WO2024051831A1 - Constitutive chimeric cytokine receptor, immune cell expressing same, and use thereof - Google Patents

Constitutive chimeric cytokine receptor, immune cell expressing same, and use thereof Download PDF

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WO2024051831A1
WO2024051831A1 PCT/CN2023/117778 CN2023117778W WO2024051831A1 WO 2024051831 A1 WO2024051831 A1 WO 2024051831A1 CN 2023117778 W CN2023117778 W CN 2023117778W WO 2024051831 A1 WO2024051831 A1 WO 2024051831A1
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seq
cells
m7cr
car
functional variant
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Chinese (zh)
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危华锋
余洲
黄丹
田纪元
徐伟
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信达细胞制药(苏州)有限公司
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • 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
    • 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
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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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
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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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

Definitions

  • the present invention relates generally to the fields of genetic engineering and cellular immunology.
  • the present invention relates to constitutively chimeric cytokine receptors for enhancing immune cell expansion and effector function, which comprise an extracellular domain and a constitutively activated IL-7R mutant, the extracellular domain is composed of effector molecules that reshape the tumor microenvironment, and the constitutively activated IL-7R mutant includes the IL-7R mutant transmembrane domain and the IL-7R intracellular structure Domain, immune cells (such as T cells) express constitutive chimeric cytokine receptors, thereby having constitutive IL-7R self-activating signals independent of exogenous cytokine activation and effector molecule efficacy on the extracellular domain. in tumor immunotherapy.
  • the invention also relates to the combination of said constitutive chimeric cytokine receptor and a chimeric antigen receptor or T cell receptor and their use.
  • adoptive cellular immunotherapy represented by chimeric antigen receptor T cells (CAR-T)
  • CAR-T chimeric antigen receptor T cells
  • CAR-T and T cell receptor (TCR) gene-modified T cells are both genetically modified cell therapy products: by collecting and activating T cells from the peripheral blood of tumor patients, using viral or non-viral vectors to mediated Gene modification allows it to carry a CAR or TCR that can specifically recognize tumor cell antigens, thus endowing T cells with tumor-specific recognition and killing functions.
  • CAR-T cells have demonstrated unprecedented clinical therapeutic effects in the treatment of relapsed and refractory hematological tumors.
  • CAR-NK CAR-modified NK cells
  • TIL tumor-infiltrating T cells
  • DNT double-negative T cells
  • iNK, iT immune cells
  • TEE tumor microenvironment
  • tumor cells produce a large number of immunosuppressive cytokines; there are a large number of suppressive immune cells such as regulatory T cells (Treg), myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) in tumors; within T cells
  • suppressive immune cells such as regulatory T cells (Treg), myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) in tumors; within T cells
  • T cells regulatory T cells (Treg), myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) in tumors; within T cells
  • T cells Source negative feedback regulatory mechanisms, such as the upregulation of expression of negative immune regulatory receptors such as PD-1, lead to functional "exhaustion".
  • the antigen expression of solid tumor cells is highly heterogeneous, and antigen deletion mutants are prone to occur under immune pressure, leading to immune escape. Therefore, selecting appropriate therapeutic targets and endowing genetically modified immune cells with the ability to persist, expand
  • optimal activation of initial T cells requires three signals.
  • the first signal provided by TCR the first signal provided by CD28,
  • the second signal provided by co-stimulatory molecules such as 41BB the third signal provided by the binding of cytokines and their receptors, where the third signal is for initial T cells to obtain optimal proliferation, differentiate into effector cells, and develop into long-acting Required for memory T cells.
  • CAR-T cells genetically modified immune cells
  • CAR-T cells can obtain the first and second signals through CAR molecules, they lack the third signal, thus affecting the expansion, survival, and function of CAR-T in the body.
  • systemic administration of exogenous cytokines can promote the in vivo expansion and function of CAR-T and TCR-T cells in animals.
  • systemic administration of cytokines has produced serious toxic side effects.
  • Gene-modified cells can be automatically modified through transgenes. Secreted cytokines also produce similar toxicity. Therefore, many studies are currently actively exploring other strategies, including expressing cytokines on the membrane surface, expressing cytokine switching receptors (CSR), or constitutively activated cytokine receptors or their fragments.
  • CSR cytokine switching receptors
  • Thomas Shum et al. designed CAR-T cells expressing a constitutively activated IL-7R (i.e., IL-7R ⁇ ) mutant (C7R) found naturally in T lymphomas that lacks the native IL-7R. Extracellular domain, but due to cysteine or proline mutations in the transmembrane region of IL-7R, a constitutive dimer is formed, which activates JAK1 kinase without relying on the extracellular domain and ligand binding. It then activates downstream STAT5 and other transcriptional effectors, regulates the expression of downstream target genes, and ultimately promotes and maintains T proliferation and survival.
  • C7R-modified CAR-T cells can repeatedly kill tumor cells while reducing functional exhaustion, and have better proliferation, survival and anti-tumor functions in the body.
  • C7R gene-modified CAR-T cells in the existing technology endow CAR-T cells with improved in vitro and in vivo expansion and survival capabilities, they lack the ability to actively transform the "unfriendly" tumor-suppressive immune microenvironment. In solid tumors characterized by severe immunosuppressive TME, these CAR-Ts are still restricted by the inhibitory TME, preventing them from functioning.
  • the inventors have developed a set of recombinant polypeptides, which are constitutively chimeric cytokine receptors, including extracellular domains and constitutively activated IL-7R mutants.
  • the extracellular domains are composed of remodeling Effector molecular composition of the tumor microenvironment.
  • the constitutive chimeric cytokine receptor of the present invention enables immune cells to utilize constitutively activated IL-7R mutants to continuously activate STAT5 signals, promote and maintain the proliferation and survival of immune cells, and also enables immune cells to acquire new cellular functions.
  • the effect of external effector molecules gives immune cells the ability to actively shape the "unfriendly" TME. By reconstructing the TME, "cold" tumors become “hot” tumors.
  • the immune cells are in a more "friendly" TME, which will be more beneficial. exert anti-tumor effects.
  • the constitutive chimeric cytokine receptor of the present invention with genetically modified immune cells (for example, CAR-T cells), it is expected that the constitutive chimeric cells will be able to activate or enhance the endogenous anti-tumor effect mechanism in the body.
  • Factor receptors and genetically modified immune cells for example, CAR-T cells
  • CAR-T cells including CAR-T cells, TCR-T, CAR-NK, genetically modified TIL, ⁇ T cells, and iNKT, produce synergistic anti-tumor effects. cells, DNT, iPSC-derived iT and iNK cells, etc.
  • the present invention provides a constitutively chimeric cytokine receptor comprising an extracellular domain and a constitutively activated IL-7R mutant.
  • the constitutively activated IL-7R mutant can continuously activate STAT5 signaling and maintain immune effector cells (e.g., T cells) independent of exogenous cytokines.
  • the extracellular domain has the ability to reshape the tumor microenvironment and stimulate Effector function of the body's endogenous anti-tumor immune response.
  • the present invention first compared 27 different constitutively activated IL-7R mutants (also referred to herein as IL7Rm or M7R) in vitro using the exogenous cytokine-dependent BaF3 cell line. It consists of the IL7R transmembrane region (IL7R-mutant(TM)) carrying different mutations (the bold part of the SEQ ID NO:20-SEQ ID NO:46 sequence in the sequence listing) and the intracellular segment of wild-type IL7R (IL7R-WT). (IL7R-wt(ICD), SEQ ID NO:19).
  • the extracellular domain tCD19 (SEQ ID NO: 17) and the 27 different M7Rs are combined to construct a constitutive chimeric cytokine receptor, which is identified by detecting the positive expression of tCD19 on the cell surface. M7R expression, the resulting constitutive chimeric cytokine receptor is also called tCD19-M7CR.
  • the experimental results of the 27 kinds of M7R in maintaining the non-exogenous cytokine-dependent survival effect of BaF3 cells in vitro showed that transducing and stably expressing 19 M7R sequences can maintain the non-exogenous cytokine-dependent survival of BaF3 cells, and the M7R gene can promote BaF3 cells proliferate in an exogenous cytokine-independent manner. And as the culture time increases, the surviving BaF3 cells are all M7R-positive cells, indicating that only BaF3 cells expressing M7R can survive without the addition of exogenous cytokines.
  • Intracellular flow cytometry staining was used to analyze IL-7R downstream signaling molecules and found that these 19 M7R molecules (IL7Rm1.1, IL7Rm1.3, IL7Rm3.1, IL7Rm4-IL7Rm19) activate and maintain STAT5 phosphorylation in BaF3 cells, and the levels It is equivalent to the addition of exogenous cytokines, indicating that these 19 M7Rs promote and maintain the non-exogenous cytokine-dependent survival of BaF3 cells by constitutively activating the STAT5 signaling pathway.
  • IL7Rm1.1, IL7Rm1.3, IL7Rm3.1, IL7Rm4-IL7Rm19 activate and maintain STAT5 phosphorylation in BaF3 cells, and the levels It is equivalent to the addition of exogenous cytokines, indicating that these 19 M7Rs promote and maintain the non-exogenous cytokine-dependent survival of BaF3 cells by constitutively activating the STAT5 signaling
  • the above 19 tCD19-M7CR containing different M7R molecules were transduced and stably expressed in primary T cells, and it was found that expression of the M7R molecules activated STAT5 signaling in T cells, compared with untransduced or T cells transduced with IL7R-WT and expressing M7R molecules have better survival ability in vitro.
  • M7R represented by IL7Rm4, IL7Rm5, IL7Rm7, and IL7Rm8 has a very significant pro-survival effect.
  • the present invention designs and constructs cells using cytokines, immune effector molecules, inhibitory molecule antagonists, or effector molecules targeting NK cell activating receptors as the constitutive chimeric cytokine receptors.
  • the extracellular domain is fused to M7R to form the constitutive chimeric cytokine receptor of the present invention (also referred to as M7CR herein).
  • the M7CR extracellular domain can be IL-12 (IL-12p40 or IL-12p70), IL15 (IL-15 or IL-15FP, the IL-15FP refers to IL-15 and IL -15R ⁇ (fusion protein selected from IL-15R ⁇ or IL-15R ⁇ (Sushi)), including IL-15/IL-15R ⁇ and IL-15R ⁇ /IL-15 fusion proteins), IL-21, IL- 18.
  • IL-12p40 or IL-12p70 IL-15 or IL-15FP
  • the IL-15FP refers to IL-15 and IL -15R ⁇ (fusion protein selected from IL-15R ⁇ or IL-15R ⁇ (Sushi)), including IL-15/IL-15R ⁇ and IL-15R ⁇ /IL-15 fusion proteins), IL-21, IL- 18.
  • the M7CR extracellular domain can also be a 4-1BB targeting molecule moiety (e.g., 4-1BB ligand (4-1BBL), anti-4-1BB antibody ( ⁇ 4-1BB)), CD40 target To molecule moieties (e.g., CD40 ligand (CD40L), anti-CD40 antibody ( ⁇ CD40)), CD83-targeting molecule moieties (e.g., anti-CD83 antibody ( ⁇ CD83)), FLT3-targeting molecule moieties (e.g., FLT3 ligand (FTL3L) ), anti-FLT3 antibody ( ⁇ FLT3)), GITR, ICOS, CD2, ICAM-1 and other immune effector molecules.
  • 4-1BB targeting molecule moiety e.g., 4-1BB ligand (4-1BBL), anti-4-1BB antibody ( ⁇ 4-1BB)
  • CD40 target To molecule moieties e.g., CD40 ligand (CD40L), anti-CD40 antibody ( ⁇ CD40)
  • the domain may also be an anti-PD-L1 antibody, an anti-CD47 molecule, an anti-IL-4 molecule, a TGF ⁇ binding molecule (e.g., anti-TGF ⁇ 1 molecule, TGF ⁇ RII), an anti-PD-1 molecule, an anti-CTLA-4 molecule, an anti-LAG-3 Molecules, anti-TIGIT molecules, anti-CD73 molecules and other antibody parts directed against inhibitory immune receptors or factors achieve the purpose of enhancing the anti-tumor immune response by antagonizing the immunosuppressive effects of inhibitory immune receptors or factors, and then interact with immune cells
  • anti-NKG2C, anti-NKG2D, anti-NKp30, anti-NKp44, anti-NKp46, etc. achieve the purpose of enhancing anti-tumor immune effects by activating endogenous NK cells, and then produce synergistic anti-tumor effects with immune cells (such as T cells).
  • immune cells such as T cells
  • the invention provides M7CR modified CAR or TCR.
  • the invention s new M7CR “arms” Tumor-targeted T cells (for example, M7CR-expressing CAR-T cells) acquire the three signals required for optimal activation of initial T cells, resulting in better T cell activation, proliferation, survival and immune effector functions, while , the effector molecules in the extracellular domain of the M7CR molecule actively remodel the "unfriendly" TME through mechanisms such as activating the body's endogenous T cells, activating APC, antagonizing immunosuppressive receptors, or activating the body's innate immune cells such as NK, promoting endogenous
  • the original anti-tumor effect mechanism ultimately produces a synergistic anti-tumor immune effect.
  • the present invention prepares a method for simultaneously expressing the CAR and the M7CR of the present invention (for example, the extracellular domain (ECD) of the M7CR is tCD19, IL-12 (p40 or p70), IL15FP (including IL-15 / IL-15R ⁇ and IL-15R ⁇ /IL-15 two forms of fusion protein, in which IL-15R ⁇ is selected from IL-15R ⁇ or IL-15R ⁇ (Sushi)), IL-21, 4-1BBL, CD40L, anti-PD- L1 nanobody (PD-L1 VHH ), M7R uses the viral vector of IL7Rm8), and M7CR-modified traditional CAR-T cells that directly target tumor antigens (such as H9.1.2 CAR targeting claudin18.2) are prepared in vitro. Or M7CR-modified "modular" PG CAR-T cells (such as 8B CAR) that target tumors mediated by P329G mutated antibodies, and their functions in
  • a bicistronic virus vector that simultaneously expresses CAR and M7CR is constructed through P2A self-cleaving peptide. T cells transduced by these viruses express CAR and M7CR of the present invention at the same time, and the expression of CAR and M7CR of the present invention has a correlation. sex.
  • M7CR-modified CAR-T cells have different effects on T cell subpopulations based on the extracellular domain (ECD) of M7CR. Group influence has varying effects. For example, IL-12-M7CR-modified CAR-T cells maintained a higher proportion of CD4 cell subsets.
  • CAR-T cells modified by M7R alone such as tCD19-M7CR, the extracellular domain is tCD19, used to examine the role of M7R
  • CPT tCD19-M7CR
  • M7R sequence comes from C7R has equivalent effects
  • M7CR-modified CAR-T cells have different effects on T cell differentiation depending on the extracellular domain (ECD) of M7CR.
  • ECD extracellular domain
  • IL-15-M7CR modified CAR-T cells have a better memory phenotype, while IL-12-M7CR modification promotes CAR-T cell differentiation.
  • the present invention conducted a more detailed study on the phenotype of H9.2.1 CAR-T cells modified by IL-15-M7CR (the M7CR extracellular ECD domain is IL15FP) targeting claudin18.2, and the results It shows that the effect of IL-15 (IL-15-M7CRin, inactivating M7R signaling) or M7R (tCD19-M7CR) alone significantly promotes the maintenance of Tscm memory cell subsets, but IL-15-M7CR has a stronger pro-Tscm effect. Memory cell maintenance effect, suggesting a synergistic effect of IL-15 and M7R signals expressed on the membrane surface.
  • the present invention studies M7CR-modified traditional CAR-T cells (e.g., H9.1.2 CAR-T cells, H9.2.1 CAR-T cells, H9.2.1 targeting claudin18.2) through in vitro killing experiments.
  • -28-L CAR-T cells or PG CAR-T cells (such as HuR968B CAR-T cells targeting claudin18.2) on the killing effect of antigen-expressing tumor cells
  • M7R alone such as tCD19-M7CR , the extracellular region is tCD19, used to examine the role of M7R.
  • the killing ability of modified CAR-T cells is significantly higher than that of unmodified CAR-T cells.
  • M7CR extracellular effector molecules are fused to form M7CR.
  • M7CR modifies CAR-T cells, it can further increase the in vitro killing function of CAR-T cells.
  • the killing capacity of traditional CAR-T cells modified with 4-1BBL-M7CR, anti-PD-L1 VHH -M7CR, IL-12-M7CR, and IL-15-M7CR is significantly higher than that of unmodified or M7R-modified CAR-T cells alone. cell.
  • the killing efficacy of IL-12-M7CR and IL-15-M7CR modified PG CAR-T cells is enhanced, especially when targeting tumor cells with low antigen expression (such as SNU-601 low ). This shows that extracellular effector molecules and M7R have a combined effect in promoting the killing effect of CAR-T cells.
  • the present invention studied the in vitro proliferation ability of M7CR-modified PG CAR-T cells under repeated stimulation of tumor cells through repeated in vitro stimulation experiments.
  • M7R alone such as tCD19-M7CR, extracellular region (tCD19, used to examine the role of M7R) modified PG CAR-T cells have better sustained proliferation ability under repeated stimulation of tumor cells.
  • the M7CR formed by fusing extracellular effector molecules on the basis of M7R can further increase the sustained proliferation ability of PG CAR-T cells in vitro.
  • M7CR-modified PG CAR-T cells such as IL-12-M7CR and IL-15-M7CR have stronger Sustained proliferation ability in vitro.
  • IL-12-M7CR-modified CAR-T cells had more CD4 + T cells under repeated tumor cell stimulation. Cytokine detection results showed that the release levels of IFN- ⁇ and TNF from IL-12-M7CR-modified CAR-T cells were significantly increased.
  • the present invention uses tumor cells expressing different levels of Claudin18.2 antigen as target cells to target traditional CAR-T cells modified by M7CR such as M7R (tCD19-M7CR), IL-12-M7CR, IL15-M7CR, etc.
  • M7CR such as M7R (tCD19-M7CR), IL-12-M7CR, IL15-M7CR, etc.
  • H9.2.1 CAR-T cells and H9.2.1-28-L CAR-T cells targeting claudin18.2 were studied on the killing function in vitro.
  • M7R alone such as tCD19-M7CR, the extracellular region is tCD19, used to examine the role of M7R
  • M7CR modification such as IL-12-M7CR, IL15-M7CR modification
  • M7CR-modified CAR-T cells such as M7R, IL-12-M7CR, IL15-M7CR, etc. maintained better in vitro sustained killing function.
  • Similar effects were observed using 2 different efficacy-to-target ratios.
  • the present invention studies the anti-tumor effect of M7CR-modified PG CAR-T cells in mice.
  • the results show that M7CR-modified CAR-T cells have stronger anti-tumor effects in vivo, and M7CR-modified CAR-T cells have stronger anti-tumor effects in vivo.
  • the proliferation ability of CAR-T cells is also stronger than that of unmodified CAR-T cells.
  • the present invention uses M7CR-modified traditional CAR-T cells (e.g., H9.2.1 CAR-T cells, H9.2.1-28-L CAR-T cells targeting claudin18.2) in mice.
  • M7CR-modified traditional CAR-T cells e.g., H9.2.1 CAR-T cells, H9.2.1-28-L CAR-T cells targeting claudin18.2
  • the anti-tumor effect in vivo was studied, and the results showed that M7CR-modified CAR-T cells had stronger anti-tumor effects in vivo, and the proliferation ability of M7CR-modified CAR-T cells was also stronger than that of unmodified CAR-T cells.
  • the invention provides nucleic acid molecules encoding the M7CR of the invention or encoding the M7CR-modified CAR or TCR of the invention, including nucleic acid molecules encoding the M7CR of the invention or encoding the M7CR-modified CAR or TCR of the invention.
  • Vector, and cells comprising the constitutive chimeric cytokine receptor M7CR or M7CR modified CAR polypeptide of the present invention, the nucleic acid molecule of the present invention, or the vector of the present invention.
  • the cells are autologous T cells or allogeneic Allogeneic T cells.
  • the invention provides a method of producing cells, such as immune effector cells, the method comprising converting a nucleic acid molecule (eg, RNA) encoding the M7CR of the invention or encoding the M7CR-modified CAR or TCR of the invention into molecule, such as an mRNA molecule), or a vector comprising a nucleic acid molecule encoding an M7CR of the invention or encoding an M7CR-modified CAR or TCR described herein introduces (eg, transduces) immune effector cells.
  • a nucleic acid molecule eg, RNA
  • the immune effector cells are T cells, NK cells, for example, the T cells are autologous T cells or allogeneic T cells, for example, the immune effector cells are T cells isolated from human PBMC, Prepared after NK cells.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an immune effector cell (e.g., T cells, NK cells), nucleic acid molecules encoding the constitutive chimeric cytokine receptors or constitutively chimeric cytokine receptor-modified CAR polypeptides of the invention, the vectors of the invention, and any combination thereof; and optionally Medicinal excipients.
  • an immune effector cell e.g., T cells, NK cells
  • nucleic acid molecules encoding the constitutive chimeric cytokine receptors or constitutively chimeric cytokine receptor-modified CAR polypeptides of the invention, the vectors of the invention, and any combination thereof; and optionally Medicinal excipients.
  • the pharmaceutical composition of the invention when the constitutive chimeric cytokine receptor-modified CAR polypeptide expressing the invention is a molecular switch-regulated CAR polypeptide, the pharmaceutical composition of the invention further includes a molecular switch, for example, a molecular switch antibody.
  • a molecular switch for example, a molecular switch antibody.
  • the present invention relates to the use of the pharmaceutical composition of the fifth aspect for treating tumors in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of the fifth aspect.
  • the present invention relates to the use of the pharmaceutical composition described in the fifth aspect in the preparation of drugs for treating cancer.
  • the present invention provides a method for treating tumors, the method comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition according to the fifth aspect.
  • Figure 1 shows the mechanism of action of T cells expressing the M7CR and/or CAR of the present invention after the M7CR and/or CAR of the present invention transduces T cells.
  • M7CR the M7CR
  • Molecules eg, cytokines, immune effector molecules, or inhibitory molecule antagonists
  • ECD extracellular domain
  • Figure 2A shows the structure of the wild-type IL7R receptor and the structure of the engineered mutant IL7R receptor.
  • Figure 2B shows that among the constructed viral expression plasmids, IL7R-tCD19 construct, IL7R-WT construct,
  • IL7Rm refers to the part composed of different mutated IL7R transmembrane regions (TM regions) and IL7R wild-type intracellular region (ICD).
  • FIG. 3A shows the results of using flow cytometry to detect the expression of tCD19 on the surface of BaF3 cells on day 4 after infection of BaF3 cells with 27 tCD19-M7CR genes containing different mutated M7R sequences.
  • BaF3 represents BaF3 cells without lentivirus infection
  • IL7R-WT represents BaF3 cells infected with lentivirus containing wild-type IL7R gene
  • IL7R-tCD19 represents BaF3 cells containing tCD19, wild-type IL7R transmembrane region and intracellular BaF3 cells were infected with lentivirus containing genes from the region
  • IL7Rm-tCD19 means BaF3 cells were infected with lentivirus containing genes from tCD19, different mutated IL7R transmembrane regions and IL7R wild-type intracellular region.
