WO2023005219A1 - Récepteur antigénique chimérique modifié par couplage d'ubiquitine et cellule immunitaire - Google Patents

Récepteur antigénique chimérique modifié par couplage d'ubiquitine et cellule immunitaire Download PDF

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WO2023005219A1
WO2023005219A1 PCT/CN2022/080608 CN2022080608W WO2023005219A1 WO 2023005219 A1 WO2023005219 A1 WO 2023005219A1 CN 2022080608 W CN2022080608 W CN 2022080608W WO 2023005219 A1 WO2023005219 A1 WO 2023005219A1
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cells
ubiquitin
car
chimeric antigen
cancer
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王皞鹏
邱士真
李文涛
王琨
陈健
宋献民
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上海科技大学
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464412—CD19 or B4
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma

Definitions

  • the invention relates to the field of biomedicine, in particular to a ubiquitin-coupled modified chimeric antigen receptor and immune cells.
  • Chimeric antigen receptor is an artificially engineered tumor-targeting antigen receptor.
  • CAR-T therapy is an adoptive immunotherapy that expresses CARs that specifically recognize antigens and transmit T cell signals on the patient's own T cells, enabling them to recognize and kill tumor cells in the patient's body.
  • the composition of CAR mainly includes: a single chain antibody (single chain antibody fragment, scFv) that can recognize and bind to a specific tumor antigen, and its specificity can realize precise recognition and "targeted attack” on tumor cells; T cell receptor (T cell The intracellular domain of the receptor (TCR) ⁇ chain can effectively activate T cells after the scFv structure recognizes tumor antigens, and the activated T cells can secrete a large amount of cytokines, and effectively kill tumor cells while rapidly proliferating themselves.
  • a single chain antibody single chain antibody fragment, scFv
  • T cell receptor T cell receptor
  • TCR T cell receptor
  • TCR The intracellular domain of the receptor (TCR) ⁇ chain can effectively activate T cells after the scFv structure recognizes tumor antigens, and the activated T cells can secrete a large amount of cytokines, and effectively kill tumor cells while rapidly proliferating themselves.
  • adding the signaling domain of co-stimulatory molecules such as CD28 or 41BB to the intracellular region of CAR can enhance the ability of CAR to activate T cells and improve the ability of CAR-T cells to activate T cells. ability to survive in vivo.
  • CAR-T therapy has achieved remarkable success in the clinical treatment of various types of tumors, especially for blood tumors.
  • CAR-T therapy still has many limitations in the treatment of solid tumors.
  • the existing optimal design strategies for CAR-T mainly include: reducing the signal of CAR by mutating and inactivating different combinations of ITAM regions in CD3 ⁇ ; mutating the co-stimulatory domain of CD28 to reduce the co-stimulatory signal of CD28; trying different structural combinations , explore the most suitable CAR structure for different targets and the lowest level of background self-activation; co-transduce transcription factors involved in the regulation of T cell proliferation and differentiation into T cells with CAR to reduce the self-activation of CAR-T cells Signaling, suppression of exhaustion of T cells, etc.
  • this type of design often needs to try CARs targeting different targets, which consumes a lot of manpower and material resources, and has the disadvantages of large errors and low universality. Therefore, a more practical and universal transformation aimed at the CAR structure itself is a more optimal solution.
  • the purpose of the present invention is to provide a ubiquitin-modified chimeric antigen receptor and immune cells to solve the problems in the prior art.
  • the present invention firstly provides a chimeric antigen receptor modification method, which includes coupling ubiquitin to the C-terminus of the chimeric antigen receptor.
  • the present invention provides a ubiquitin-modified chimeric antigen receptor, the ubiquitin-modified chimeric antigen receptor comprises a transmembrane domain, an intracellular domain and an extracellular domain, and the C-terminus of the intracellular domain is coupled with ubiquitin .
  • the present invention provides an isolated polynucleotide comprising nucleotides encoding the ubiquitin-modified chimeric antigen receptor.
  • the present invention provides a nucleic acid construct comprising the isolated polynucleotide.
