuals of the same species.

[0105] As used in this application, “HLA molecule-specific match” means that T cells can be activated only when the HLA molecules of the DC and the T cell are identical or similar; or that the DC can activate only T cells that are identical or similar to its HLA molecules. It should be noted that, here, HLA molecules do not refer to all HLA molecules identical or similar to the HLA molecule, but rather to HLA molecules that play a role in the process of activating specific T cells.

[0106] As used in this application, “ SONGS-I “Class genes” refer to a type of human major histocompatibility complex (MHC) gene that plays an important role in immune response and immune recognition processes. SONGS-I The products encoded by “class genes” are HLA-I class molecules (sometimes referred to as HLA-I class proteins), and these molecules are primarily involved in the presentation of endogenous antigens, helping the immune system recognize its own cells and cells infected by external pathogens. Specifically, HLA-I class molecules can bind to intracellularly synthesized antigen peptides, such as viral protein antigens synthesized within the cell after a virus infects it; after binding to HLA-I class molecules, these antigen peptides are transported to the cell surface for recognition by CD8 T lymphocytes, and CD8 + T cells recognize these antigen-peptide-HLA-I class molecular complexes through their T cell receptors (TCRs), initiate a cellular immune response, and kill virus-infected cells. SONGS-I “Class genes” mainly include three types, and each HLA-A gene, HLA-B gene, HLA-C It is a gene, and its coding products are HLA-A, HLA-B, and HLA-C molecules, respectively. The binding specificity of HLA-A, HLA-B, and HLA-C molecules to different antigen peptides differs, for example, HLA-A molecules may have a high affinity for specific viral antigen peptides, whereas HLA-B molecules may have a stronger binding ability to other antigen peptides depending on the structural characteristics of the antigen binding groove. HLA-A gene, HLA-B gene, HLA-C Genes possess a high degree of polymorphism, that is, in a population, HLA-A gene, HLA-B gene, HLA-C Genes have various different allele forms, and different individuals are different HLA-A Two individuals that possess genes and have the same genes HLA-A Alleles may also differ, and specific alleles of a particular individual can be detected and classified through specific technical solutions, and specific alleles are referred to as “gene subtypes.” In this application, “ SONGS-I "Class gene" is HLA-A gene, HLA-B gene, HLA-C It includes three genes, but all HLA-A gene, HLA-B Genes and HLA-C It should be noted that this does not refer to genes, and unless otherwise specified, “ SONGS-I "Class gene" is HLA-A gene, HLA-B Genes and HLA-C It may refer to any one of the genes or a combination thereof, and a person skilled in the art may determine this based on the context.

[0107] As used in this application, “Peripheral Blood Mononuclear Cells (PBMCs)” is a general term for cells with a single nucleus present in peripheral blood, and these cells include lymphocytes (T cells, B cells, NK cells) and other monocytes. Mature DCs can be obtained from monocytes through a series of in vitro induction cultures and are referred to as MoDCs (monocyte-derived DCs, MODCs).

[0108] As used in this application, “knockout,” “gene knockout,” and also “gene removal” and “gene knock-out” refer to the removal, inactivation, or reduction of expression of a specific gene within a cell genome, and the “specific gene” is also referred to as a “target gene,” “target gene,” etc.

[0109] As used in this application, “original DC” refers to an initial cell for producing a general-purpose DC, which may be a natural DC, or an optimized DC such as a DC that can be immortalized after purification or a DC that can be immortalized after gene editing.

[0110] As used in this application, “immortalization” means having infinite proliferation ability, and under appropriate culture conditions, immortalized cells, unlike normal somatic cells, do not enter the senescence and death phase after a certain number of divisions but continuously perform cell division, thereby enabling an incessant increase in the number of cells.

[0111] As used in this application, “target antigen-specific T cell” refers to a T cell capable of recognizing a target antigen.

[0112] As used in this application, “negative expression” means that an expected signal or marker does not appear in the cell, which indicates that the content of the detected target substance (e.g., protein, gene, antigen, etc.) in the cell is extremely low or non-existent. For example HLA-A It is a DC with negative gene expression, that is, a DC with very low or absent HLA-A molecule content.

[0113] As used in this application, CRISPR / Cas (Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR-associated proteins) is a gene editing technology, and you may refer to the reagent supplier's manual.

[0114] As used in this application, gRNA (guide RNA), i.e., guide RNA, is a core component of a CRISPR / Cas gene editing system and can realize editing of a specific gene region by guiding a Cas protein (e.g., Cas9) to a target DNA sequence.

[0115] As used in this application, “restriction antigen peptide” refers to an antigen peptide that binds to a specific major histocompatibility complex (MHC) molecule during the immune response process, is presented to and recognized by T lymphocytes via the MHC molecule, and there exists a certain restriction relationship between such antigen peptide and the MHC molecule, namely that only a specific antigen peptide can bind to a specific MHC molecule, and only a specific T cell receptor (TCR) can recognize such antigen peptide-MHC complex. Likewise, the said MHC molecule is referred to as the restriction MHC molecule of the antigen. For example, the HLA-A0201 restriction antigen peptide refers to a specific antigen peptide that binds to HLA-A0201; and HLA-A0201 is referred to as the restriction HLA-A molecule of the antigen peptide.

[0116] As used in this application, “antigen epitope” refers to a specific chemical group that determines antigen specificity in an antigen molecule. In this application, the CMV epitope is a specific region in a cytomegalovirus (CMV) antigen molecule recognized by immune cells; and the Mart-1 epitope is a specific region in a melanoma cell-associated antigen (Mart-1 antigen) molecule recognized by immune cells.

[0117] As used in this application, “reintroduction” means the reintroduction of a target gene. For example, HLA-A0201 The reintroduction of [it] into universal DCs through gene introduction technologies such as gene editing, viruses, or mRNA. HLA-A0201 It means re-expressing the HLA-A0201 molecule by introducing a gene.

[0118] As used in this application, “ HLA-ABC KO DC(s)” is HLA-A gene, HLA-B Genes and HLA-C It refers to DCs with negative gene expression, that is, the said DCs do not produce HLA-A, HLA-B, and HLA-C molecules, and are also referred to as HLA-ABC negative cells in terms of protein molecules. Similarly, HLA-A0201 KO DC is HLA-A0201 It refers to a DC with negative gene expression, meaning that the said DC does not produce the HLA-A0201 molecule.

[0119] As used in this application, “HLA-ABC + DCs” is HLA-A gene, HLA-B Genes and HLA-C It refers to a DC in which at least one gene is positively expressed, that is, at least one of HLA-A, HLA-B, and HLA-C molecules is present in the said DC.