  • Figure 3B shows that after the lentivirus containing the tCD19-M7CR gene of different mutated M7R sequences infected BaF3 cells, culturing the BaF3 cells infected with each lentivirus without adding exogenous mIL-3 can provide sustained activation of IL7R signals and promote BaF3 Plot of results of cell growth with different mutated M7R sequences.
  • the meanings of each icon in the figure are the same as in Figure 3A.
  • Figure 3C shows BaF3 cells were infected with lentiviruses containing tCD19-M7CR genes with different mutated M7R sequences, exogenous mIL-3 was not added starting from day 3, and BaF3 expressing CD19 + was detected on days 3 and 11. The result of cell percentage, the higher the percentage of BaF3 cells expressing CD19 + , the more BaF3 cells survive.
  • Figure 3D shows that after infecting BaF3 cells with lentiviruses containing the tCD19-M7CR gene of different mutated M7R sequences, the BaF3 cells infected with each lentivirus were cultured without adding exogenous mIL-3, and the surviving BaF3 cells were counted. result.
  • Parental BaF3 with IL3 means that BaF3 cells without virus infection are cultured with medium containing IL3
  • parental BaF3 w/o IL3 means that BaF3 cells without virus infection are cultured with medium without IL3 nourish.
  • Figure 4 shows the results of infecting BaF3 cells with lentiviruses containing tCD19-M7CR genes containing different mutated M7R sequences, and then staining BaF3 cells with anti-pSTAT5 antibodies to detect the basal phosphorylation level of STAT5 in the cells.
  • ISO means staining with an anti-STAT5 isotype control antibody
  • +IL3 means adding IL3 to BaF3 cells that have not been infected with lentivirus to stimulate the activation of STAT5 in the cells, as a positive control;
  • without IL3 means not using IL3 to stimulate the cells without lentivirus. Lentivirus invasion stained BaF3 cells.
  • Figure 5 shows the results of using flow cytometry to detect the expression of tCD19 on the surface of T cells 48 hours after infecting T cells with lentivirus containing the tCD19-M7CR gene of different mutated M7R sequences.
  • UNT represents T cells without lentivirus infection
  • IL7R-WT represents T cells infected with lentivirus containing wild-type IL7R gene
  • IL7R-tCD19 represents T cells containing tCD19, wild-type IL7R transmembrane region and intracellular IL7Rm-tCD19 indicates that T cells are infected with lentivirus containing genes from tCD19, different mutated IL7R transmembrane regions and IL7R wild-type intracellular region.
  • FIG. 6 shows that after infecting T cells with lentivirus containing the tCD19-M7CR gene of different mutated M7R sequences, without adding exogenous IL-2 stimulation, the T cells were stained with anti-pSTAT5 antibodies to detect the basal phosphate of STAT5 in the cells. level results.
  • UNT represents T cells without lentivirus infection
  • IL7Rm-tCD19 represents T cells infected with lentivirus containing genes of tCD19, different mutations of IL7R transmembrane region and IL7R wild-type intracellular region.
  • Figure 7A shows that after infecting T cells with lentiviruses containing tCD19-M7CR genes containing different mutated M7R sequences, without adding exogenous IL-2 stimulation, the number of T cells expressing tCD19-M7CR was counted over time. the result of.
  • Figure 7B shows the number of T cells expressing tCD19-M7CR over time after infecting T cells with lentivirus containing the tCD19-M7CR gene of different mutated M7R sequences without adding exogenous IL-2 stimulation. Change multiple.
  • FIG. 8 shows the structure of M7CR-modified CAR, where M7CR contains extracellular domains ECD and IL7Rm.
  • the N-terminus of the M7CR is connected to the C-terminus of different CAR polypeptides through P2A, thereby forming an M7CR-modified CAR.
  • Figure 9A shows the expression levels of CAR and M7CR on day 9 after infecting T cells with lentivirus containing H9.1.2 CAR genes containing different M7CR modifications.
  • Figure 9B shows the proportion of CD4 and CD8 positive cells.
  • “UNT” represents T cells without lentivirus infection
  • “H9.1.2” represents H9.1.2 CAR-T cells
  • the others are tCD19-M7CR, tCD19-M7CR(CPT), IL-15/IL- 15R ⁇ -M7CR (marked as IL-15-M7CR in the figure), IL-12-P70-M7CR (marked as IL-12-M7CR in the figure), IL-21-M7CR, CD40L-M7CR, 4-1BBL-M7CR, Anti-PD-L1VHH-M7CR modified H9.1.2 CAR-T cells.
  • Figure 9C shows the expression levels of CAR and M7CR on day 9 after infecting T cells with lentivirus containing HuR968B CAR genes containing different M7CR modifications.
  • Figure 9D shows the proportion of CD4 and CD8 positive cells.
  • “UNT” represents T cells without lentivirus infection
  • “8B” represents HuR968B CAR-T cells, and the others are tCD19-M7CR, tCD19-M7CR(CPT), IL-15/IL-15R ⁇ -M7CR( IL-15-M7CR in the figure), IL-12-P70-M7CR (IL-12-M7CR in the figure), IL-21-M7CR, CD40L-M7CR, 4-1BBL-M7CR, anti-PD-L1 VHH -M7CR modified HuR968B CAR-T cells.
  • Figure 9E shows the results of flow cytometric detection of the phenotypes of total T cells, CD4 + and CD8 + T in unmodified H9.1.2 CAR-T samples and M7CR-modified H9.1.2 CAR-T samples.
  • “UNT” indicates T cells without lentivirus infection
  • “H9.1.2” indicates H9.1.2 CAR-T cells
  • the rest are M7CR-modified H9.1.2 CAR-T cells (the markings in the figure are the same as in Figure 9A ).
  • Figure 9F shows the results of flow cytometric detection of the phenotypes of total T cells, CD4 + and CD8 + T in unmodified HuR968B CAR-T samples and M7CR-modified HuR968B CAR-T samples.
  • “UNT” indicates T cells without lentivirus infection
  • “8B” indicates HuR968B CAR-T cells
  • the rest are M7CR-modified HuR968B CAR-T cells (the markings in the figure are the same as in Figure 9C).
  • Figure 9G and Figure 9H show the phenotypes of total T cells, CD4 + and CD8 + T in unmodified H9.2.1-218 CAR-T samples and M7CR-modified H9.2.1-218 CAR-T samples by flow cytometry The results of technical testing.
  • Figure 9G shows CAR T cells prepared from PBMC of donor 15, and
  • Figure 9H shows CAR T cells prepared from PBMC of donor 17.
  • H9.2.1-tCD19-M7CR represents tCD19-M7CR modified H9.2.1-218 CAR-T cells;
  • H9.2.1-IL-15-M7CR represents IL-15-M7CR modified H9.2.1-218 CAR-T cells.
  • H9.2.1in-IL15-M7CR indicates the loss of CAR structure and function in H9.2.1-IL-15-M7CR cells
  • H9.2.1-IL15-M7CRin indicates the intracellular structure of M7CR in H9.2.1-IL-15-M7CR cells (M7R) loss of function
  • H9.2.1-sIL15 represents the combination of H9.2.1-218 CAR-T cells and soluble IL15
  • H9.2.1-IL-12-M7CR represents IL-12-p70-M7CR modified H9.2.1 CAR -T cells (the H9.2.1 CAR sequence is shown in SEQ ID NO: 100)
  • H9.2.1-28-IL-15-M7CR represents H9 modified by IL-15-M7CR and whose costimulatory domain is CD28.
  • 2.1-28 CAR-T cells, 8E5 represents the CT041 product from CARsgen Company (the CAR sequence is shown in SEQ ID NO: 188).
  • Figure 9I and Figure 9J show that in CAR-T cells prepared from PBMC of donors 15 and 17, respectively, M7R can continuously provide activation signals and activate downstream signaling pathways.
  • H9.2.1-tCD19-M7CR represents tCD19-M7CR modified H9.2.1 CAR-T cells
  • H9.2.1-IL-12-M7CR represents IL-12-p70-M7CR modified H9.2.1 CAR-T cells.
  • H9.2.1in-IL12-M7CR indicates the loss of CAR structure and function in H9.2.1-IL-12-M7CR cells
  • H9.2.1-IL12-M7CRin indicates the intracellular structure of M7CR in H9.2.1-IL-12-M7CR cells ( M7R) loss of function
  • H9.2.1-sIL12 represents a combination of H9.2.1 CAR-T cells and soluble IL12.
  • Figure 10A shows the detection of the number of molecules of CLDN18.2 on the cell surface of DANG-G18.2, SNU-601 high , and SNU-601 low by Qufikit.
  • the dark part represents ISO
  • the light part represents positive cells.
  • Figure 10B shows the expression level of CLDN18.2 in DANG18.2 cells, NUGC-4 cells, SNU-620 cells, PANC-1 cells, SNU-601 cells, and Hup-T4 cells as target cells.
  • ISO in the figure represents the isotype.
  • Antibody control, K562 is CLDN18.2 negative control cells.
  • FIG 11A, Figure 11B and Figure 11C show that unmodified H9.1.2 CAR-T cells or H9.1.2 CAR-T cells modified with different M7CR were co-incubated with tumor target cells DAN-G18.2, respectively, in E:T The killing effect of each CAR-T cell on target cells when the ratio is 1:1, 1:3, and 1:10 respectively.
  • PC represents positive control (Positive control, target cells are treated with lysis solution to lyse all target cells);
  • NT represents T cells without lentivirus infection;
  • Tuor cell only represents DAN -G18.2 cell line;
  • H9.1.2 indicates H9.1.2 CAR-T cells, and the rest are M7CR-modified H9.1.2 CAR-T cells (the markings in the figure are the same as in Figure 9A).
  • Figure 11D shows that unmodified HuR968B CAR-T cells or HuR968B CAR-T cells modified with different M7CRs and P329G mutation-containing A6 antibodies (2nM) were co-incubated with target cells SUN-601 high or SUN-601 low , respectively.
  • :T is 1:1, the killing effect of each CAR-T cell on target cells.
  • PC positive control (Positive control, target cells are treated with lysis solution to lyse all target cells);
  • NT represents T cells that have not been infected by lentivirus;
  • 8B represents unmodified HuR968B CAR-T cells, and the rest are M7CR-modified HuR968B CAR-T cells (the labels in the figure are the same as in Figure 9C).
  • FIG 12A, Figure 12C and Figure 12E show that unmodified HuR968B CAR-T cells or HuR968B CAR-T cells modified with different M7CR and P329G mutation-containing A6 antibody (2nM) were co-incubated with the target cell SUN-601 high , respectively.
  • E:T is 2:1
  • “8B” represents unmodified HuR968B CAR-T cells, and the rest are M7CR-modified HuR968B CAR-T cells (the meaning of the marks in the figure is the same as in Figure 9C).
  • FIG 12B, Figure 12D and Figure 12F show that unmodified HuR968B CAR-T cells or HuR968B CAR-T cells modified with different M7CR and P329G mutant A6 antibody (2nM) were co-incubated with the target cell SUN-601 high , respectively.
  • E:T is 2:1
  • CAR + in HuR968B CAR-T cells Fold changes in the proportion of cells and the percentage of CAR + cells.
  • “8B” represents unmodified HuR968B CAR-T cells, and the rest are M7CR-modified HuR968B CAR-T cells (the meaning of the marks in the figure is the same as in Figure 9C).
  • Figure 13A shows representative flow cytometry results of CD4 + and CD8 + T cell numbers in each group after the first and third rounds of stimulation in Figures 12A-12F.
  • Figure 13B shows the statistical results of the proportion of CD4 + and CD8 + T cells in each group of Figure 13A.
  • FIG 13C shows that unmodified HuR968B CAR-T cells or HuR968B CAR-T cells modified with different M7CRs and P329G mutation-containing A6 antibodies (2nM) were co-incubated with the target cell SUN-601 high , and the E:T was 2: At 1 hour, the target cells SUN-601 high were repeatedly stimulated for multiple rounds, and the BD TM Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit II was used to detect the cytokines in the culture supernatant.
  • “8B” indicates HuR968B CAR-T cells, and the rest are M7CR-modified HuR968B CAR-T cells (the meaning of the marks in the figure is the same as in Figure 9C).
  • Figures 14A-14D show that in target cells with different expression levels of CLDN18.2, the killing effect of CAR-T cells increases as the expression level of CLDN18.2 increases.
  • H9.2.1-tCD19-M7CR represents tCD19-M7CR modified H9.2.1 CAR-T cells
  • H9.2.1-IL-12-M7CR represents IL-12-M7CR modified H9.2.1 CAR-T cells
  • H9 .2.1in-IL12-M7CR indicates the loss of CAR structure and function in H9.2.1-IL-12-M7CR cells
  • H9.2.1-IL-12-M7CRin indicates the intracellular structure of M7CR in H9.2.1-IL-12-M7CR cells ( M7R) loss of function
  • H9.2.1-sIL12 represents a combination of H9.2.1 CAR-T cells and secretes soluble IL12.
  • Figure 14E shows the co-incubation of unmodified H9.2.1 CAR-T cells or IL-12-M7CR-modified CAR-T cells with the target cell Hup-T4 at E:T of 1:1 and 1:5, respectively. , using the target cell Hup-T4 to repeatedly stimulate three rounds, and the killing effect of each CAR-T cell on the target cell. After three rounds of continuous killing experiments, IL-12-M7CR modified CAR-T cells still had a better killing effect on target cells, while unmodified H9.2.1 CAR-T cells or M7R-modified H9.2.1 alone In multiple rounds of killing, the killing effect of CAR-T cells gradually weakens as the number of rounds increases.
  • H9.2.1 represents H9.2.1 CAR-T cells
  • H9.2.1-tCD19-M7CR represents tCD19-M7CR modified H9.2.1 CAR-T cells
  • H9.2.1-IL-12-M7CR represents IL-12- M7CR modified H9.2.1 CAR-T cells.
  • Figure 15 shows the changes in tumor burden in mice detected by the IVIS imaging system in a gastric cancer abdominal metastasis model constructed by intraperitoneal injection of luciferase-expressing NUGC-4 cells.
  • Figure 16 shows the anti-tumor effect of PG CAR-T cells expressing constitutive chimeric cytokine receptors in mice.
  • IL-12-M7CR-modified PG CAR-T cells labeled 8B-IL12-M7CR CAR-T in the figure
  • tCD19- M7CR-modified PG CAR-T cells labeleled 8B-M7R CAR-T in the figure
  • Figure 17 shows the expansion levels of PG CAR-T cells expressing constitutively chimeric cytokine receptors in mice.
  • IL-12-M7CR modified PG CAR-T cells (labeled 8B-IL12-M7CR CAR-T in the figure) from day 7 to day 28 after administration of PG CAR-T cells and A6 antibody and tCD19-M7CR modified PG CAR-T cells (labeled 8B-M7R CAR-T in the figure) have higher expansion levels.
  • Figure 18 shows the anti-tumor effect of H9.2.1 CAR-T cells expressing constitutive chimeric cytokine receptors in mice.
  • IL-12-M7CR-modified CAR T cells (labeled IL12-M7CR-H9.2.1 CAR-T in the figure) had the strongest anti-tumor effect, followed by tCD19-M7CR-modified CAR-T cells (labeled M7R-H9.2.1-CAR-T in the figure), unmodified H9.2.1 CAR T cells have the weakest anti-tumor effect in vivo.
  • Figure 19 shows the anti-tumor effect of traditional CAR-T cells expressing constitutive chimeric cytokine receptors in mice.
  • IL-12-M7CR-modified CAR-T cells (labeled IL12-M7CR-H9.2.1 CAR-T in the figure) and tCD19-M7CR-modified increased over time after administration of CAR-T cells.
  • CAR-T cells (labeled M7R-H9.2.1 CAR-T in the figure) have better anti-tumor effects in vivo.
  • Figure 20 shows the expansion levels of conventional CAR-T cells expressing constitutive chimeric cytokine receptors in mice.
  • IL-12-M7CR-modified CAR-T cells labeleled IL12-M7CR-H9.2.1 CAR-T in the figure
  • tCD19- M7CR-modified CAR-T cells labeleled M7R-H9.2.1 CAR-T in the figure
  • Figure 21 shows the structure of IL-15-M7CR modified CAR, where M7CR contains IL-15ECD and IL7Rm8.
  • the N-terminus of the M7CR is connected to the C-terminus of the CAR polypeptide through P2A, thereby forming an M7CR-modified CAR.
  • Figure 22 shows a statistical histogram of amplification folds on day 9 of CAR-T preparation.
  • H9.1.2 indicates H9.1.2 CAR-T cells
  • H9.2.1in-IL15-M7CR indicates the loss of CAR structure and function in H9.2.1-IL-15-M7CR cells
  • H9.2.1-IL15-M7CRin indicates H9.2.1 -IL-15-M7CR cells have a loss of intracellular structure and function of M7CR
  • H9.2.1-sIL15 represents the H9.2.1 CAR-T expressing secreted sIL-15
  • 8E5 represents the CT041 product from CARsgen.
  • Figure 23A shows CAR or M7CR expression in prepared CAR-T cells.
  • Figure 23A is a representative flow scatter plot, and
  • Figure 23B is a statistical histogram.
  • Figure 23C and Figure 23D show the ratio of CD4 and CD8, Figure 23C is a representative flow scatter plot, and Figure 23D is a statistical histogram. “NT” indicates T cells without lentivirus infection.
  • Figure 23E and Figure 23F show the differentiation phenotype of each CAR-T cell on the 7th and 9th days of preparation.
  • Figure 23E is a representative flow cytometry scatter plot
  • Figure 23F is a statistical histogram.
  • Figure 23G and Figure 23H show the expression of intracellular phosphorylated STAT5 in each CAR-T cell on the 9th day of preparation.
  • Figure 23G is a representative flow cytometry diagram
  • Figure 23H is a statistical histogram.
  • Figure 24A and Figure 24C show the CD25 and CD69 expression results after culture of each CAR-T cell and PANC1 target cell.
  • Figure 24A is a representative flow cytometry scatter plot
  • Figure 24C is a statistical histogram of CD25 + CD69 + cells.
  • Figure 24B and Figure 24D show the CD25 and CD69 expression results after culture of each CAR-T cell and Hup-T4 target cell.
  • Figure 24B is a representative flow cytometry scatter plot
  • Figure 24D is a statistical histogram of CD25 + CD69 + cells.
  • Figure 25A shows the concentrations of IL-2, IFN- ⁇ and TNF ⁇ in the supernatant of each CAR-T cell and PANC1 target cell after culture for 24 hours.
  • Figure 25B shows the concentrations of IL-2, IFN- ⁇ and TNF ⁇ in the supernatant of each CAR-T cell and Hup-T4 target cell after culture for 24 hours.
  • Figure 26A shows representative flow cytometry plots of CAR + and CAR- cell proliferation after 5 days of co-culture with different CAR-T cells and PANC-1 cells from 2 donors. Baseline is the direct detection result after CAR-T cell labeling.
  • Figure 26B shows representative flow cytometry plots of CAR + and CAR- cell proliferation after co-culture of different CAR-T cells and SUN620 cells from 2 donors for 5 days. Baseline is the direct detection result after CAR-T cell labeling.
  • Figure 27A shows the in vivo anti-tumor effects of 3 different doses of H9.2.1-IL-15-M7CR CAR-T cells or H9.2.1-CD28-IL-15-M7CR CAR-T cells in the intraperitoneal NUGC-4 model, mice Abdominal tumor burden is monitored directly by in vivo imaging.
  • Figure 27B shows a statistical graph of tumor burden. “NT” indicates T cells without lentivirus infection.
  • Figure 27C shows changes in body weight of treated mice.
  • Figure 27D and Figure 27E respectively show the expansion of total T cells and CAR-T cells in mice over time, expressed as the number of cells per 100 ⁇ l of mouse peripheral blood.
  • Figure 28 shows the cytokine-M7CR modified CAR structure.
  • Different cytokines are directly connected as the extracellular domain (ECD) and M7Rm8 (SEQ ID NO: 34) to construct a constitutive chimeric cytokine receptor M7CRm8; then through P2A
  • the N-terminus of M7CRm8 is connected to the C-terminus of the H9.2.1 CAR polypeptide to form a cytokine-M7CR modified H9.2.1 CAR.
  • tCD19-M7CR served as a control molecule.
  • Figure 29A shows the amplification curves of various CAR-T derived from donor 16 over time from day 1 to day 9.
  • H9.1.2 represents H9.1.2 CAR-T cells
  • H9.1.2-218 represents H9.1.2-218 CAR-T cells
  • the rest are H9.1.2 CAR- modified by tCD19-M7CR and different cytokines-M7CR. T cells.
  • Figure 29B shows the amplification curves of various CAR-T derived from donors 6, 11, and 17 over time from day 1 to day 9.
  • Figure 29C shows a statistical histogram of amplification folds on day 9 of preparation of various CAR-Ts derived from donors 6, 11, and 17.
  • Figures 30A and 30B show representative flow scatter plots and statistical plots of CAR and/or ECD expression as M7CR at days 7 and 9, respectively, after donor 16-derived CAR-T.
  • Figure 30C and Figure 30D show representative flow scatter plots and statistical graphs of CAR expression on day 9 of CAR-T cells derived from donors 6, 11, and 17, respectively. “NT” indicates T cells without lentivirus infection.
  • Figure 30E shows representative flow scatter plots of CD4 and CD8 subpopulation ratios on days 7 and 9 of donor 16-derived CAR-T cell preparation.
  • Figure 30F and Figure 30G respectively show representative flow scatter plots and statistical diagrams of the proportions of CD4 and CD8 subpopulations on day 9 of preparation of CAR-T cells derived from donors 6, 11, and 17.
  • Figure 30H and Figure 30I show representative flow cytometry scatter plots and CD45RA + CCR7 + cell proportion statistical diagrams of the differentiation phenotype of donor 16-derived CAR-T cells on days 7 and 9 of preparation, respectively.
  • CD45RA + CCR7 + represents naive T cells or stem memory T cells (TN/TSCM)
  • CD45RA-CCR7 + represents central memory T cells (TCM)
  • CD45RA - CCR7 - represents effector memory T cells (TEM)
  • CD45RA + CCR7 - represents the effector T cell (Teff) subset.
  • Figure 30J and Figure 30K respectively show representative flow scatter plots and statistical diagrams of the differentiation phenotypes of CAR-T cells derived from donors 6, 11, and 17 on day 9 of preparation.
  • Figure 30L and Figure 30M respectively show representative flow cytometry and statistical diagrams (expressed as mean fluorescence intensity (MFI)) of CD45RA expression in CAR-T cells derived from donors 6, 11, and 17 on day 9 of preparation.
  • MFI mean fluorescence intensity
  • Figure 30N and Figure 30O respectively show representative flow cytometry and statistical diagrams (expressed as MFI) of CCR7 expression in CAR-T cells derived from donors 6, 11, and 17 on day 9 of preparation.