  • the present invention provides an immune cell, which includes the nucleic acid construct or the exogenous polynucleotide integrated in the genome, or can express the ubiquitin-modified chimeric antigen receptor.
  • the immune cells are selected from CAR-T cells, CAR-NK cells, CAR-macrophages or CAR-TIL cells.
  • the present invention also provides a preparation method of the immune cells, the preparation method comprising introducing the nucleic acid construct or the polynucleotide into immune effector cells or stem cells producing immune effector cells.
  • the immune effector cells are selected from T cells, NK cells, macrophages or TIL cells.
  • the present invention also provides the use of the immune cells in the preparation of medicines for treating cancer or immune diseases.
  • the present invention also provides a treatment method for tumors or immune diseases, the treatment method comprising administering a therapeutically effective amount of the immune cells to a subject.
  • the ubiquitin-conjugated modified chimeric antigen receptor and immune cells of the present invention have the following beneficial effects: provide a method for the optimization and transformation of CAR-T, especially for CAR-T that is not capable of proliferating in solid tumors Provides solutions to the best problems.
  • the optimization and transformation scheme of CAR with high self-activation level which is simple and applicable to different targets, has the advantages of simple application, high repeatability, good universality, high degree of optimization, and good anti-tumor activity. Suitable for all kinds of current CAR-T cell production technologies, especially suitable for CAR-T therapeutic products developed for solid tumor targets. , Excessive exhaustion and other problems provide a new solution.
  • Figure 1 shows the detection of the self-activation level of CAR-T cell lines targeting different targets in the human T lymphoma cell line Jurkat, using the expression of indicator proteins such as CD69 and ICOS to measure the activation level of T cells In this case, the background self-activation level of these cell lines was evaluated.
  • the left panel shows the detected expression level of CD69, and the right panel shows the detected ICOS expression level.
  • CARs targeting different targets used in a large number of clinical and preclinical trials lead to high levels of background self-activation of T cells.
  • FIG. 2-1 to Figure 2-3 show that CD19-28WT-CAR (nucleotide sequence such as SEQ ID NO.12, amino acid sequence such as SEQ ID NO.28) and a CD19-targeting CAR C-terminal coupling Wild-type ubiquitin (wtUb) (nucleotide sequence such as SEQ ID NO.13, amino acid sequence such as SEQ ID NO.29) or monoubiquitin (monoUb) (nucleotide sequence such as SEQ ID NO.14, amino acid sequence such as The CD19-28wtUb-CAR and CD19-28MonoUb-CAR obtained from SEQ ID NO.30) were expressed in Jurkat T cell line (Fig. 2-1) or primary human T cells (Fig.
  • Figure 3-1 to Figure 3-4 show that after expressing a variety of monoubiquitin-coupled CD28 CARs targeting different targets in the Jurkat cell line, the effect of the modification on the background self-activation level of CAR-T was tested to verify this Universality of the program.
  • GD2 nucleotide sequence such as SEQ ID NO.15, amino acid sequence such as SEQ ID NO.31, the results as shown in Figure 3-2
  • GPC3 Nucleotide sequence such as SEQ ID NO.16, amino acid sequence such as SEQ ID NO.32, the results are shown in Figure 3-3
  • CD19 and CD22 as the dual target (nucleotide sequence such as SEQ ID NO.33, Amino acid sequence such as SEQ ID NO.34, results shown in Figure 3-4) the expression level of monoubiquitin-coupled CAR on the cell membrane, and the background self-activation signal of the corresponding CAR-T cells (with CD19 and CD22 as the
  • SEQ ID NO.35 The nucleotide sequence of the dual-target WT-CAR is shown in SEQ ID NO.35, and the amino acid sequence is shown in SEQ ID NO.36).
  • Figure 4-1 to Figure 4-4 show the detection of wild-type targets (ie CD19-28WT-CAR T cells, GD2-28WT-CAR T cells, GPC3-28WT-CAR T cells, GPC3-28WT-CAR T cells, CD19 ⁇ 22WT-CAR T cells) and CAR-T cells coupled with monoubiquitin (i.e. CD19-28MonoUb-CAR T cells, GD2-28MonoUb-CAR T cells, GPC3-28MonoUb-CAR T cells, CD19 ⁇ 22MonoUb-CAR T cells, CD19 ⁇ 22MonoUb-CAR T cells differentiation of T cells).