[0120] As used in this application, flow cytometry (FCM) is a technology for rapidly and quantitatively analyzing and classifying cells or microparticles (e.g., bacteria, viruses, etc.), and may refer to the manuals of instrument or reagent suppliers.

[0121] As used in this application, “gene-edited DC” means a gene-edited DC (UDC mix).

[0122] As used in this application, “UDC981 HLA-A0201” refers to UDC981 expressing the HLA-A0201 molecular subtype.

[0123] 1. 실시예

[0124] The present embodiment provides a method for manufacturing a general-purpose DC and a manufactured general-purpose DC.

[0125] In a method for manufacturing a general-purpose DC, the method is of the original DC SONGS-I The step of knocking out class genes - SONGS-I Class genes are HLA-A gene, HLA-B Genes and / or HLA-C It is a gene -; and SONGS-I It includes a step of screening DCs in which the class gene is negatively expressed, i.e., universal DCs.

[0126] In this embodiment, the original DC is DC0502, the DC is an immortalized DC, and is deposited with the General Microbiology Center of the Chinese Commission on Depositary Use of Microorganisms (CGMCC), with a deposit date of December 25, 2024, a deposit address of No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and a deposit number of CGMCC No. 46259; the immortalized DC HLA-A Gene subtypes are HLA-A0201 and HLA-A6801 is; HLA-B Gene subtypes are HLA-B0801 and HLA-B1507 And; HLA-C Gene subtypes are HLA-C0303 and HLA-C0702 am.

[0127] In this embodiment, SONGS-I Class genes are HLA-A gene, HLA-B Genes and HLA-C It is a gene, that is, the original DC HLA-A gene, HLA-B Genes and HLA-C Knock out genes.

[0128] In this embodiment, the original DC SONGS-I Knockout class genes is done using the CRISPR / Cas gene editing method. SONGS-I It involves knocking out class genes, whereas the CRISPR / Cas gene editing method is the original DC's SONGS-I A step of designing one or more sgRNAs that bind to a class gene; and introducing the sgRNA into the original DC using a CRISPR-Cas system, SONGS-I It includes a step of knocking out class genes.

[0129] In the example, the nucleotide sequence of the sgRNA is denoted as SEQ ID NO.1 (CGGCTACTACAACCAGAGCG), and the sgRNA is the original DC's HLA-A A pair of alleles of a gene HLA-A0201 and HLA-A6801 ; HLA-B A pair of alleles of a gene HLA-B0801 and HLA-B1507 ; and HLA-C A pair of alleles of a gene HLA-C0303 and HLA-C0702 It can simultaneously target and can simultaneously knock out three pairs of alleles, thus having very high gene editing efficiency; the above sgRNA was synthesized by Nanjing Genscript Biotech Co., Ltd. (GenScript).

[0130] In the examples, the step of introducing sgRNA into the original DC using the CRISPR-Cas system is specifically,

[0131] Collect the logarithmic growth DC0502 in a 15 mL centrifuge tube, centrifuge at 400 g for 5 min, remove the supernatant, add 10 mL of phosphate-buffered saline (PBS), invert the centrifuge tube to gently wash, and centrifuge at 400 g for 5 min;

[0132] The supernatant is aspirated and removed, the cell precipitate is resuspended using PBS and counted; 1×10⁶ according to a standard electroporation protocol 6 Prepare the cell count for cells / test; centrifuge at 400g for 5 min, remove the supernatant, resuspend the cell precipitate at 50μL / test using Gene Pulser electroporation buffer equilibrated at room temperature (purchased from BIO-RAD, USA, Product No.: 1652676), mix gently, aspirate the cell suspension, and add it to a sterile 1.5mL EP tube;

[0133] In accordance with the instructions for use of GenCRISPR™ Cas9 v1.2 (purchased from Nanjing Genscript Biotech Co., Ltd., Product No.: Z03702), GenCRISPR™ Cas9 v1.2 and sgRNA were added to a sterile EP tube with electroporation buffer in a molar ratio of 1:3; the mixture was incubated at room temperature for 30 minutes to form an RNP complex;

[0134] Absorb the cell suspension in the EP tube into the RNP complex, mix thoroughly, absorb the mixture, and transfer it to a sterile electroporation cuvette;

[0135] BIO-RAD Electroporation System: RNP complex electroporation was performed using the Gene Pulser Xcell eukaryotic electroporation system (No. 165-2661), and the electroporation conditions were as follows: pulse type, square wave; voltage, 200V; pulse, 2ms;

[0136] After electroporation, the cells were immediately aspirated into a pre-warmed cell medium, the cell medium being XVIVO15 (LONZA, 04-418Q) + 400 IU / mL IL-2 (FourRingsBiology, National Drug Approval No. S10970015), and the culture plate was placed in a 37°C incubator and cultured continuously to obtain the UDC mix.

[0137] In this embodiment, SONGS-I Screening of DCs negatively expressing class genes was performed using a magnetic bead negative selection method, and purified SONGS-I Screening DCs with negative expression of the class gene, HLA-ABC It was recorded as KO DC.

[0138] In this embodiment, cell separation is performed using the Miltenyi MACS® Separation System, and the operation steps refer to the manual for Anti-PE MicroBeads (miltenyi biotec, Order no. 130-048-801) and are specifically as follows.

[0139] Cells were collected after amplification of the UDC mix obtained by culturing following gene editing, and labeled with the primary antibody: PE anti-human HLA-A, B, C Antibody (Biolegend, 311406); 100 μL each of 1 × 10⁶ 8 Add 5 μL of PE anti-human HLA-A, B, C Antibody to the cells / mL cell suspension, mix thoroughly, and incubate for 30 min in a dark room at 4°C;

[0140] Wash the labeled cells, resuspend the cell suspension using the buffer prepared according to the instructions, centrifuge at 300g for 6 min, and repeat the washing once;

[0141] Aspirate and completely remove the supernatant, and 1×10 7Add 20 μL of Anti-PE MicroBeads per cell, mix thoroughly, and incubate in a 4°C refrigerator for 15 minutes;

[0142] Wash, centrifuge at 300g for 6 min, and;

[0143] The supernatant is aspirated to completely remove it, and the cell precipitate is resuspended with 500μL of buffer;

[0144] Place the magnetic rack in the biosafety cabinet, install the classification LD column on the magnetic rack, and place the collection tube below the classification column;

[0145] Before sorting the cells, add 2 mL of buffer to the sorting column to ensure sufficient infiltration, and allow the buffer to pass through the sorting column and into the collection tube; once all the MACS buffer has been introduced, replace it with a new collection tube;