  • Figure 31 shows CAR structures modified with chimeric receptor M7CR of different signal intensities. The N-terminus of tCD19-M7CR with different signal strengths is connected to the C-terminus of the H9.2.1 CAR polypeptide through P2A, thereby forming a tCD19-M7CR modified H9.2.1 CAR.
  • tCD34-M7CR with different signal strengths is connected to the C-terminus of BB2121 CAR polypeptide through P2A to form a tCD34-M7CR modified BB2121 CAR.
  • tCD19-M7CR(CPT) and tCD34-M7CR(CPT) are positive controls.
  • Figure 32 shows the expansion kinetics of H9.2.1 and BB2121 derived from donor 15 and M7CR-modified CAR-T cells with different signal intensities from day 1 to day 9.
  • “CAR alone” in the picture refers to H9.2.1 CAR-T cells or BB2121 CAR-T cells, and the others are M7CR-modified CAR-T cells.
  • Figures 33A and 33B respectively show representative flow cytometry diagrams of CAR expression levels of H9.2.1 and BB2121 derived from donor 15 and M7CR-modified CAR-T cells with different signal intensities.
  • Figure 33C shows a statistical diagram of the positive rate of CAR expression.
  • Figure 34A and Figure 34B respectively show representative flow cytometry diagrams of CD4 and CD8 cell subsets in H9.2.1 and BB2121 derived from donor 15 and their M7CR-modified CAR-T cells with different signal intensities.
  • Figure 34C shows a statistical graph of CD4 and CD8 cell subset proportions.
  • Figures 35A and 35B show representative flow scatter plots of the differentiation phenotype expression of H9.2.1 and BB2121 derived from donor 15 and their M7CR-modified CAR-T cells with different signal intensities, respectively.
  • Figure 35C shows a statistical graph of the proportion of CD45RA+CCR7+ cells.
  • Figure 36A and Figure 36B respectively show representative statistical diagrams of the expression of intracellular phosphorylated STAT5 (pSTAT5) in CAR-T cells derived from donor 15 on the 5th day of preparation and in CAR-T cells after cryopreservation and recovery.
  • pSTAT5 intracellular phosphorylated STAT5
  • “CAR alone” in the picture refers to H9.2.1 CAR-T cells or BB2121 CAR-T cells, and the others are M7CR-modified H9.2.1 or BB2121 CAR-T cells.
  • Figure 37A shows a representative flow scatter diagram of CD25 and CD69 expression after culture of H9.2.1 derived from donor 15 and M7CR-modified CAR-T cells with different signal intensities and NUGC-4 target cells.
  • Figure 37B shows CD25+CD69+ cells.
  • Proportion chart. “NT” represents T cells without lentivirus infection
  • “CAR alone” represents H9.2.1 CAR-T cells
  • others represent M7CR-modified H9.2.1 CAR-T cells
  • 8E5 represents control CAR-T cells.
  • Figure 38 shows a statistical histogram showing the killing of NUGC-4 target cells by H9.2.1 derived from donor 15 and its M7CR-modified CAR-T cells with different signal intensities.
  • Figure 39 shows the concentrations of IL-2, IFN- ⁇ and TNF ⁇ in the supernatant of H9.2.1 derived from donor 15 and M7CR-modified CAR-T cells with different signal intensities and NUGC-4 target cells co-cultured for 24 hours.
  • Figure 40A shows the expansion kinetics of H9.2.1 and M7CR-modified CAR-T cells derived from donors 13, 16, and 17 with different signal intensities from day 1 to day 9, and Figure 40B shows the CAR at harvest on day 9 -T cell expansion fold.
  • NT represents T cells without lentivirus infection
  • CAR alone represents H9.2.1 CAR-T cells
  • 8E5 represents control CAR-T cells.
  • Figure 41A shows representative flow cytometry diagrams of CAR and tCD19 expression in H9.2.1 and M7CR-modified CAR-T cells with different signal intensities derived from donors 13, 16, and 17, and Figure 41B shows a statistical diagram of the positive rate of CAR expression.
  • Figure 42A shows representative flow scatter plots of CD4 and CD8 cell subsets in H9.2.1 and M7CR-modified CAR-T cells derived from donors 13, 16, and 17 with different signal intensities.
  • Figure 42B shows a statistical graph of CD4 and CD8 cell subset proportions.
  • Figure 43A shows a representative flow scatter diagram of the differentiation phenotype expression of H9.2.1 derived from donors 13, 16, and 17 and its M7CR-modified CAR-T cells with different signal intensities.
  • Figure 43B shows a statistical graph of CD45RA + CCR7 + cell proportions.
  • Figure 43C and Figure 43D respectively show representative flow cytometry and statistical diagrams (expressed as MFI) of CD45RA expression in H9.2.1 derived from donors 13, 16, and 17 and their M7CR-modified CAR-T cells with different signal intensities.
  • Figure 43E and Figure 43F respectively show representative flow cytometry and statistical diagrams (expressed as MFI) of CCR7 expression in H9.2.1 derived from donors 13, 16, and 17 and their different signal intensity M7CR-modified CAR-T cells.
  • Figure 45A shows the anti-tumor effect of donor 15-derived H9.2.1 and its different signal intensity M7CR modified CAR-T cells in the NUGC4 abdominal tumor model, and mouse abdominal tumor growth was monitored by weekly imaging. “NT” indicates T cells without lentivirus infection.
  • Figure 45B shows a statistical graph of tumor burden in treated mice.
  • Figure 45C shows changes in body weight of treated mice.
  • Figure 45D and Figure 45E respectively show the expansion of total T cells and CAR-T cells in mice over time, expressed as the number of cells per 100 ⁇ l of mouse peripheral blood.
  • Figure 46A shows the anti-tumor effects of donor 17-derived BB2121 and its M7CR(8) modified CAR-T cells in the subcutaneous H929 tumor model. “CAR alone” is the group administered BB2121 CAR-T cells.
  • Figure 46B shows changes in body weight of treated mice.
  • FIG. 47 shows the structure diagram of TGF ⁇ RII-M7CR CAR.
  • the N-terminus of chimeric receptors such as TGF ⁇ RII-M7CR, dnTGF ⁇ RII, TGF ⁇ RII-CD28 and TGF ⁇ RII-41BB is connected to the C-terminus of H9.2.1 or H9.2.1-28 CAR polypeptide through P2A are connected to form a CAR modified by chimeric receptors such as TGF ⁇ R-M7CR.
  • Figure 48 shows CAR-T cells derived from H9.2.1 and its dnTGF ⁇ RII, TGF ⁇ RII-CD28, and TGF ⁇ RII-M7CR modifications derived from donors 5, 10, and 18, and H9.2.1-28 and its dnTGF ⁇ RII, TGF ⁇ RII-BB, and TGF ⁇ RII-M7CR modifications. Kinetics of CAR-T cell expansion over time.
  • Figure 49A shows a representative flow scatter plot of CAR and TGF ⁇ RII expression as ECD in each CAR-T cell in Figure 48.
  • Figure 49B shows a statistical histogram of the CAR positivity rate of each CAR-T cell in Figure 48.
  • Figures 50A and 50B respectively show the representative flow scatter plots and CD4 and CD8 subpopulation ratio statistical diagrams of CD4 and CD8 subpopulations in each CAR-T cell in Figure 48.
  • Constant IL-7R mutant refers to a mutant IL-7R produced by mutations in the transmembrane region of the wild-type IL-7 receptor alpha chain (IL7R ⁇ ), which can activate ligands independent of wild-type IL7R ⁇ . Upon binding, dimerization occurs and the downstream STAT5 signaling pathway is activated.
  • autologous refers to any substance derived from the same individual to whom the substance is later reintroduced.
  • allogeneic refers to any substance derived from a different animal of the same species as the individual into which the substance is introduced. Two or more individuals are said to be allogeneic to each other when the genes at one or more loci are not identical. In some aspects, allogeneic agents from individuals of the same species can be genetically dissimilar enough for antigenic interaction to occur.
  • xenogeneic refers to a graft derived from an animal of a different species.
  • apheresis refers to an art-recognized extracorporeal method by which blood from a donor or patient is removed from the donor or patient and passed through a device that separates selected specific components and return the remainder to the donor or patient's circulation, for example, by retransfusion. Therefore, in the context of "single sample”, it refers to a sample obtained using apheresis.
  • immune effector cells refers to cells involved in an immune response, such as in promoting an immune effector response.
  • immune effector cells include T cells, eg, alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.
  • immune effector function refers to, for example, a function or response of an immune effector cell that enhances or promotes an immune attack on a target cell.
  • immune effector functions or responses refer to properties of T cells or NK cells that promote killing of target cells or inhibit the growth or proliferation of target cells.
  • primary stimulation and costimulation are examples of immune effector functions or responses.
  • effector function refers to a specialized function of a cell.
  • the effector function of T cells may be, for example, cytolytic activity or auxiliary activity, including secretion of cytokines.
  • T cell activation refers to one or more cellular responses of T lymphocytes, in particular cytotoxic T lymphocytes, selected from: proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity and activation Expression of markers.
  • the chimeric antigen receptor of the present invention can induce T cell activation. Suitable assays for measuring T cell activation are described in the Examples and are known in the art.
  • lentivirus refers to a genus of the family Retroviridae. Lentiviruses are unique among retroviruses in their ability to infect non-dividing cells; they can deliver significant amounts of genetic information to host cells, making them one of the most efficient methods of gene delivery vectors. HIV, SIV and FIV are examples of lentiviruses.
  • lentiviral vector refers to a vector derived from at least a portion of a lentiviral genome, including in particular self-inactivating lentiviral vectors as provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009).
  • Other examples of lentiviral vectors that may be used clinically include, but are not limited to, lentiviral vectors from Oxford BioMedica Gene delivery technology, LENTIMAX TM vector system from Lentigen, etc.
  • Non-clinical types of lentiviral vectors are also available and known to those skilled in the art.
  • tumor and cancer are used interchangeably herein to encompass both solid and liquid tumors.
  • cancer and “cancerous” refer to a physiological disorder in mammals in which cell growth is unregulated.
  • neoplastic refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • Claudins is a type of integrin membrane protein that exists in epithelial and endothelial tight junctions and is an important component of tight junctions. It was discovered by Shoichiro Tsukita et al. in 1998. The family has 24 members. The human Claudin 18 gene has two alternative exons 1, resulting in two proteins, Claudin 18.1 (also referred to as “CLDN18.1” in this article) and Claudin 18.2 (also referred to as "CLDN18.2” in this article) Isoforms, both of which have about 50 amino acids in the first extracellular domain There are only 7 amino acid residue differences in sequence.
  • Claudin 18.2 There is a significant difference in the expression of Claudin 18.2 in cancer tissues and normal tissues. This may be due to the fact that the CREB binding site in the promoter region of Claudin 18.2 is highly methylated in CpG in normal tissues, while the level of CpG methylation in the process of cell canceration decrease, and then CREB participates in activating the transcription of Claudin18.2.
  • Tumor immune escape refers to the process by which tumors escape immune recognition and clearance.
  • tumor immunity is “cured” when such evasion is attenuated, and tumors are recognized and attacked by the immune system.
  • Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.
  • binding means that the binding is selective for the antigen and can be distinguished from undesired or non-specific interactions.
  • the ability of an antibody to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA), SPR or biofilm layer interference techniques, or other conventional binding assays known in the art.
  • stimulation refers to a primary response induced by the binding of a stimulatory molecule (e.g., the TCR/CD3 complex) to its corresponding ligand, which primary response thereby mediates a signaling event, such as, but not limited to, via the TCR/CD3 complex body signal transduction.
  • a stimulatory molecule e.g., the TCR/CD3 complex
  • Stimulation can mediate the expression of certain molecular changes, such as down-regulation of TGF- ⁇ and/or reorganization of cytoskeletal structure.
  • the term "stimulatory molecule” refers to a molecule expressed by a T cell that provides a primary cytoplasmic signaling sequence that modulates the TCR complex in a stimulatory manner in at least some aspect of the T cell signaling pathway.
  • Primary activation In one embodiment, the primary signal is initiated, for example, by binding of a TCR/CD3 complex to a peptide-loaded MHC molecule and results in the mediation of a T cell response, including but not limited to proliferation, activation, differentiation, and the like.
  • CD3 ⁇ is defined as the protein provided by GenBan accession number BAG36664.1 or its equivalent
  • CD3 ⁇ stimulatory signaling domain is defined as the amino acid residues from the cytoplasmic domain of the CD3 ⁇ chain that are sufficient to functionally propagates the initial signal necessary for T cell activation.
  • the cytoplasmic domain of CD3 ⁇ comprises residues 52 to 164 of GenBank accession number BAG36664.1 or as a functional ortholog thereof from a non-human species (e.g., mouse, rodent, equivalent residues of monkeys, apes, etc.).
  • the "CD3 ⁇ stimulating signal domain” is the sequence provided in SEQ ID NO: 12 or a variant thereof.
  • costimulatory molecule refers to a corresponding binding partner on a cell that specifically binds to a costimulatory ligand thereby mediating a costimulatory response (such as, but not limited to, proliferation) of the cell.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an effective immune response.
  • Costimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activated NK cell receptors, OX40 , CD40, GITR, 4-1BB (ie CD137), CD27 and CD28.
  • the "costimulatory molecule” is CD28, 4-1BB (ie, CD137).
  • the costimulatory signaling domain refers to the intracellular part of the costimulatory molecule.
  • 4-1BB refers to a TNFR superfamily member having the amino acid sequence provided as GenBank accession number AAA62478.2 or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.) ; and "4-1BB costimulatory signaling domain” is defined as amino acid residues 214-255 of GenBank accession number AAA62478.2 or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.) .
  • the "4-1BB costimulatory domain” is the sequence provided as SEQ ID NO: 11 or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.).
  • signaling pathway refers to the biochemical relationships between various signaling molecules that play a role in propagating signals from one part of a cell to another part of the cell.
  • the extracellular domain When referring to the extracellular domain of a constitutively chimeric cytokine receptor, the extracellular domain may be a cytokine, and the "cytokine" is released by a cell population and acts as an intercellular mediator on A general name for a protein from another cell.
  • cytokines are lymphokines, monokines, interleukins (IL), such as IL-1, IL-1 ⁇ , IL-2, IL-3, IL-4, IL-5, IL-6, IL- 8.
  • the "cytokine” that is the extracellular domain of the constitutive chimeric cytokine receptor of the invention is selected from any of IL-12 (e.g., IL-12p40 or IL-12p70), IL15 (e.g., IL-15 or IL-15FP, the IL-15FP refers to the fusion protein of IL-15 and IL-15R ⁇ (selected from IL-15R ⁇ or IL-15R ⁇ (Sushi)), including IL-15/IL-15R ⁇ and IL -15R ⁇ /IL-15 two forms of fusion protein)), IL-21, IL-18, IL-9, IL-23, IL-36 ⁇ and IFN ⁇ 2b.
  • IL-12 e.g., IL-12p40 or IL-12p70
  • IL15 e.g., IL-15 or IL-15FP
  • the IL-15FP refers to the fusion protein of IL-15 and IL-15R ⁇ (selected from IL-15R ⁇ or IL-15R ⁇ (Sushi
  • the extracellular domain may be an immune effector molecule, and the "immune effector molecule" may be selected from: (i) enhancing antigen presentation (e.g. , tumor antigen presentation); (ii) molecules that enhance effector cell responses (e.g., activate and/or mobilize B cells and/or T cells).
  • enhancing antigen presentation e.g. , tumor antigen presentation
  • enhance effector cell responses e.g., activate and/or mobilize B cells and/or T cells.
  • the "immune effector molecule” is, for example, the following molecules or their agonists: GITR, OX40, ICOS, SLAM (e.g., SLAMF7), HVEM, LIGHT, CD2, CD27, CD28, CDS, ICAM1, LFA-1 (CD11a/CD18 ), ICOS (CD278), 4-1BB (CD137), CD30, CD40, BAFFR, CD7, CD160, B7-H3 or CD83.
  • GITR e.g., OX40, ICOS, SLAM (e.g., SLAMF7), HVEM, LIGHT, CD2, CD27, CD28, CDS, ICAM1, LFA-1 (CD11a/CD18 ), ICOS (CD278), 4-1BB (CD137), CD30, CD40, BAFFR, CD7, CD160, B7-H3 or CD83.
  • the "immune effector molecule" that is the extracellular domain of the constitutive chimeric cytokine receptor of the invention is selected from any 4-1BB targeting molecule moiety (e.g., 4-1BB ligand, anti- 4-1BB antibody), CD40 targeting molecule moieties (e.g., CD40 ligand, anti-CD40 antibody), CD83 targeting molecule moieties (e.g., anti-CD83 antibody), FLT3 ligand, GITR, ICOS, CD2 and ICAM1.
  • 4-1BB targeting molecule moiety e.g., 4-1BB ligand, anti- 4-1BB antibody
  • CD40 targeting molecule moieties e.g., CD40 ligand, anti-CD40 antibody
  • CD83 targeting molecule moieties e.g., anti-CD83 antibody
  • FLT3 ligand GITR, ICOS
  • the extracellular domain may be an inhibitory molecule antagonist, and the "inhibitory molecule antagonist" is an agent that reduces tumor immunosuppression.
  • inhibitory molecules include, but are not limited to, PD-1, PD-L1, CD47, TIM-3, IL-4, TGF ⁇ , LAG-3, VISTA, B7-H4, CTLA-4, CD73 or TIGIT.
  • the "inhibitory molecule antagonist" of the extracellular domain of the constitutive chimeric cytokine receptor of the invention is selected from the group consisting of any anti-PD-L1 molecule, anti-CD47 molecule, and anti-IL-4 molecule , TGF ⁇ binding molecules (eg, anti-TGF ⁇ 1 molecules, TGF ⁇ RII), anti-PD-1 molecules, anti-CTLA-4 molecules, anti-LAG-3 molecules, anti-TIGIT molecules, and anti-CD73 molecules.
  • TGF ⁇ binding molecules eg, anti-TGF ⁇ 1 molecules, TGF ⁇ RII
  • anti-PD-1 molecules eg, anti-CTLA-4 molecules, anti-LAG-3 molecules, anti-TIGIT molecules, and anti-CD73 molecules.
  • the extracellular domain When referring to the extracellular domain of a constitutively chimeric cytokine receptor, the extracellular domain may be an effector molecule targeting an NK cell activating receptor, and "targeting an NK cell activating receptor” "Effector molecules" are a class of molecules that can activate NK cells after binding to NK cell activating receptors.
  • the NK cell activating receptors include, but are not limited to, NKG2C, NKG2D, NKp30, NKp44 and NKp46 on NK cells.
  • the "effector molecule targeting NK cell activating receptors" as the extracellular domain of the constitutive chimeric cytokine receptor of the invention is selected from the group consisting of targeting NK cell activating receptors NKG2C, NKG2D , NKp30, NKp44 and NKp46 molecules, such as anti-NKG2C, anti-NKG2D, anti-NKp30, anti-NKp44, and anti-NKp46, obtain enhanced anti-tumor immune effects by activating endogenous NK cells.
  • antibody is used in the broadest sense herein to refer to proteins containing antigen-binding sites, encompassing natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies ( For example, bispecific antibodies), single-chain antibodies, intact antibodies, and antibody fragments.
  • Antibody fragment or "antigen-binding fragment” are used interchangeably herein to refer to a molecule, distinct from an intact antibody, that contains a portion of an intact antibody and binds the antigen to which the intact antibody binds.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single chain Fv, single chain Fab, diabody.
  • scFv refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are optionally Continuously with the help of flexible short peptide linkers ligated and capable of expression as a single-chain polypeptide in which the scFv retains the specificity of the intact antibody from which it was derived.
  • a scFv may have a VL variable region and a VH variable region in any order (eg, relative to the N-terminus and C-terminus of the polypeptide), the scFv may comprise a VL-linker-VH or may comprise VH-joint-VL.
  • a “complementarity determining region” or “CDR region” or “CDR” or “hypervariable region” is an antibody variable domain that is hypervariable in sequence and forms a structurally defined loop (a “hypervariable loop") and/or A region containing antigen contact residues ("antigen contact points").
  • CDRs are mainly responsible for binding to antigenic epitopes.
  • the CDRs of the heavy and light chains are generally referred to as CDR1, CDR2 and CDR3 and are numbered sequentially starting from the N-terminus.
  • the CDRs located within the variable domain of the antibody heavy chain are called CDR H1, CDR H2, and CDR H3, while the CDRs located within the variable domain of the antibody light chain are called CDR L1, CDR L2, and CDR L3.
  • the precise amino acid sequence boundaries of each CDR in a given light chain variable region or heavy chain variable region amino acid sequence can be determined using any one or a combination of many well-known antibody CDR assignment systems, including For example: Chothia based on the three-dimensional structure of antibodies and the topology of CDR loops (Chothia et al.
  • CDRs may also be determined based on having the same Kabat number position as a reference CDR sequence (eg, any of the CDRs exemplified herein).
  • a reference CDR sequence eg, any of the CDRs exemplified herein.
  • reference is made to antibody variable regions and specific CDR sequences (including heavy chain variable region residues) reference is made to the numbering positions according to the Kabat numbering system.
  • CDRs vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding.
  • the minimal overlapping region can be determined, thus providing the "minimum binding unit" for antigen binding.
  • the smallest binding unit may be a subportion of a CDR.
  • the remainder of the CDR sequence can be determined from the structure of the antibody and protein folding. Therefore, variants of any CDR given herein are also contemplated by the present invention.
  • the amino acid residues of the minimal binding unit can remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia or AbM can be replaced by conserved amino acid residues.
  • variable region refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen.
  • the variable domains of the heavy and light chains of natural antibodies generally have similar structures, with each domain containing four conserved framework regions (FR) and three complementarity determining regions (CDR). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co. p. 91 (2007)).
  • FR conserved framework regions
  • CDR complementarity determining regions
  • Fc region is used herein to define the C-terminal region of an immunoglobulin heavy chain, which region contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carbonyl terminus of the heavy chain.
  • the C-terminal lysine (Lys447) of the Fc region may or may not be present.
  • the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, which is also known as the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.
  • the term “Functional variant” refers to a polypeptide having substantial or significant sequence identity or similarity with a polypeptide encoded by a nucleic acid sequence of the invention, which functional variant retains the biological activity of a polypeptide encoded by a nucleic acid sequence of the invention.
  • Functional variants may, for example, comprise at least one conservative amino acid substitution in the amino acid sequence of the polypeptide encoded by the nucleic acid sequence of the invention.
  • conservative sequence modification and “conservative sequence change” are used interchangeably and refer to amino acid modifications or changes that do not significantly affect or alter the biological activity of the polypeptide containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the polypeptides of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative substitution is an amino acid substitution in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art.
  • isolated nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. Isolated nucleic acid includes nucleic acid molecules that are contained in cells that normally contain the nucleic acid molecule, but that are present extrachromosomally or at a chromosomal location that is different from its native chromosomal location.
