WO2025065472A1 - Lymphocyte t modifié et son utilisation - Google Patents

Lymphocyte t modifié et son utilisation Download PDF

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Publication number
WO2025065472A1
WO2025065472A1 PCT/CN2023/122454 CN2023122454W WO2025065472A1 WO 2025065472 A1 WO2025065472 A1 WO 2025065472A1 CN 2023122454 W CN2023122454 W CN 2023122454W WO 2025065472 A1 WO2025065472 A1 WO 2025065472A1
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Prior art keywords
cells
bcl6
expression
cell
tumor
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English (en)
Chinese (zh)
Inventor
董晨
孙勤利
倪凌
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Bennu Biotherapeutics Shanghai Co Ltd
Renji Hospital
Shanghai Jiao Tong University
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Bennu Biotherapeutics Shanghai Co Ltd
Renji Hospital
Shanghai Jiao Tong University
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Application filed by Bennu Biotherapeutics Shanghai Co Ltd, Renji Hospital, Shanghai Jiao Tong University filed Critical Bennu Biotherapeutics Shanghai Co Ltd
Priority to PCT/CN2023/122454 priority Critical patent/WO2025065472A1/fr
Publication of WO2025065472A1 publication Critical patent/WO2025065472A1/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to the field of medicine, and in particular, to a modified T cell and application thereof.
  • T prog stem cell-like T cell subpopulations
  • T term terminally differentiated T cell subpopulations
  • NR4A1 is a transcription factor associated with T cell activation and death. It is highly expressed in T ex cells and promotes T cell exhaustion and inhibits T cell function by inhibiting the function of c-Jun. In T ex cells in chronic LCMV virus infection and tumor models, continuous antigen stimulation and NFAT activation induce high levels of TOX, which regulates T cell exhaustion and maintenance at the transcriptional and epigenetic levels.
  • TCF1 is a transcription factor highly expressed in naive and memory CD8 + T cells, and is also a characteristic molecule of T prog cells.
  • cytokines In addition to transcription factors, cytokines also play a very important role in the exhaustion of CD8 + T cells.
  • IL-27 and IL-2 are key cytokines that induce the expression of co-inhibitory molecules in T ex cells.
  • IL-27 can significantly improve the survival of TCF1 + T prog cells; in tumor models, IL-27 can significantly enhance the anti-tumor ability of T cells.
  • Type I interferon significantly enhances the development of T term cells by antagonizing TCF1.
  • IL-10 and IL-21 significantly enhance the development and effector function of T term cells by activating the STAT3 signaling pathway, thereby enhancing the anti-tumor ability of T cells.
  • T cell differentiation is regulated by transcription factors and cytokines and then transform T cells to provide support and assistance for anti-tumor immunotherapy.
  • the present invention aims to solve one of the technical problems in the related art at least to a certain extent.
  • the present invention provides a modified T cell and its application, wherein the modified T cell of the present invention can improve its stemness and ability to maintain T prog or promote the development of T term state without reducing the number of T prog cells, thereby improving its ability to kill tumors, and can provide support and assistance for anti-tumor immunotherapy.
  • BCL6 is a transcription factor of the BTB/POZ zinc finger protein family and a lineage transcription factor for follicular helper CD4 + T cells (Tfh cells) and germinal center B cells. Previous studies have reported that BCL6 is important for the development of memory CD8 + T cells in mouse models of acute viral infection and protein immunization. In a model of chronic LCMV virus infection, BCL6 is highly expressed in TCF1 + CXCR5 + T prog cells, which can enter the B cell follicles of lymphoid tissues.
  • BCL6 can be highly expressed in tumor-specific CD8 + T cells in tumor models.
  • tumor tissues generally do not have lymphoid structures, the aggregation areas of antigen-presenting cells contain many TCF1 + T prog cells. Therefore, the inventors speculate that tumor-infiltrating T prog cells may have different generation and maintenance mechanisms from T prog cells in chronic infection models.
  • the inventors further studied the relationship between BCL6 and T prog cells in a tumor model.
  • the inventors found that when BCL6 in T cells is overexpressed, it will damage the T cells themselves.
  • the inventors unexpectedly found that upregulating BCL6 in T cells will not only not damage the T cells themselves, but also increase the proliferation of T prog cells and improve their ability to kill tumors.
  • the inventors also unexpectedly found that simply increasing the number of copies of the BCL6 encoding gene in T cells to achieve overexpression of BCL6 cannot achieve this type of technical effect. This type of effect can only be achieved by regulating the expression regulation mechanism of the BCL6 encoding gene.
  • the combination of missing CD8 + T cells and anti-PD-1 can significantly promote the expansion of tumor-infiltrating T prog cells, producing a more effective anti-tumor effect.
  • the present invention proposes a T cell.
  • the expression of a given protein in the T cell is changed, and the given protein is suitable for regulating the expression of BCL6 in the T cell.
  • the T cell of the present invention can maintain the T prog state, enhance its T cell stemness and maintain the long-term survival of T cells, and can improve its tumor-killing ability, thereby contributing to the development of a new type of anti-tumor immunotherapy for the treatment and prevention of cancer.
  • the T cell of the present invention can promote the development of the T term state without reducing the number of T prog cells.
  • the T cell has a strong instantaneous killing ability for early tumors containing fewer tumor cells and can be used to treat early cancer.
  • the present invention provides a pharmaceutical composition.
  • the pharmaceutical composition comprises an agent and the T cells described in the first aspect of the present invention.
  • the agent is used to regulate the IL-2-STAT5 pathway and/or the TGF-beta-SMAD2 pathway so as to regulate the expression of BCL6 in T cells.
  • the T cells described in the first aspect can maintain the T prog state, enhance their T cell stemness and maintain the long-term survival of T cells, and can improve their ability to kill tumors.
  • the use of a pharmaceutical composition containing the above-mentioned agent or T cells can produce a lasting and effective anti-tumor immune response, which is helpful for the development of new anti-tumor immunotherapy to treat and prevent cancer.
  • the T cells described in the first aspect can promote the development of the T term state without reducing the number of T prog cells, and the T cells have a strong instantaneous killing ability for early tumors containing fewer tumor cells. Therefore, the use of a pharmaceutical composition containing the above-mentioned agent or T cells can be used to treat early cancer.
  • the present invention provides a use of the T cell described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention in the preparation of a drug, wherein the drug is used to treat or prevent cancer.
  • the T cell or pharmaceutical composition of the present invention can effectively kill tumors, and is helpful for the development of new anti-tumor immunotherapy for the treatment or prevention of cancer.
  • the present invention provides a drug combination.
  • the drug combination includes an immune checkpoint inhibitor and the T cell described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention.
  • the drug combination through the combined administration of the above two drugs, it has a higher tumor killing ability, can effectively kill tumor cells, inhibit tumor growth or reduce tumor volume, and can effectively prevent and treat tumors or cancer.
  • the present invention provides a method for treating cancer.
  • the method comprises: administering the T cells described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention to a patient.
  • the T cells described in the first aspect and the pharmaceutical composition described in the second aspect both have a high tumor killing ability, which can effectively kill tumor cells, inhibit tumor growth or reduce tumor volume. Therefore, the method of the present invention can effectively treat and prevent cancer.
  • the present invention provides a method for upregulating the content of stem-like cells in immune cells.
  • the method comprises: administering to a patient the T cells described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect.
  • the aforementioned T cells are in the stem-like T cell subpopulation (T prog ) stage, and the proportion of stem-like cells in immune cells can be increased by adding the T cells. Since T prog cells are in a relatively quiescent state and have a high level of proliferation potential, the immune cells obtained by this method have a strong tumor cell killing ability and can effectively treat and prevent cancer.
  • the present invention provides a method for inhibiting the transformation of T cells into T term cells.
  • the method comprises upregulating the expression of BCL6 in the T cells; the T cells comprise CD4 + T cells and CD8 + T cells.
  • upregulating the BCL6 protein in T cells can maintain the T cells in a stem cell-like cell (T prog ) state, thereby inhibiting the transformation of T cells into T term cells (terminally differentiated T cells). Therefore, the method helps to maintain the T prog state of T cells, resulting in a more durable and effective anti-tumor ability.
  • Figure 1 shows the expression of BCL6 at the transcription and protein levels and the flow cytometric graph showing the expression of BCL6-RFP in CD8 + T cells in Example 4.
  • the ordinate of the flow cytometric graph in Figure 1 indicates the amount of cells expressing BCL6-RFP.
  • Figure 2 is a result diagram comparing the functional differences between BCL6 and BLIMP1 in Example 6 of the present invention.
  • Figure 2A is a DNA binding heat map and Venn diagram centered on the binding sites of BCL6 and BLIMP1
  • Figure 2B is a Venn diagram of genes bound by BCL6 and BLIMP1
  • Figure 2C is the expression of characteristic genes of BCL6 + and TIM-3 + CD8 + T cells in the transcriptome of OT-I cells overexpressing PRDM1 and the control group
  • Figure 2D is a Gene Ontology (GO) analysis result diagram of the top 20 significant biological signaling pathways enriched by genes upregulated and downregulated by PRDM1 overexpression.