[0146] A cell suspension labeled with Anti-PE Microbeads is sieved through a 40 μm mesh to allow the cells to form a single-cell suspension;

[0147] The sieved cell suspension is added to the sorting column, and when the droplets flowing along the sorting column have almost stagnated, 2 mL of buffer is added to the sorting column again to wash, and the cell suspension flowing into the collection tube, i.e. HLA-ABC Collect KO DCs;

[0148] When the droplet flowing along the sorting column has nearly stagnated, remove the sorting column from the magnetic rack and insert it into a new collection tube, add 3 mL of buffer back to the sorting column, connect a booster pump to the inlet of the sorting column, pump the liquid inside the sorting column into the new collection tube, and the cell suspension is HLA-ABC + DCs and;

[0149] Classified HLA-ABC molecule-negative UDCs were inoculated into a cell culture plate, the cell medium was XVIVO15 (LONZA, 04-418Q) + 400 IU / ml IL-2 (Sahwan Bio, National Drug Approval No. 10970015), and the culture plate was placed in a 37℃ incubator and cultured continuously.

[0150] This embodiment also includes magnetic bead classification after HLA-ABC Provides sympathy for KO DCs.

[0151] In this embodiment, in the DC classified by magnetic bead technology HLA-ABC KO DC and HLA-A0201 By detecting the ratio of KO DC, HLA-ABC KO DC and HLA-A0201 The purity of KO DC was confirmed, and the detection results are shown in Figure 1.

[0152] As can be seen from the right side of the arrow in Fig. 1, the 2nd round Anti-PE MicroBeads negative selected gene-edited DCs account for a 100% ratio HLA-ABC KO DC and 100% ratio HLA-A0201 It is KO DC, and the purified gene-edited DC was named UDC.

[0153] The cell morphology images of DCs before and after gene editing are shown in Figure 2, and it can be seen that the gene-edited DC (UDC mix) has a morphology that matches that of the wild-type DC (DC0502).

[0154] 1. 실시예

[0155] This embodiment provides for the detection of gene editing efficiency of the CRISPR / Cas gene editing method of Example 1.

[0156] Specifically, while detecting DC0502 HLA-A gene, HLA-B Genes and HLA-C It is the efficiency of performing gene editing on three genes (each gene contains two alleles, i.e., a total of six genes).

[0157] In this embodiment, gene editing efficiency was first detected for the entire UDC mix obtained by culturing after electroporation using the HLA-ABC antibody (Biolegend, 311406). HLA-ABC After detecting KO efficiency, regarding the UDC mix obtained by culture after electroporation using the HLA-A0201 antibody (BD bioscience, 567739) SONGS- A0 Single gene editing efficiency was detected. Specifically:

[0158] DC0502 was cultured for 48 h after electroporation, and the gene-edited electroporated UDC mix and non-gene-edited DC0502 were collected in EP tubes, and HLA-ABC and HLA-A0201 molecules on the cell surface were stained according to the standard staining steps of flow cytometry.

[0159] The flow cytometry detection results are shown in Fig. 1, and as can be seen from the left side of the arrow in Fig. 1, the UDC mix obtained by culture after electroporation was 15% HLA-ABC molecular negative and 55.2% HLA-A0201 molecular negative, and the CRISPR / Cas gene editing method of Example 1, specifically the designed target sgRNA, HLA-A gene, HLA-B Genes and HLA-C It can simultaneously knock out genes and demonstrates high gene editing efficiency.

[0160] 3. 실시예

[0161] This embodiment provides additional verification and screening of a purified gene-edited DC (UDC) single clone.

[0162] The selection target of this embodiment is genotype HLA-A gene, HLA-B Genes and HLA-C It is a homozygous background UDC monoclone with three pairs of alleles completely knocked out.

[0163] Purified gene-edited DCs were collected and seeded into 96-well plates at a density of 0.5 to 1 cell / well for subcloning culture; the medium was XVIVO15 (LONZA, 04-418Q) + 400 IU / ml IL-2 (Sahwan Bio, National Drug Approval No. 10970015); after 2 to 3 weeks of culture and amplification, the expression of HLA-A, HLA-B, and HLA-C molecules of the DC monoclones and HLA-A gene, HLA-B Genes and HLA-C The gene was identified.

[0164] Seven single clones numbered 1-2, 1-3, 1-9, 1-10, 1-11, 1-19, and 1-20 were selected from dozens of UDC single clones to verify the expression of HLA-A, HLA-B, and HLA-C molecules. Flow cytometry samples were prepared according to standard flow cytometry staining procedures using 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), analyzed using a flow cytometry analyzer (Beckman), and plotted using FlowJo software (BD).

[0165] At the same time, cell genomic DNA is extracted, and through sequencing methods, its HLA-A gene, HLA-B Genes and HLA-C The gene sequence was detected.

[0166] The results are shown in Figures 3 to 5.

[0167] Figure 3 shows a purified gene-edited DC (UDC) HLA-A gene, HLA-B Genes and HLA-CThis is an overlaid histogram obtained by flow cytometry analysis of selected homozygous background UDC monoclones in which three pairs of alleles of the gene were completely knocked out, where the histogram of the first row of each group's histogram is the DC0502 negative control, the histogram of the second row of each group's histogram is the UDC mix positive control, and the third histogram of each group's histogram is the HLA-I class molecular expression intensity of each monoclone, where the third row of the first group corresponds to the HLA-A molecular expression intensity, the histogram of the third row of the second group corresponds to the HLA-B molecular expression intensity, and the histogram of the third row of the third group corresponds to the HLA-ABC molecular expression intensity. The results show that in the seven single clones 1-2, 1-3, 1-9, 1-10, 1-11, 1-19, and 1-20, the HLA-A, HLA-B, and HLA-ABC molecules of the single clones 1-11, 1-19, and 1-20 are at background levels.

[0168] As shown in Figure 4, analysis of the average fluorescence intensity of HLA-A, HLA-B, and HLA-ABC molecules revealed that HLA-A, HLA-B, and HLA-ABC molecules of the single clones 1-11, 1-19, and 1-20 exhibited negative expression.

[0169] Sequencing was performed on UDC single clones 1-19, and the results are as shown in Fig. 5.