  • wild-type targets ie CD19-28WT-CAR T cells, GD2-28WT-CAR T cells, GPC3-28WT-CAR T cells, GPC3-28WT-CAR T cells, CD19 ⁇ 22MonoUb-CAR T cells, CD19 ⁇ 22MonoUb-CAR T cells differentiation
  • the differentiation status of T cells can be divided into CD45RA+CD62L+ (Stem cell memory T cell, T SCM ), CD45RA-CD62L+ (Central memory T cell, T CM ), CD45RA- CD62L- (Effector memory T cell, T EM ), and CD45RA+CD62L- (Effector T cell, T EFF ), the differentiation level increases successively, and the proliferation and differentiation ability decrease successively.
  • Figure 4-1 is CAR-T cells targeting CD19
  • Figure 4-2 is CAR-T cells targeting GD2
  • Figure 4-3 CAR-T cells targeting GPC3
  • Figure 4-4 shows CAR-T cells targeting both CD19 and CD22, the left figure in each figure is the detected CD4, and the right figure is the detected CD8), coupled with single
  • the modification of ubiquitin can effectively slow down the differentiation of T cells, so that more CAR-T cells remain in the TSCM state, which also implies that CAR-T cells coupled with monoubiquitin should have better persistence. proliferation and tumor killing ability.
  • Figure 5 shows the in vitro detection of the proliferation of GD2-targeting wild-type and monoubiquitin-coupled CAR-T cells under continuous stimulation of target cells.
  • the same amount of GD2-28WT-CAR and GD2-28MonoUb-CAR T cells were given the same amount of target cells (irradiated) to stimulate.
  • live cell counts were performed every 2 days to record cell proliferation.
  • the results of this experiment showed that in the presence and continuous stimulation of target cells, monoubiquitin-coupled CAR-T cells had lower background self-activation levels and cell differentiation degrees, and had better sustained proliferation capabilities.
  • Figure 6-1 to Figure 6-2 show the in vivo tumor killing experiments in tumor-bearing mice, verifying the functional differences between wild-type and monoubiquitin-coupled CAR-T cells targeting CD19.
  • the tumor-bearing mice were randomly divided into 3 groups, and the same amount of normal T cells not transfected with CAR, CD19 WT CAR-T cells and CD19 MonoUb CAR-T cells were given to the mice by tail vein injection respectively.
  • Imaging equipment in vivo imaging system, IVIS
  • IVIS in vivo imaging system
  • wild-type CAR-T cells have limited ability to control tumors, while CAR-T cells coupled with monoubiquitin can still effectively control tumor growth to a certain extent.
  • tumor clearance can be achieved ( Figure 6-1), which effectively improves the survival rate of tumor-bearing mice ( Figure 6-2), and its efficacy is significantly better than that of wild-type CAR-T cells.
  • Figure 7-1 to Figure 7-2 show the detection of CAR-T cell proliferation, differentiation and exhaustion in tumor-bearing mice.
  • the results showed that under the stimulation of tumor cells in vivo, CAR-T cells coupled with monoubiquitin still had a higher proportion of stem cell memory T cells (T SCM ) with higher sustained proliferation ability (Figure 7-1, The left picture shows CD4 CAR-T cells in the bone marrow, and the middle picture shows CD8 CAR-T cells in the bone marrow); and correspondingly, the number of CAR-T cells coupled with monoubiquitin is also significantly more than that of wild-type CAR-T cells ( Figure 7-1, the right figure is the count of CAR-T cells in the spleen), and the indicative proteins of its cell exhaustion (PD1, LAG3, TIM3, that is, Figure 7-2, the left figure is the CD4 CAR-T cells in the bone marrow, and the right figure The expression level of CD8 CAR-T cells in bone marrow is also significantly lower, which may
  • the present invention provides an optimized transformation scheme of CAR with high self-activation level that is simple and applicable to different targets.