  • FACS fluorescence-activated cell sorting
  • Such instruments include the FACS Star Plus, FACScan, and FACSort instruments from Becton Dickinson (Foster City, CA), the Epics C from Coulter Epics Division (Hialeah, FL), and the MoFlo from Cytomation (Colorado Springs, Colorado).
  • pharmaceutically acceptable excipient refers to diluents, adjuvants (such as Freund's adjuvant (complete and incomplete)), excipients, buffers or stabilizers, etc., which are administered with the active substance.
  • treating means slowing, interrupting, retarding, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Desired therapeutic effects include, but are not limited to, preventing the emergence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis.
  • a “therapeutically effective amount” means an amount effective to achieve the desired therapeutic result, at the required doses and for the required period of time.
  • the therapeutically effective amount may vary depending on factors such as disease state, age, sex and weight of the individual.
  • a “therapeutically effective amount” preferably inhibits a measurable parameter (eg, tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, relative to an untreated subject. 60% or 70% and still more preferably at least about 80% or 90%.
  • the ability of a compound to inhibit a measurable parameter eg, cancer
  • the present invention relates to constitutive chimeric cytokine receptors that can continuously activate STAT5 signaling, maintain exogenous cytokine-independent survival of immune effector cells (eg, T cells), and have effector molecules that reshape the tumor microenvironment.
  • the constitutive chimeric cytokine receptor of the present invention includes:
  • said (i) extracellular domain of the constitutive chimeric cytokine receptor of the invention is selected from the group consisting of cytokines, immune effector molecules, inhibitory molecule antagonists, or NK cell-targeted activating receptors effector molecules.
  • the cytokine may be IL-12 (IL-12-P40 or IL-12-P70), IL15 (IL-15 or IL-15FP, the IL-15FP refers to the fusion protein of IL-15 and IL-15R ⁇ (selected from IL-15R ⁇ or IL-15R ⁇ (Sushi)), including IL-15/IL-15R ⁇ and IL-15R ⁇ /IL-15 two forms of fusion protein), IL-21, IL-18, IL-9, IL-23, IL-36 ⁇ , IFN ⁇ 2b and other cytokines, using immune cells genetically modified by these cytokines It has enhanced immune effector function and anti-tumor effect.
  • the immune effector molecule may be a 4-1BB targeting molecule moiety (e.g., 4-1BB ligand (4-1BBL), anti-4-1BB antibody ( ⁇ 4-1BB)), CD40-targeting molecule moiety (e.g., CD40 ligand (CD40L), anti-CD40 antibody ( ⁇ CD40)), CD83-targeting molecule moiety (e.g., anti- CD83 antibody ( ⁇ CD83)), FLT3 ligand (FTL3L), GITR, ICOS, CD2, ICAM1, etc.
  • these immune effector molecules are recognized by receptors on the surface of professional antigen-presenting cells (APC) such as dendritic cells (DC) in the body.
  • APC professional antigen-presenting cells
  • DC dendritic cells
  • the inhibitory molecule antagonist may be an anti-PD-L1 molecule, an anti-CD47 molecule, or an anti-CD47 molecule.
  • IL-4 molecules, TGF ⁇ binding molecules e.g., anti-TGF ⁇ 1 molecules, TGF ⁇ RII
  • anti-PD-1 molecules e.g., anti-CTLA-4 molecules, anti-LAG-3 molecules, anti-TIGIT molecules, anti-CD73 molecules, etc.
  • target inhibitory immune receptors Or the antibody part of the factor, for example, anti-PD-L1 VHH , achieves the purpose of enhancing the anti-tumor immune response by antagonizing the immunosuppressive effect of inhibitory immune receptors or factors.
  • the (i) extracellular domain of the constitutive chimeric cytokine receptor of the invention is selected from activating receptors targeting NK cell surface expression such as NKG2C, NKG2D, NKp30, NKp44, NKp46, etc.
  • the molecular parts, such as anti-NKG2C, anti-NKG2D, anti-NKp30, anti-NKp44, anti-NKp46 and other antibody parts, achieve the purpose of enhancing the anti-tumor immune effect by activating endogenous NK cells.
  • the (ii) constitutively activated IL-7R mutant of the constitutively chimeric cytokine receptor of the invention comprises any one selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:28, SEQ ID NO:30 to the amino acid sequence shown in SEQ ID NO:45, preferably, the constitutively activated IL-7R mutant includes any one selected from SEQ ID NO:30, SEQ ID NO :31, the amino acid sequence shown in SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:44, most preferably, the constitutively activated IL-7R mutant includes the amino acid sequence shown in SEQ ID NO:34 Amino acid sequence.
  • the constitutive chimeric cytokine receptor of the present invention is a constitutive dimer that can activate IL-7R independently of the binding of IL-7R to its ligand and independent of combination with a common ⁇ signal chain ( ⁇ c).
  • Intracellular signaling activates JAK1 kinase, which in turn phosphorylates downstream STAT5 and other transcriptional effectors, regulates the expression of downstream target genes, and ultimately promotes and maintains T proliferation and survival.
  • the constitutive chimeric cytokine receptor of the present invention reshapes the tumor microenvironment by including the (i) extracellular domain as an effector molecule on the basis of (ii) the constitutively activated IL-7R mutant. .
  • the constitutive chimeric cytokine receptor of the present invention and the chimeric antigen receptor (CAR) polypeptide are co-expressed in T cells
  • the constitutive chimeric cytokine receptor contains an extracellular domain and a constitutively activated IL -7R mutant, which passed the group
  • the activated IL-7R mutant continuously activates STAT5 signaling, promotes and maintains the proliferation and survival of immune cells, and gives immune cells new extracellular effector molecule effects through the extracellular domain, making it a modified immune system.
  • CAR-T cells have the ability to actively shape the "unfriendly” tumor microenvironment (TME), turning “cold” tumors into “hot” tumors by remodeling the TME, and their modified immune cells (e.g., CAR -T cells) in a more "friendly” TME will be more conducive to exerting anti-tumor effects.
  • TME tumor microenvironment
  • CAR -T cells modified immune cells
  • the CAR polypeptide is a traditional CAR polypeptide that directly targets one or more cancer-associated antigens.
  • the cancer-associated antigen also known as "tumor antigen” is selected from one or more of the following: CD19; CD20; CD22; CD24; CD30; CD123; CD171; CD33 epidermal growth factor receptor variant III (EGFRvIII ); ganglioside G2 (GD2); TNF receptor family member B cell maturation (BCMA); prostate-specific membrane antigen (PSMA); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 ( TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin 13 receptor subunit ⁇ -2 (IL-13Ra2 or CD213A2); Mesothelin; interleukin 11 receptor alpha
  • the cancer-associated antigen directly targeted by a traditional CAR polypeptide comprising a signal peptide, a cancer-associated antigen-binding domain, a transmembrane domain, a covalent Stimulatory signaling domain and main signaling domain.
  • the encoded cancer-associated antigen binding domain of the CAR polypeptide comprises an antibody, antibody fragment, scFv, Fv, Fab, (Fab')2, single domain antibody (SDAB), VH Or VL domain, or Camelidae VHH domain.
  • the transmembrane domain of the CAR polypeptide comprises a transmembrane domain selected from the group consisting of the alpha, beta, or zeta transmembrane domain of a T cell receptor, CD28, CD3 ⁇ , CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1(CD11a, CD18), ICOS(CD278), 4-1BB( CD137), GITR, CD40, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80(KLRF1), CD160, CD19, IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f , ITGAD, CD11d, ITGAE, CD103, ITGAL
  • the transmembrane domain of the CAR polypeptide comprises the amino acid sequence of the CD8 transmembrane domain having one, two, or three amino acid modifications of SEQ ID NO: 8. In one embodiment, the transmembrane domain comprises the sequence of SEQ ID NO: 8.
  • the cancer-associated antigen binding domain is connected to the transmembrane domain by a hinge region.
  • the hinge region comprises the amino acid sequence of the CD8 hinge, e.g., SEQ ID NO:7, or a sequence with one, two, or three amino acid modifications to SEQ ID NO:7.
  • the CAR polypeptide comprises an intracellular signaling domain, such as a primary signaling domain and/or a costimulatory signaling domain.
  • the intracellular signaling domain comprises a primary signaling domain.
  • the intracellular signaling domain comprises a costimulatory signaling domain.
  • the intracellular signaling domain includes a primary signaling domain and a costimulatory signaling domain.
  • the primary signaling domain comprises a functional signaling structure of a protein selected from the group consisting of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , common FcR ⁇ (FCER1G), FcR ⁇ (Fc ⁇ R1b), CD79a, CD79b, Fc ⁇ RIIa, DAP10, and DAP12 area.
  • a functional signaling structure of a protein selected from the group consisting of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , common FcR ⁇ (FCER1G), FcR ⁇ (Fc ⁇ R1b), CD79a, CD79b, Fc ⁇ RIIa, DAP10, and DAP12 area.
  • the primary signaling domain of the CAR polypeptide comprises a functional signaling domain of CD3 ⁇ .
  • the CD3 ⁇ primary signaling domain may comprise 1, 2 or 3 amino acid modifications having the amino acid sequence of SEQ ID NO:12. In some embodiments, the primary signaling domain comprises the sequence of SEQ ID NO: 12.
  • the intracellular signaling domain of a CAR polypeptide comprises a primary signaling domain and a costimulatory signaling domain.
  • the costimulatory signaling domain comprises a functional signaling domain of a protein selected from one or more of the following: CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD1, ICOS , Lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligand specifically binding to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM(LIGHTR), SLAMF7 , NKp80(KLRF1), CD160, CD19, CD4, CD8 ⁇ , CD8 ⁇ , IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, IT
  • the costimulatory signaling domain of the CAR polypeptide comprises 1, 2, or 3 amino acid modifications having the amino acid sequence of SEQ ID NO: 11.
  • the encoded costimulatory signaling domain comprises the sequence of SEQ ID NO: 11.
  • the CAR further comprises a signal peptide sequence.
  • the signal peptide sequence comprises the sequence of SEQ ID NO: 1.
  • the cancer-associated antigen binding domain of the CAR polypeptide has a binding affinity K D for the cancer-associated antigen of 10 ⁇ 4 M to 10 ⁇ 8 M.
  • the conventional CAR polypeptide comprises a conventional CLDN18.2 CAR polypeptide.
  • a traditional CLDN18.2 CAR polypeptide comprises:
  • H9.1.2 antibody scFv sequence that specifically binds to CLDN18.2 molecules, which includes a heavy chain variable region and a light chain variable region,
  • the heavy chain variable region includes a CDR H1 represented by the Kabat numbered amino acid sequence SYNIH (SEQ ID NO: 106), or a variant of the CDR H1 with no more than 2 amino acid changes or no more than 1 amino acid change.
  • the amino acid sequence LNRGQSLDY (SEQ ID NO: 108 ), or a variant of the CDR H3 with no more than 2 amino acid changes or no more than 1 amino acid change
  • the light chain variable region includes the amino acid sequence KSSQSLFNAGNQRNYLT (SEQ ID NO: The CDR L1 shown in 109), or a variant of the CDR L1 with no more than 2 amino acid changes or no more than 1 amino acid change
  • the CDR L2 shown with the amino acid sequence WASTRES (SEQ ID
  • Variants of CDR L2 with no more than 2 amino acid changes or no more than 1 amino acid change and CDR L3 shown in the amino acid sequence QNNYIYPLT (SEQ ID NO: 111), or with no more than 2 amino acid changes of the CDR L3 or Variants with no more than 1 amino acid change;
  • amino acid change is the addition, deletion or substitution of amino acids
  • the heavy chain variable region comprises or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 14 Identity sequence
  • the light chain variable region comprises SEQ ID NO:13 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;
  • Transmembrane region which is selected from the CD8 transmembrane domain or a variant thereof having 1-5 amino acid modifications, for example, the sequence shown in SEQ ID NO:8 or having 1-2 amino acids Modified variants;
  • Costimulatory signal domain which is selected from the 4-1BB costimulatory domain or a variant thereof with 1-5 amino acid modifications, for example, the sequence shown in SEQ ID NO: 11 or a variant thereof with 1 -2 amino acid modified variants;
  • Stimulating signal domain which is a CD3 ⁇ signaling domain or a variant thereof with 1-10 amino acid modifications, for example, the sequence shown in SEQ ID NO: 12 or a sequence with 1-10, 1 -5 amino acid modified variants
  • the traditional CLDN18.2 CAR polypeptide also includes a signal peptide sequence located at the N-terminus, for example, the signal peptide sequence shown in SEQ ID NO: 1,
  • a traditional CLDN18.2 CAR polypeptide includes, for example, the H9.1.2 CAR (SEQ ID NO: 16) described herein.
  • a conventional CLDN18.2 CAR polypeptide comprises:
  • H9.2.1 antibody scFv sequence that specifically binds to CLDN18.2 molecules, which contains a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region includes a CDR H1 represented by the Kabat numbered amino acid sequence SYNIH (SEQ ID NO: 112), or a variant of the CDR H1 with no more than 2 amino acid changes or no more than 1 amino acid change.
  • the amino acid sequence LNRGNALDY (SEQ ID NO: 114 ), or a variant of the CDR H3 with no more than 2 amino acid changes or no more than 1 amino acid change
  • the light chain variable region includes the amino acid sequence KSSQSLFQSGNQRNYLT (SEQ ID NO: The CDR L1 shown in 115), or a variant of the CDR L1 with no more than 2 amino acid changes or no more than 1 amino acid change
  • the CDR L2 shown with the amino acid sequence WASTRES (SEQ ID
  • amino acid change is the addition, deletion or substitution of amino acids
  • the heavy chain variable region comprises or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 99 Sequence identity, and the light chain variable region contains or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or Sequences with 99% identity;
  • Transmembrane region which is selected from the CD8 transmembrane domain or a variant thereof with 1-5 amino acid modifications, for example, the sequence shown in SEQ ID NO:8 or SEQ ID NO:147 or its Variants with 1-2 amino acid modifications;
  • Costimulatory signal domain which is selected from the group consisting of 4-1BB costimulatory domain or a variant thereof with 1-5 amino acid modifications, for example, the sequence shown in SEQ ID NO: 11 or a variant thereof with 1 - A variant with 2 amino acid modifications; or it is selected from the CD28 costimulatory domain or a variant thereof with 1-5 amino acid modifications, for example, the sequence shown in SEQ ID NO: 143 or the sequence thereof Variants with 1-2 amino acid modifications;
  • Stimulating signal domain which is a CD3 ⁇ signaling domain or a variant thereof with 1-10 amino acid modifications, for example, the sequence shown in SEQ ID NO: 12 or a sequence with 1-10, 1 -5 amino acid modified variants
  • the traditional CLDN18.2 CAR polypeptide also includes a signal peptide sequence located at the N-terminus, for example, the signal peptide sequence shown in SEQ ID NO: 1,
  • a traditional CLDN18.2 CAR polypeptide comprises, for example, a H9.2.1 CAR (SEQ ID NO: 100) described herein.
  • a conventional CLDN18.2 CAR polypeptide includes, for example, the H9.2.1-218 CAR (SEQ ID NO: 144) described herein.
  • a conventional CLDN18.2 CAR polypeptide comprises, for example, the H9.2.1-28-L CAR (SEQ ID NO: 142) described herein.
  • the CAR polypeptide is a molecular switch-regulated CAR polypeptide, which does not directly target one or more cancer-associated antigens, but targets one or more cancer-associated antigens through a "molecular switch” .
  • a CAR molecule is constructed that can specifically bind to the Fc containing the P329G mutant Domain-specific antibodies do not bind to antibodies that do not contain the P329G mutated Fc domain, whereby immune effector cells (e.g., T cells, NK cells) expressing the CAR are associated with targeted cancers as a "molecular switch” P329G mutated antibody combination of the antigen for the treatment of tumors.
  • the molecular switch-regulated CLDN18.2 CAR polypeptide comprises:
  • a humanized anti-P329G mutation scFv sequence wherein the scFv sequence includes the following sequence that is capable of specifically binding to an antibody Fc domain containing a P329G mutation, but is unable to specifically bind to an unmutated parent antibody Fc domain:
  • CDR L The light chain complementarity determining region (CDR L) 1 shown in the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 121), or a variant of the CDR L1 with no more than 2 amino acid changes or no more than 1 amino acid change;
  • amino acid change is the addition, deletion or substitution of an amino acid
  • the heavy chain variable region comprises or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 9 Sequence identity
  • the light chain variable region comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical sequence
  • Hinge region which is selected from the CD8 hinge region (SEQ ID NO:7), or a hinge region with at least 80% sequence identity;
  • Transmembrane region which is selected from the CD8 transmembrane domain or a variant thereof having 1-5 amino acid modifications, for example, the sequence shown in SEQ ID NO:8 or having 1-2 amino acids Modified variants;
  • Costimulatory signal domain which is selected from the 4-1BB costimulatory domain or a variant thereof with 1-5 amino acid modifications, for example, the sequence shown in SEQ ID NO: 11 or a variant thereof with 1 -2 amino acid modified variants;
  • Stimulating signal domain which is a CD3 ⁇ signaling domain or a variant thereof with 1-10 amino acid modifications, for example, the sequence shown in SEQ ID NO: 12 or a sequence with 1-10, 1 -5 amino acid modified variants.
  • the molecular switch-regulated CLDN18.2 CAR polypeptide also includes a signal peptide sequence located at the N-terminus, for example, the signal peptide sequence shown in SEQ ID NO:1.
  • a molecular switch-regulated CLDN18.2 CAR polypeptide comprises, for example, a HuR968B CAR (SEQ ID NO: 15) described herein
  • the P329G mutant antibody targeting cancer-associated antigen as a "molecular switch” includes a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region includes a heavy chain variable region according to Kabat numbering
  • the light chain variable region includes the CDR L1 shown according to the amino acid sequence RASQSISSWLA (SEQ ID NO: 12
  • amino acid change is the addition, deletion or substitution of amino acids
  • the heavy chain variable region comprises or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 130 Sequence identity, and the light chain variable region contains or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical sequence.
  • CAR-T cells expressing a molecular switch-regulated CAR polypeptide are combined with a P329G mutated anti-CLDN18.2 antibody (e.g., a P329G mutated HB37A6PG Ab,
  • a P329G mutated anti-CLDN18.2 antibody e.g., a P329G mutated HB37A6PG Ab
  • A6 antibody also called A6 antibody in this article (see Chinese Application No. 202111416497.1), shows the ability to continuously kill tumor cells and maintains the specificity of the CAR molecule.
  • T cells expressing molecular switch-regulated CAR polypeptides interact with tumor cells.
  • the T cells expressing the molecular switch-regulated CAR polypeptide be activated, proliferate, secrete effector cytokines, and produce a killing effect on tumor cells expressing or overexpressing CLDN18.2 .
  • the constitutive chimeric cytokine receptor can be constructed on one construct,
  • the CAR polypeptide is constructed in another On one construct, the two constructs are co-introduced into immune effector cells for expression.
  • a nucleic acid encoding a constitutively chimeric cytokine receptor-modified CAR polypeptide is constructed on a nucleic acid construct, the constitutively chimeric cytokine receptor-modified CAR polypeptide comprising a protein located at the N-terminus or C of the CAR polypeptide. end of the constitutive chimeric cytokine receptor of the present invention, and there is a self-cleaving peptide between the constitutive chimeric cytokine receptor and the CAR polypeptide, so that the nucleic acid construct generates a constitutive chimeric cytokine receptor of the present invention connected by the self-cleaving peptide.
  • Chimeric cytokine receptors and CAR polypeptides do not require any external cleavage activity to cleave the polypeptide produced by the nucleic acid construct into separate constitutive chimeric cytokine receptors and separate CAR polypeptides.
  • the "self-cleaving peptide” refers to a peptide that functions such that when a fusion polypeptide is produced that includes a first polypeptide, a self-cleaving peptide, and a second polypeptide from the N-terminus to the C-terminus, the fusion polypeptide is cleaved. into unique and discrete first and second polypeptides without the need for any external cleavage activity.
  • the self-cleaving peptide may be a 2A self-cleaving peptide from foot-and-mouth virus or cardiovirus.
  • the self-cleaving peptide is P2A shown in SEQ ID NO: 3 or a variant thereof with 1-5 amino acid modifications.
  • the constitutively chimeric cytokine receptor-modified CAR polypeptides of the invention extend from the N-terminus to the C-terminus
  • nucleic acid molecules, vectors and expression cells encoding the constitutive chimeric cytokine receptor of the present invention or encoding the constitutively chimeric cytokine receptor-modified CAR polypeptide of the present invention
  • the present invention provides nucleic acid molecules encoding the constitutive chimeric cytokine receptor of the present invention or encoding the CAR polypeptide modified by the constitutive chimeric cytokine receptor of the present invention.
  • the nucleic acid molecules are provided as DNA constructs.
  • the DNA construct encoding the constitutive chimeric cytokine receptor of the invention includes from the N-terminus to the C-terminus a polynucleotide encoding a signal peptide, encoding a polynucleotide composed of an effector molecule with the ability to remodel the tumor microenvironment.
  • polynucleotide encoding a hinge region between the polynucleotide encoding the extracellular domain and the polynucleotide encoding the IL-7R mutant transmembrane domain and the IL-7R intracellular domain, so
  • the hinge region is, for example, the Flag Tag shown in SEQ ID NO: 6 or a functional variant thereof.
  • the signal peptide comprises the sequence of SEQ ID NO: 2 or a functional variant thereof.
  • the polynucleotide encoding the IL-7R mutant transmembrane domain and the IL-7R intracellular domain comprises encoding any one selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 28 , SEQ ID NO:30 to the polynucleotide of the amino acid sequence shown in SEQ ID NO:45, preferably, include coding for any one selected from the group consisting of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ The polynucleotide of the amino acid sequence shown in ID NO:34 and SEQ ID NO:44, most preferably, includes a polynucleotide encoding the amino acid sequence shown in SEQ ID NO:34.
  • the extracellular domain consisting of effector molecules that remodel the tumor microenvironment is a cytokine, which may be, for example, IL-12 (IL-12-P40 or IL-12-P70), IL15 (IL-15 or IL-15FP, the IL-15FP refers to the fusion protein of IL-15 and IL-15R ⁇ (selected from IL-15R ⁇ or IL-15R ⁇ (Sushi)), including IL-15/IL-15R ⁇ and IL-15R ⁇ /IL-15 two forms of fusion proteins), IL-21, IL-18, IL-9, IL-23, IL-36 ⁇ , IFN ⁇ 2b and other cytokines, which are expressed by immune cells (such as T cells)
  • the M7CR gene containing cytokines has enhanced immune effector function and anti-tumor effect.