  • GO Gene Ontology
  • Figure 3 is a result diagram comparing the functional differences between BCL6 and BLIMP1 in Example 6 of the present invention.
  • Figure 3A is a correlation analysis of the gene expression levels regulated by overexpression of BCL6 and PRDM1
  • Figure 3B is a Venn diagram of genes directly regulated by BLIMP1
  • Figure 3C is a Venn diagram of characteristic genes of BCL6 + and TIM-3 + CD8 + T cells regulated by BLIMP1, as well as a gene regulatory network diagram centered on BCL6 and BLIMP1.
  • Figure 4 is a result diagram of the molecular mechanism of BCL6 and BLIMP1 in the differentiation process of CD8 + T cells in tumors in Example 6 of the present invention.
  • Figure 4A is a ChIP-seq and ATAC-seq diagram of the combination of BCL6 and BLIMP1 at specific gene sites
  • Figure 4B is a ChIP-seq diagram of the combination of BCL6 and BLIMP1 at specific gene sites.
  • Figure 5 is a result diagram showing that BCL6 and BLIMP1 play an antagonistic role in the differentiation process of CD8 + T cells in tumors in Example 6 of the present invention.
  • Figure 5A shows the expression of specific genes in CD8 + T cells activated in vitro
  • Figure 5B shows the expression of LY108 and TIM-3, wherein the abscissa of the flow cytometry graph in Figure 5B indicates the amount of cells expressing TIM-3, and the ordinate indicates the amount of cells expressing LY108.
  • Figure 6 is a result diagram showing that BCL6 and BLIMP1 play an antagonistic role in the differentiation process of CD8 + T cells in tumors in Example 6 of the present invention.
  • Figure 6A shows the expression of BCL6-RFP, BLIMP1-EYFP, LY108 and TIM-3 in OT-I cells, wherein the abscissa in Figure 6A indicates the amount of cells expressing BLIMP1-EYFP, and the ordinate indicates the amount of cells expressing BCL6-RFP, LY108 and TIM-3.
  • Figure 6B is a UMAP diagram of the expression of specific genes
  • Figure 6C is a violin diagram of the expression level of specific genes in CD8 + T cell subsets.
  • Figure 7 is a result diagram of the molecular mechanism of the induction of BLIMP1 expression in CD8 + T cells in Example 6 of the present invention.
  • Figure 7A shows the expression of TIM-3 and BLIMP1-EYFP in CD8 + T cells activated in vitro, wherein the abscissa in Figure 7A indicates the amount of cells expressing BLIMP1-EYFP and the ordinate indicates the amount of cells expressing TIM-3.
  • Figure 7B shows the expression of CD25, TIM-3 and TCF1 in CD8 + T cells activated in vitro, wherein the abscissa in Figure 7B indicates the amount of cells expressing TIM-3 and the ordinate indicates the amount of cells expressing CD25.
  • Figure 7C shows the expression of LY108 and Granzyme B in CD8 + T cells activated in vitro, wherein the abscissa in Figure 7C indicates the expression amounts of LY108 and Granzyme B, respectively.
  • FIG8 is a result diagram of the regulatory effect of BLIMP1 on CD8 + T cells in tumors in Example 6 of the present invention.
  • FIG8A is the expression of BCL6 and TCF1 in OT-I cells, wherein the abscissa in FIG8A indicates the amount of cells expressing CD8, and the ordinate indicates the amount of cells expressing TCF1 and BCL6.
  • FIG8B is the expression of Granzyme B, IFN- ⁇ and TNF- ⁇ in OT-I cells, wherein the abscissa in FIG8B indicates the amount of cells expressing TNF- ⁇ , and the ordinate indicates the amount of cells expressing IFN- ⁇ and Granzyme B.
  • Figure 9 is a result diagram of the regulatory effect of BLIMP1 on CD8 + T cells in tumors in Example 6 of the present invention.
  • Figure 9A shows the expression of T-BET, TCF1 and BCL6 in OT-I cells, wherein the abscissa in Figure 9A indicates the amount of cells expressing T-BET, and the ordinate indicates the amount of cells expressing TCF1 and BCL6.
  • Figure 9B shows the ratio of LY108 + and TIM-3 + cells in tumor-infiltrating OT-I cells
  • Figure 9C shows the dilution of CTV in OT-I cells in the spleen, wherein the abscissa in Figure 9C indicates the amount of cells expressing CTV.
  • Figure 10 is a result diagram of the expression and function of BLIMP1 in the process of immune response of CD8 + T cells to endogenous tumor antigens in Example 6 of the present invention.
  • Figure 10A shows the expression of BLIMP1-EYFP in CD8 + T cells infiltrating tumors and draining lymph nodes, wherein the abscissa in Figure 10A indicates the amount of cells expressing BLIMP1-EYFP and the ordinate indicates the amount of cells expressing CD44.
  • Figure 10B shows the expression of TCF1 and Granzyme B in tumor-infiltrating CD8 + T cells, wherein the abscissa in Figure 10B indicates the amount of cells expressing BLIMP1-EYFP and the ordinate indicates the amount of cells expressing CD44.
  • Figure 11 is a graph showing the results of regulating the anti-tumor function of T cells by overexpression of BCL6 in Example 7 of the present invention.
  • Figure 11A is a growth curve of E.G7 tumors
  • Figure 11B is the expression of LY108 and TIM-3 in OT-I cells, wherein the abscissa in Figure 11B indicates the amount of cells expressing TIM-3, and the ordinate indicates the amount of cells expressing LY108.
  • Figure 11C is the number of OT-I cells, LY108 + and TIM-3 + OT-I cells infiltrating the tumor per unit weight.
  • Figure 12 is a result diagram showing that anti-PD-1 treatment in Example 7 of the present invention cannot significantly expand BCL6-overexpressing T prog cells and produce effective tumor control effects.
  • Figure 12A is a schematic diagram of adoptive delivery of OT-I cells, E.G7 tumor inoculation and anti-PD-1 treatment, and Figures 12B and C are growth curves of E.G7 tumors.
  • Figure 13 is a result diagram showing that the treatment with anti-PD-1 in Example 7 of the present invention cannot significantly expand T prog cells overexpressing BCL6 and produce effective tumor control effects.
  • Figure 13A shows the expression of TCF1 and TIM-3 in OT-I cells, wherein the abscissa in Figure 13A indicates the amount of cells expressing TIM-3, and the ordinate indicates the amount of cells expressing TCF1.
  • Figure 13B shows the number of OT-I cells, TCF1 + and TIM-3 + OT-I cells infiltrating E.G7 tumors per unit
  • Figure 13C shows the ratio of Granzyme B + cells, as well as the number of Granzyme B + OT-I cells infiltrating E.G7 tumors per unit weight, wherein the abscissa in Figure 13C indicates the amount of cells expressing TIM-3, and the ordinate indicates the amount of cells expressing Granzyme B cell amount.
  • Figure 14 is a result diagram of the combination of PRDM1 deficiency in tumor-specific CD8 + T cells and anti-PD-1 in Example 7 of the present invention.
  • 14A is a schematic diagram of adoptive delivery of OT-I cells, E.G7 tumor inoculation and anti-PD-1 treatment
  • Figure 14B is a growth curve of E.G7 tumor
  • Figure 14C is the expression of TCF1 and TIM-3 in OT-I cells, the number of OT-I cells, TCF1 + and TIM-3 + OT-I cells infiltrating per unit E.G7 tumor, wherein the abscissa in Figure 14C indicates the amount of cells expressing TIM-3, and the ordinate indicates the amount of cells expressing TCF1.
  • Figure 15 is a result diagram of the combination of PRDM1 deficiency in tumor-specific CD8 + T cells and anti-PD-1 in Example 7 of the present invention.
  • Figure 15A shows the ratio of Granzyme B + OT-I cells and the number of Granzyme B + OT-I cells infiltrating per tumor, wherein the abscissa in Figure 15A indicates the amount of cells expressing TIM-3 and the ordinate indicates the amount of cells expressing Granzyme B.
  • Figure 15B shows the expression of IFN- ⁇ and TNF- ⁇ in OT-I cells, wherein the abscissa in Figure 15B indicates the amount of cells expressing TNF- ⁇ and the ordinate indicates the amount of cells expressing IFN- ⁇ .
  • first and second are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as “first” and “second” may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality” is two or more.
  • the term "pharmaceutical composition” generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical field. All methods include the step of combining the active ingredient (T cell) with a carrier that constitutes one or more accessory ingredients. Typically, the composition is prepared by uniformly and fully combining the active compound with a liquid carrier, a finely divided solid carrier, or both.
  • the term "pharmaceutically acceptable amount” or “pharmaceutically acceptable dose” refers to a dose that is suitable for humans and/or mammals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), that is, a substance with a reasonable benefit/risk ratio.
  • it can be "effective amount” or "effective dose”, wherein “effective amount” or “effective dose” refers to an amount that can produce a function or activity on humans and/or animals and can be accepted by humans and/or animals.
  • the term "pharmaceutically acceptable excipient” may include any solvent, diluent or other liquid excipient, etc., suitable for a specific target dosage form. Except for any conventional excipients incompatible with the compounds of the present invention, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope of the present invention.