  • a ubiquitin protein mutant (monoubiquitin, MonoUb for short) is coupled to the C-terminus of CARs targeting different targets to obtain MonoUb-CAR. Therefore, the present invention firstly provides a chimeric antigen receptor modification method, which includes coupling ubiquitin to the C-terminus of the chimeric antigen receptor.
  • Ubiquitin is a small protein present in all eukaryotes (most eukaryotic cells). Ubiquitin consists of 76 amino acids with a molecular weight of about 8.451kDa. Its main function is to mark proteins that need to be broken down so that they can be degraded by the 26S proteasome. For membrane proteins, especially some special ubiquitin modification types may be involved in the down-regulation, transport and vesicle sorting of membrane proteins, as well as the regulation of their signal transmission. Ubiquitin as referred to in this application refers to any wild-type ubiquitin or mutant ubiquitin from any eukaryotic cellular source, including mammals such as primates (e.g.
  • Ubiquitin is a very conserved protein. Almost all eukaryotic cells express ubiquitin since yeast, and its amino acid sequence is basically identical (yeast and human ubiquitin have only one amino acid difference).
  • the type of ubiquitin is wild-type ubiquitin or mutant ubiquitin.
  • Wild-type ubiquitin can be modified by intracellular endogenous ubiquitin but cannot be used as a substrate to modify other proteins, so it can form ubiquitin chains.
  • the ubiquitin is mutant ubiquitin. Because the mutant ubiquitin cannot be modified by endogenous ubiquitin, and the mutant ubiquitin cannot be used as a substrate to ubiquitinate other proteins, the mutant ubiquitin is monoubiquitin, so the mutant ubiquitin can also be Known as mutant monoubiquitin or monoubiquitin.
  • the ubiquitin is coupled to the C-terminus of the chimeric antigen receptor through a linker peptide.
  • the connecting peptides described in the present application may be commonly used connecting peptides in the art.
  • the connecting peptide is, for example, GSGGSG, GSGGSGG GSGGSGGG or GGGGSGGG or GGSGGGSGGGSAAA.
  • the present invention also provides a ubiquitin-modified chimeric antigen receptor, which includes a transmembrane domain, an intracellular domain, and an extracellular domain, and the C-terminus of the intracellular domain is coupled with ubiquitin white.
  • the ubiquitin-modified chimeric antigen receptor is obtained by the engineering method.
  • the transmembrane domain may include transmembrane domains of protein molecules such as CD8 ⁇ , CD28, and DAP10.
  • the amino acid sequence of CD8 ⁇ may include the following sequence: IYIWAPLAGTCGVLLLSLVITLYC.
  • NM_001145873 for the sequence of CD8 ⁇
  • NM_006139 for the sequence of CD28.
  • the intracellular domain may comprise a co-stimulatory domain and/or a signaling domain.
  • the intracellular domain may include one or a combination of several of the following: signal transduction domains of protein molecules such as 4-1BB, CD28, OX40, ICOS, CD3 ⁇ , and DAP10.
  • amino acid sequence of 4-1BB includes as follows:
  • the amino acid sequence of the CD3 ⁇ includes as follows:
  • the intracellular domain sequentially includes CD28 and CD3 ⁇ from the N-terminus to the C-terminus.
  • the chimeric antigen receptor includes an extracellular domain, a transmembrane domain, and an intracellular domain sequentially from the N-terminus to the C-terminus.
  • the chimeric antigen receptor includes a single-chain antibody, a transmembrane domain, and an intracellular domain in sequence from the N-terminus to the C-terminus.
  • the chimeric antigen receptor includes a single-chain antibody, a CD8 ⁇ transmembrane region, a 4-1BB co-stimulatory domain, and a CD3 ⁇ signaling domain sequentially from the N-terminus to the C-terminus.
  • the chimeric antigen receptor comprises a single-chain antibody, a CD28 transmembrane region, a CD28 co-stimulatory domain, and a CD3 ⁇ signaling domain sequentially from the N-terminus to the C-terminus.