  • the cytokine is IL-15 set forth in SEQ ID NO: 47 or a functional variant thereof; IL-15FP (IL15/IL15R ⁇ (Sushi) fusion protein) set forth in SEQ ID NO: 48; or Its functional variant; IL-15FP (IL15/IL15R ⁇ fusion protein) shown in SEQ ID NO: 140 or its functional variant; IL-15FP (IL15R ⁇ (Sushi)/IL15 fusion protein) shown in SEQ ID NO: 141 or functional variants thereof; SEQ IL-12-P70 or its functional variant shown in ID NO: 49; IL-12-p40 or its functional variant shown in SEQ ID NO: 50; IL-21 or its functional variant shown in SEQ ID NO: 51 Functional variant; IL-9 shown in SEQ ID NO: 52 or a functional variant thereof; IL-18 shown in SEQ ID NO: 53 or a functional variant thereof; IL-23 shown in SEQ ID NO: 54 or Its functional variant; IL-36 ⁇ shown in SEQ ID NO: 55
  • the extracellular domain consisting of effector molecules that remodel the tumor microenvironment is an immune effector molecule, which may be, for example, a 4-1BB targeting molecule moiety (e.g., a 4-1BB ligand (4-1BBL), anti-4-1BB antibody ( ⁇ 4-1BB)), CD40-targeting molecule moiety (e.g., CD40 ligand (CD40L), anti-CD40 antibody ( ⁇ CD40)), CD83-targeting molecule moiety (e.g., anti- CD83 antibody ( ⁇ CD83)), FLT3 ligand (FTL3L), GITR, ICOS, CD2, ICAM1, etc.
  • these immune effector molecules are recognized by receptors on the surface of professional antigen-presenting cells (APC) such as dendritic cells (DC) in the body.
  • APC professional antigen-presenting cells
  • DC dendritic cells
  • the immune effector molecule is 4-1BBL or a functional variant thereof shown in SEQ ID NO: 57; CD40L or a functional variant thereof shown in SEQ ID NO: 58; SEQ ID NO: 59 FLT3L shown in SEQ ID NO: 60 or its functional variant; ICOS shown in SEQ ID NO: 60 or its functional variant; GITR shown in SEQ ID NO: 61 or its functional variant; ICAM-1 shown in SEQ ID NO: 62 Or its functional variant; CD2 or its functional variant shown in SEQ ID NO: 63; Anti-4-1BB or its functional variant shown in SEQ ID NO: 64; Anti-CD40 or its functional variant shown in SEQ ID NO: 65 Its functional variant; anti-CD83 shown in SEQ ID NO: 66 or its functional variant.
  • the extracellular domain consisting of effector molecules that remodel the tumor microenvironment is an inhibitory molecule antagonist, which may be, for example, an anti-PD-L1 molecule, an anti-CD47 molecule, an anti- IL-4 molecules, TGF ⁇ binding molecules (e.g., anti-TGF ⁇ 1 molecules, TGF ⁇ RII), anti-PD-1 molecules, anti-CTLA-4 molecules, anti-LAG-3 molecules, anti-TIGIT molecules, anti-CD73 molecules, etc.
  • an inhibitory molecule antagonist which may be, for example, an anti-PD-L1 molecule, an anti-CD47 molecule, an anti- IL-4 molecules, TGF ⁇ binding molecules (e.g., anti-TGF ⁇ 1 molecules, TGF ⁇ RII), anti-PD-1 molecules, anti-CTLA-4 molecules, anti-LAG-3 molecules, anti-TIGIT molecules, anti-CD73 molecules, etc.
  • target inhibitory immune receptors Or the antibody part of the factor, by antagonizing the immunosuppressive effect of inhibitory immune receptors or factors, achieves the purpose of enhancing the anti-tumor immune response, and then produces a synergistic anti-tumor effect with immune cells (such as T cells).
  • immune cells such as T cells
  • the inhibitory molecule antagonist is an anti-TGF ⁇ molecule set forth in SEQ ID NO: 67 or a functional variant thereof; a TGF ⁇ RII ECD set forth in SEQ ID NO: 198 or a functional variant thereof; SEQ ID NO : TGF ⁇ RII represented by 199 or a functional variant thereof; anti-PD-L1 VHH represented by SEQ ID NO: 68 or a functional variant thereof; anti-CD47 molecule represented by SEQ ID NO: 69 or a functional variant thereof; SEQ ID The anti-IL-4 molecule shown in NO: 70 or its functional variant; the anti-PD-1 molecule shown in SEQ ID NO: 71 or its functional variant; the anti-CTLA-4 molecule shown in SEQ ID NO: 72 or Its functional variant; the anti-LAG-3 molecule shown in SEQ ID NO: 73 or its functional variant; the anti-TIGIT molecule shown in SEQ ID NO: 74 or its functional variant; the anti-LAG-3 molecule shown in SEQ ID NO: 75 CD
  • the extracellular domain consisting of an effector molecule that remodels the tumor microenvironment is an effector molecule targeting an NK cell activating receptor
  • the effector molecule targeting an NK cell activating receptor may be Target the molecular parts of activating receptors expressed on the surface of NK cells such as NKG2C, NKG2D, NKp30, NKp44, NKp46, etc., for example, anti-NKG2C, anti-NKG2D, anti-NKp30, anti-NKp44, anti-NKp46 and other antibody parts, by activating endogenous NK cells achieve the purpose of enhancing anti-tumor immune effects and then produce synergistic anti-tumor effects with immune cells (such as T cells).
  • the NK cell activating molecule is anti-NKG2D or a functional variant thereof set forth in SEQ ID NO: 76; anti-NKG2C or a functional variant thereof set forth in SEQ ID NO: 77; SEQ ID NO: 78
  • a DNA construct encoding a constitutively chimeric cytokine receptor-modified CAR polypeptide of the invention includes from the N-terminus to the C-terminus a polynucleotide encoding a constitutively chimeric cytokine receptor, encoding a self-cleaving peptide polynucleotides and polynucleotides encoding CAR polypeptides.
  • a DNA construct encoding a constitutively chimeric cytokine receptor modified CAR polypeptide of the invention includes from N-terminus to C-terminus a polynucleotide encoding a CAR polypeptide, a polynucleotide encoding a self-cleaving peptide and polynucleotides encoding constitutively chimeric cytokine receptors.
  • the polynucleotide encoding a constitutively chimeric cytokine receptor is as described above.
  • the polynucleotide encoding a self-cleaving peptide is, for example, a polynucleotide encoding P2A shown in SEQ ID NO: 3 or a variant thereof with 1-5 amino acid modifications.
  • the polynucleotide encoding a CAR polypeptide may be a polynucleotide encoding any CAR polypeptide known in the art.
  • the CAR polypeptide is a traditional CAR polypeptide that directly targets one or more of the cancer-associated antigens described above. In some embodiments, the CAR polypeptide is a traditional CAR polypeptide that directly targets CLDN18.2, which includes a signal peptide, a cancer-associated antigen binding domain, a transmembrane domain, and a costimulatory signaling structure from the N-terminus to the C-terminus. domain and the main signaling domain.
  • the traditional CAR polypeptide includes from N-terminus to C-terminus: the CD8 signal peptide shown in SEQ ID NO:1 or a variant thereof with 1-5 amino acid modifications; the CD8 signal peptide shown in SEQ ID NO:13 VL-(G 4 S) n peptide linker shown - VH shown in SEQ ID NO: 14, wherein "n" is an integer from 1 to 10, such as an integer from 2 to 4, such as SEQ ID NO: 4, SEQ ID The sequence shown in NO:5; the CD8 hinge region shown in SEQ ID NO:7 or its variant with 1-5 amino acid modifications; the transmembrane domain shown in SEQ ID NO:8 or its variant with 1-5 Variants with amino acid modifications; costimulatory signaling domain shown in SEQ ID NO:11 or variants thereof with 1-5 amino acid modifications; main signaling domain shown in SEQ ID NO:12 or variants thereof with Variants with 1-5 amino acid modifications.
  • the conventional CAR polypeptide is a H9.1.2
  • the traditional CAR polypeptide includes from N-terminus to C-terminus: the CD8 signal peptide shown in SEQ ID NO: 1 or a variant thereof with 1-5 amino acid modifications; the CD8 signal peptide shown in SEQ ID NO: 98 VL-(G 4 S) n peptide linker shown - VH shown in SEQ ID NO: 99, wherein "n" is an integer from 1 to 10, such as an integer from 2 to 4, such as SEQ ID NO: 4, SEQ ID The sequence shown in NO:5; the CD8 hinge region shown in SEQ ID NO:7 or its variant with 1-5 amino acid modifications; the transmembrane domain shown in SEQ ID NO:8 or its variant with 1-5 Variants with amino acid modifications; costimulatory signaling domain shown in SEQ ID NO:11 or variants thereof with 1-5 amino acid modifications; main signaling domain shown in SEQ ID NO:12 or variants thereof with Variants with 1-5 amino acid modifications.
  • the conventional CAR polypeptide is a
  • the traditional CAR polypeptide includes from N-terminus to C-terminus: the CD8 signal peptide shown in SEQ ID NO:1 or a variant thereof with 1-5 amino acid modifications; the CD8 signal peptide shown in SEQ ID NO:98
  • the VL-linker shown in - the VH shown in SEQ ID NO: 99, for example, the linker is the sequence shown in SEQ ID NO: 145; the CD8 hinge region shown in SEQ ID NO: 147 or it has 1-5 Amino acid modified variants; the transmembrane domain shown in SEQ ID NO:148 or its variants having 1-5 amino acid modifications; the costimulatory signaling domain shown in SEQ ID NO:11 or its variants having 1- Variants with 5 amino acid modifications; the main signaling domain shown in SEQ ID NO: 12 or variants thereof with 1-5 amino acid modifications.
  • the traditional CAR polypeptide is a H9.2.1-218 CAR having the amino acid sequence set forth in SEQ ID NO: 144.
  • the traditional CAR polypeptide includes from N-terminus to C-terminus: as shown in SEQ ID NO:1 CD8 signal peptide or a variant thereof with 1-5 amino acid modifications; VL-linker shown in SEQ ID NO:98-VH shown in SEQ ID NO:99, for example, the linker is shown in SEQ ID NO:145 The sequence shown; the CD8 hinge region shown in SEQ ID NO: 147 or its variant with 1-5 amino acid modifications; the transmembrane domain shown in SEQ ID NO: 148 or its variant with 1-5 amino acid modifications Variant; the costimulatory signaling domain shown in SEQ ID NO:143 or a variant thereof having 1-5 amino acid modifications; the main signaling domain shown in SEQ ID NO:12 or a variant thereof having 1-5 Amino acid modified variants.
  • the conventional CAR polypeptide is a H9.2.1-28-L CAR having the amino acid sequence set forth in SEQ ID NO:142.
  • the CAR polypeptide is a molecular switch-regulated CAR polypeptide.
  • the CAR polypeptide targets the cancer-associated antigen by combining it with an antibody against the cancer-associated antigen of the P329G mutation (the P329G mutation is also referred to as "PG") as a molecular switch, from the N-terminus to The C-terminus contains signal peptide, anti-PG antibody scFv sequence, transmembrane domain, costimulatory signaling domain and main signaling domain.
  • the molecular switch-regulated CAR polypeptide includes from the N-terminus to the C-terminus: the CD8 signal peptide shown in SEQ ID NO: 1 or a variant thereof with 1-5 amino acid modifications; SEQ ID NO: Anti-PG antibody VH shown in 9-(G 4 S) n peptide linker-anti-PG antibody VL shown in SEQ ID NO: 10, wherein "n" is an integer from 1 to 10, such as an integer from 2 to 4, such as Sequences shown in SEQ ID NO:4 and SEQ ID NO:5; GGGGS hinge; transmembrane domain shown in SEQ ID NO:8 or variants thereof with 1-5 amino acid modifications; SEQ ID NO:11 The co-stimulatory signaling domain shown in SEQ ID NO: 12 or a variant thereof having 1-5 amino acid modifications; the main signaling domain shown in SEQ ID NO: 12 or a variant thereof having 1-5 amino acid modifications.
  • the molecular switch-regulated CAR polypeptide is a HuR
  • a DNA construct encoding a constitutively chimeric cytokine receptor modified CAR polypeptide of the invention comprises encoding any of SEQ ID NO:80-SEQ ID NO:95, SEQ ID NO:101, SEQ ID Polynucleotides of the amino acid sequences of NO:104, SEQ ID NO:133, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138 and functional variants thereof.
  • the invention also provides a vector into which the DNA construct of the invention is inserted.
  • a vector into which the DNA construct of the invention is inserted.
  • the vector may be suitable for replication and integration in eukaryotic organisms.
  • Common cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters for regulating expression of the desired nucleic acid sequence.
  • retroviruses provide convenient platforms for gene delivery systems.
  • the selected genes can be inserted into the vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to the subject's cells in vivo or ex vivo.
  • retroviral systems are known in the art.
  • lentiviral vectors are used.
  • Retroviral vectors may also be, for example, gamma retroviral vectors.
  • a gamma retroviral vector may, for example, comprise a promoter, a packaging signal ( ⁇ ), a primer binding site (PBS), one or more (e.g., two) long terminal repeats (LTRs) and a transgene of interest, e.g., the codebook
  • Gamma retroviral vectors can lack viral structural genes such as gag, pol and env.
  • a promoter capable of expressing the transgene of the invention in mammalian T cells is the EF1a promoter.
  • the native EF1a promoter drives expression of the alpha subunit of the elongation factor-1 complex, which is responsible for enzymatic delivery of aminoacyl tRNA to ribosomes.
  • the EF1a promoter has been widely used in mammalian expression plasmids and has been shown to efficiently drive expression of transgenes cloned into lentiviral vectors. See, eg, Milone et al., Mol. Ther. 17(8):1453–1464 (2009).
  • CMV immediate early cytomegalovirus
  • constitutive promoter sequences may also be used, including, but not limited to, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) Long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to the actin promoter , myosin promoter, elongation factor-1 ⁇ promoter, hemoglobin promoter and creatine kinase promoter. Additionally, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention.
  • the invention provides methods of expressing the DNA constructs of the invention in mammalian immune effector cells (eg, mammalian T cells or mammalian NK cells) and immune effector cells generated thereby.
  • mammalian immune effector cells eg, mammalian T cells or mammalian NK cells
  • a source of cells eg, immune effector cells, eg, T cells or NK cells
  • T cells can be obtained from numerous sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • T cells can be obtained from blood components collected from a subject using any technique known to those skilled in the art, such as Ficoll TM isolation.
  • cells from the individual's circulating blood are obtained by apheresis.
  • Apheresis products generally contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • cells collected by apheresis can be washed to remove the plasma fraction and to place the cells in a suitable buffer or culture medium for subsequent processing steps.
  • cells are washed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • T cell subsets such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated through positive or negative selection techniques.
  • anti-CD3/anti-CD28 e.g. M-450 CD3/CD28T
  • the time period is between about 30 minutes and 36 hours or longer. Longer incubation times can be used to isolate T cells in any situation where small numbers of T cells are present, such as for isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or from immunocompromised individuals.
  • TILs tumor-infiltrating lymphocytes
  • Enrichment of T cell populations through a negative selection process can be accomplished using a combination of antibodies directed against surface markers unique to the negatively selected cells.
  • One method is to sort and/or select cells by means of negative magnetic immunoadhesion or flow cytometry, which uses cells present on the negatively selected cells. Mixture of monoclonal antibodies to surface markers.
  • the immune effector cells may be allogeneic immune effector cells, such as T cells or NK cells.
  • the cells may be allogeneic T cells, e.g., allogeneic that lack functional T cell receptor (TCR) and/or human leukocyte antigen (HLA) (e.g., HLA class I and/or HLA class II) expression.
  • TCR T cell receptor
  • HLA human leukocyte antigen
  • a T cell lacking a functional TCR can, for example, be engineered so that it does not express any functional TCR on its surface; engineered so that it does not express one or more subunits that constitute a functional TCR (e.g., engineered ized such that it does not express or displays reduced expression of TCR ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ and/or TCR ⁇ ); or engineered such that it produces very few functional TCRs on its surface.
  • a T cell described herein can, for example, be engineered such that it does not express functional HLA on its surface.
  • T cells described herein can be engineered such that cell surface expression of HLA (e.g., HLA class I and/or HLA class II) is downregulated.
  • HLA downregulation can be achieved by reducing or eliminating beta-2 microglobulin (B2M) expression.
  • T cells may lack functional TCR and functional HLA, e.g., HLA class I and/or HLA class II.
  • the effector function of the immune effector cells co-expressing CAR and the constitutive chimeric cytokine receptor of the present invention obtained after in vitro proliferation can be tested as described in the Examples.
  • the invention provides pharmaceutical compositions comprising immune effector cells (e.g., T cells, NK cells) that express the constitutively chimeric cytokine receptors of the invention, cells encoding said constitutively chimeric cytokine receptors, Nucleic acid molecules encoding cytokine receptors, vectors comprising nucleic acid molecules encoding said constitutively chimeric cytokine receptors, and any combination thereof; and optionally pharmaceutically acceptable excipients.
  • immune effector cells e.g., T cells, NK cells
  • the invention provides a pharmaceutical composition
  • an immune effector cell e.g., T cell, NK cell
  • an immune effector cell selected from the group consisting of expressing a constitutively chimeric cytokine receptor modified CAR polypeptide of the invention, encoding The nucleic acid molecule of the constitutively chimeric cytokine receptor modified CAR polypeptide, a vector comprising the nucleic acid molecule encoding the constitutively chimeric cytokine receptor modified CAR polypeptide, and any combination thereof; and optionally can medical supplements.
  • the CAR polypeptide is a molecular switch-regulated CAR polypeptide
  • the pharmaceutical composition further includes a molecular switch, such as an antibody molecular switch.
  • the immune effector cells are prepared from autologous T cells or allogeneic T cells, for example, the immune effector cells are prepared from T cells isolated from human PBMCs.
  • composition of the present invention can be formulated according to conventional methods (for example, Remington’s Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, U.S.A.).
  • Pharmaceutically acceptable excipients may include, for example, surfactants, excipients, colorants, flavors, preservatives, stabilizers, buffers, suspending agents, isotonic agents, binders, disintegrants, lubricants, and flow promoters. , flavoring agents, etc.
  • other commonly used carriers may also be suitably used, such as light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carboxymethylcellulose calcium, carboxymethylcellulose sodium, hydroxypropyl Cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain fatty acid triglycerides, polyoxyethylene hardened castor oil 60, white sugar, carboxylic acid Methyl cellulose, corn starch, inorganic salts, etc. are used as carriers, but are not limited to these.
  • compositions of the invention are used to treat cancer, such as cancers that express or overexpress CLDN 18.2.
  • the present invention provides the aforementioned pharmaceutical composition of the present invention for use in treating tumors (eg, cancer) in a subject.
  • the invention also relates to a method of treating a tumor (eg, cancer) in a subject, comprising administering to said subject an effective amount of a pharmaceutical composition of the invention.
  • the tumor is cancer.
  • tumors, such as cancers, described herein include, but are not limited to, solid tumors, hematologic cancers, soft tissue tumors, and metastatic lesions.
  • the pharmaceutical composition of the invention is used to treat a cancer that expresses or overexpresses CLDN 18.2 in a subject and is capable of reducing the severity of at least one symptom or indication of cancer or inhibiting cancer cell growth.
  • the invention provides methods of treating cancer (e.g., cancers that express or overexpress CLDN 18.2) in a subject, comprising administering to an individual in need thereof a therapeutically effective amount of a pharmaceutical composition of the invention.
  • the present invention provides the use of the aforementioned pharmaceutical composition of the present invention in the preparation of medicaments for treating cancer (eg, cancer expressing or overexpressing CLDN18.2).
  • cancer eg, cancer expressing or overexpressing CLDN18.2.
  • compositions of the present invention may also be administered to individuals whose cancer has been treated with one or more prior therapies and has subsequently relapsed or metastasized.
  • the pharmaceutical composition of the present invention can be administered to a subject at an appropriate dose.
  • the dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, the dosage for any given patient depends on many factors, including the patient's weight, body surface area, age, the specific compound to be administered, sex, time and route of administration, general health, and concurrent medications to be administered. Other medications administered.
  • administration of a pharmaceutical composition of the present invention to an individual with cancer results in complete disappearance of the tumor.
  • administration of a pharmaceutical combination of the invention to an individual with cancer results in a reduction in tumor cells or tumor size of at least 85% or greater.
  • Tumor reduction can be measured by any method known in the art, such as X-ray, positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), cytology, histology, or molecular genetics analyze.
  • Example 1.1 Construction of lentiviral vector for expressing M7R gene
  • IL-7 binds to its wild-type receptor IL-7 receptor ⁇ chain (IL7R ⁇ ) and induces heterodimerization of the latter with the common ⁇ signal chain, activating downstream JAK/STAT signaling.
  • IL7R ⁇ wild-type receptor IL-7 receptor ⁇ chain
  • the mutation may induce self-dimerization of the mutated IL7R, thereby enabling the self-dimerization of the mutated IL7R without relying on IL-7 binding.
  • IL7R constitutively activates the downstream STAT5 signaling pathway.
  • a viral expression plasmid was constructed for expressing chimeric receptors tCD19-M7CR (also called IL7Rm-tCD19 in this specification) containing different IL7R mutations (IL7Rm or M7R) and tCD19.
  • tCD19- M7CR consists of the same extracellular domain (ECD) composed of truncated CD19 (tCD19, SEQ ID NO:17) and different IL7R mutants (also referred to as IL7Rm or M7R in the text), the IL7Rm (SEQ ID NO:20 -SEQ ID NO:46) consists of the IL7R transmembrane region (IL7R-mutant(TM)) carrying different mutations (see the bold part of the sequence) and the wild-type IL7R intracellular segment (IL7R-wt(ICD), SEQ ID NO :19) composition.
  • ECD extracellular domain
  • IL7Rm SEQ ID NO:20 -SEQ ID NO:46
  • IL7R-mutant(TM) IL7R transmembrane region
  • ICD wild-type IL7R intracellular segment
  • the 27 IL7Rm are named IL7Rm1.1, IL7Rm1.2, IL7Rm1.3, IL7Rm1.4, IL7Rm2.1, IL7Rm2.2, IL7Rm2.3, IL7Rm2.4, IL7Rm3.1, IL7Rm3.2, IL7Rm4, respectively.
  • IL7Rm5 IL7Rm5, IL7Rm6, IL7Rm7, IL7Rm8, IL7Rm9, IL7Rm10, IL7Rm11, IL7Rm12, IL7Rm13, IL7Rm14, IL7Rm15, IL7Rm16, IL7Rm17, IL7Rm18, IL7Rm19, IL7Rm20 (see SEQ ID NO:20-SEQ ID NO:46 in the sequence listing), and their corresponding tCD19-M7CRs are named IL7Rm1.1-tCD19, IL7Rm1.2-tCD19, and IL7Rm1 respectively.
  • the IL7R-tCD19 construct contains the tCD19 extracellular domain and the wild-type IL7R transmembrane region and intracellular segment (SEQ ID NO: 96), IL7R- The WT construct contains the complete wild-type IL7R chain (SEQ ID NO: 18).
  • Example 1.2 Construction of BaF3 cell lines stably expressing different M7Rs
  • BaF3 (purchased from Nanjing Kebai Biotechnology Co., Ltd.) is a mouse-derived pre-B lymphocyte that relies on exogenously added mouse IL-3 (mIL-3) cytokine (R&D system) for survival.