  • the term "administration" refers to the introduction of a predetermined amount of a substance into a patient by some suitable means.
  • the T cells or pharmaceutical compositions of the present invention can be administered by any common route as long as it can reach the desired tissue.
  • Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present invention is not limited to these exemplified modes of administration.
  • the T cells or pharmaceutical compositions of the present invention are administered by intravenous injection.
  • treatment refers to obtaining a desired pharmacological and/or physiological effect.
  • the effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and/or may be therapeutic in terms of partially or completely curing a disease and/or adverse effects caused by the disease.
  • Treatment covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease.
  • Treatment covers any medication that administers T cells or pharmaceutical compositions to an individual to treat, cure, alleviate, improve, mitigate or inhibit an individual's disease, including but not limited to administering a drug containing the T cells described herein to an individual in need.
  • the present invention provides a T cell, a pharmaceutical composition, use of a T cell or a pharmaceutical composition in preparing a drug, a drug combination, a method for treating cancer, a method for upregulating the content of stem-like cells in immune cells, and a method for inhibiting the transformation of T cells into T term cells.
  • a T cell is provided.
  • the expression of a given protein in the T cell is changed, and the given protein is suitable for regulating the expression of BCL6 in the T cell.
  • the T cell of the present invention can maintain the T prog state, enhance its T cell stemness and maintain the long-term survival of T cells, and can improve its ability to kill tumors, thereby contributing to the development of new anti-tumor immunotherapy for the treatment and prevention of cancer.
  • the T cell of the present invention can promote the development of the T term state without reducing the number of T prog cells.
  • the T cell has a strong instantaneous killing ability for early tumors containing fewer tumor cells and can be used to treat early cancer.
  • the above-mentioned T cell may further include at least one of the following technical features:
  • the T cells are modified T cells.
  • the T cells are derived from T cells isolated from a healthy individual or a patient.
  • the patient may be a tumor patient or a patient with other diseases.
  • the patient is a tumor patient.
  • the T cell is at least one of a CD8 + T cell and a CD4 + T cell, preferably a CD8 + T cell that specifically recognizes tumor cells.
  • CD8 + T cells that specifically recognize tumor cells refers to CD8 + T cells that are CD44 positive (CD44 + ).
  • flow cytometry is used to detect CD8 + T cells that specifically recognize tumor cells, and CD44 + CD8 + T cells (CD44-positive CD8 + T cells) are determined as CD8 + T cells that specifically recognize tumor cells.
  • the given protein is suitable for regulating the expression of BCL6 via the IL-2-STAT5 pathway and/or the TGF-beta-SMAD2 pathway.
  • the expression of BCL6 in the T cells is upregulated.
  • the T cells can maintain the T prog state, enhance their T cell stemness and maintain long-term survival of T cells, and improve their ability to kill tumors, thereby contributing to the development of new anti-tumor immunotherapy for the treatment and prevention of cancer.
  • the T cells before modification are T cells isolated from tumor-infiltrated individuals; "the expression of BCL6 is upregulated” means that the expression level of BCL6 protein in the T cells of the present invention is increased compared to the isolated T cells.
  • the expression of the given protein in the T cells is altered so as to inhibit the IL-2-STAT5 pathway and/or activate the TGF-beta-SMAD2 pathway.
  • the given protein includes at least one downstream protein on the IL-2-STAT5 pathway, and the given protein is down-regulated.
  • the given protein includes at least one selected from the following: PRDM1 or its homologous family members.
  • the term "the expression of the given protein is downregulated" means that the expression level of the given protein in the transformed T cells is reduced compared to that in the T cells before transformation.
  • the T cells before transformation are T cells isolated from tumor-infiltrated individuals; "the expression of the given protein is downregulated” means that the expression level of the given protein in the T cells of the present invention is reduced compared to the isolated T cells.
  • the expression of the given protein is downregulated by at least one of gene silencing, gene editing, small molecule inhibitors, and antibody drugs.
  • the small molecule inhibitor includes at least one of the small molecule inhibitors of STAT5a/b, STAT3, STAT1, and STAT4.
  • the antibody drug includes at least one of the blocking antibodies of IL-2, IL-2R ⁇ , IL-2 ⁇ and IL-2R ⁇ .
  • the gene silencing or gene editing includes at least one of CRISPR-Cas9 virus and non-virus, shRNA, and siRNA.
  • the PRDM1 gene in the T cells is knocked down.
  • the PRDM1 gene in the T cells is knocked out.
  • the expression of BCL6 in the T cells is downregulated.
  • the T cells can promote the development of the T term state without reducing the number of T prog cells, and the T cells have a strong instantaneous killing ability against early tumors containing fewer tumor cells, and can be used to treat early cancer.
  • the expression of BCL6 is downregulated means that the expression level of BCL6 protein in the transformed T cells is reduced compared with that in the T cells before transformation.
  • the T cells before transformation are T cells isolated from tumor-infiltrated individuals; "the expression of BCL6 is downregulated” means that the expression level of BCL6 protein in the T cells of the present invention is reduced compared to the isolated T cells.
  • the expression of the given protein in the T cells is altered so as to activate the IL-2-STAT5 pathway and/or inhibit the TGF-beta-SMAD2 pathway.
  • the given protein includes at least one downstream protein on the IL-2-STAT5 pathway, and the expression of the given protein is upregulated.
  • the term "the expression of the given protein is upregulated" means that the expression level of the given protein in the transformed T cells is increased compared to that in the T cells before transformation.
  • the T cells before modification are T cells isolated from tumor-infiltrated individuals; "the expression of the given protein is upregulated” means that the expression level of the given protein in the T cells of the present invention is increased compared to the isolated T cells.
  • the given protein includes at least one selected from the following: PRDM1 or its homologous family members.
  • the up-regulation of the expression of the given protein is achieved by at least one of gene transfection, promoter enhancer, and auxiliary expression factor.
  • the promoter enhancer includes at least one of a CMV promoter enhancer, an EF1 ⁇ promoter enhancer, a CD2 promoter enhancer, and an IL-2 promoter enhancer.
  • the auxiliary expression factor includes at least one of T cell activating factor, NF- ⁇ B, CD28 co-stimulatory factor, and IL-2 cytokine.
  • the gene transfection is achieved by at least one of liposome, calcium phosphate and PEI.
  • the PRDM1 gene in the T cells is upregulated.
  • the T cell is at least one of a CAR-T cell, a TIL cell and a TCR-T cell.
  • the expression of immune checkpoint-related proteins in the T cells is downregulated.
  • the term "the expression of immune checkpoint-related proteins is downregulated” means that the expression level of immune checkpoint-related proteins is reduced.
  • the expression of immune checkpoint-related proteins in the transformed T cells is increased.
  • immune checkpoint-related proteins is downregulated
  • gene silencing or gene editing systems CRISPR-Cas9 viruses and non-viruses, shRNA, siRNA
  • small molecule inhibitors and antibody drugs immunosorbent-associated antigen antibodies
  • the immune checkpoint-related protein includes at least one of PD-1, CTLA-4, LAG-3, TIM-3, TIGIT and VISTA.
  • the present invention provides a pharmaceutical composition.
  • the pharmaceutical composition includes an agent, the agent is used to regulate the IL-2-STAT5 pathway and/or the TGF-beta-SMAD2 pathway to regulate the expression of BCL6 in T cells; or the T cells described in the first aspect of the present invention.
  • the T cells described in the first aspect can maintain the T prog state, enhance their T cell stemness and maintain the long-term survival of T cells, and can improve their ability to kill tumors. Therefore, the use of a pharmaceutical composition containing the above T cells can achieve a lasting and effective anti-tumor immune response, which is helpful for the development of new anti-tumor immunotherapy to treat and prevent cancer.
  • the T cells described in the first aspect can promote the development of the T term state without reducing the number of T prog cells, and the T cells have a strong instantaneous killing ability for early tumors containing fewer tumor cells. Therefore, the use of a pharmaceutical composition containing the above reagents or T cells can be used to treat early cancer.
  • the agent is used to inhibit the IL-2-STAT5 pathway.
  • the agent inhibits the IL-2-STAT5 pathway by down-regulating the expression of a given protein, wherein the given protein comprises at least one selected from the following: PRDM1 or a member of its homologous family.
  • the reagent includes at least one of small molecule inhibitors, antibody drugs, CRISPR-Cas9 viruses and non-viruses, TALEN, ZFN, shRNA, and siRNA.
  • the reagent is used to activate the TGF-beta-SMAD2 pathway, and the reagent includes a TGF-beta-SMAD2 pathway activator and a TGF-beta-SMAD3 pathway activator.
  • the agent is used to activate the IL-2-STAT5 pathway.
  • the agent activates the IL-2-STAT5 pathway by upregulating the expression of a given protein, wherein the given protein comprises at least one selected from the following: PRDM1 or a member of its homologous family.
  • the reagent includes at least one of liposome, calcium phosphate and PEI.