[0170] HLA-A0201 The nucleotide sequence of a partial gene fragment is ACCCTGCGCGGCTACTACAACCAGAGCGAGGCCGG (SEQ ID NO.8), and the nucleotide sequence of the said fragment after gene editing is ACCCTGCGCGGCTACTACAACCAGAGGCCGG (SEQ ID NO.9);

[0171] HLA-A6801Part of the gene's nucleotide sequence is ACCCTGCGCGGCTACTACAACCAGAGCGAGG (SEQ ID NO. 10), and after gene editing, the nucleotide sequence of the said fragment is ACCCTGCGCGGCTACTACAACCAGAAGCGAGG (SEQ ID NO. 11);

[0172] HLA-B0801 Part of the gene's nucleotide sequence is AACCTGCGCGGCTACTACAACCAGAGCGAGG (SEQ ID NO. 12), and the nucleotide sequence of the fragment after gene editing is AACCTGCGCGGCTACTACAACCAGAAGCGAGG (SEQ ID NO. 13);

[0173] HLA-B1507 Part of the gene's nucleotide sequence is AACCTGCGCGGCTACTACAACCAGAGCGAGGCC (SEQ ID NO. 14), and the nucleotide sequence of the fragment after gene editing is AACCTGCGCGGCTACTACAACCAGCGAGGCC (SEQ ID NO. 15);

[0174] HLA-C0303 Part of the gene's nucleotide sequence is AACCTGCGCGGCTACTACAACCAGAGCGAGGCCA (SEQ ID NO. 16), and the nucleotide sequence of the fragment after gene editing is AACCTGCGCGGCTACTACAACCAGAAGCGAGGCCA (SEQ ID NO. 17);

[0175] HLA-C0702 Part of the gene's nucleotide sequence is AACCTGCGCGGCTACTACAACCAGAGCGAGGAC (SEQ ID NO.18), and the nucleotide sequence of the fragment after gene editing is AACCTGCGCGGCTACTACAACCAGAAGCGAGGAC (SEQ ID NO.19);

[0176] dental HLA-A gene, HLA-B Genes and HLA-C Three pairs of alleles of the gene are completely knocked out, and HLA-ABCIndicates that it is KO DC.

[0177] The above flow cytometry and sequencing results show that UDC monoclones 1-19 HLA-A gene, HLA-B Genes and HLA-C It indicates that the gene is a homozygous organism with three pairs of alleles completely knocked out, and the preferred UDC monoclone 1-19 is named UDC981 and is deposited with the General Microbiology Center of the Chinese Commission on Depositary Use of Microorganisms (CGMCC) on December 25, 2024, at the deposit address No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 46260.

[0178] 4. 실시예

[0179] This embodiment provides the detection of the proliferation ability of the original DC (DC0502) and UDC single clone 1-19 (UDC981).

[0180] Collect and count DC0502 and UDC981 respectively, and 5×10 5 Inoculate into T25 culture flasks according to cell density (cells / mL), total initial cell count: 5×10 6 Cells, 10 mL medium: XVIVO15 (LONZA, 04-418Q) + 400 IU / ml IL-2 (Sahwan Bio, National Drug Approval No. 10970015) was added. The color of the medium and cell status were observed daily; when the medium turned yellow, half was replaced for sampling and counting, and fresh medium was added simultaneously to maintain a cell density of 5–8 × 10⁶ 5 The cells / mL level was adjusted, and subcultures and counts were performed for 30 days.

[0181] The results of the amplification culture of DC0502 and UDC981 cells are shown in Figure 6, and after culturing for 30 days, the amplification multiplier reached 18,000 times, indicating that DC0502 and UDC981 can be continuously subcultured and amplified, thus possessing immortalization properties.

[0182] 5. 실시예

[0183] The present embodiment provides detection and identification of UDC monoclone 1-19 (UDC981) cell phenotypes.

[0184] Phenotypic identification of UDC981 cells was performed through a comprehensive evaluation of the expression of lineage markers on the DC surface, DC maturation markers, and functionally relevant molecules such as antigen-presenting molecules, co-stimulatory molecules, and molecules related to T cell activation.

[0185] The effects of gene editing on the phenotype and related functional molecules of DCs were verified through comprehensive expression detection of UDC981 phenotypes and co-stimulatory molecules. The specific steps are as follows.

[0186] UDC monoclonal UDC981 and DC0502 cells were collected and added to 1.5 mL EP tubes, respectively. Flow cytometry samples were prepared according to the standard staining procedure for flow cytometry, analyzed using a flow cytometer (Beckman), and the data were plotted using FlowJo software (BD). Antibody reagents used are: PE anti-human CD4 (Biolegend, 300441), FITC anti-human CD19 (Biolegend, 363008), PE anti-human CD56 (Biolegend, 362508), BV421 anti-human CD14 (Biolegend, 325628), 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 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 the corresponding allogeneic control (Biolegend), and the results are as shown in FIGS. 7 to 12.

[0187] As can be seen from Figs. 7 to 12, by comparing the phenotypes of UDC981 and DC0502 and the expression intensity of co-stimulatory molecules, DCs encoding HLA-I class molecules through gene editing HLA-A gene, HLA-B Genes and HLA-C After knocking out the gene, it was verified that the antigen-presenting HLA-I class classical molecules HLA-A, HLA-B, and HLA-C were not expressed, while other DC surface molecules still maintained the phenotype and ability to express co-stimulatory molecules consistent with DC WT (DC0502).

[0188] 6. 실시예

[0189] The present example provides the expression of an HLA-I class molecule after UDC monoclone 1-19 (UDC981) is reintroduced into the gene of an HLA-I class molecule.

[0190] The antigen-presenting ability of DCs is one of the core capabilities of specialized antigen-presenting cells, and DCs present endogenous antigens to CD8 through classical molecules of the HLA-I class, such as HLA-A, HLA-B, or HLA-C molecules. + Presented to and recognized by the TCR on the surface of T cells, CD8 + We stimulated T cell activation to initiate a specific immune cell response. We verified whether UDC981 could achieve stimulation and activated proliferation of antigen-specific cells of different HLA-I class subtypes by knocking out genes expressing HLA-A, HLA-B, and HLA-C molecules, and by reintroducing genes encoding different types of HLA-I class molecules through different gene introduction pathways that do not express HLA-A, HLA-B, and HLA-C molecules, lack antigen-presenting ability themselves, and otherwise.

[0191] In this embodiment, reintroducing different HLA-I class molecules into UDC981 includes incorporating genes encoding different HLA-I class molecules into the UDC981 genome using a lentiviral method based on UDC981.