  • mIL-3 mouse IL-3
  • R&D system cytokine
  • Example 1.1 In order to identify whether the tCD19-M7CR gene constructed in Example 1.1 can continuously activate STAT5 signals after being introduced into the BaF3 cell line, different tCD19-M7CR genes were transferred into BaF3 cells through lentivirus. According to whether the BaF3 cells can produce IL-3 Growth-independent screening of M7R genes with sustained activation of STAT5.
  • the specific experimental steps are as follows. Lenti-X-293T cells (Takara Company) (3 ⁇ 10 5 cells) in the logarithmic growth phase were seeded into a 6-well plate.
  • the lentivirus-containing supernatant was used to infect BaF3 cells for 24 hours, and then conventionally cultured in RPMI 1640 complete medium containing mIL-3 (R&D systems, 403-ML) for 48 hours.
  • Example 1.2 take the BaF3 cells infected with the lentivirus prepared in Example 1.2, wash them once with FACS buffer, resuspend the BaF3 cells with FACS buffer, add LIVE/DEAD Fixable Dead Cell Stain (Thermo, L34963), PE-CD19 antibody (BD Company, 555413), incubate at 4°C for 30 to 45 minutes. Then, the cells were washed once with FACS buffer, and the cells were resuspended in FACS buffer, and the expression of tCD19 on the cell surface was detected by flow cytometry.
  • LIVE/DEAD Fixable Dead Cell Stain Thermo, L34963
  • PE-CD19 antibody BD Company, 555413
  • each group of BaF3 cells selected with mIL-3-independent growth was added to a 24-well plate at the same number of cells (i.e., 5 ⁇ 10 5 cells/well), and each group of cells was analyzed by a cell counter. Count, record cell proliferation, and draw a growth curve.
  • the proportion of tCD19 + cells in BaF3 cells increased, and the remaining groups of BaF3 cells could not maintain survival after culture without the addition of exogenous mIL-3, indicating that IL7Rm1.1, IL7Rm1.3, IL7Rm3.1, IL7Rm4, IL7Rm5, IL7Rm6, IL7Rm7, IL7Rm8,
  • M7R genes such as IL7Rm1.1, IL7Rm1.3, IL7Rm3.1, IL7Rm4 ⁇ 12, IL7Rm14 ⁇ 18, etc. can promote the proliferation of BaF3 cells in an mIL-3-independent manner.
  • ISO refers to staining with an anti-STAT5 isotype control antibody
  • +IL3 refers to adding IL3 to uninfected BaF3 cells to stimulate cell STAT5 activation as a positive control
  • without IL3 refers to uninfected cells without IL3 stimulation.
  • BaF3 cells were stained with anti-pSTAT5 antibody to detect the basal phosphorylation level of STAT5 in the cells.
  • BaF3 cells that have not been infected with lentivirus can only detect STAT5 phosphorylation signals when exogenous mIL-3 is added to stimulate them; while expressing IL7Rm1.1, IL7Rm1.3, and IL7Rm3 .1.
  • STAT5 phosphorylation was detected in BaF3 cells of IL7Rm4, IL7Rm5, IL7Rm6, IL7Rm7, IL7Rm8, IL7Rm9, IL7Rm10, IL7Rm11, IL7Rm12, IL7Rm14, IL7Rm15, IL7Rm16, IL7Rm17, and IL7Rm18 genes without the addition of exogenous mIL-3 stimulation. signal, indicating that these M7R genes can constitutively activate the STAT5 signaling pathway.
  • M7R sequences with constitutive activation function were selected (IL7Rm1.1, 1.3, 3.1, 4-12, 14-18 respectively).
  • tCD19-M7CR genes containing IL7Rm1.1, 1.3, 3.1, 4-12, respectively.
  • the tCD19-M7CR gene 14-18 years old is transferred into T cells through lentivirus, and the M7R gene with the function of continuously activating STAT5 in T cells is screened based on whether the T cells can produce IL-2-independent growth.
  • the lentivirus packaging steps are the same as those described in Example 1.2, and lentiviral supernatants containing tCD19-M7CR genes containing different M7R sequences (IL7Rm1.1, 1.3, 3.1, 4-12, and 14-18 respectively) are obtained.
  • Lentivirus expressing different tCD19-M7CR genes was used to infect activated human T cells (see Table 1 for PBMC information) to obtain T cells stably expressing different tCD19-M7CR genes. Specific steps are as follows.
  • T cell sorting and activation steps Add recombinant human interleukin-2 for injection (National Drug Approval No. S20040020) to TexMACS GMP Medium (Miltenyi Biotec, 170-076-309) to prepare T cells with an IL-2 concentration of 200IU/ml. Cell culture medium.
  • Pan T Cell Isolation Kit human (Miltenyi, 130-096-535) was used to sort the PBMC of each donor after recovery to obtain T cells, and the T cells were resuspended to a certain concentration using T cell culture medium. density (for example, cell density 1 ⁇ 10 6 cells/mL) and add TransAct (Miltenyi, 130-111-160) for activation; on the first day, separate a certain amount of cells and continue culturing without adding lentivirus.
  • density for example, cell density 1 ⁇ 10 6 cells/mL
  • TransAct TransAct
  • This part of the cells is not Transduced cells (UNT cells, un-transduced T cells), add the supernatant of lentivirus containing different tCD19-M7CR genes to the remaining cells and pipet the T cells evenly; centrifuge on the second day to remove the supernatant containing lentivirus. Resuspend T cells in fresh IL-2-containing T cell medium. No manipulation was performed on UNT cells. After culturing for 48 hours in a 5% CO2 cell incubator at 37°C, use LIVE/DEAD Fixable Dead Cell Stain (Thermo, L34963), PE-CD19 (BD, 555413), and AF647-p-STAT5 (BD, 562076) antibody combination for detection.
  • PBMC cells from donor 3 were used, and 48 hours after the virus infected T cells, the expression of tCD19 on T cells was detected.
  • the results are shown in Figure 5. Different proportions of tCD19 expression were detected in human T cells infected with different tCD19-M7CR genes, IL7R-tCD19 genes containing wild-type IL7R ⁇ transmembrane region and intracellular segment, while untransfected (UNT) No tCD19 expression was detected in T cells and T cells expressing wild-type IL7R ⁇ (IL7R-WT). It shows that the M7R gene plays a role in T cells successfully expressed in cells.
  • PBMC cells of donor 3 After the virus infects the T cells, culture them for 5 days (from day 0 counting activation until detection for a total of 5 days) and then detect the phosphorylation level of STAT5 in the T cells.
  • untransfected T cells UNT
  • STAT5 phosphorylation signals instead of expressing the wild-type IL7R transmembrane and intracellular segments (control IL7R-tCD19), STAT5 phosphorylation signals were detected in T cells, indicating that these M7R genes can also constitutively activate the STAT5 signaling pathway in human T cells.
  • T cells expressing IL7R-tCD19 continue to expand in vitro under the stimulation of exogenous IL-2. 2 weeks, after which the cells stopped expanding and maintained for about 1 week, and then the number of cells dropped significantly. Without the addition of exogenous IL-2 stimulation, the number of T cells expressing IL7R-tCD19 continued to decrease, and all cells died after 2 weeks.
  • T cells expressing IL7R-tCD19 Compared with T cells expressing IL7R-tCD19, the number of T cells expressing IL7Rm4, 5, 7 and 8 constructs could be maintained for 1 week without the addition of exogenous IL-2, and began to slowly decrease after 1 week until 4 weeks. All cells died, and these in vitro experiments showed that expression of the M7R gene promoted the survival of T cells and had the ability to maintain T cell survival.
  • M7CR genes were designed, in which the extracellular domain (ECD) and M7R (the fusion IL-7R mutant consisting of the transmembrane and intracellular signaling regions of the IL-7R ⁇ mutant) were directly connected.
  • ECD extracellular domain
  • M7R the fusion IL-7R mutant consisting of the transmembrane and intracellular signaling regions of the IL-7R ⁇ mutant
  • Constitutive chimeric cytokine receptor M7CR the N-terminus of M7CR is then connected to the C-terminus of different CAR polypeptides through P2A to form an M7CR-modified CAR.
  • the ECD located at the N-terminus of M7R includes but is not limited to tCD19 (SEQ ID NO: 17), sIL-15 (SEQ ID NO: 47), IL-15/IL-15R ⁇ (SEQ ID NO: 140) , IL-15/IL-15R ⁇ (Sushi)(SEQ ID NO:48), IL-15R ⁇ (Sushi)/IL-15(SEQ ID NO:141), IL-12-P70(SEQ ID NO:49), IL-12-p40 (SEQ ID NO: 50), IL-21 (SEQ ID NO: 51), IL-9 (SEQ ID NO: 52), IL-18 (SEQ ID NO: 53), IL-23 ( SEQ ID NO: 54), IL-36 ⁇ (SEQ ID NO: 55), IFN ⁇ 2b (SEQ ID NO: 56), 4-1BBL (SEQ ID NO: 57), CD40L (SEQ ID NO: 58), FLT3L (SEQ ID NO: 59), ICOS (SEQ ID NO: 59),
  • M7CR molecules tCD19-M7CR, sIL-15-M7CR, IL-15/IL-15R ⁇ -M7CR, IL15/IL15R ⁇ (Sushi)-M7CR, IL15R ⁇ (Sushi)/IL15- M7CR, IL-12-P70-M7CR, IL-12-p40-M7CR, IL-21-M7CR, IL-9-M7CR, IL-18-M7CR, IL-23-M7CR, IL-36 ⁇ -M7CR, IFN ⁇ 2b- M7CR, 4-1BBL-M7CR, CD40L-M7CR, FLT3L-M7CR, ICOS-M7CR, GITR-M7CR, ICAM-1-M7CR, CD2-M7CR, anti-4-1BB-M7CR, anti-CD40-M7CR, anti-CD83-M7CR , anti-TGF ⁇ -M7CR,
  • ECDs are connected to M7Rm8 to construct tCD19-M7CR shown in SEQ ID NO: 171, IL-12-M7CR shown in SEQ ID NO: 172, IL-15-M7CR shown in SEQ ID NO: 173, SEQ IL-21-M7CR shown in ID NO: 174, IL-12-p40-M7CR shown in SEQ ID NO: 175, IL-9-M7CR shown in SEQ ID NO: 176, and IL-9-M7CR shown in SEQ ID NO: 177 IL-18-M7CR, IL-23-M7CR shown in SEQ ID NO: 178, IL-36 ⁇ -M7CR shown in SEQ ID NO: 179.
  • Figure 1 shows the mechanism of action of T cells expressing the constructed M7CR after the constructed M7CR transduces T cells.
  • H9.2.1-28-IL-15-M7CR (SEQ ID NO: 137), H9.2.1-P2A-IL15R ⁇ (Sushi)/IL-15-M7CR (hereinafter also referred to as H9.2.1-IL-15-M7CR) ( SEQ ID NO:138) protein.
  • the HuR968B CAR polypeptide includes a signal peptide (CD8-SP) (SEQ ID NO: 1) derived from CD8 (SEQ ID NO: 1), an anti-PG antibody VH (SEQ ID NO:9), G4S linker (SEQ ID NO:5), anti-PG antibody VL (SEQ ID NO:10), GGGGS hinge, CD8-derived transmembrane domain (CD8TMD) (SEQ ID NO:8), source Costimulatory signaling domain from 4-1BB (4-1BB CSD) (SEQ ID NO: 11) and stimulatory signaling domain from CD3 ⁇ (CD3 ⁇ SSD) (SEQ ID NO: 12).
  • CD8-SP signal peptide
  • CD8-SP signal peptide
  • CD8-SP signal peptide
  • CD8-SP signal peptide
  • CD8-SP signal peptide
  • CD8-SP signal peptide
  • CD8-SP signal peptide
  • SEQ ID NO: 1 derived from CD8
  • the H9.1.2 CAR molecule includes CD8-SP (SEQ ID NO:1), H9.1.2-VL (SEQ ID NO:13), (G4S) 3 connector (SEQ ID NO:4), H9.1.2-VH (SEQ ID NO:14), CD8 hinge (SEQ ID NO:7), CD8TMD (SEQ ID NO:8), 4-1BB CSD ( SEQ ID NO:11) and CD3 ⁇ SSD (SEQ ID NO:12).
  • the CAR polypeptide is connected to the M7CR molecule through P2A (SEQ ID NO: 3).
  • the H9.2.1 CAR molecule includes CD8-SP (SEQ ID NO:1), H9.2.1-VL (SEQ ID NO:98), (G4S) 3 connector (SEQ ID NO:4), H9.2.1-VH (SEQ ID NO:99), CD8 hinge (SEQ ID NO:7), CD8TMD (SEQ ID NO:8), 4-1BB CSD ( SEQ ID NO:11) and CD3 ⁇ SSD (SEQ ID NO:12).
  • the H9.2.1-218 CAR molecule includes CD8-SP (SEQ ID NO:1), H9.2.1-VL (SEQ ID NO:98) from N-terminus to C-terminus ), 218 linker sequence (SEQ ID NO:145), H9.2.1-VH (SEQ ID NO:99), CD8 long hinge (SEQ ID NO:146), CD8TMD extension (SEQ ID NO:147), 4-1BB CSD (SEQ ID NO:11) and CD3 ⁇ SSD (SEQ ID NO:12).
  • the CAR polypeptide is connected to the M7CR molecule through P2A (SEQ ID NO: 3).
  • H9.2.1-28 CAR molecule contains CD8-SP (SEQ ID NO:1), H9.2.1-VL (SEQ ID NO: 98), 218 linker sequence (SEQ ID NO:145), H9.2.1-VH (SEQ ID NO:99), CD8 long hinge (SEQ ID NO:146), CD8TMD extension (SEQ ID NO:147), CD28CSD ( SEQ ID NO:143) and CD3 ⁇ SSD (SEQ ID NO:12).
  • the CAR polypeptide is connected to the M7CR molecule through P2A (SEQ ID NO: 3).
  • H9.1.2-P2A-tCD19-M7CR molecule contains H9.1.2 CAR (SEQ ID NO:16), P2A (SEQ ID NO: 3) and tCD19-M7CR (SEQ ID NO: 171).
  • H9.1.2-P2A-tCD19-M7CR (CPT) molecule contains H9.1.2 CAR (SEQ ID NO:16), P2A (SEQ ID NO: 3) and tCD19-M7CR(CPT) (SEQ ID NO: 182).
  • H9.1.2-P2A-IL-12-M7CR molecule contains H9.1.2 CAR (SEQ ID NO:16), P2A (SEQ ID NO:16) from N-terminus to C-terminus. NO: 3) and IL-12-P70-M7CR.
  • H9.1.2-P2A-IL-15-M7CR molecule contains H9.1.2 CAR (SEQ ID NO:16), P2A (SEQ ID NO:16) from N-terminus to C-terminus. NO: 3) and IL-15/IL-15R ⁇ -M7CR.
  • H9.1.2-P2A-IL-21-M7CR molecule contains H9.1.2 CAR (SEQ ID NO:16), P2A (SEQ ID NO:3) and IL-21-M7CR (SEQ ID NO:174).
  • H9.1.2-P2A-CD40L-M7CR molecule contains H9.1.2 CAR (SEQ ID NO:16), P2A (SEQ ID NO: 3) and CD40L-M7CR (SEQ ID NO: 183).
  • H9.1.2-P2A-4-1BBL-M7CR molecule contains H9.1.2 CAR (SEQ ID NO:16), P2A (SEQ ID NO:16) from N-terminus to C-terminus. NO: 3) and 4-1BBL-M7CR (SEQ ID NO: 184).
  • H9.1.2-P2A-anti-PD-L1 VHH -M7CR molecule contains H9.1.2 CAR (SEQ ID NO:16), P2A ( SEQ ID NO: 3) and anti-PD-L1 VHH -M7CR (SEQ ID NO: 185).
  • the 8B-P2A-tCD19-M7CR molecule contains HuR968B CAR (SEQ ID NO:15), P2A (SEQ ID NO:3) and tCD19 from N-terminus to C-terminus -M7CR (SEQ ID NO: 171).
  • 8B-P2A-tCD19-M7CR (CPT) molecule contains HuR968B CAR (SEQ ID NO:15), P2A (SEQ ID NO:3) from N-terminus to C-terminus ) and tCD19-M7CR(CPT) (SEQ ID NO: 182).
  • 8B-P2A-IL-15-M7CR molecule contains HuR968B CAR (SEQ ID NO:15), P2A (SEQ ID NO:3) from N-terminus to C-terminus and IL-15/IL-15R ⁇ -M7CR.
  • 8B-P2A-IL-12-M7CR molecule contains HuR968B CAR (SEQ ID NO:15), P2A (SEQ ID NO:3) from N-terminus to C-terminus and IL-12-P70-M7CR.
  • the 8B-P2A-IL-21-M7CR molecule contains HuR968B CAR (SEQ ID NO:15), P2A (SEQ ID NO:3) from N-terminus to C-terminus and IL-21-M7CR (SEQ ID NO: 174).
  • the 8B-P2A-CD40L-M7CR molecule contains HuR968B CAR (SEQ ID NO:15), P2A (SEQ ID NO:3) and CD40L from N-terminus to C-terminus -M7CR (SEQ ID NO: 183).
  • 8B-P2A-4-1BBL-M7CR molecule contains HuR968B CAR (SEQ ID NO:15), P2A (SEQ ID NO:3) from N-terminus to C-terminus and 4-1BBL-M7CR (SEQ ID NO: 184).
  • 8B-P2A-anti-PD-L1 VHH -M7CR molecule contains HuR968B CAR (SEQ ID NO:15), P2A (SEQ ID NO: 3) and anti-PD-L1 VHH -M7CR (SEQ ID NO: 185).
  • all M7CR molecules comprise GM-CSFRa-SP (SEQ ID NO: 2), ECD and M7R from N-terminus to C-terminus, optionally at the ECD A Flag Tag is connected between the C terminus and the N terminus of M7R (hereinafter exemplified as M7R are IL7Rm4 represented by SEQ ID NO:30, IL7Rm5 represented by SEQ ID NO:31, IL7Rm7 represented by SEQ ID NO:33, and SEQ ID NO:33).
  • IL7Rm8 shown in ID NO: 34 and IL7Rm18 shown in SEQ ID NO: 44 ECD selected from tCD19 (SEQ ID NO: 17), IL-12-P70 (SEQ ID NO: 49), IL-15/IL -15R ⁇ (SEQ ID NO: 140), IL-21 (SEQ ID NO: 51), 4-1BBL (SEQ ID NO: 57), CD40L (SEQ ID NO: 58), anti-PD-L1 VHH (SEQ ID NO :68).
  • IL-12-P70-M7CR, IL-15/IL-15R ⁇ -M7CR and IL-21-M7CR also contain Flag Tag (SEQ ID NO: 6) between the ECD sequence and the M7R sequence.
  • the M7R part of tCD19-M7CR, IL-12-P70-M7CR, IL-15/IL-15R ⁇ -M7CR, IL-21-M7CR, 4-1BBL-M7CR, CD40L-M7CR, anti-PD-L1 VHH -M7CR molecule is IL7Rm8 (SEQ ID NO:34), tCD19-M7CR(CPT) is used as a control, and its M7R part is IL7Rm(CPT) (SEQ ID NO:97) (IL7Rm(CPT) is the M7R molecule used as a control).
  • the N-terminus of the IL7Rm8 sequence of the M7R part of the anti-PD-L1 VHH -M7CR molecule also includes the "ESKYGPPCPPCP" sequence.
  • the H9.2.1-P2A-tCD19-M7CR molecule includes H9.2.1 CAR (SEQ ID NO:100), P2A (SEQ ID NO:3) and tCD19-M7CR from the N-terminus to the C-terminus. (SEQ ID NO: 171).
  • the H9.2.1-P2A-IL-12-M7CR molecule includes H9.2.1 CAR (SEQ ID NO: 100), P2A (SEQ ID NO: 3) and IL from the N end to the C end. -12-M7CR (SEQ ID NO: 172).
  • the H9.2.1-P2A-IL-12-M7CRin molecule (SEQ ID NO: 102) includes H9.2.1 CAR (SEQ ID NO: 100), P2A (SEQ ID NO: 3) and IL from the N-terminus to the C-terminus.
  • IL-12-M7CRin (SEQ ID NO: 181), where IL-12-M7CRin means that on the basis of IL-12-M7CR, the IL-7 receptor intracellular Box1 domain (amino acids 1060-1071, the sequence is The PIVWPPSLPDHKK shown in SEQ ID NO:132 was deleted and Y1239F, Y1246F (using IL7R ⁇ (P16871-1) as a reference) point mutations were simultaneously designed to inactivate the intracellular M7R signal.
  • the H9.2.1in-P2A-IL-12-M7CR molecule (SEQ ID NO: 103) contains H9.2.1in CAR (SEQ ID NO: 153), P2A (SEQ ID NO: 3) from the N end to the C end.
  • H9.2.1in represents the deletion of the two intracellular 4-1BB and CD3 domains of the H9.2.1 CAR (SEQ ID NO: 100) molecule (to inactivate the CAR Molecular intracellular signal), and the "KRGR" sequence is added to the C terminus.
  • the H9.2.1-P2A-sIL-12 molecule includes H9.2.1 CAR (SEQ ID NO: 100), P2A (SEQ ID NO: 3) and sIL-12 from the N-terminus to the C-terminus.
  • sIL-12 represents a gene composed of GM-CSFR ⁇ -SP (SEQ ID NO:2) and IL-12-p70 (SEQ ID NO:49) that can express exocrine soluble IL-12.
  • the M7R part of the above-mentioned M7CR molecule is IL7Rm8 (SEQ ID NO: 34).
  • the H9.2.1-P2A-IL-15-M7CR molecule (SEQ ID NO: 136) contains H9.2.1-218 CAR (SEQ ID NO: 144), P2A (SEQ ID NO: 3) from the N end to the C end. and IL-15-M7CR (SEQ ID NO: 173).
  • the H9.2.1-P2A-IL-15-M7CRin molecule (SEQ ID NO: 135) contains H9.2.1-218 CAR (SEQ ID NO: 144), P2A (SEQ ID NO: 3) from the N end to the C end.
  • IL-15-M7CRin SEQ ID NO: 180
  • IL-15-M7CRin means that on the basis of IL-15-M7CR, the IL-7 receptor intracellular Box1 domain (amino acids 1060-1071 ( 272-280 (using IL7R ⁇ as reference (P16871-1))), the sequence is PIVWPSLPDHKK shown in SEQ ID NO: 132), delete and design Y1239F, Y1246F point mutations to inactivate the intracellular M7R signal.
  • H9.2.1in-P2A-IL-15-M7CR molecule contains H9.2.1-218in CAR (SEQ ID NO: 186), P2A (SEQ ID NO: 3) from the N end to the C end. ) and IL-15-M7CR, where H9.2.1-218in CARin means that the two intracellular 4-1BB and CD3 domains of H9.2.1-218 (SEQ ID NO: 144) are deleted to inactivate the intracellular signal of the CAR molecule .