  • the reagent is used to inhibit the TGF-beta-SMAD2 pathway, and the reagent includes a TGF-beta-SMAD2 pathway inhibitor and a TGF-beta-SMAD3 pathway inhibitor.
  • the pharmaceutical composition further comprises: an immune checkpoint blocker.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutical composition is a combined drug or a drug kit.
  • immune checkpoint blockers in the present invention should be understood in a broad sense, including but not limited to pharmaceutical agents, treatment methods, etc. As long as they can block the immune checkpoints, they are all within the scope of protection of the present invention.
  • the immune checkpoint blocker includes at least one of PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, LAG-3 antibody, TIM-3 antibody, TIGIT antibody, and VISTA antibody.
  • the immune checkpoint blocker can be further achieved by the following methods, including but not limited to tyrosine kinase inhibitors, co-stimulatory molecule monoclonal antibodies, epigenetic therapy drugs, chemotherapeutic drugs, radiotherapy preparations, vaccines, PDCD1 knockout therapy, PD1, PDL1 antibodies, or viral vector-based gene knockout methods.
  • the immune checkpoint blocker further includes CAR-T, CAR-NK, tumor vaccine, oncolytic virus vaccine, TLR7/8 agonist, anti-CD47 or IL-2 receptor agonist.
  • the immune checkpoint blocker is formulated to be suitable for simultaneous administration with the T cells or separate administration.
  • the present invention provides a use of the T cell described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention in the preparation of a drug, wherein the drug is used to treat or prevent cancer.
  • the T cell or pharmaceutical composition of the present invention can effectively kill tumors, and is helpful for the development of new anti-tumor immunotherapy for the treatment or prevention of cancer.
  • the cancer includes at least one selected from the following: gastric cancer, lung cancer, pancreatic cancer, liver cancer, breast cancer, cervical cancer, skin cancer, prostate cancer, melanoma, thyroid cancer, uterine fibroids, lymphoma, esophageal cancer, intestinal cancer, bone marrow cancer, nasal cancer, bone cancer, head and neck cancer, oral cancer, kidney cancer, and Kaposi's sarcoma.
  • the present invention provides a drug combination.
  • the combined drug comprises an immune checkpoint inhibitor and the T cell described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention.
  • the combined administration of the above two drugs it has a higher tumor killing ability, can effectively kill tumor cells, inhibit tumor growth or reduce tumor volume, and can effectively prevent and treat tumors or cancer.
  • the present invention provides a method for treating cancer.
  • the method comprises: administering the T cells described in the first aspect of the present invention and the pharmaceutical composition described in the second aspect of the present invention to a patient.
  • the T cells described in the first aspect and the pharmaceutical composition described in the second aspect both have a high tumor killing ability, which can effectively kill tumor cells, inhibit tumor growth or reduce tumor volume. Therefore, the method of the present invention can effectively treat and prevent cancer.
  • it further includes administering at least one of PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, LAG-3 antibody, TIM-3 antibody, TIGIT antibody, and VISTA antibody to the patient simultaneously or sequentially.
  • the active ingredients of the present invention may vary with the mode of administration and the severity of the disease to be treated.
  • the selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials).
  • the factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's weight, the patient's immune status, the route of administration, etc. For example, depending on the urgency of the treatment condition, several divided doses may be given per day, or the dose may be reduced proportionally.
  • the present invention provides a method for up-regulating the content of stem-like cells in immune cells.
  • the method comprises: administering to a patient the T cells described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect; the expression of BCL6 in the T cells is up-regulated.
  • the aforementioned T cells are in the stem-like T cell subpopulation (T prog ) stage, and the proportion of stem-like cells in immune cells can be increased by adding the T cells. Since T prog cells are in a relatively quiescent state and have a high level of proliferation potential, according to an embodiment of the present invention, the immune cells obtained by the method have a strong tumor cell killing ability and can effectively treat and prevent cancer or tumors.
  • the present invention provides a method for inhibiting the transformation of T cells into T term cells.
  • the method comprises upregulating the expression of BCL6 in the T cells; the T cells comprise CD4 + T cells and CD8 + T cells.
  • upregulating the BCL6 protein in T cells can maintain the T cells in a stem cell-like cell (T prog ) state, thereby inhibiting the transformation of T cells into T term cells (terminally differentiated T cells). Therefore, according to an embodiment of the present invention, the method helps to maintain the T prog state of T cells, resulting in a more durable and effective anti-tumor ability.
  • the up-regulation of the expression of BCL6 in the T cells is achieved by down-regulating the expression of a given protein in the T cells, and the given protein is suitable for regulating the expression of BCL6 through the IL-2-STAT5 pathway; or the up-regulation of the expression of BCL6 in the T cells is achieved by contacting the T cells with an agent, and the agent is used to activate the TGF-beta-SMAD2 pathway.
  • the reagents include a TGF-beta-SMAD2 pathway activator and a TGF-beta-SMAD3 pathway activator.
  • the given protein includes at least one selected from the following: PRDM1 or its homologous family members.
  • the downregulation of the expression level of a given protein in the T cells is achieved by at least one of gene silencing or gene editing, small molecule inhibitors and antibody drugs.
  • the gene silencing or gene editing includes at least one of CRISPR-Cas9 virus and non-virus, shRNA, and siRNA.
  • the up-regulation of the expression of BCL6 in the T cells is achieved by knocking out the PRDM1 gene in the T cells.
  • the knocking out of the PRDM1 gene in the T cells is achieved by at least one of CRISPR-Cas9, shRNA and siRNA.
  • CRISPR-Cas9 technology is used to knock out PRDM1 in OT-I cells, and the T cells after PRDM1 knockout are combined with anti-PD-1 to detect the killing ability of E.G7 tumors in CD45.1 receptor mice.
  • Example 1 BCL6 is selectively highly expressed in tumor stem-like CD8 + T cells
  • dimensionality reduction and cluster analysis were performed on single-cell RNA-seq data of head and neck cancer and melanoma patients (GEO number: GSE103322 and GSE120575).
  • the dimensionality reduction and cluster analysis software was Seurat (version 3.2.2), Find Neighborsand, Find Clustersfunction and Feature Plotfunction.
  • CD8 + T cells were identified by the expression of CD3E, CD8B, CD4 and FOXP3 molecules, and two major cell populations were obtained.
  • TCF7 stem-like TCF1 + P14 cells infiltrating mouse B16-GP33 tumors
  • TCF7 CCR7 and IL7R
  • the other cell subset significantly overexpressed the characteristic genes of terminally differentiated TCF1 - P14 cells infiltrating mouse B16-GP33 tumors, including GZMB, PRF1, HAVCR2, PRDM1 (encoding BLIMP1 protein), PDCD1 and TOX, and was therefore identified as a T term cell subset.
  • mice C57BL/6 mice were divided into two groups, 8 in each group.
  • E.G7 tumor cells and B16-OVA tumor cells were subcutaneously inoculated in the two groups of mice, respectively.
  • tumor-draining lymph nodes and spleens of the mice were removed.
  • Tumor-infiltrating lymphocytes were isolated by density gradient centrifugation using 30% and 70% Percoll (GE) solutions, and single-cell suspensions were obtained by grinding tumor-draining lymph nodes and spleens, thereby obtaining isolated CD8 + T cells.
  • GE Percoll
  • naive OT-I cells that can specifically recognize OVA 257-264 were adoptively transferred into C57BL/6 recipient mice (8 mice), and then E.G7 tumor cells were inoculated one day later.
  • the spleen and lymph nodes were then removed from the CD45.1OT-I mice, and the CD8 + T cells were isolated and purified using the Dynabeads TM FlowComp TM MouseCD8Kit (Invitrogen) kit.
  • the CD25 - CD44lowCD62Lhigh naive OT-I cells were labeled with flow cytometry antibodies, and then the naive OT-I cells were sorted using a FACSAria (BD) flow cytometer to obtain isolated naive OT-I cells, and then the method described in step 2 of this example was used for experiments and detection.
  • the results showed that from day 11 to day 20, BCL6 continued to be highly expressed in OT-I cells infiltrated by E.G7 tumors.
  • BCL6 was selectively expressed in T prog cells, but not in T term cells.
  • BCL6 + CD8 + T cells BCL6 + CD8 + T
  • BCL6-RFP - TIM-3 + cells TIM-3 is a marker molecule for T term cells
  • Magzolreagent Magen
  • BCL6 + CD8 + T cells are T prog cells, which further indicates that BCL6 is selectively highly expressed in tumor-infiltrating T prog cells.
  • step 5 of this example the inventors adoptively transferred CFSE-labeled naive CD45.2OT-I cells into CD45.1 recipient mice (8 mice), and subcutaneously inoculated E.G7 tumor cells one day later.
  • the method described in step 2 of this example was used for experiments and detection, and then the expression of BCL6 in OT-I cells infiltrating the tumors and TDLN (tumor-draining lymph nodes) of the recipient mice was analyzed.
  • Naive CD8 + T cells from BCL6 CreERT2x Rosa26 tdTomato OT-I mice were adoptively transferred into CD45.1 recipient mice (8 mice), and then E.G7 tumor cells were inoculated one day later and Tamoxifen (T5648; Sigma-Aldrich) was intraperitoneally injected once a day at a concentration of 0.2 mg/g body weight on days 7-9. Experiments and detections were performed using the method described in step 2 of this example.