[0192] In this embodiment, UDCs constructed with HLA-A0201, HLA-A1101, and HLA-A2402 molecular reintroductions were selected according to the most common types of human HLA-A molecular subtypes, respectively, and the amino acid sequences of the HLA-A0201, HLA-A1101, and HLA-A2402 molecules are denoted by SEQ ID NO.2 to SEQ ID NO.4, and their coding genes were synthesized by Nanjing Genscript Biotech Co., Ltd.:

[0193] HLA-A0201 molecule:

[0194] MAVMAPRTLVLLLSGALALTQTWAGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYL EGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEPSSQPTIPIVGIIAGLVLFGAVITGAVVAAVMWRRKSSDRKGGSYSQAASSDSAQGSDVSLTACKV(SEQ ID NO.2);

[0195] HLA-A1101 molecule:

[0196] MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRGYYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYL EGRCVEWLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWELSSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSSDRKGGSYTQAASSDSAQGSDVSLTACKV(SEQ ID NO.3);

[0197] HLA-A2402 molecule:

[0198] MAVMAPRTLVLLLSGALALTQTWAGSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIALRYYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRAYL EGTCVDLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEPSSQPTVPIVGIIAGLVLLGAVITGAVVAAVMWRRNSSDRKGGSYSQAASSDSAQGSDVSLTACKV(SEQ ID NO.4).

[0199] As an example, incorporating genes encoding different HLA-I class molecules into the UDC981 genome using a lentiviral approach includes viral vector construction and lentiviral packaging, specifically,

[0200] Genes encoding HLA-A0201, HLA-A1101, and HLA-A2402 molecules are inserted into the commercially available pCDH-CMV-MCS-EF1 lentivirus vector and transformed into Stbl3 competent cells; after accurate sequencing, the plasmids are extracted and purified using an endotoxin-free plasmid extraction kit;

[0201] 293T cells are cultured in 10 cm cell culture dishes, and plasmid transfection is initiated when the volume does not exceed 80%; the target plasmid is mixed with the packaging plasmid psPAX2, pMD2.G, and the transfection reagent PEI (polyethyleneimine) in a mass ratio of 3:2:1 in serum-free DMEM medium; the mixture containing a gene lentivirus plasmid encoding HLA-A0201, HLA-A1101, or HLA-A2402 molecules is added to the 293T cells and incubated at 37°C; the cell culture supernatant is collected at 48 h and 72 h, ultracentrifuged and concentrated to collect virus particles loaded with the target gene, and the viral titer is measured;

[0202] Collect and count UDC981 cells, 5×10 5 Cells were dispensed into four wells of a 6-well plate according to cells / mL, and 2 mL / well of the quantified cell suspension was added; lentiviruses containing genes encoding HLA-A0201, HLA-A1101, or HLA-A2402 molecules were used to infect each experimental well of UDC981 according to MOI=50, and after 3 weeks of culture and amplification of the infected UDC981, HLA-ABC positive cells were separated using the Miltenyi MACS® Separation System, and the operation steps were referred to in the instructions for Anti-PE MicroBeads (miltenyi biotec, Order no.130-048-801).

[0203] The results are as shown in Fig. 13, and as can be seen from Fig. 13, UDC981 HLA-A0201, UDC981 HLA-A1101, and UDC981 HLA-A2402 were obtained, with an HLA-A positivity rate of 95% after positive classification and purification.

[0204] The above results indicate that UDC981 can re-express HLA-I class molecules after being reintroduced into genes encoding HLA-I class molecules.

[0205] 7. 실시예

[0206] This embodiment verifies the antigen-presenting ability of UDC981 HLA-A0201, that is, verifies whether it can stimulate the proliferation of antigen-specific T cells that match the HLA-A0201 molecule.

[0207] Stimulating the proliferation of antigen-specific T cells of the HLA-A0201 molecular subtype includes the following:

[0208] UDC981 HLA-A0201 loads an HLA-A0201 molecular subtype restriction antigen peptide; the antigen epitope of the antigen peptide is an antigen epitope recognized by target antigen-specific T cells;

[0209] DCs loaded with antigen peptides are co-cultured with a T cell population, and specific T cells capable of recognizing the antigen epitope are activated and amplified.

[0210] In this embodiment, the T cell population is PBMC (Bokang Biotechnology (Shandong) Co., Ltd.), the HLA-A subtype is HLA-A0201, and the cells were resuscitated prior to use. In this embodiment, resuscitation includes the following: removing PBMCs rested for 2 hours from the incubator, centrifuging at 600g for 5 min, aspirating and removing the supernatant, adding Optivitro (EXCEL, TE000-N022) + 2% Clin-SFM (Beijing Kelin'en Biotechnology Co., Ltd., 1007) medium to the centrifuge tube to resuspend the cells, and adjusting the density of the cell suspension to 2×10⁶ 6 It was adjusted to cells / mL and inoculated at 1mL / well in a 24-well plate.

[0211] In this embodiment, UDC981 HLA-A0201 is HLA-A0201 This is a general-purpose DC introduced, specifically as described in Example 6.

[0212] In this embodiment, the antigen peptide is a CMV epitope and a Mart-1 epitope, and is an HLA-A molecular restriction antigen peptide.

[0213] In this embodiment, loading the antigen peptide of UDC981 HLA-A0201 comprises the following:

[0214] Cell suspensions of DC0502 (positive control), UDC981 (negative control), and UDC981 HLA-A0201 were added to centrifuge tubes and centrifuged at 600g for 5 min; the supernatant was aspirated and removed, and each tube was resuspended in culture medium. The cell medium was XVIVO15 (LONZA, 04-418Q) + 400 IU / ml IL-2 (Sahwan Biotech, National Drug Approval No. 10970015), and the density of the cell suspension was 1.0 × 10⁻⁶ 6Cells were adjusted to cells / mL and inoculated into a 48-well plate; each cell of DC0502, UDC981, and UDC981 HLA-A0201 was dispensed into four culture wells; CMV-A0201, Mart-1-A0201 antigen peptides (amino acid sequences indicated by SEQ ID NO.5 and SEQ ID NO.6, synthesized by Nanjing Genscript Biotech Co., Ltd.) (10 μg / μL) and a blank control were added to each culture well at a 1:1000 ratio, respectively, and the cell culture plates were incubated in a 37°C incubator for 4 hours to obtain three types of DCs loaded with antigen peptides.

[0215] In this embodiment, stimulating the proliferation of antigen-specific T cells by co-culturing DCs loaded with antigen peptides with a T cell population comprises the following:

[0216] Different DCs loaded with antigen peptides and corresponding blank control DCs were taken, counted, and the number of DCs was added to the PBMC culture wells at a DC:PBMC ratio of 1:100. The DC / PBMC co-culture plates were placed in a 37°C, 5% CO2 incubator and cultured for about 14 days, during which time liquid was replenished or amplification operations were performed every 2 days.