  • the chimeric antigen receptor includes a single-chain antibody, a CD8 ⁇ transmembrane region, an OX40 co-stimulatory domain, and a CD3 ⁇ signaling domain sequentially from the N-terminal to the C-terminal.
  • the chimeric antigen receptor comprises a single-chain antibody, a CD8 ⁇ transmembrane region, an ICOS co-stimulatory domain, and a CD3 ⁇ signaling domain sequentially from the N-terminal to the C-terminal.
  • the chimeric antigen receptor comprises a single-chain antibody, a CD8 ⁇ transmembrane region, a 4-1BB co-stimulatory domain, and a CD3 ⁇ signaling domain sequentially from the N-terminus to the C-terminus.
  • the chimeric antigen receptor comprises a single-chain antibody, a CD28 transmembrane region, a CD28 co-stimulatory domain, an OX40 co-stimulatory domain, and a CD3 ⁇ signaling domain from the N-terminus to the C-terminus.
  • the chimeric antigen receptor includes CD8 ⁇ signal peptide, myc tag, scFv, extracellular domain composed of CD8 ⁇ hinge, transmembrane domain of CD8 ⁇ , CD28 and CD3 ⁇ in sequence from N-terminal to C-terminal Intracellular domains organized in tandem.
  • the ubiquitin-modified chimeric antigen receptor that is, the CD3 ⁇ of any one of the above chimeric antigen receptors is connected with ubiquitin through a linker peptide (or linker).
  • the single-chain antibody of the present invention is not specifically limited, and can be selected from any single-chain antibody.
  • Examples of single-chain antibodies used in the present invention include any one or more of CD19 scFv, GD2 scFv, GPC3 scFv, Her2 scFv, CSPG4 scFv, EGFR scFv, Meso scFv, TRBC1 scFv, CD133 scFv, and BCMA scFv.
  • the chimeric antigen receptor is a multi-target chimeric antigen receptor.
  • the ubiquitin referred to in this application is selected from wild-type ubiquitin or mutant ubiquitin.
  • amino acid sequence of wild-type ubiquitin is shown in SEQ ID NO.17.
  • the ubiquitin is selected from mutant ubiquitin.
  • the amino acid sequence of the mutant ubiquitin selected in the present invention is shown in SEQ ID NO.18.
  • the present invention provides an isolated polynucleotide comprising nucleotides encoding the ubiquitin-modified chimeric antigen receptor.
  • polynucleotides encoding the transmembrane domain, intracellular domain and extracellular domain can all be selected from polynucleotides in the prior art.
  • the polynucleotide encoding the ubiquitin-modified chimeric antigen receptor sequentially includes nucleotides encoding the CD8 ⁇ signal peptide, myc tag, scFv, and the ectodomain composed of the CD8 ⁇ hinge, Nucleotides encoding the transmembrane domain of CD8 ⁇ , nucleotides encoding the intracellular domain composed of CD28, CD3 ⁇ , and ubiquitin in tandem.
  • the nucleotide sequence of wild-type ubiquitin is shown in SEQ ID NO.1.
  • the ubiquitin is selected from mutant ubiquitin.
  • the polynucleotide sequence encoding mutant ubiquitin selected in the present invention is shown in SEQ ID NO.2.
  • the present invention provides a nucleic acid construct comprising the isolated polynucleotide.
  • nucleic acid construct refers to an artificially constructed nucleic acid segment that can be introduced into cells or tissues, and the nucleic acid construct is a non-viral vector or a viral vector.
  • the viral vectors are lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, and baculoviral vectors.
  • the nucleic acid construct is a lentiviral vector, and the lentiviral vector includes a vector backbone, that is, an empty vector and an expression framework.
  • the empty vector includes various expression-controlling elements, including a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element and a reporter gene.
  • the vector may also contain an origin of replication.
  • the empty vector is, for example, pHR-hEF1 ⁇ -IRES-EGFP empty vector.
  • the expression framework is the isolated polynucleotide.
  • vector refers to a nucleic acid fragment or polynucleotide fragment used to introduce or transfer one or more nucleic acids or one or more polynucleotides into a target cell or tissue.