  • Example 2 BCL6 does not regulate the generation of tumor Tprog cells, but is essential for their long-term maintenance
  • naive WT OT-I cells and BCL6 -/- OT-I cells were adoptively transferred into CD45.1 recipient mice (8 mice), and E.G7 tumor cells were inoculated one day later. The experiment was carried out using the method described in Step 2 of Example 1.
  • the BCL6 -/- OT-I cells infiltrating the E.G7 tumor still contained a group of T prog cells, and the number of T prog cells infiltrating the tumor per unit weight was not affected by the loss of BCL6, indicating that BCL6 is not necessary for the production of T prog cells in the early development of the tumor.
  • loss of BCL6 led to a significant decrease in the proportion of TCF1 + T prog cells and the expression level of EOMES in tumor-infiltrating OT-I cells, while a significant increase in the proportion of CX3CR1 + T term cells and the expression level of T-bet.
  • BCL6 plays different roles in CD8 + T cells in tumor and chronic viral infection models. It is essential for the generation of T prog cells in chronic viral infection models but does not affect the generation of T prog cells in short-term tumor models.
  • WT OT-I cells and BCL6 -/- OT-I cells were isolated from the E.G7 tumor of the mice in step 1 of this example by the method of step 3 of example 1, and then equal amounts of OT-I cells were adoptively transferred into a second Tcrbd -/- recipient mouse (8 mice) inoculated with E.G7 tumors, and experiments and detection were performed using the method described in step 2 of example 1.
  • step 1 and step 2 were replaced by B16-OVA tumor cells.
  • the loss of BCL6 led to a significant increase in the number of tumor-infiltrating T term cells on day 14, but a significant decrease on day 22, indicating that although the loss of BCL6 temporarily promoted the development of T term cells, the T term cells had a strong instantaneous killing ability against early tumors containing fewer tumor cells.
  • T prog cells infiltrating TDLN were identified as memory CD8 + T cells, and were able to continuously migrate into the tumor to exert a sustained anti-tumor effect.
  • BCL6 is also highly expressed in T prog cells in TDLN, but their differentiation and number are not affected by the loss of BCL6. Therefore, it is urgent to verify the role of BCL6 in the TDLN model. The specific steps are as follows:
  • WT OT-I cells and BCL6 -/- LY108 + TIM-3 - OT-I cells were isolated from the TDLN of the mice described in step 1 of this example by the method of step 3 of example 1, and then adoptively transferred in equal amounts into Tcrbd -/- recipient mice (8 mice) that had received CD4 + T cells, and inoculated with E.G7 tumor cells one day later. Then, on the 16th day, the recipient mice were experimented and tested by the method described in step 2 of example 1.
  • BCL6 was persistently expressed in tumor- and TDLN-infiltrating T prog cells. Loss of BCL6 resulted in a significant impairment in the ability of OT-I cells to control tumors in secondary recipient mice. Loss of BCL6 resulted in the inability to maintain tumor-infiltrating TCF1 + T prog cells and a significant reduction in the number of tumor-infiltrating OT-I cells, with both T prog and T term cells significantly reduced. However, in TDLN, loss of BCL6 did not affect the proportion and number of TCF1 + T prog cells.
  • WT CD8 + T cells and BCL6 -/- naive CD8 + T cells (BCL6 knockout in naive WT CD8 + T cells using CRISPR-Cas9 is called BCL6 -/- ) were isolated and purified from CD45.1WT mice and CD45.2BCL6fl/fl Cd8aCre mice by the method of step 2 of Example 1, and then the WT CD8 + T cells and BCL6 -/- naive CD8 + T cells were adoptively transferred into Tcrbd -/- recipient mice (8 mice) respectively and CD4 + T cells at the same time. Then, B16BL6, EL4, HEPA1-6 and MC38 tumor cells were subcutaneously inoculated one day later, and experiments and detection were performed using the method described in step 2 of Example 1.
  • Example 3 BCL6 transcriptionally regulates the differentiation of CD8 + T cells in tumors
  • the inventors used the method described in step 2 of Example 1 to obtain isolated CD8 + T cells. Next, they were activated in vitro using a flat-bottomed plate combined with anti-CD3 (BioXCell, clone145-2C11, 5 ⁇ g/ml) and anti-CD28 (BioXCell, clone37.51, 1 ⁇ g/ml) antibodies to obtain in vitro activated CD8 + T cells. Next, BCL6-HA (NM_001348026.1) was cloned into a retroviral vector expressing IRES-GFP.
  • the retroviral plasmid and the pCL-ECO packaging plasmid were first transfected into the Phoenix cell line using the calcium transfer method, and then the viral supernatant was collected 48 hours later, and the viral supernatant was used to infect in vitro Activated CD8 + T cells were used to obtain in vitro activated CD8 + T cells that overexpressed BCL6-HA. Subsequently, about 1 ⁇ 10 7 in vitro activated CD8 + T cells that overexpressed BCL6-HA were collected and then cross-linked and fixed with paraformaldehyde at a final concentration of 1%.
  • ChIP experiments were performed according to the instructions of ActiveMotif's ChIP assay kit (53035).
  • the obtained DNA fragments were sent to BGI genomics for library construction and high-throughput sequencing to obtain ChIP-seq data of in vitro activated CD8 + T cells that overexpressed BCL6-HA.
  • the sequencing data were analyzed using bowtie2, picardMarkDuplicates, MACS2, ChIPseeker, bedtoolsintersect, Deeptools, computeMatrix and HOMER to obtain genome-wide binding results of in vitro activated CD8 + T cells that overexpressed BCL6-HA.
  • BCL6 had similar sequence binding characteristics in different cell types.
  • BCL6 significantly bound to 5949 DNA sites and 2931 genes.
  • KEGG analysis results showed that BCL6-bound genes were significantly enriched in multiple T cell-related biological pathways, including T cell differentiation, activation, proliferation and adhesion.
  • 42.58% and 39.89% of BCL6-bound DNA sites were distributed in introns and distal intergenic regions, and only 12.96% of BCL6 binding sites were distributed in promoter regions.
  • more than 70% of BCL6 binding sites were distributed in regions more than 10kb away from the transcription start site (TSS).
  • the isolated CD45.1OT-I cells were obtained by the method described in step 3 of Example 1, and then BCL6 (NM_001348026.1) was cloned into a retroviral vector expressing IRES-GFP and CD45.1OT-I cells overexpressing BCL6 were obtained by the method in step 1 of this example.
  • the inventors co-adopted CD45.1OT-I cells overexpressing BCL6 and CD45.2OT-I cells in the control group into Tcrbd -/- recipient mice (8 mice) inoculated with E.G7 tumors.
  • the inventors cut the E.G7 tumors with scissors and transferred them to DMEM digestion solution supplemented with 1 mg/ml collagenaseD and 10U/ml DNaseI, digested them at 37°C for 40 minutes, and then used Percoll gradient (GE Healthcare) solution for density gradient centrifugation to separate and purify the donor OT-I cells.
  • the library construction and high-throughput sequencing of the OT-I cell transcriptome were sent to BGI Genomics for further analysis.
  • the cDNA library was sequenced by Illumina HiSeq 4000 of BGI Genomics to obtain RNA-seq sequencing data of BCL6.
  • the sequenced data were analyzed by GSEA using TrimGalorev 0.4.4, bowtie2, featureCounts and other software.
  • GSEA results show that overexpression of BCL6 can induce OT-I cells to show significant transcriptome characteristics of BCL6 + TIM-3 - T prog cells.
  • the inventor integrated and analyzed the ChIP-seq data and RNA-seq data of BCL6, and obtained 180 genes directly induced by BCL6 and 156 genes directly inhibited by BCL6.
  • 49 genes directly induced by BCL6 are characteristic genes of BCL6 + T prog cells (Id3, Cxcr5, Tgfbr3, Ccr6, etc.)
  • 49 genes inhibited by BCL6 are characteristic genes of TIM-3 + T term cells (PRDM1, Havcr2, Cxcr6, Gzmb, etc.).
  • the inventors integrated and analyzed the ChIP-seq data of TCF1 reported in the literature and the RNA-seq data of P14 cells overexpressing Tcf7 in mice infected with LCMV cl13 virus, and obtained 224 genes directly induced by TCF1 and 294 genes directly inhibited by TCF1. Then, by integrating and analyzing the genes directly regulated by BCL6 and TCF1, the inventors obtained 19 genes that were commonly upregulated by them (Cxcr5, Xcl1, Ikzf2, etc.), and 20 genes that were commonly inhibited by them (PRDM1, Gzmb, Prf1, Entpd1, Cxcr6, etc.).
  • the inventors used the method described in step 3 of Example 1 to obtain isolated OT-I cells, and then used the method in step 1 of this example to obtain in vitro activated OT-I cells. Subsequently, the inventors cloned BCL6 (NM_001348026.1) and Tcf7 (NM_001313981.1) into a retroviral vector expressing IRES-GFP and used the method in step 1 of this example to obtain in vitro activated CD45.1 OT-I cells that overexpressed BCL6 and Tcf7 (also known as TCF1).