[0217] During the culture process, the proliferation rate of antigen-specific T cells stimulated by UDC981 HLA-A0201 loaded with an antigen peptide was detected, and the detection method comprises the following steps:

[0218] DC / PBMC loaded with antigen peptides were co-cultured for day 7 and day 9 to collect cells from different sample groups, added to 1.5 mL EP tubes respectively, and collected by centrifugation at 400 g for 5 min;

[0219] Remove the supernatant, resuspend the cell precipitate in 50 μL of FACS buffer (purchased from Beijing Tianjingsha Gene Technology Co., Ltd), add a 1 μL / EP tube of Human TruStain FcX to the sample tube, and incubate for 15 min in a dark room at room temperature;

[0220] After the incubation is finished, Tetramer-CMV-A0201 and Tetramer-Mart-1 A0201 corresponding to the loading of different antigen peptides are added to each tube, a 0.5 μL / EP Tetramer tube is added, and incubation is performed at 37°C for 30 min in a dark room;

[0221] After the incubation was completed, cells were washed with 200μL of FACS buffer, collected by centrifugation at 400g for 5 min, the supernatant was aspirated and removed, and flow cytometry samples were prepared according to the standard staining steps for flow cytometry; the PBMC surface-labeled molecular antibodies used were Alexa Fluor 700 anti-human CD3, APC anti-human CD4, Brilliant Violet 510™ anti-human CD8, and PE / Cyanine7 anti-human CD56 (NCAM);

[0222] Analysis was performed using a flow cytometer (Beckman), and the flow cytometry data was analyzed using Beckman CytExpert software. The results are shown in Figures 14 and 15, where Figure 14 is with the addition of the CMV-A0201 antigen and Figure 15 is with the addition of the Mart-1-A0201 antigen. The statistical results are shown in Tables 1 and 2.

[0223]

[0224] As can be seen by connecting Figures 14 and 15 with Tables 2 and 3,

[0225] At days 7 and 9 of co-culture between UDC981 (negative control) and PBMC, both showed CMV-tetramer-tetramer positive and Mart-1-tetramer-specific CD8 + T cells were not stimulated, which is because UDC981 lacks antigen-presenting ability due to a deficiency in HLA-A molecules, and the results are consistent with expectations;

[0226] CMV-tetramer-positive specific CD8 stimulated at day 7 and day 9 of co-culture between DC0502 (HLA-A0201 positive) and PBMC + T cell ratios are 1.06%, 1.7%; Mart-1-tetramer-positive specific CD8 + The T cell ratios are 0.36% and 0.49%;

[0227] CMV-tetramer-positive specific CD8 stimulated at day 7 and day 9 of co-culture of UDC981 HLA-A0201 and PBMC + T cell ratios are 1.27%, 2.98%; Mart-1-tetramer-positive specific CD8 + T cell ratios are 0.53% and 1.09%; (Calculation method: Antigen peptide group Tetramer positive ratio - Mock group Tetramer positive ratio).

[0228] The above example shows that UDC981 HLA-A0201 is a different type of antigen-specific CD8, similar to wild-type DC0502 (HLA-A0201 positive). + It was shown that it could stimulate T cells, and the positivity rate gradually increased over time from day 7 to day 9. After reintroducing the HLA-A0201 molecule based on UDC981, UDC981 HLA-A0201 had antigen presentation ability consistent with wild-type DC0502 (HLA-A0201), and its effect was superior to that of wild-type DC0502.

[0229] 8. 실시예

[0230] This embodiment verifies the antigen-presenting ability of UDC981 HLA-A2402, that is, verifies whether it can stimulate the activation and proliferation of antigen-specific T cells that match the HLA-A2402 molecule.

[0231] Referring to Example 7, in this example, the T cell population is PBMC (Bokang Biotechnology (Shandong) Co., Ltd.) with an HLA-A subtype of HLA-A2402, and was resuscitated prior to use. In this example, resuscitation includes the following: removing PBMC that has been rested for 2 hours from the incubator, centrifuging at 600 g for 5 min, aspirating and removing the supernatant, resuspending the cells by adding Optivitro (EXCEL, TE000-N022) + 2% Clin-SFM (Beijing Kelin'en Biotechnology Co., Ltd., 1007) medium to the centrifuge tube, and adjusting the density of the cell suspension to 2×10⁶ 6 It was adjusted to cells / mL and inoculated at 1mL / well in a 24-well plate.

[0232] In this embodiment, UDC981 HLA-A2402 is HLA-A2402 It is a general-purpose DC to which is introduced, specifically as described in Example 6. The original DC (DC0502) of the UDC981 HLA-A2402 HLA-A Gene subtypes are HLA-A0201 and HLA-A6801 is, that is, a general-purpose DC different from the original DC HLA-A Gene subtypes were introduced.

[0233] In this embodiment, the antigen peptide is a CMV epitope and is an HLA-A2402 molecular restriction antigen peptide.

[0234] In this embodiment, loading the antigen peptide of UDC981 HLA-A2402 comprises the following:

[0235] Cell suspensions of DC-0801 and DC-0802 (both containing HLA-A2402 and HLA-A1101 molecular phenotypes, positive controls) and UDC981 HLA-A2402, developed by our company, were each added to centrifuge tubes and centrifuged at 600g for 5 min. The supernatant was aspirated and removed, and each tube was resuspended in the respective cell medium. The cell medium was XVIVO15 (LONZA, 04-418Q) + 400 IU / ml IL-2 (Sahwan Bio, National Drug Approval No. 10970015), and the density of the cell suspension was 1.0 × 10⁻⁶ 6 Cells were adjusted to cells / mL and inoculated into a 48-well plate, and each cell of DC-0801, DC-0802, and UDC981 HLA-A2402 was dispensed into three culture wells; CMV-A2402 antigen peptide (amino acid sequence indicated by SEQ ID NO.7, synthesized by Nanjing Genscript Biotech Co., Ltd.) (10 μg / μL) and a blank control were added to each culture well at a 1:1000 ratio, respectively, and the cell culture plates were incubated in a 37°C incubator for 4 hours to obtain four types of DCs loaded with antigen peptides.

[0236] In this embodiment, stimulating the proliferation of antigen-specific T cells by co-culturing DCs loaded with antigen peptides with a T cell population includes the following.

[0237] Different DCs loaded with antigen peptides and corresponding blank control DCs were taken, counted, and the number of DCs was added to the PBMC culture wells at a DC:PBMC ratio of 1:100. The DC / PBMC co-culture plates were placed in a 37°C, 5% CO2 incubator and cultured for about 14 days, during which time liquid was replenished or amplification operations were performed every 2 days.