  • vectors are used to introduce foreign DNA into another cell or tissue.
  • the vector may contain a bacterial resistance gene for growth in bacteria and a promoter for expressing a protein of interest in an organism.
  • DNA can be produced in vitro by PCR or any other suitable technique or techniques known to those skilled in the art.
  • the present invention provides an immune cell, which includes the nucleic acid construct or the exogenous polynucleotide integrated in the genome, or is capable of expressing the ubiquitin-modified chimeric antigen receptor.
  • the immune cells are selected from CAR-T cells, CAR-NK cells, CAR-macrophages or CAR-TIL cells.
  • the present invention also provides a preparation method of the immune cells, the preparation method comprising introducing the nucleic acid construct or the polynucleotide into immune effector cells or stem cells producing immune effector cells.
  • the immune effector cells are selected from T cells, NK cells, macrophages or TIL cells.
  • the present invention also provides the use of the immune cells in the preparation of medicines for treating cancer or immune diseases.
  • Cancer in this application refers to any medical condition that is mediated by the growth, proliferation or metastasis of tumor or malignant cells and causes solid tumors and non-solid tumors such as leukemia.
  • Tumor in the present invention refers to the solid substance of tumor and/or malignant cells.
  • the cancer is, for example, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer , thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer; leukemia, lymphoma, myeloma, mycoses fungoids (mycoses fungoids), Merkel cell carcinoma and other hematological malignancies, Such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocytic-rich large B-cell lymphoma, EBV-positive and negative PTLD, and EBV-associated diffuse large B-cell lymphoma lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T cell lympho
  • the immune diseases are, for example, systemic lupus erythematosus (SLE), autoimmune diabetes, psoriasis, vitiligo, scleroderma, and rheumatoid arthritis.
  • SLE systemic lupus erythematosus
  • autoimmune diabetes for example, systemic lupus erythematosus (SLE)
  • psoriasis vitiligo
  • scleroderma rheumatoid arthritis
  • Treatment or “therapy” for a condition includes preventing or alleviating a condition, reducing the rate at which a condition occurs or develops, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition , to reduce or terminate symptoms associated with a condition, to produce a complete or partial reversal of a condition, to cure a condition, or a combination of the above.
  • treating or “therapy” can refer to inhibiting or slowing the growth, reproduction, or metastasis of tumor or malignant cells, or some combination thereof.
  • treatment or “therapy” includes eradicating all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying tumor progression, or some combination of the above.
  • the present invention also provides a treatment method for tumors or immune diseases, the treatment method comprising administering a therapeutically effective amount of the immune cells to a subject.
  • the cancer is, for example, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer , thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer; leukemia, lymphoma, myeloma, mycoses fungoids (mycoses fungoids), Merkel cell carcinoma and other hematological malignancies, Such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocytic B-rich lymphoma, EBV-positive and negative PTLD, and EBV-associated diffuse large B-cell lymphoma ( DLBCL), plasmablastic lymphoma, extranodal NK/T cell lymphoma, nas
  • the immune diseases are, for example, systemic lupus erythematosus (SLE), autoimmune diabetes, psoriasis, vitiligo, scleroderma, rheumatoid arthritis.
  • SLE systemic lupus erythematosus
  • autoimmune diabetes for example, systemic lupus erythematosus (SLE)
  • psoriasis vitiligo
  • scleroderma rheumatoid arthritis
  • the "therapeutically effective amount” or “effective dose” in the present invention refers to the dose or concentration of a drug that can effectively treat the disease or state associated with the antigen of the chimeric antigen receptor.
  • the therapeutically effective amount is at the dosage or concentration, the antibody or antigen-binding compound can eliminate all or part of the tumor, inhibit or slow down tumor growth, inhibit Mediating the growth or proliferation of cells in a cancerous state, inhibiting tumor cell metastasis, alleviating any symptoms or markers associated with a tumor or cancerous state, preventing or delaying the development of a tumor or cancerous state, or some combination of the above.