  • the inventors used CTV to label the OT-I cells overexpressing BCL6 or Tcf7 obtained in step 3 (2) of the above example and the OT-I cells of the control group, and then adoptively transferred them into C57BL/6 recipient mice (8 mice). The experiment was carried out using the method described in step 2 of example 1 and the results were analyzed.
  • Tcf7-deficient CD45.2OT-I cells referred to as Tcf7 -/- OT-I cells
  • BCL6-overexpressing Tcf7 -/- OT-I cells and CD45.1WT OT-I cells were adoptively transferred into CD45.1 recipient mice (8 mice) 7 days after inoculation with E.G7 tumors, and experiments and detections were performed using the method described in step 2 of Example 1.
  • the inventors used the method in step 3 (2) of Example 3 to overexpress Tcf7 in BCL6 -/- OT-I cells, and then adoptively transferred the BCL6 -/- OT-I cells overexpressing Tcf7 and CD45.1WT OT-I cells into CD45.1 recipient mice (8 mice) inoculated with E.G7, and conducted the experiment using the method described in step 2 of Example 1, and then analyzed it after 12 days.
  • Tcf7 significantly improves the survival of OT-I cells in a BCL6-independent manner.
  • the inventors analyzed the ChIP-seq data of BCL6 and TCF1 obtained in step 3 (2) of this example.
  • the results showed that TCF1 significantly bound to the promoter region of BCL6, but BCL6 did not bind to the gene locus of Tcf7.
  • BCL6 significantly bound to the gene locus of Havcr2, a characteristic gene of T term
  • TCF1 did not bind to the gene locus of Havcr2, a characteristic gene of T term .
  • BCL6 acts as a downstream molecule of TCF1, significantly inhibiting the differentiation of T prog cells into T term cells, but does not regulate the survival ability of cells.
  • RNA-seq data of BCL6 obtained in step 3 of this embodiment.
  • Overexpression of BCL6 resulted in a significant decrease in the expression of Gzmb (abbreviation of Granzyme B), Prf1 and Fasl at the transcriptional level.
  • overexpression of BCL6 resulted in a significant decrease in the proportion of tumor-infiltrating Granzyme B + OT-I cells, and at the same time resulted in a significant decrease in the expression level of Granzyme B in T term cells.
  • overexpression of BCL6 did not affect the expression level of IFN- ⁇ , but slightly enhanced the expression level of TNF- ⁇ .
  • overexpression of BCL6 resulted in a significant decrease in the proportion of Caspase-3 + cells, indicating that the apoptosis level of OT-I cells was significantly reduced.
  • BCL6 transcriptionally inhibits the differentiation of T prog cells into T term cells in tumors. BCL6 significantly inhibited the proliferation of T cells, while TCF1 did not.
  • TGF- ⁇ /SMAD2 signaling pathway induces BCL6 expression in CD8 + T cells
  • step 2 of Example 1 The inventors used the method described in step 2 of Example 1 to obtain isolated CD8 + T cells, and then activated them in vitro with a flat-bottomed plate combined with anti-CD3 (BioXCell, clone145-2C11, 5 ⁇ g/ml) and anti-CD28 (BioXCell, clone37.51, 1 ⁇ g/ml) antibodies to obtain in vitro activated BCL6-RFPCD8 + T cells, and IL-2, IL-6, IL-10, IL-12, IL-12, anti-IL-2, anti-IFN- ⁇ and TGF- ⁇ were added simultaneously for stimulation.
  • anti-CD3 BioXCell, clone145-2C11, 5 ⁇ g/ml
  • anti-CD28 BioXCell, clone37.51, 1 ⁇ g/ml
  • Tgfbr2 -/- CD8 + T cells were activated and stimulated in vitro with anti-CD3/28 and TGF- ⁇ , and the experiment was performed using the method in step 1 of this embodiment. The results showed that the loss of Tgfbr2 resulted in TGF- ⁇ being unable to induce high expression of BCL6 at the RNA and protein levels.
  • TGF- ⁇ stimulation significantly inhibited the expression of T-bet, but did not affect the expression of TCF1.
  • Tgfbr2 led to a significant increase in the number of tumor-infiltrating OT-I cells per unit weight, among which the number of T term cells increased significantly, while the number of T prog cells did not change significantly.
  • the loss of Tgfbr2 led to a significant increase in the proportion of IFN- ⁇ + and Granzyme B + cells.
  • the loss of Tgfbr2 led to a significant increase in the number of Granzyme B + OT-I cells per unit tumor infiltration and a significant increase in the proportion of Ki-67 + proliferating cells.
  • TGF- ⁇ signaling pathway significantly inhibits the development of tumor-infiltrating T term cells, but has no significant effect on the production of T prog cells, and that the TGF- ⁇ signaling has a significant inhibitory effect on the effector function and proliferation activity of tumor-infiltrating T term cells.
  • the inventors first adoptively transferred in vitro activated WT and Tgfbr2 -/- OT-I cells into CD45.1 recipient mice 8 days after inoculation with E.G7 tumors, and performed experiments and tests using the method of step 3 of Example 1, and then analyzed the recipient mice on days 16 and 24.
  • the results showed that on days 16 and 24, the loss of Tgfbr2 led to a significant decrease in the proportion of BCL6 + TCF1 + T prog cells, while the proportion of TCF1 - TIM-3 + T term cells increased significantly.
  • the loss of Tgfbr2 led to the gradual disappearance of the T prog cell subpopulation.
  • step 4 (1) of this example were counted by flow cytometry to obtain the number of OT-I cells in the tumor.
  • the results showed that the loss of Tgfbr2 led to a significant increase in the number of tumor-infiltrating OT-I cells on day 16, whereas the loss of Tgfbr2 led to a significant decrease in the number of OT-I cells on day 24.
  • the loss of Tgfbr2 led to a significant increase in the number of both T prog and T term cells on day 16, whereas their numbers were significantly decreased on day 24.
  • the inventors analyzed the transcriptome sequencing data of WT and Tgfbr2 -/- P14 cells in the disclosed chronic LCMV clone13 virus infection model (GEO number: GSE209577), and obtained the differentially expressed genes of WT and Tgfbr2 -/- P14 cells as the characteristic expression genes of WT and Tgfbr2 -/- P14 cells. Then GSEA was used to compare the expression of the characteristic expression genes of WT and Tgfbr2 -/- P14 cells in the transcriptomes of BCL6 + and TIM-3 + CD8 + T cells.
  • the GSEA analysis results showed that the characteristic genes of WT P14 cells in the LCMV cl13 model were significantly enriched in the transcriptome of BCL6 + CD8 + T cells, while the characteristic genes of Tgfbr2 -/- P14 cells were significantly enriched in the transcriptome of TIM-3 + CD8 + T cells. These results indicate that in the early stages of tumor development, TGF- ⁇ signaling significantly inhibits the proliferation of tumor-infiltrating T cells and the differentiation of T prog cells, but it is indispensable for the long-term maintenance of T prog cells.
  • the overexpression of BCL6 resulted in a significant decrease in the number of tumor-infiltrating Tgfb2 -/- OT-I cells, which was significantly less than the number of WT and Tgfbr2 -/- OT-I cells.
  • the overexpression of BCL6 in Tgfbr2 -/- OT-I cells resulted in the number of LY108 + T prog cells returning to the number of T prog cells in WT OT-I cells, but resulted in a significant decrease in the number of TIM-3 + T term cells.
  • overexpression of BCL6 also led to a significant decrease in the number of Granzyme B + Tgfbr2 -/- OT-I cells.
  • Example 5 IL-2/STAT5 signaling pathway inhibits the expression of BCL6 in CD8 + T cells and antagonizes the TGF- ⁇ signaling pathway
  • CD8 + T cells were cultured in vitro, and naive CD8 + T cells were activated for three days using a flat-bottomed plate combined with anti-CD3 (5 ⁇ g/ml) and anti-CD28 (1 ⁇ g/ml) antibodies, and IL-2 was added to stimulate them.
  • the expression level of BCL6 protein in CD8 + T cells was detected by flow cytometry, and the expression of BCL6 gene at the transcriptional level was detected by fluorescence real-time quantitative PCR.
  • the results showed that the expression levels of RNA and protein of BCL6 in Il2ra -/- CD8 + T cells cultured in vitro were significantly increased.
  • the expression level of TCF1 was significantly increased, while the expression level of T-bet was significantly decreased.
  • T prog cells Tcf7, Id3, Slamf6 and etc.
  • PRDM1, Id2, Havcr2 and etc. characteristic genes of T prog cells
  • the loss of Il2ra caused a significant decrease in the proportion and number of tumor-infiltrating OT-I cells, among which the number of T term cells decreased by about 21 times, while the number of T prog cells decreased by only about 3 times.
  • This result shows that the loss of Il2ra significantly impairs the development of T term cells, but has a relatively small effect on T prog cells.
  • the ability of OT-I cells lacking Il2ra to control tumors was significantly impaired.