[0238] During the culture process, the proliferation rate of antigen-specific T cells stimulated by UDC981 HLA-A0201 loaded with antigen peptides was detected, and the detection method includes the following:

[0239] DC / PBMC loaded with antigen peptides were co-cultured for day 9 and day 11 to collect cells from different sample groups, added to 1.5 mL EP tubes respectively, and collected by centrifugation at 400 g for 5 min;

[0240] Remove the supernatant, resuspend the cell precipitate in 50 μL FACS buffer, add 1 μL / EP tube of Human TruStain FcX to the sample tube, and incubate for 15 min in a dark room at room temperature;

[0241] After the incubation is finished, Tetramer-CMV-A2402 corresponding to the antigen peptide loading is added to each tube, 0.5 μL / EP of Tetramer is added to the tube, and incubation is performed at 37°C for 30 min in a dark room;

[0242] After the incubation was completed, cells were washed with 200μL of FACS buffer, collected by centrifugation at 400g for 5 min, the supernatant was aspirated and removed, and flow cytometry samples were prepared according to the standard staining steps for flow cytometry; the PBMC surface-labeled molecular antibodies used were Alexa Fluor 700 anti-human CD3, APC anti-human CD4, Brilliant Violet 510™ anti-human CD8, and PE / Cyanine7 anti-human CD56 (NCAM);

[0243] 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.

[0244]

[0245] As can be seen from Fig. 16 and Table 3,

[0246] CMV-tetramer-positive specific CD8 stimulated by the antigen peptide at days 9 and 11 of co-culture of two wild-type DCs (including HLA-A2402 subtype) DC-0801 and DC-0802 and PBMCs + The T cell ratios are 0.63%, 0.15%, and 2.47%, 2.15%, respectively;

[0247] CMV-tetramer-positive specific CD8 antigen peptide stimulated at day 9 and day 11 of co-culture of UDC981 HLA-A2402 and PBMC + T cell ratios are 4.9% and 9.78%, and antigen peptide CMV-tetramer positive-specific CD8 + The T cell ratio on day 11 is about twice that of day 9 with time extension, indicating that UDC981 has a very strong antigen-presenting ability after HLA-A2402 is reintroduced.

[0248] In the above experiment, the stimulating ability of UDC981 HLA-A2402 on antigen-specific CD8+ T cells is significantly stronger than that of wild-type DC (HLA-A2402 subtype) DC-0801 and DC-0802. This is because UDC981 HLA-A2402 is a monotype of HLA-A2402, whereas DC-0801 and DC-0802 are heterozygotes of HLA-A2402 and HLA-A1101, and UDC981 HLA-A2402 is more specific for antigen-specific T cell amplification.

[0249] 9. 실시예

[0250] This example verifies the immunogenic response of UDC981 HLA-A11 to homologous and heterologous PBMCs.

[0251] T cells are stimulated to proliferate when in contact with allogeneic somatic cells that are incompatible with HLA-I. To investigate whether single-allergic HLA-I matching can reduce immune stimulation against allogeneic T cells, the immunogenicity of UDC981 with reintroduced HLA-I molecules was evaluated through T cell proliferation experiments. Specifically, the following was performed: Allogeneic PBMCs (Bokang Biotechnology (Shandong) Co., Ltd.) possessing single-allergic HLA-A*11:01 but incompatible with other alleles were labeled with CFSE. The staining procedure is as follows: The HLA-A subtype is HLA-A02:HLA-A11 PBMC (Bokang Biotechnology (Shandong) Co., Ltd.), and it is resuscitated prior to use. Resuscitation includes the following: PBMCs rested for 2 hours are removed from the incubator, centrifuged at 600g for 5 min, and the supernatant is aspirated and removed. The cells are resuspended by adding Optivitro (EXCEL, TE000-N022) + 2% Clin-SFM (Beijing Kelin'en Biotechnology Co., Ltd., 1007) medium to the centrifuge tube, and the density of the cell suspension is set to 1×10⁶ 7 The concentration was adjusted to cells / mL. A CFSE Cell Division Tracker Kit (Biolegend, GYS0004) was added to the cell suspension to achieve a final concentration of 5 μM; additionally, a portion of the cells was aspirated and used as a control without staining. The stained cell suspension was placed in a 37°C incubator and incubated in a dark room for 15 min.

[0252] Five times the volume of pre-warmed medium (1640+10% FBS) was added to the stained cell suspension and incubated for 5 minutes to remove excess free dye.

[0253] Centrifuge the cell suspension at 600g for 10 min, resuspend the cell precipitate using preheated medium (1640 + 10% FBS + 20 IU IL-2), and set the cell density to 1×10⁶ 6 The concentration was adjusted to cells / mL. Stained PBMCs and unstained PBMCs were inoculated into a 6-well plate, and 2 mL / well of cell suspension was added. Wild-type DC0502 with HLA-A subtypes HLA-A02:HLA-A68 was added to the CFSE-labeled PBMC culture wells at a ratio of 200:1, and UDC981 HLA-A11 (specifically HLA-A1101, whose amino acid sequence is indicated by SEQ ID NO.3, whose coding gene was synthesized by Nanjing Genscript Biotech Co., Ltd., and UDC981 HLA-A11 construction was referenced in Example 6) was added to other wells; PBMC-only culture wells (CFSE-labeled, unlabeled) were set up, and a positive control well was set up: 2 μL of T Cell TransAct™ (Miltenyi, 130-128-758) was added.

[0254] Co-cultured for 6 days, and CD3 by flow cytometry (FACS) + The CFSE fluorescence intensity of T cell subgroups was detected. 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 17.

[0255] According to the results, CD3 proliferating in the wild-type DC0502 group (DC0502 WT) with HLA-I subtype mismatch + The T cell percentage was high at 27.57%; in the UDC981 HLA-A11 group, the percentage decreased to 4.86%, reaching background levels (unstimulated CD3 +It was found to be close to cells (2.62%). This indicates that UDC981, which matches a single allele HLA-I, can reduce non-antigen-specific immune stimulation against allogeneic T cells.

[0256] As can be seen from the above, the universal DC and the method of manufacturing and application thereof provided in this application can prevent gene-edited modified DCs from expressing HLA-A, HLA-B, and HLA-C molecules, and the sequencing results from the obtained UDC single clone, HLA-A , HLA-B , HLA-C This indicates that mutations have occurred in the corresponding regions of all six alleles. The modified UDC can re-express all subtype HLA-I class molecules through gene editing, viral vectors, and mRNA technologies, thereby bypassing the HLA-I class molecule restriction of the DC itself and amplifying any HLA-I restricted antigen-specific T cells as an APC. Simultaneously, allogeneic immunogenicity tests of UDC981 performed after HLA-I molecule reintroduction indicate that UDC981 not only possesses antigen presentation and specific immune activation functions in practical applications but can also prevent the core risk of allogeneic immune rejection. The low non-specific T cell activation characteristics of UDC981 cells provide a key foundation for realizing safe allogeneic universal application. In conjunction with antigen peptide loading or mRNA antigen expression technologies, it can be developed into a DC vaccine product for any HLA-I type.