  • monoUb-CAR can effectively promote the endocytosis and degradation of CAR, greatly reduce the expression of CAR on the cell surface, realize a substantial decrease in the background self-activation level of immune cells such as CAR-T cells, and significantly reduce the over-differentiation of immune cells such as T cells. Speed, degree of functional exhaustion, etc.
  • monoUb-CAR significantly enhances the sustained proliferation ability of CD28 CAR-T cells in vitro and in vivo under target cell stimulation conditions, and effectively improves the tumorigenicity of CAR-T cells in tumor-bearing mice. killing ability, thereby improving the survival rate of mice.
  • the present invention compares the anti-tumor effects of CD28 monoUb CAR-T and CD28 WT CAR-T in vivo on a tumor mouse model.
  • CD28 monoUb CAR-T has a stronger proliferative response and longer-lasting proliferative ability; in terms of cell differentiation phenotype, whether in the spleen, blood, or tumor, the engineered CAR-T has accumulated more Stem cell memory T cells (Stem cell memory T cells, T SCM ) and reduced differentiation to terminal effector T cells. Therefore, the modified CAR-T can more effectively infiltrate and kill tumor tissues, and at the same injection dose of T cells, the modified CAR-T can more effectively control the development of tumors.
  • Stem cell memory T cells Stem cell memory T cells
  • Embodiment 1 Vector construction of CAR
  • the antigen-specific single-chain antibody (scFv) sequences of CD19, GD2, and GPC3 CAR used in the present invention are respectively from the clinically used FMC63, 14g2A, and GPC3 sequences; the dual-target CAR is the integration of specific antibodies targeting CD19 and CD22
  • the integration method refer to the report in the article "CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted CAR immunotherapy".
  • the extracellular segment structure of CAR is composed of CD8 ⁇ signal peptide sequence, myc tag sequence, scFv sequence, and CD8 ⁇ hinge sequence in series; the transmembrane sequence is the transmembrane region sequence of CD8 ⁇ ; the intracellular segment structure is composed of human CD28 intracellular segment sequence in series Sequence composition of human CD3 ⁇ intracellular segment.
  • the ubiquitin-modified chimeric antigen receptor connects ubiquitin to CD3 ⁇ through a linker peptide in the intracellular domain.
  • the above scFv amino acid sequences were converted into base sequences after codon optimization, and synthesized by a third-party company (genscript).
  • the base sequences of all CARs in the present invention were finally cloned into the pHR-hEF1 ⁇ -IRES-EGFP vector (derived from addgene) by Gibson connection.
  • Example 2 Human primary T cell culture and lentivirus infection
  • Human primary T cells were obtained from healthy informed volunteers. Primary T cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin sulfate, 1 mM sodium pyruvate, non-essential amino acids, and 55 ⁇ M 2-mercaptoethanol (The above reagents were purchased from Gibco). In order to maintain the proliferation of T cells, 100 U/ml of hIL-2 (Sigma-Aldrich) was added to the culture medium.
  • hIL-2 Sigma-Aldrich
  • Preparation of lentivirus Resuspend Lenti-X 293T cells (TaKaRa#632180) in DMEM medium (Gibco#11995-065) containing 10% fetal bovine serum and without antibiotics, and make 6.5 ⁇ 10 5 cells/well The density was inoculated in 6-well cell culture plate (Corning#CLS3516) and cultured for 24 hours.
  • liposome transfection system (Mirus#2300), mix 500ng lentiviral packaging plasmid pCMVdR8.92 (Addgene#8455) and 50ng pMD2.G (Addgene#12259) with 500ng lentiviral plasmid to be packaged, according to liposome After mixing the operation steps of the transfection instructions, add Lenti-X 293T cells. Discard the liposome-containing medium after 16-18 hours, and add an appropriate amount of fresh medium; collect the cell supernatant after 48 hours, concentrate directly or by ultracentrifugation, and store in a -80°C refrigerator for later use.