  • the ability of OT-I cells lacking Il2ra to produce IFN- ⁇ and Granzyme B and the expression level of KI-67 were significantly reduced, indicating that their proliferation and effector functions were significantly impaired.
  • step 6 (2) of Example 4 the inventors used CD8 + T cells treated with IL-2 in vitro to perform a ChIP experiment of STAT5.
  • the experimental method was referred to step 6 (2) of Example 4, except that 10U/ml IL-2 was used instead of 2ng/ml hTGF- ⁇ for stimulation for 30 minutes.
  • the ChIP-qPCR results showed that STAT5 significantly bound to the promoter region of the BCL6 gene, but not to the exon region of the BCL6 gene.
  • the addition of exogenous IL-2 significantly enhanced the binding of STAT5 protein to the promoter region of the BCL6 gene.
  • RNA-seq results in step 2 of this example were subjected to GSEA analysis.
  • the results of GSEA analysis showed that in mice infected with LCMV cl13, the characteristic genes in WT P14 cells were significantly highly expressed in OT-I cells not treated with IL-2, while the characteristic genes in Tgfbr2 -/- P14 cells were significantly highly expressed in OT-I cells treated with IL-2.
  • STAT5 and SMAD2 bind to the same promoter site of the BCL6 gene, but produce opposite transcriptional regulatory effects; the binding amount of STAT5 at the promoter site of the BCL6 gene is significantly decreased in CD8 + T cells treated with TGF- ⁇ in vitro, and at the same time, the binding amount of SMAD2 at the promoter site of the BCL6 gene is significantly decreased in CD8 + T cells treated with IL-2; in CD8 + T cells treated with TGF- ⁇ in vitro, the secretion of IL-2 and the expression level of CD25 are significantly decreased, indicating that TGF- ⁇ may indirectly regulate the expression of BCL6 by inhibiting the IL-2 signaling pathway.
  • Example 6 BCL6 and BLIMP1 play antagonistic roles in the differentiation of CD8 + T cells in tumors
  • BLIMP1 (encoded by PRDM1) is one of the downstream effector proteins of the IL-2-STAT5 pathway.
  • PRDM1 is one of the downstream effector proteins of the IL-2-STAT5 pathway.
  • BLIMP1 significantly inhibits the expression of BCL6 and antagonizes the function of BCL6; in the acute infection model, BLIMP1 significantly inhibits the formation of memory CD8 + T cells; in the chronic viral infection model, BLIMP1 significantly inhibits the development of Tfc cells. Therefore, this example needs to verify the expression and function of BLIMP1 in the tumor model. The specific steps are as follows:
  • the inventors analyzed the ChIP-seq data of BLIMP1-Biotin in CD8 + T cells (GEO number: GSE79339) and obtained the binding genes of BLIMP1 in the genome of mouse CD8 + T cells.
  • the ChIP-seq data of BCL6 obtained in step 1 of Example 3 and the ChIP-seq data of BLIMP1 obtained in this example, the inventors found that BCL6 and BLIMP1 had significantly different binding spectra, as shown in Figure 2A. However, these two transcription factors bind to a large number of the same target genes together, and more than half of the genes bound by BCL6 are also bound by BLIMP1, as shown in Figure 2B.
  • the inventors isolated and purified the co-adoptively delivered PRDM1 (encoding BLIMP1 protein) overexpression and control group OT-I cells from the E.G7 tumor, and then obtained RNA-seq data of PRDM1 overexpression by the method described in step 2 of Example 3 (the difference was that PRDM1 (NM_007548.4) was cloned into a retroviral vector expressing IRES-BFP instead of overexpressing BCL6 and TCF1), and performed GSEA analysis on the RNA-seq data.
  • GSEA results showed that overexpression of PRDM1 caused OT-I cells to exhibit significant transcriptome characteristics of BCL6 - TIM-3 + T term cells, as shown in Figure 2C.
  • Genes induced by PRDM1 overexpression to be differentially expressed were significantly enriched in multiple biological signaling pathways, including T cell activation, differentiation, and adhesion, as shown in Figure 2D.
  • the common genes induced by BCL6 and PRDM1 overexpression to be differentially expressed showed a significant negative correlation, as shown in Figure 3A.
  • the inventor further integrated and analyzed the BLIMP1ChIP-seq data obtained in step 1 of the embodiment and the RNA-seq data of PRDM1 overexpression, and obtained 133 BLIMP1 directly induced expression genes and 132 BLIMP1 directly inhibited expression genes, as shown in Figure 3B.
  • 27 BLIMP1 directly induced expression genes are characteristic genes of TIM-3 + T term cells (Havcr2, Cd244a, Il2ra, Cd38, etc.)
  • 41 BLIMP1 directly inhibited expression genes are characteristic genes of BCL6 + T prog cells (Tcf7, Id3, Cxcr5, Ccr7, etc.), as shown in Figure 3C. Therefore, BCL6 and BLIMP1 are key transcription factors for regulating the differentiation of T prog and T term cells.
  • the inventors analyzed the ChIP-seq data and ATAC-seq data (GEO number: GSE122713) of BCL6 obtained in step 1 of this example.
  • the ChIP-seq results showed that in CD8 + T cells, BCL6 directly bound to multiple sites of the PRDM1 gene, distributed in introns and distal intergenic regulatory regions.
  • BCL6 and BLIMP1 bind to different sites of the characteristic genes Il7r, Sell and Ccr7 of T prog cells and regulate their expression inversely.
  • BCL6 and BLIMP1 may co-regulate the expression of target genes in a variety of ways, including direct binding to different sites of the same gene, competitive binding to the same site of the gene, or indirectly inducing the expression of downstream genes by inhibiting the expression of each other.
  • BCL6 and BLIMP1 directly bind to multiple sites of the Ifng gene (Interferon-gamma gene), they do not regulate the expression of the Ifng gene, as shown in Figures 3C and 4B.
  • the inventors obtained in vitro activated CD8 + T cells that simultaneously overexpressed BCL6 and PRDM1 by the method of step 1 of Example 3, and extracted the total RNA from the CD8 + T cells using Magzolreagent (Magen). Then, cDNA was obtained using the M-MLV reverse transcription kit (Invitrogen). Then, the expression level of specific genes was detected using the SYBR real-time kit (Bio-Rad Laboratories). The results showed that BCL6 and PRDM1 mutually regulated the transcription of genes such as Tcf7, Cxcr5, Id3 and Havcr2, as shown in Figure 5A.
  • the inventors obtained OT-I cells that overexpressed both BCL6 and PRDM1 by the method of step 3 (2) of Example 3, and co-adoptively transferred the OT-I cells that overexpressed both BCL6 and PRDM1 and the OT-I cells of the control group into Tcrbd -/- recipient mice (8 mice) inoculated with E.G7 tumors, and conducted experiments by the method described in step 2 of Example 1.
  • the results showed that overexpression of BCL6 and PRDM1 mutually regulated the proportion of T prog and T term cells, as shown in Figure 5B.
  • BLIMP1 was significantly overexpressed in tumor-infiltrating OT-I cells after 11 and 20 days.
  • BLIMP1-EYFP was specifically expressed in TIM-3 + T term cells, but not in LY108 + T prog cells.
  • BLIMP1-EYFP was also expressed in TIM-3+ T term cells, but not in LY108+ T prog cells.
  • PRDM1 was significantly highly expressed in tumor-infiltrating CD8 + T cells, and the expression of PRDM1 was mutually exclusive with the expression of TCF7 and BCL6, as shown in Figure 6B and C.
  • step 4 Verify the molecular mechanism by which BLIMP1 expression is induced in CD8 + T cells.
  • BLIMP1 is a downstream transcription factor of the IL-2 signaling pathway. Therefore, the inventors first activated the naive BLIMP1-EYFP CD8+ T cells described in step 3(3) of this example in vitro with anti-CD3/28 by implementing the method of step 1 of step 3, and cultured WT CD8 + T cells and PRDM1 -/- CD8 + T cells in vitro by the method of step 2 of example 1, and continuously stimulated them with IL-2 for 6 days. Then, flow cytometry was used to detect the expression level of BLIMP1 in BLIMP1-EYFP CD8 + T cells.
  • the control group was CD8 + T cells obtained without the addition of IL-2, which is hereinafter referred to as control CD8+ T cells.
  • BLIMP1 is hardly expressed in CD8 + T cells activated for 3 days.
  • BLIMP1-EYFP was significantly highly expressed in TIM-3 + CD8 + T cells.
  • CD8 + T cells without IL-2 stimulation BLIMP1-EYFP was not significantly expressed.
  • under continuous IL-2 stimulation more than 60% of CD8 + T cells differentiated into TIM-3 + cells; in CD8 + T cells without IL-2 stimulation, less than 10% of CD8 + T cells expressed TIM-3.
  • the inventors adoptively transferred the naive WT and PRDM1 -/- OT-I cells described in step 4 of this example into Tcrbd -/- recipient mice (8 mice) at a ratio of 1:1, and then inoculated E.G7 tumor cells one day later.
  • the experiment was performed using the method described in step 2 of example 1 and the experimental results were analyzed.