[0257] Depository Name: General Microbiology Center, China Microbial Species Preservation and Management Committee Accession Number: CGMCC46259 Date of Deposit: 2024-12-25 Depository Name: General Microbiology Center, China Microbial Species Preservation and Management Committee Trustee Number: CGMCC46260 Date of Deposit: 2024-12-25

Claims

Claim 1 In the case of a general-purpose DC, the general-purpose DC HLA-I The class gene is knocked out, and the above HLA-I Class genes are HLA-A gene, HLA-B Genes and / or HLA-C Universal DC characterized by containing a gene. Claim 2 In paragraph 1, the above HLA-I Class genes are HLA-A gene, HLA-B Genes and HLA-C Universal DC characterized by containing a gene. Claim 3 A general-purpose DC according to claim 1 or 2, characterized in that the general-purpose DC is an immortalized general-purpose DC. Claim 4 In paragraph 3, the above immortalized universal DC is UDC981, is deposited with the General Microbiology Center of the Chinese Commission on Depositary Use of Microorganisms (CGMCC), the deposit date is December 25, 2024, the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 46260, characterized by the universal DC. Claim 5 A universal DC cell population, wherein the cell population comprises a universal DC according to any one of claims 1 to 4. Claim 6 In a method for manufacturing a general-purpose DC, the original DC HLA-I Step of knocking out class genes - above HLA-I Class genes are HLA-A gene, HLA-B Genes and / or HLA-C Includes the gene; the original DC of the above HLA-I The step of knocking out class genes of the original DC using homologous recombination methods, CRISPR / Cas gene editing methods, or transcription factor-like effector nuclease methods HLA-I Includes knocking out class genes -; and HLA-I A method for manufacturing a universal DC, characterized by including the step of screening a DC in which a class gene is negatively expressed, i.e., a universal DC. Claim 7 In claim 6, the above-mentioned CRISPR / Cas gene editing method is the original DC's HLA-I A step of designing one or more sgRNAs that bind to a class gene; and introducing the sgRNA into the original DC using a CRISPR-Cas system, HLA-I A method for manufacturing a universal DC, characterized by including a step of knocking out a class gene. Claim 8 In Clause 7, the above original DC HLA-A Gene subtypes are HLA-A0201 and / or HLA-A6801 and; and / or HLA-B Gene subtypes are HLA-B0801 and / or HLA-B1507 is; and / or HLA-C Gene subtypes are HLA-C0303 and / or HLA-C0702 A method for manufacturing a universal DC, characterized in that the nucleotide sequence of the sgRNA is represented by SEQ ID NO.

1. Claim 9 A method for manufacturing a general-purpose DC, characterized in that, in any one of claims 6 to 8, the original DC is an immortalized DC. Claim 10 A method for manufacturing a general-purpose DC according to claim 9, characterized in that the immortalized DC is DC0502, is deposited with the General Microbiology Center of the Chinese Commission on Depositary Use of Microorganisms (CGMCC), the deposit date is December 25, 2024, the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 46259. Claim 11 A method for amplifying target antigen-specific T cells, comprising the step of obtaining an HLA-I class molecular subtype of a T cell population limited to a target antigen, wherein the HLA-I class molecule includes an HLA-A molecule, an HLA-B molecule, and / or an HLA-C molecule; and encoding the HLA-I class molecular subtype in the universal DC. HLA-I Step of introducing class genes; HLA-I A method for amplifying target antigen-specific T cells, characterized by comprising: a step of loading an HLA-I class molecular subtype restriction antigen peptide into a DC into which a class gene has been introduced, wherein the antigen epitope of the antigen peptide is an antigen epitope recognized by target antigen-specific T cells; and a step of co-culturing the DC loaded with the antigen peptide with a T cell population and activating and amplifying specific T cells capable of recognizing the antigen epitope. Claim 12 A method for amplifying target antigen-specific T cells according to claim 11, characterized in that the antigen peptide is derived from a tumor or a virus. Claim 13 In Article 11 or Article 12, the above HLA-I A method for amplifying target antigen-specific T cells, characterized in that the method of introducing class genes includes gene editing, viral, or mRNA methods. Claim 14 A method for amplifying target antigen-specific T cells according to claim 13, characterized in that the T cell population comprises PBMCs. Claim 15 A reagent for amplifying target antigen-specific T cells, characterized by comprising a universal DC according to any one of claims 1 to 4 or a universal DC cell population according to claim 5. Claim 16 In item 15, encoding the HLA-I class molecular subtype of the above-mentioned target antigen-specific T cell HLA-I A reagent characterized by further including class genes, wherein the HLA-I class molecule includes HLA-A molecules, HLA-B molecules and / or HLA-C molecules. Claim 17 In the modified DC, the original of the modified DC HLA-I Class genes are knocked out, and at the same time, exogenous HLA-I Class genes are introduced, but the above exogenous HLA-I Class genes are a single subtype HLA-I It is a class gene and exogenous HLA-I Class genes are HLA-A gene, HLA-B Gene or HLA-C Includes genes; the above original HLA-I Class genes are at least exogenous HLA-I Class genes and homologous HLA-I A modified DC characterized by containing a class gene. Claim 18 sgRNA, characterized in that the nucleotide sequence of the sgRNA is represented by SEQ ID NO.

1. Claim 19 cell HLA-I In the application of sgRNA according to claim 18 in the knockout of class genes, the above HLA-I Class genes are HLA-A gene, HLA-B Genes and / or HLA-C Includes a gene; the cell of the above HLA-A Gene subtypes are HLA-A0201 and / or HLA-A6801 is; and / or HLA-B Gene subtypes are HLA-B0801 and / or HLA-B1507 and; and / or HLA-C Gene subtypes are HLA-C0303 and / or HLA-C0702 Application characterized by being. Claim 20 In claim 19, the application is characterized in that the cell comprises a DC or T cell. Claim 21 In Clause 20, the application is characterized in that the above DC is an immortalizing DC. Claim 22 The application according to claim 21, characterized in that the immortalized DC is DC0502, is deposited with the General Microbiology Center of the Chinese Commission on Depositary Use of Microorganisms (CGMCC), the deposit date is December 25, 2024, the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 46259. Claim 23 Immortalization DC, designated as DC0502, is deposited with the General Microbiology Center of the Chinese Commission on Depositary Use of Microorganisms (CGMCC), with a deposit date of December 25, 2024, a deposit address of No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and deposit number CGMCC No. 46259.

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