  • the H9.2.1-P2A-sIL-15 molecule (SEQ ID NO:139) includes H9.2.1-218 CAR (SEQ ID NO:144), P2A (SEQ ID NO:3) and sIL from the N-terminus to the C-terminus.
  • sIL-15 represents a gene composed of GM-CSFR ⁇ -SP (SEQ ID NO: 2) and IL-15 (SEQ ID NO: 47) and capable of expressing exocrine soluble IL-15.
  • the H9.2.1-P2A-IL15R ⁇ (Sushi)/IL-15-M7CR molecule includes H9.2.1-218 CAR (SEQ ID NO:144), P2A (SEQ ID NO: 3) and IL15R ⁇ (Sushi)/IL-15-M7CR (SEQ ID NO: 187).
  • the M7R part of the above-mentioned M7CR molecule is IL7Rm8 (SEQ ID NO: 34).
  • the H9.2.1-CD28-P2A-IL-12-M7CR molecule (SEQ ID NO: 133) includes H9.2.1-28 from the N-terminus to the C-terminus. CAR (SEQ ID NO:142), sequence "RAKR", P2A (SEQ ID NO:3) and IL-12-M7CR (SEQ ID NO:172).
  • the H9.2.1-CD28-P2A-IL-15-M7CR molecule (SEQ ID NO: 137) contains H9.2.1-28 CAR (SEQ ID NO: 142), the sequence "RAKR", and P2A from the N-terminus to the C-terminus. (SEQ ID NO: 3) and IL-15-M7CR (SEQ ID NO: 173).
  • the M7R portion of the above M7CR molecule is IL7Rm8 (SEQ ID NO: 34).
  • M7CR molecules contain GM-CSFR ⁇ -SP (SEQ ID NO: 2), ECD and M7R (Herinafter, IL7Rm4 shown in SEQ ID NO:30, IL7Rm5 shown in SEQ ID NO:31, IL7Rm7 shown in SEQ ID NO:33, IL7Rm8 shown in SEQ ID NO:34 and SEQ ID NO: IL7Rm18 shown in 44), ECD is selected from tCD19 (SEQ ID NO: 17), IL-12-P70 (SEQ ID NO: 49), IL-15/IL-15R ⁇ (Sushi) (SEQ ID NO: 48), IL-15R ⁇ (Sushi)/IL-15 (SEQ ID NO: 141).
  • tCD19-M7CR (SEQ ID NO: 171), IL-12-M7CR (SEQ ID NO: 172), IL-15-M7CR (SEQ ID NO: 173) and IL-15R ⁇ (Sushi)/IL-15-M7CR ( SEQ ID NO: 187)
  • the M7R part of the molecule is IL7Rm8 (SEQ ID NO: 34)
  • tCD19-M7CR (CPT) is used as a control
  • its M7R part is IL7Rm (CPT) (SEQ ID NO: 97)
  • IL7Rm (CPT) is M7R molecule as control).
  • the above-mentioned synthesized DNA fragment was inserted into the pRKN lentiviral expression vector (Genewise Company) downstream of the EF1 ⁇ promoter, and the EGFR sequence in the original vector was replaced to obtain the corresponding expression plasmid (synthesized by Genewise Company).
  • the expression plasmid obtained in Example 4.1 was combined with the structural plasmid pMDLg/pRRE (Addgene, 12251, purchased from Biowind), regulatory plasmid pRSV-rev (Addgene, 12253, purchased from Biowind) and envelope plasmid pMD2G (Addgene, 12259, Lenti-X-293T cells (Takara Company) were transfected with PEI transfection method at a mass ratio of 3:3:2:2 (purchased from Biowind). After 16 hours of transfection, the cells were replaced with 2% fetal bovine serum (FBS).
  • FBS fetal bovine serum
  • Fresh DEME culture medium continue culturing for 48 hours, collect the cell supernatant, centrifuge to remove cell debris, add PEG8000 and incubate at 4°C for 16-64 hours to concentrate the virus, centrifuge again and discard the supernatant, use T cell culture medium (TexMACs) Resuspend the virus pellet to obtain lentivirus concentrate, aliquot and freeze at -80°C.
  • Digest Lenti-X-293T cells (Takara Company) and resuspend them in DMEM medium containing 8 ⁇ g/ml Polybrene (Sigma, H9268-5G) and add them to a 24-well plate. Add different volumes of lentivirus concentrate obtained above and culture for 72 hour, transduction of 293T cells was performed.
  • the transduced 293T cells were digested, stained with Biotin-SP-conjugated anti-Human IgG, F(ab')2-specific (Jackson ImmunoResearch, 109-066-006) and APC-Streptadvidin (BioLegend, 405207), and Cell flow cytometry was used to detect the proportion of APC-positive cells.
  • the virus titer (TU/ml) was calculated from the starting cell volume, virus volume and positive cell proportion.
  • Example 4.3 Obtaining T cells and lentiviral transduction
  • H9.1.2 refers to H9.1.2 CAR-T cells, and the others are tCD19-M7CR, tCD19-M7CR (CPT), IL-15/IL-15R ⁇ -M7CR (marked as IL-15-M7CR in the figure) , IL-12-M7CR, IL-21-M7CR, CD40L-M7CR, 4-1BBL-M7CR, anti-PD-L1 VHH -M7CR modified H9.1.2 CAR-T cells.
  • CPT tCD19-M7CR
  • CPT tCD19-M7CR
  • IL-15/IL-15R ⁇ -M7CR marked as IL-15-M7CR in the figure
  • IL-12-M7CR IL-21-M7CR
  • CD40L-M7CR 4-1BBL-M7CR
  • anti-PD-L1 VHH -M7CR modified H9.1.2 CAR-T cells.
  • CAR peptide and M7CR were detected by FACS on day 9.
  • the CAR peptide could be expressed in all CAR-T cells.
  • the proportion of CAR + cells in H9.1.2 was approximately 26%, while CAR in tCD19-M7CR, tCD19-M7CR(CPT), IL-15-M7CR, IL-12-M7CR, IL-21-M7CR, CD40L-M7CR, 4-1BBL-M7CR and anti-PD-L1 VHH -M7CR group
  • the proportions of + cells are: 12.14%, 11.87%, 6.81%, 6.37%, 18.75%, 12.97%, 11.13%, 10.62%.
  • the proportions of CD4 and CD8 positive cells in cells expressing H9.1.2 CAR were 59.9% and 36.3% respectively; in tCD19-M7CR, tCD19-M7CR(CPT), IL-15-M7CR, IL-
  • the proportions of CD4 + cells in the 12-M7CR, IL-21-M7CR, CD40L-M7CR, 4-1BBL-M7CR and anti-PD-L1 VHH -M7CR groups were respectively: 55.9%, 62.5%, 60.2%, 56.1%, 62 %, 65.7%, 60.5% and 46.5%; the proportions of CD8 + cells were: 36.3%, 33.5%, 26.9%, 38%, 31.5%, 29.8%, 32.7% and 48.2% respectively.
  • FIG. 9C shows the expression levels of CAR and M7CR on the 9th day after preparing CAR-T.
  • “8B” represents HuR968B CAR-T cells, and the rest For tCD19-M7CR, tCD19-M7CR (CPT), IL-15/IL-15R ⁇ -M7CR (marked as IL-15-M7CR in the figure), IL-12-M7CR, IL-21-M7CR, CD40L-M7CR, 4 -1BBL-M7CR, anti-PD-L1 VHH -M7CR modified HuR968B CAR-T cells.
  • tCD19-M7CR, tCD19-M7CR(CPT), IL-15-M7CR, IL-12-M7CR, IL-21-M7CR, CD40L-M7CR, 4-1BBL-M7CR and anti-PD-L1 VHH -M7CR molecules in each group All are expressed, and the expression efficiencies are: 6.6%, 6.81%, 1.43%, 3.59%, 4.95%, 2.97%, 1.03%, and 7.45% respectively. This shows that both CAR polypeptides and M7CR molecules can be expressed, and there is a certain correlation between the expression of CAR and each M7CR molecule.
  • the proportions of CD4 and CD8 positive cells in cells expressing 8B CAR were 33.2% and 61.6% respectively; in tCD19-M7CR, tCD19-M7CR (CPT), IL-15-M7CR, IL-12
  • the proportions of CD4 + cells in -M7CR, IL-21-M7CR, CD40L-M7CR, 4-1BBL-M7CR and anti-PD-L1 VHH -M7CR groups were: 31.1%, 32.4%, 29.8%, 38.2%, 32.4% respectively , 32.2%, 32.2% and 32.2%; the proportions of CD8 + cells were: 64.3%, 63.2%, 65.1%, 55.7%, 63.5%, 63.3%, 63.5% and 61.1% respectively.
  • CD45RA + CCR7 + represents naive T cells or stem memory T cells (TN/TSCM)
  • CD45RA-CCR7 + represents central memory T cells ( TCM)
  • CD45RA - CCR7 - represents effector memory T cells (TEM)
  • CD45RA + CCR7 - represents effector T cells (Teff) subsets
  • most CAR-T cells are TN/TSCM and TCM cells.
  • the total T cells, CD4 + and CD8 + T phenotypes are shown in Figure 9G and Figure 9H .
  • CAR-T cells were found to The proportion of total T cells, CD4 + and CD8 + T cells TCM and TN subset cells in the sample increased; while the proportion of total T cells, CD4 + and CD8 + T cells in the IL-12-M7CR modified CAR-T cell sample occurred During differentiation, the proportion of TCM and TN subpopulation cells decreased, while the proportion of TEM and Teff subpopulation cells increased. This shows that different ECD structures will have different effects on the phenotype of CAR-T cells.
  • the expression level of intracellular p-STAT5 was detected by FACS to study the activation of STAT5.
  • the experimental steps are as follows. Take 1E6 T cells, wash them once with PBS, and resuspend them in serum-free RPMI1640 medium overnight. The next day, UNT cells were stimulated with IL-2 (200UI/mL) for 20 minutes and then washed once with FACS buffer. Then add AF647-p-STAT5 (BD, 562076) antibody for intracellular staining. The specific steps are the same as Example 1.5.
  • UNT increased the level of p-STAT5 under the stimulation of IL-2, but p-STAT5 did not increase in H9.2.1 CAR-T cells regardless of CAR+ or CAR-.
  • CAR+ cells expressing M7R the average expression level of p-STAT5 increased.
  • H9.2.1-IL-12-M7CRin with an inactivating mutation of M7R the expression level of p-STAT5 did not increase.
  • DAN-G18.2 and SNU-601 cells (1E5) were placed in a 96-well V-bottom plate, and the supernatant was discarded. Then add FACS buffer for resuspension and washing, and centrifuge again to remove the supernatant.
  • FACS buffer for resuspension and washing, and centrifuge again to remove the supernatant.
  • Use FACS buffer to prepare a saturated mouse anti-human CLDN18.2 mixture and add 100 ⁇ L of the above mixture into the well. Add 100 ⁇ L of FACS buffer to the well. Incubate in the dark for 30 minutes at 4°C. Centrifuge at 300g for 5 minutes and discard the supernatant.
  • the number of cell surface molecules was calculated through Qufikit quantitative analysis.
  • the numbers of CLDN18 molecules on the surface of SNU-601high, SNU-601low, and DANG-18.2high were: 87427, 24360, and 655891, respectively.
  • Antibodies were used to label CLDN18.2 on the surface of different target cells, and then flow cytometry was used to detect the expression level of CLDN18.2 on the target cell surface. Specifically, the A6 antibody was used to incubate the target cells with the target cells for 30 minutes at 4°C; then, after washing once with FACS buffer, the cells were incubated with the APC-labeled anti-human Fc antibody for 30 minutes at 4°C; finally, after washing once with FACS buffer, the cells were plated. Machine detection. As shown in Figure 10B, the peak diagram represents the expression level of CLDN18.2 in each cell type, and the expression level was quantified by calculating the MFI.
  • CLDN18.2 expression is higher in DANG18.2 and NUGC-4 cells
  • SNU-601 and Hup-T4 are medium expression
  • SNU-620 and PANC-1 are low expression
  • ISO is the isotype antibody control
  • K562 is CLDN18 .2 Negative control cells.
  • the xCELLigence RTCA MP instrument (Agilent company) was used to dynamically detect the killing of target cells by CAR-T cells in real time. Add 50 ⁇ L of culture medium to the E-Plates plate. After the instrument reads the baseline value, add 50 ⁇ L of tumor target cells, and then place them in the machine to dynamically monitor the cell growth. Resuscitate the UNT cells and CAR-T cells prepared in Example 4.3 (T cells are from PBMC cells of donor 5 and donor 13), and place them in a 37°C cell culture incubator overnight. The next day, add CAR-T into the E-Plates wells of the corresponding group according to the E:T ratio required for the experiment.
  • Mutated A6 antibody uses the VH/VL domain of the P329G mutated A6 antibody to bind to tumor target cells and the Fc-terminal P329G mutation to bind to the extracellular binding region of HuR968B CAR-T cells, thereby activating HuR968B CAR-T cells or M7CR-modified HuR968B CAR- T thin Targeted tumor killing function of cells.
  • the xCELLigence RTCA MP instrument system dynamically monitors the killing of target cells by CAR-T cells for 48-96 hours.
  • H9.1.2 CAR-T cells or H9.1.2 CAR-T cells modified with different M7CR were co-incubated with the tumor target cell DAN-G18.2.
  • the E:T ratio was 1:1 and 1 respectively. :3.
  • the killing effect of tCD19-M7CR modified H9.1.2 CAR-T cells is equivalent and better than that of H9.1.2 CAR-T cells.
  • the killing effect of CAR-T cells in other groups is similar to that of H9.1.2 CAR-T cells are basically equivalent.
  • A6 antibody 2nM
  • the killing effect of IL-12-M7CR modified HuR968B CAR-T cells on SNU-601 high and SNU-601 low is better than that of unmodified HuR968B CAR-T, tCD19-M7CR modified HuR968B CAR-T cells and other M7CR modified cells.
  • HuR968B CAR-T cells, and this effect is more obvious in SNU-601 low .
  • HuR968B CAR-T cells in all groups proliferated under the stimulation of target cells, and from days 7 to 21, IL-15/IL-15R ⁇ -M7CR (marked in Figure 12A
  • the HuR968B CAR-T cells in the IL-15-M7CR) and IL-12-M7CR modified groups continued to expand, and the proliferation multiples were higher than those in the unmodified HuR968B CAR-T cell group and tCD19-M7CR modified HuR968BCAR-T cells.
  • the CAR-T cells in the IL-12-M7CR modified group had the highest cumulative expansion fold after 21 days of stimulation (approximately 44 times). The above shows that IL-12-M7CR modification can improve the proliferation ability of CAR-T cells under repeated stimulation of target cells.
  • Figure 13A shows representative flow cytometry results of the numbers of CD4 + and CD8 + T cells in each group after the first and third rounds of stimulation of target cells in Figures 12A to 12F.
  • the ratio of CD4 + and CD8 + T cells in each group was basically maintained at 1:2 at the beginning of the experiment.
  • IL-12-M7CR modification was able to increase the ratio of CD4 + and CD8 + T cells (approximately 1:1), while the proportion of CD8 + T cells in other groups increased significantly and the proportion of CD4 + T cells decreased significantly.
  • IL-12-M7CR modification can promote the expansion of CD4 + T cells under the stimulation of target cells.
  • Figure 13B shows the statistical results of the proportion of CD4 + and CD8 + T cells in each group in Figure 13A.
  • IL-12-M7CR modification can increase the ratio of CD4 + and CD8 + T cells to about 1:1, while the ratio of CD8 + T cells in other groups increased significantly, and the ratio of CD4 + T cells increased significantly. The ratio is significantly reduced.
  • Cytokines were detected using BD TM Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit II. Mix the Capture Beads in an equal volume and plate at 25 ⁇ L/well. Add an equal volume of supernatant or supernatant dilution or standard from the repeated stimulation experiment with in vitro tumor cells. After mixing, add 25 ⁇ L of equal volume of human Th1/Th2PE detection reagent and incubate at room temperature in the dark for 3 hours. Wash twice with wash buffer and resuspend, and calculate the cytokine concentration through the MFI value of the PE channel of the flow cytometer.
  • CBA Cytometric Bead Array
  • the levels of IL-2, IFN- ⁇ , and TNF cytokines in the supernatant 24 h after the addition of effector cells in the first round of stimulation were detected by CBA. It was found that the IL-2 level in the supernatant of the IL-15/IL-15R ⁇ -M7CR (labeled IL-15-M7CR in Figure 13C) modified group ( ⁇ 2000pg/mL) was the lowest, IL-12-M7CR modified and tCD19-M7CR (CPT) modified group (2000-4000pg/mL) followed by unmodified HuR968B CAR group, tCD19-M7CR, IL-21-M7CR, CD40L-M7CR, 4-1BBl-M7CR and ⁇ PD-L1VHH-M7CR modified HuR968B The CAR group (>6000pg/mL) was higher.
  • the level of IFN- ⁇ in the supernatant of the IL-12-M7CR modified group was the highest, while the levels of IFN- ⁇ in the supernatant of other groups were all lower than 5000pg/mL.
  • the level of TNF in the supernatant of the IL-12-M7CR modified group was the highest, while the TNF levels in the supernatant of other groups were all at lower levels.
  • Example 9 In vitro killing effect of IL-12-M7CR/IL-15-M7CR modified H9.2.1 CAR-T cells
  • H9.2.1-IL12-M7CR represents IL-12-P70 modified H9.2.1 CAR-T cells
  • H9.2.1in-IL12-M7CR represents the 4-1BB costimulatory domain and The CD3 ⁇ signaling domain is deleted, thereby achieving the purpose of loss of CAR structure and function
  • H9.2.1-IL12-M7CRin means that the M7CR intracellular Box1 domain is deleted, and Y449F, Y456F (using IL7R ⁇ (P16871-1) as a reference) mutation is introduced To achieve the purpose of inactivating the intracellular structure and function of M7CR
  • H9.2.1-sIL12 represents the combination of H9.2.1 CAR-T cells and soluble IL12.
  • 2.1 CAR-T cells are equivalent to H9.2.1-IL-12-M7CRin CAR-T cells and H9.2.1-sIL12 CAR-T cells, and IL-12-M7CR-modified H9.2.1 CAR-T cells have the strongest killing effect. This shows that IL-12-P70 and M7R have a combined effect in promoting the killing effect of CAR-T cells.
  • the Xcelligence instrument was used to detect the killing effect of CAR-T cells on the target cell Hup-T4.
  • mice purchased from Viton Lever
  • IL-12-M7CR molecules can promote the anti-tumor effect of PG CAR-T cells in vivo. and proliferation ability.
  • the specific experimental method is as follows. NOG mice (purchased from Viton Lever) were selected. On Day-7, the mice were intraperitoneally injected with NUGC-4-Gluc cells (1 ⁇ 10 6 each) to create a model.
  • mice Through the small animal in vivo imaging system The mouse modeling conditions were tested, and the animals were divided into groups (5 animals in each group) when the tumor burden was 1 ⁇ 10 9 p/s (the calculated value and unit for the number of photons generated by the tumor in the IVIS imaging system) on Day 0. . Each mouse was then given 5 ⁇ 10 5 CAR-T cells via tail vein injection. The number of UNT cells given to the UNT group was consistent with the total number of T cells infused into the mice with the lowest CAR positivity rate. Mice burden, and the number of CAR-T cells in peripheral blood, were imaged weekly.
  • the M7CR-expressing CAR-T cells constructed based on PG CAR-T cells were injected into the model mice through the tail vein, and 0.3 mg/kg was injected at the same time.
  • Antibody containing P329G mutation A6 found through small animal in vivo imaging, increased IL-12-M7CR and tCD19-M7CR modified PG CAR-T over time The cells have better anti-tumor effects in vivo, and as shown in Figure 17, PG CAR-T cells have higher expansion levels from days 7 to 28, while unmodified PG CAR-T cells have It has weak anti-tumor effect and poor amplification ability in vivo.
  • the above in vivo results show that M7CR modification can promote the expansion of PG CAR-T cells and improve the anti-tumor effect of CAR-T cells in vivo.
  • the M7CR-expressing CAR-T cells constructed based on traditional CAR-T cells are passed After tail vein injection into model mice, it was found through small animal in vivo imaging that IL-12-M7CR and tCD19-M7CR-modified CAR-T cells had better anti-tumor effects in vivo as time went by, and on Day 13 , IL-12-M7CR modified CAR-T cells can completely eliminate tumors in model mice. As shown in Figure 19, this figure is a quantitative statistics of IVIS images.
  • H9.2.1 CAR-T cells H9.2.1 CAR-T cells
  • M7R H9.2.1 CAR-T cells and IL-12-M7CR CAR- T cells have good anti-tumor effects in model mice.
  • M7R modification can improve the anti-tumor effect of H9.2.1 CAR-T cells
  • IL-12 modification can further improve the anti-tumor effect of M7R H9.2.1 CAR-T cells.
  • Figure 20 the number of total human T cells and CAR-T cells in the peripheral blood of mice was detected by flow cytometry. After a single injection of CAR-T cells, the number increased with time.
  • the extracellular domain (ECD) of different molecules and M7Rm8 (SEQ ID NO: 34) were directly connected to construct a constitutive chimeric cytokine receptor M7CRm8; then the N-terminus of M7CRm8 was connected to H9 through P2A.
  • the C-terminus of the 2.1-218 CAR (SEQ ID NO: 144) or H9.2.1-28 CAR (SEQ ID NO: 142) or H9.2.1 CAR (SEQ ID NO: 100) polypeptide is connected to form an M7CR-modified CAR.
  • the sequence of the constructed H9.2.1-tCD19-M7CR is shown in SEQ ID NO: 101.
  • the sequence of the constructed H9.2.1-IL-12-M7CR is shown in SEQ ID NO: 104.
  • the constructed H9.2.1 -The sequence of IL-15-M7CRin is shown in SEQ ID NO: 135.
  • the sequence of the constructed H9.2.1in-IL-15-M7CR is shown in SEQ ID NO: 134.
  • the constructed H9.2.1-IL- The sequence of 15-M7CR is shown in SEQ ID NO: 136, and the sequence of the constructed H9.2.1-28-IL-15-M7CR is shown in SEQ ID NO: 137.
  • ECD includes tCD19 (SEQ ID NO: 17), IL-15/IL-15R ⁇ (Sushi) (SEQ ID NO: 48), IL-12-P70 (SEQ ID NO: 49); the above differences ECD and M7Rm8 are connected to form tCD19-M7CR (SEQ ID NO: 171), IL-12-M7CR (SEQ ID NO: 172), and IL-15-M7CR (SEQ ID NO: 173).
  • control molecules were designed, including IL-15-M7CRin (SEQ ID NO: 180) with missing intracellular signal of M7Rm8, H9.2.1-218in CAR (SEQ ID NO: 186) with missing intracellular signal, and secreted sIL-15. H9.2.1-sIL-15 (SEQ ID NO: 139).