  • Lentiviral infection of primary T cells Use magnetic beads (Life Technologies #11132D) coated with anti-human CD3 and anti-human CD28 antibodies to activate T cells, mix T cells with magnetic beads 1:3, add to the preparation after 24 hours of culture Infect with a good lentivirus; after 18 hours, the medium containing the virus solution was discarded and replaced with fresh T cell complete medium. After T cells were stimulated by magnetic beads for 4-5 days, the magnetic beads were removed, and the cell density was adjusted to 0.8-1*10 ⁇ 6/ml with T cell complete medium, and fresh T cell complete medium was supplemented every 2 days.
  • FACS buffer phosphate buffered saline PBS + 2% fetal bovine serum
  • FACS buffer phosphate buffered saline PBS + 2% fetal bovine serum
  • Flow cytometry data were acquired by a BD LSR Fortessa machine (BD bioscience) and analyzed using FlowJo software (Tree Star). Antibodies used in flow cytometry are listed below.
  • Example 4 In vitro killing function detection of CAR-T based on flow cytometry
  • K562 target cells expressing CD19 and mCherry fluorescence After mixing double-positive K562 target cells expressing CD19 and mCherry fluorescence with K562 non-target cells not expressing CD19 and mCherry fluorescence at a ratio of 1:1, they were mixed with CAR-T cells at a certain ratio of effector cells: target cells and co-incubated 24 hours. Cells were cultured in complete T cell medium without IL-2.
  • CD19-28WT-CAR and CD19-28Mono-CAR T cells were counted, they were mixed with irradiated target cells Nalm6 3:1, and the cells were resuspended in IL-2-free T cell complete medium to a cell density of 1 *10 ⁇ 6/ml; Count the viable cells every 2 days to calculate the amount of cell proliferation, and adjust the cell density to 1*10 ⁇ 6/ml with the complete medium of T cells without IL-2; when the cells obtained by the viable cell count When the density is less than or equal to 1*10 ⁇ 6/ml, it indicates that the cells are no longer proliferating and the experiment is terminated.
  • Example 6 Mouse tumor model and CAR-T cell function detection
  • NSG mice were first inoculated with 1 ⁇ 106 B lymphoma cells Nalm6 expressing the firefly luciferase gene in the tail vein; Treat NSG mice with 2 ⁇ 10 6 CAR-T cells by injection; detect the intensity of firefly luciferase carried by mouse tumor cells every week through a small animal in vivo imaging system to reflect the load of tumor cells, To track the development of tumors in the body.
  • the specific implementation of the small animal in vivo imaging system includes: giving the firefly luciferase substrate (D-luciferin sodium salt) to tumor-bearing mice by intraperitoneal injection, and the substrate dosage is 0.15 mg/g mouse body weight; 10 minutes later After the substrate was fully circulated to the whole body of the mouse, the mouse was anesthetized with 2.5%-3.5% isoflurane gas and then imaged. Bioluminescence imaging was performed with an IVIS Spectral Imaging System (Perkin Elmer), and fluorescence quantitative data were acquired with an in vivo imaging software (Perkin Elmer).
  • Example 7 Detection of CAR-T cell proliferation, differentiation and exhaustion in tumor-bearing mice
  • NSG mice 5- to 8-week-old combined immunodeficiency mice.
  • NSG mice were first inoculated with 2 ⁇ 106 B lymphoma cells Nalm6 in the tail vein; after the target cells grew in vivo for 4 days, they were injected through the tail vein.
  • 2 ⁇ 10 6 CAR-T cells were administered to NSG mice; 3 mice inoculated with CD19-28 WT and CD19-28 MonoUb CAR-T cells were sacrificed 7 days, 14 days, and 21 days later, and their spleens and hind limbs were collected.

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Abstract

L'invention concerne un récepteur antigénique chimérique modifié par couplage d'ubiquitine et une cellule immunitaire. L'ubiquitine est couplée à une extrémité C-terminale du récepteur antigénique chimérique, et est appropriée pour diverses technologies actuelles de production de cellules CAR-T, et particulièrement appropriée pour des produits thérapeutiques CAR-T développés contre des cibles de tumeurs solides.
PCT/CN2022/080608 2021-07-29 2022-03-14 Récepteur antigénique chimérique modifié par couplage d'ubiquitine et cellule immunitaire WO2023005219A1 (fr)

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