  • PRDM1 overexpression resulted in a significant decrease in the proportion of LY108 + T prog cells and the proportion of TCF1 + and BCL6 + cells in tumor-infiltrating OT-I cells, while the proportion of TIM-3 + T term cells was significantly increased.
  • the loss of PRDM1 had no significant effect on the number of tumor-infiltrating OT-I cells, as shown in Figure 9A.
  • the number of T prog cells increased significantly, while the number of T term cells decreased significantly.
  • Overexpression of PRDM1 resulted in a slight decrease in the number of tumor-infiltrating OT-I cells, among which the number of T prog cells decreased significantly, while the number of T term cells increased significantly, as shown in Figure 9B.
  • the in vivo proliferation results showed that overexpression of PRDM1 did not significantly affect the CTV signal in OT-I cells on days 2 and 3, as shown in Figure 9C, indicating that BLIMP1 had no significant regulatory effect on cell proliferation.
  • the inventors first inoculated B16BL6, EL4, HEPA1-6 and MC38 tumors into BLIMP1-EYFP mice, then isolated CD8 + T cells using the method described in step 2 of Example 1 and performed flow cytometry experiments, and then analyzed the experimental results on day 15.
  • the naive WT CD8 + T cells and PRDM1 -/- CD8 + T cells and CD4 + T cells in step 4 of this example were adoptively transferred into Tcrbd -/- recipient mice (8 mice), and tumor cells were inoculated one day later.
  • the experiment was performed using the method described in step 2 of Example 1 and the experimental results were analyzed.
  • Example 7 Loss of PRDM1 in tumor-specific CD8 + T cells significantly enhances the therapeutic effect of tumor control
  • T prog cells In cancer patients, the number of T prog cells is positively correlated with better clinical prognosis and responsiveness to immune checkpoint blockade therapy. Increasing the number of T prog cells by overexpressing Tcf7 can significantly increase the proliferation of CD8 + T cells after PD-1/PD-L1 blockade and therapeutic immunotherapy, resulting in better tumor control. Therefore, considering the important role of BCL6 and BLIMP1 in T cell differentiation, it is urgent to verify whether BCL6 overexpression and PRDM1 deficiency will enhance the therapeutic effect of anti-PD-1. The specific steps are as follows:
  • the inventors adoptively transferred equal amounts of BCL6-overexpressing OT-I cells or control OT-I cells (the preparation method of the two OT-I cells is shown in Implementation 3, step 3 (2)) into Tcrbd -/- recipient mice (8 mice) inoculated with E.G7 tumors, and performed experiments and tests using the method described in Example 1, step 2. The test results are shown in FIG11A .
  • the inventors adoptively transferred equal amounts of in vitro activated BCL6-overexpressing OT-I cells and control OT-I cells (the preparation method of the two OT-I cells is shown in Implementation 3, step 3 (2)) into CD45.1 recipient mice (8 mice) inoculated with E.G7 tumors for 7 days, and injected anti-PD-1 antibodies every 2-3 days starting from the 3rd day after the adoptive transfer, for a total of 3 injections (schematic diagram as shown in FIG12A ), and performed experiments and detection using the method described in Example 1, step 2.
  • the overexpression of BCL6 resulted in a significant decrease in the number of Granzyme B + OT-I cells infiltrating the unit tumor, and did not increase significantly after anti-PD-1 treatment, as shown in Figure 13C. Therefore, anti-PD-1 treatment cannot effectively promote the proliferation and differentiation of OT-I cells overexpressing BCL6, and therefore cannot induce a more effective anti-tumor effect. Therefore, in the tumor model, although the overexpression of BCL6 can effectively maintain the characteristics of T prog cells, it cannot effectively improve the anti-tumor effect and cannot produce a combined effect with anti-PD-1 because it significantly inhibits the proliferation and cytotoxicity of T cells.
  • the inventors adoptively transferred equal amounts of in vitro activated CD45.2WT and PRDM1 -/- OT-I cells (the preparation method of the two OT-I cells is shown in Implementation 3, step 3 (2), the difference being that CRISPR-Cas9 was used to knock out PRDM1 instead of overexpressing BCL6 and TCF1) into CD45.1 recipient mice (8 mice) inoculated with E.G7 tumors, and performed experiments and detection using the method described in Example 1, step 2.
  • the inventors adopted equal amounts of in vitro activated CD45.2WT and PRDM1 -/- OT-I cells (the preparation method of the two OT-I cells is shown in Implementation 3, step 3 (2), the difference being that CRISPR-Cas9 was used to knock out PRDM1 instead of overexpressing BCL6 and TCF1) into CD45.1 recipient mice (8 mice) inoculated with E.G7 tumors, and then intraperitoneally injected anti-PD-1 antibodies starting from 3 days later, for a total of 3 injections (schematic diagram as shown in FIG14A ), and performed experiments and detection using the method described in Example 1, step 2.
  • the loss of PRDM1 and anti-PD-1 treatment can produce a more effective tumor control effect, and is significantly better than the effect of adoptive delivery of WT OT-I cells and the combination of anti-PD-1, as shown in Figure 14B.
  • the loss of PRDM1 significantly increased the proportion of TCF1 + T prog cells and significantly increased the number of TCF1 + T prog cells infiltrating per tumor, while anti-PD-1 treatment further increased the number of TCF1 + T prog cells infiltrating per tumor.
  • anti-PD-1 treatment significantly increased the number of T term cells infiltrating per tumor and reached the number of the WT OT-I group, as shown in Figure 14C.
  • anti-PD-1 treatment also significantly increased the number of T term cells infiltrating per tumor.
  • Granzyme B + PRDM1 -/- OT-I cells were significantly increased, as shown in Figure 15A.
  • the combination of PRDM1 deficiency and anti-PD-1 significantly increased the ability of OT-I cells to produce IFN- ⁇ and TNF- ⁇ , as shown in Figure 15B. Therefore, the loss of PRDM1 in tumor-specific CD8 + T cells significantly promoted the expansion of T prog cells and significantly enhanced their response to anti-PD-1, resulting in better tumor control.

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Abstract

La présente invention concerne un lymphocyte T, l'expression d'une protéine donnée dans le lymphocyte T étant modifiée, et la protéine donnée étant appropriée pour réguler l'expression de BCL6 dans le lymphocyte T. Le lymphocyte T est au moins l'un des suivants : un lymphocyte T CD8+ et un lymphocyte T CD4+, de préférence un lymphocyte T CD8+ reconnaissant spécifiquement les cellules tumorales. La protéine donnée est appropriée pour réguler l'expression de BCL6 au moyen de la voie IL-2-STAT5 et/ou de la voie TGF-bêta-SMAD2. L'expression de BCL6 dans le lymphocyte T est renforcée. L'expression de la protéine donnée dans le lymphocyte T est modifiée. La protéine donnée comporte au moins une protéine en aval sur la voie IL-2-STAT5, et l'expression de la protéine donnée est réduite. La protéine donnée comprend au moins un élément choisi parmi PRDM1 ou un élément de sa même famille.
PCT/CN2023/122454 2023-09-28 2023-09-28 Lymphocyte t modifié et son utilisation Pending WO2025065472A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102191244A (zh) * 2010-03-18 2011-09-21 上海交通大学医学院附属瑞金医院 一组能有效下调PRDM1β表达的siRNA分子及其应用
CN102770455A (zh) * 2009-08-03 2012-11-07 迈阿密大学 用于体内扩增调节性t细胞的方法
CN111601882A (zh) * 2017-11-22 2020-08-28 拉霍拉敏感及免疫学研究所 工程化的免疫细胞的应用和生产
CN113286811A (zh) * 2018-07-30 2021-08-20 南加利福尼亚大学 改善过继性细胞疗法的效力和安全性
CN113766919A (zh) * 2019-04-05 2021-12-07 2赛文缇生物公司 制造抗bcma car t细胞
CN114929262A (zh) * 2019-08-21 2022-08-19 得克萨斯大学体系董事会 用于过继细胞疗法的免疫细胞
CN116583607A (zh) * 2020-08-13 2023-08-11 耶鲁大学 用于工程改造和选择具有期望表型的car t细胞的组合物和方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102770455A (zh) * 2009-08-03 2012-11-07 迈阿密大学 用于体内扩增调节性t细胞的方法
CN102191244A (zh) * 2010-03-18 2011-09-21 上海交通大学医学院附属瑞金医院 一组能有效下调PRDM1β表达的siRNA分子及其应用
CN111601882A (zh) * 2017-11-22 2020-08-28 拉霍拉敏感及免疫学研究所 工程化的免疫细胞的应用和生产
CN113286811A (zh) * 2018-07-30 2021-08-20 南加利福尼亚大学 改善过继性细胞疗法的效力和安全性
CN113766919A (zh) * 2019-04-05 2021-12-07 2赛文缇生物公司 制造抗bcma car t细胞
CN114929262A (zh) * 2019-08-21 2022-08-19 得克萨斯大学体系董事会 用于过继细胞疗法的免疫细胞
CN116583607A (zh) * 2020-08-13 2023-08-11 耶鲁大学 用于工程改造和选择具有期望表型的car t细胞的组合物和方法

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