  • H9.2.1 CAR (SEQ ID NO: 100), H9.2.1-218 CAR (SEQ ID NO: 144), H9.2.1-28 CAR (SEQ ID NO: 142), H9.2.1-tCD19-M7CR (SEQ ID NO:101), H9.2.1-IL-15-M7CRin (SEQ ID NO:135), H9.2.1in-IL-15-M7CR (SEQ ID NO:134), H9.2.1-IL-15-M7CR (SEQ ID NO:136), H9.2.1-sIL-15 (SEQ ID NO:139), H9.2.1-IL-12-M7CR (SEQ ID NO:104), H9 .2.1-28-IL-15-M7CR (SEQ ID NO:137) and control 8E5 CAR (SEQ ID NO:188).
  • the synthesized DNA fragment was inserted into the pRKN lentiviral expression vector (Genewise Company) downstream of the EF1 ⁇ promoter, and the EGFR sequence in the original vector was replaced to obtain the corresponding expression plasmid (synthesized by Genewise Company).
  • Example 12.2. Obtaining T cells and lentiviral transduction
  • the lentivirus preparation method is the same as Example 4.2, and the preparation method of CAR-T or M7CR-modified CAR-T cells is the same as Example 4.3.
  • Figure 22 shows the statistical histogram of amplification folds on the 9th day of preparation of CAR-T or M7CR-modified CAR-T cells. It can be seen that T cells derived from different donors The amplification status of each CAR-T prepared from the cells is different, and the overall amplification factor is between about 20-80 times, which meets the experimental needs.
  • Figure 23A and Figure 23B show the expression of CAR or M7CR in the prepared CAR-T cells.
  • Figure 23A is a representative flow cytometry scatter plot
  • Figure 23B is a statistical histogram of CAR-positive cells.
  • the proportion of CAR + cells in the H9.2.1 group was approximately 56%, while H9.2.1-tCD19-M7CR, H9.2.1-IL-15-M7CRin, H9.2.1 in-IL-15-M7CR, H9.2.1-IL-15-M7CR, H9.2.1-28-IL-15-M7CR, H9.2.1-sIL-15, H9.2.1-IL12-M7CR, 8E5 group
  • the proportions of CAR + cells were: 38.6%, 7.99%, 24.2%, 26.1%, 34.5%, 17.6%, 3.17%, 59.8%.
  • Figure 23C and Figure 23D show that after using donor 15's T cells to transduce H9.1.2 CAR, the proportions of CD4 and CD8 positive cells in cells expressing H9.1.2 CAR were 38.4% and 55.8% respectively; in H9.2.1- tCD19-M7CR, H9.2.1-IL-15-M7CRin, H9.2.1in-IL-15-M7CR, H9.2.1-IL-15-M7CR, H9.2.1-28-IL-15-M7CR, H9.2.1 The proportions of CD4 + cells in -sIL-15, H9.2.1-IL12-M7CR, and 8E5 groups were: 37.8%, 31.4%, 37.1%, 38.3%, 39.4%, 29.7%, 53.6%, and 41.3% respectively; CD8 + The cell proportions were: 56.5%, 63.9%, 59.1%, 57.3%, 56.3%, 64.9%, 40.2% and 55%.
  • the proportions of CD4 and CD8 positive cells in cells expressing H9.1.2 CAR were 18.6% and 73.6% respectively; in H9.2.1-tCD19-M7CR, H9.2.1 -IL-15-M7CRin, H9.2.1in-IL-15-M7CR, H9.2.1-IL-15-M7CR, H9.2.1-28-IL-15-M7CR, H9.2.1-sIL-15, H9.
  • the proportions of CD4 + cells in the 2.1-IL12-M7CR and 8E5 groups were: 18.9%, 19%, 24.4%, 23.5%, 27.2%, 18%, 29.7% and 22.7% respectively; the proportions of CD8 + cells were: 72.5% respectively. , 72.7%, 68.3%, 67.5%, 63.8%, 73%, 64.2% and 72%.
  • CD45RA + CCR7 + represents initial T cells or stem memory T cells (TN/TSCM)
  • CD45RA-CCR7 + Represents central memory T cells (TCM)
  • CD45RA - CCR7 - represents effector memory T cells (TEM)
  • CD45RA + CCR7 - represents effector T cells (Teff) subsets
  • most CAR-T cells are TN/TSCM, TCM cell.
  • the expression level of intracellular p-STAT5 was detected by FACS to study the activation of STAT5 signal.
  • the experimental steps are as follows. Take 1E6 CAR-T cells, wash them once with PBS, and resuspend them in serum-free RPMI1640 medium overnight. The next day, cells were taken out and washed once with FACS buffer. Then add AF647-p-STAT5 (BD, catalog number 562076) antibody for intracellular staining. The specific steps are the same as Example 2.
  • H9.2.1 CAR-T cells 8E5 CAR-T cells or H9.2.1 CAR-T cells with different M7CR modifications were compared with PANC-1 with low expression in CLDN18.2 or HUP-T4 with high expression in CLDN18.2 in E:T Co-incubate culture at 1:1. After 24 hours, 100 ⁇ L of supernatant was collected for subsequent cytokine detection experiments (Example 14).
  • T cells upregulate CD25 and CD69 expression after activation, so CD25 + CD69 + represents a population of activated T cells.
  • CD25 + CD69 + represents a population of activated T cells.
  • H9.2.1-IL-15-M7CR, H9.2.1-28-IL-15-M7CR, and H9.2.1-IL12-M7CR significantly increased activation of H9.2.1 CAR-T cells compared with H9.2.1, indicating that IL-15-M7CR, IL -12-M7CR can promote the activation of CAR-T after low antigen stimulation.
  • H9.2.1-tCD19-M7CR H9.2.1-IL-15-M7CRin, H9.2.1in-IL-15-M7CR, H9.2.1-IL-
  • the proportions of CD25 + CD69 + cells in the 15-M7CR, H9.2.1-28-IL-15-M7CR, H9.2.1-sIL-15, H9.2.1-IL12-M7CR, and 8E5 groups were: 19.9% and 14.1% respectively. , 2.08%, 1.49%, 16.8%, 24.6%, 8.93%, 34.1% and 27.5%.
  • H9.2.1-IL12-M7CR significantly increased the activation of CAR-T cells compared with H9.2.1, indicating that IL-12-M7CR can promote the activation of CAR-T after medium and high antigen stimulation.
  • Example 13 The cell culture supernatant collected in Example 13 was used to detect cytokines using BD TM Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit II.
  • CBA Cytometric Bead Array
  • the experimental procedures were the same as those in Example 8.
  • the levels of IL-2, IFN- ⁇ and TNF ⁇ cytokines in the supernatant after CAR-T activation were detected by CBA.
  • the results are shown in Figures 25A and 25B.
  • CAR-T in each group increased cytokine secretion after co-incubation with HUP-T4 for 24 hours.
  • CAR-T cells derived from donor 15 and donor 17 cells expressing the same CAR molecule showed similar amounts of cytokine secretion after co-incubation with tumor cells, indicating high reproducibility of the experiment.
  • H9.2.1-IL-15-M7CR H9.2.1-28-IL-15-M7CR
  • H9.2.1-IL12-M7CR CAR- The secretion of IL-2, IFN- ⁇ and TNF ⁇ by T cells increased significantly, indicating that IL-15-M7CR and IL-12-M7CR can promote the secretion of effector cytokines by CAR-T cells.
  • H9.2.1in-IL-15-M7CR CAR-T cells secrete very few cytokines, indicating that IL-15-M7CR increases CAR-T cytokine secretion dependent on CAR signaling.
  • NT cells were first used to adjust the positive rate of each CAR-T to be consistent, and then the CAR-T cells were labeled with CTV (Thermo, C34557), and the CTV-labeled CAR-T cells were compared with CLDN18.2 low PANC-1-expressing cells and CLDN18.2 high-expressing SNU620 cells were co-incubated and cultured according to 1:1E:T. The medium was changed every 2 days. The cells were harvested after 5 days, washed once with FACS buffer (PBS+2% FBS), and resuspended.
  • CTV Thermo, C34557
  • FACS buffer containing LIVE/DEAD Fixable Dead Cell Stain (Thermo, L34965) and Biotin-SP-conjugated AffiniPure F(ab)2Fragment Goat Anti-human IgG, F(ab)2fragment specific, stain for 30 minutes at room temperature, and wash for two Second, APC-Strep (Biolegend, 405207) antibody was used as the second staining reagent for detection, staining at 4°C for 30 to 45 minutes; cells were washed twice and resuspended in FACS buffer, and detected with a flow cytometer.
  • mice were selected, and NUGC-4-Gluc cells were intraperitoneally injected into the mice on Day-7 to create a model.
  • the mouse modeling was detected through a small animal in vivo imaging system.
  • the tumor load was 1 ⁇ 10 9 p/
  • the animals were divided into groups (5 in each group), and 1 ⁇ 10 6 , 1 ⁇ 10 5 , and 1 ⁇ 10 4 H9.2.1-IL-15-M7CR CAR-T cells or H9 were administered to the mice via tail vein injection.
  • .2.1-CD28-IL-15-M7CR CAR-T cells control mice were given NT cells (the dose was consistent with the total number of T cells in the 1 ⁇ 10 6 CAR-T cell group).
  • the tumor burden of mice was detected through weekly imaging, and peripheral blood was collected weekly to detect CAR-T amplification in vivo.
  • H9.2.1-IL-15-M7CR CAR-T cells or H9.2.1-CD28-IL-15-M7CR CAR-T cells produced significant anti-tumor effects after infusion 7 and showed dose dependent.
  • the body weight of the mice treated in the high-dose group decreased significantly 2 weeks after CAR-T reinfusion. It is speculated that the expression of CLDN18.2 in the normal gastric epithelial cells of the mice resulted in "on-target" toxicity.
  • H9.2.1-IL-15-M7CR and H9.2.1-CD28-IL-15-M7CR CAR-T cells significantly expanded in mice in a dose-dependent manner.
  • cytokine-M7CR modified H9.2.1 CAR As shown in Figure 28, different cytokines are directly connected as extracellular domains (ECD) and M7Rm8 (SEQ ID NO: 34) to construct a constitutive chimeric cytokine receptor M7CRm8; then the N-terminus of M7CRm8 is connected to H9 through P2A .2.1 The C-terminus of the CAR polypeptide is connected to form the cytokine-M7CR modified H9.2.1 CAR.
  • ECD extracellular domains
  • M7Rm8 SEQ ID NO: 34
  • H9.2.1 CAR has the amino acid sequence shown in SEQ ID NO: 100; H9.2.1-tCD19-M7CR has the amino acid sequence shown in SEQ ID NO: 101; H9.2.1-IL-18-M7CR has SEQ ID NO: 148 The amino acid sequence shown; H9.2.1-IL-9-M7CR has the amino acid sequence shown in SEQ ID NO: 149; H9.2.1-IL-36-M7CR has the amino acid sequence shown in SEQ ID NO: 150; H9.
  • 2.1-IL-23-M7CR has the amino acid sequence shown in SEQ ID NO: 151; H9.2.1-IL12-p40-M7CR has the amino acid sequence shown in SEQ ID NO: 152; H9.2.1-IL-12-M7CR has The amino acid sequence shown in SEQ ID NO: 104; H9.2.1-218 CAR has the amino acid sequence shown in SEQ ID NO: 144.
  • the cytokine ECD located at the N-terminus of M7Rm8 includes but is not limited to IL-18 (SEQ ID NO: 53), IL-9 (SEQ ID NO: 52), IL-36 ⁇ (SEQ ID NO: 55) , IL-23 (SEQ ID NO: 54), IL-12-P70 (SEQ ID NO: 49), IL-12-P40 (SEQ ID NO: 50).
  • IL18-M7CR SEQ ID NO: 177
  • IL9-M7CR SEQ ID NO: 176
  • IL36 ⁇ -M7CR SEQ ID NO: 179
  • IL36- M7CR IL23-M7CR
  • IL12-M7CR SEQ ID NO: 172
  • IL12-p40-M7CR SEQ ID NO: 175
  • H9.2.1-CAR (SEQ ID NO:100), H9.2.1-tCD19-M7CR (SEQ ID NO:101), H9.2.1-IL8-M7CR (SEQ ID NO :148), H9.2.1-IL9-M7CR (SEQ ID NO:149), H9.2.1-IL36-M7CR (SEQ ID NO:150), H9.2.1-IL23-M7CR (SEQ ID NO:151), H9 .2.1-p40-M7CR (SEQ ID NO:152), H9.2.1-IL12-M7CR) (SEQ ID NO:104), H9.2.1-218-CAR (SEQ ID NO:153).
  • H9.2.1-IL-18-M7CR molecule (SEQ ID NO:148) includes the H9.2.1CAR molecule (SEQ ID NO:100), P2A (SEQ ID NO: 3), SP (SEQ ID NO: 2) and IL-18-M7CR (SEQ ID NO: 177).
  • the H9.2.1-IL-9-M7CR molecule (SEQ ID NO: 149) includes the H9.2.1 CAR molecule (SEQ ID NO: 100), P2A (SEQ ID NO: 3), SP (SEQ ID NO: 2) and IL-9-M7CR (SEQ ID NO: 176).
  • the H9.2.1-IL-36-M7CR molecule includes the H9.2.1 CAR molecule (SEQ ID NO:100), P2A (SEQ ID NO:3), SP (SEQ ID NO: 2) and IL36 ⁇ -M7CR (SEQ ID NO: 179).
  • the H9.2.1-IL-23-M7CR molecule includes the H9.2.1 CAR molecule (SEQ ID NO:100), P2A (SEQ ID NO:3), SP (SEQ ID NO: 2) and IL-23-M7CR (SEQ ID NO: 178).
  • the H9.2.1-IL12-p40-M7CR molecule includes the H9.2.1 CAR molecule (SEQ ID NO:100), P2A (SEQ ID NO:3), SP (SEQ ID NO: 2) and IL-12p40-M7CR (SEQ ID NO: 175); the H9.2.1-IL-12-M7CR molecule (SEQ ID NO: 104) contains the H9.2.1-BB-L CAR molecules (SEQ ID NO:100), P2A (SEQ ID NO:3), SP (SEQ ID NO:2) and IL-12-M7CR (SEQ ID NO:172).
  • M7CR molecules include GM-CSFR ⁇ -SP (SEQ ID NO: 2), cytokine ECD and M7R from the N-terminus to the C-terminus (hereinafter, the related construction of this embodiment body, IL7Rm8) shown in SEQ ID NO: 34 is exemplified as M7R.
  • the above-mentioned synthesized DNA fragment was inserted into the pRKN lentiviral expression vector (Genewise Company) downstream of the EF1 ⁇ promoter, and the EGFR sequence in the original vector was replaced to obtain the corresponding expression plasmid (synthesized by Genewise Company).
  • Example 18.2. Obtaining T cells and lentiviral transduction
  • the lentivirus preparation method is the same as Example 4.2, and the CAR-T preparation method is the same as Example 4.3.
  • T cells from donor 16 and donors 6, 11, and 17, Figure 29A and Figure 29B show the expansion of the prepared CAR-T cells from day 1 to day 9, and Figure 29C shows donor 6,
  • the statistical histogram of the expansion fold of CAR-T cells from 11,17 on the 9th day of preparation shows that there are differences in the expansion of CAR-T cells prepared from T cells from different donors.
  • the overall expansion fold is between 20 and 20 -80 times to meet the needs.
  • Example 18.2 Take an appropriate amount of CAR-T cells obtained from the above Example 18.2.
  • the staining procedure is as shown in Example 4.4. After the staining is completed, the cells are washed twice and resuspended in FACS buffer, and detected with a flow cytometer.
  • Figure 30A and Figure 30B show the cytokine expression of CAR and/or ECD on days 7 and 9 after CAR-T cells derived from donor 16.
  • CAR polypeptide expression was detected by FACS on day 9. The CAR polypeptide could be expressed in all CAR-T cells.
  • T cells from donor 16 the proportion of CAR + cells in the H9.2.1 group was approximately 39.8%, and H9.2.1- The proportion of CAR + cells in the 218 group was approximately 41.2%. It can be seen that the selection of connectors G4SLinker and 218Linker has little impact on the CAR + rate.
  • H9.2.1-IL18-M7CR, H9.2.1-IL9-M7CR, H9.2.1-IL23-M7CR, H9.2.1-p40-M7CR The proportions of CAR+ cells in the H9.2.1-IL36-M7CR group were: 20.2%, 19.5%, 13.6%, 19.0%, and 21.1% respectively.
  • the expression of extracellular cytokines in H9.2.1-IL23-M7CR and H9.2.1-p40-M7CR cells was also detected using PE-labeled IL-12P40 antibody.
  • Figure 30C and Figure 30D show the CAR expression on day 9 after CAR-T cells derived from donors 6, 11, and 17.
  • the proportion of CAR + cells in the H9.2.1 group was approximately 54.6%, while H9.2.1-tCD19-M7CR, H9.2.1-IL12-M7CR, H9.2.1-
  • the proportions of CAR+ cells in the IL9-M7CR, H9.2.1-IL18-M7CR, H9.2.1-IL23-M7CR, H9.2.1-IL36-M7CR, and H9.2.1-p40-M7CR groups were: 28.5% and 14.7% respectively. , 27.3%, 25.3%, 5.84%, 13.7%, 14.3%.
  • the differentiation phenotype of CAR-T cells derived from donors 6, 11, and 17 on day 9 of preparation was detected, and compared with NT, H9.2.1 and H9.2.1-IL12-M7CR, H9.2.1- tCD19-M7CR, H9.2.1-IL18-M7CR, H9.2.1-IL9-M7CR, H9.2.1-IL23-M7CR, H9.2.1-p40-M7CR, H9.2.1-IL36-M7CR maintain a higher ratio of TN/TSCM cell.
  • the differentiation phenotype of CAR-T cells derived from donors 6, 11, and 17 on day 9 of preparation was detected, and compared with NT, H9.2.1 and H9.2.1-IL12-M7CR, H9.2.1- tCD19-M7CR, H9.2.1-IL18-M7CR, H9.2.1-IL9-M7CR, H9.2.1-IL23-M7CR, H9.2.1-p40-M7CR, H9.2.1-IL36-M7CR maintained higher CD45RA expression.
  • a viral expression plasmid was constructed for expressing chimeric receptor M7CR containing different signal strengths.
  • tCD19 and IL7R mutations constitute the chimeric receptor tCD19-M7CR
  • tCD34 and IL7R mutations constitute the chimeric receptor tCD34-M7CR.
  • tCD19-M7CR or tCD34-M7CR It consists of an extracellular domain (ECD) composed of the same tCD19 (SEQ ID NO:17) or tCD34 (SEQ ID NO:189) and different IL7R mutants (also called IL7Rm or M7R in the text).
  • the four selected IL7Rm Able to deliver signals of different strengths (see Example 4.5).
  • the N-terminus of tCD19-M7CR with different signal strengths is connected to the C-terminus of the H9.2.CAR polypeptide through P2A to form a tCD19-M7CR modified H9.2.1 CAR.
  • the N-terminus of tCD34-M7CR with different signal strengths is connected to the C-terminus of BB2121 CAR polypeptide through P2A to form a tCD34-M7CR modified BB2121CAR.
  • the four IL7Rm are IL7Rm5 (SEQ ID NO:31), IL7Rm7 (SEQ ID NO:33), IL7Rm8 (SEQ ID NO:34), IL7Rm18 (SEQ ID NO:44), and their corresponding tCD19-M7CR
  • tCD19-M7CR(7)(SEQ ID NO:191) tCD19-M7CR(8)
  • tCD19-M7CR SEQ ID NO:171 in the text
  • tCD19-M7CR(CPT) were used as positive controls.
  • tCD34-M7CR The corresponding tCD34-M7CR are named tCD34-M7CR(5)(SEQ ID NO:194), tCD34-M7CR(7)(SEQ ID NO:195), tCD34-M7CR(8)(SEQ ID NO: 196), tCD34-M7CR(18) (SEQ ID NO:197), tCD34-M7CR(CPT) as a positive control (SEQ ID NO:193).
  • H9.2.1CAR (SEQ ID NO:100), tCD19-M7CR (CPT) (SEQ ID NO:154), H9.2.1-tCD19-M7CR (5) (SEQ ID NO:155), H9.2.1-tCD19-M7CR(7)(SEQ ID NO:156), H9.2.1-tCD19-M7CR(8)(SEQ ID NO:101), H9.2.1-tCD19-M7CR(18 ))(SEQ ID NO:157), BB2121-CAR(SEQ ID NO:158), BB2121-tCD34-M7CR(CPT)(SEQ ID NO:159), BB2121-tCD34-M7CR(5))(SEQ ID NO :160), BB2121-tCD34-M7CR(7)(SEQ ID NO:161), BB2121-tCD34-M7CR(8)(SEQ ID NO:162), BB2121-tCD34-M7CR(18)(SEQ ID NO:
  • H9.2.1 CAR molecule SEQ ID NO: 100
  • BB2121-CAR molecule SEQ ID NO: 158 polypeptides are connected to the M7CR molecule through P2A (SEQ ID NO: 3) respectively.
  • the H9.2.1-tCD19-M7CR (CPT) molecule (SEQ ID NO: 154) includes the H9.2.1 CAR molecule (SEQ ID NO: 100) and P2A (SEQ ID NO: 3) from the N-terminus to the C-terminus. , SP (SEQ ID NO: 2) and tCD19-M7CR (CPT) (SEQ ID NO: 182).
  • the H9.2.1-tCD19-M7CR(5) molecule (SEQ ID NO:155) includes the H9.2.1 CAR molecule (SEQ ID NO:100) and P2A (SEQ ID NO:3) from the N-terminus to the C-terminus. , SP (SEQ ID NO:2) and tCD19-M7CR(5) (SEQ ID NO:190).
  • the H9.2.1-tCD19-M7CR (7) molecule (SEQ ID NO: 156) includes the H9.2.1 CAR molecule (SEQ ID NO: 100) and P2A (SEQ ID NO: 3) from the N end to the C end. , SP (SEQ ID NO:2) and tCD19-M7CR(7) (SEQ ID NO:191).
  • the H9.2.1-tCD19-M7CR (8) molecule (SEQ ID NO: 101) includes the H9.2.1 CAR molecule (SEQ ID NO: 100) and P2A (SEQ ID NO: 3) from the N end to the C end. , SP (SEQ ID NO:2) and tCD19-M7CR(8) (SEQ ID NO:171).
  • the H9.2.1-tCD19-M7CR (18) molecule includes the H9.2.1 CAR molecule (SEQ ID NO: 100) and P2A (SEQ ID NO: 3) from the N end to the C end. , SP (SEQ ID NO:2) and tCD19-M7CR(18) (SEQ ID NO:192).
  • the BB2121-tCD34-M7CR (CPT) molecule (SEQ ID NO: 159) includes the BB2121CAR molecule (SEQ ID NO: 158), P2A (SEQ ID NO: 3), SP (SEQ ID NO: 3) from the N end to the C end. NO: 2) and tCD34-M7CR(CPT) (SEQ ID NO: 193).
  • the BB2121-tCD34-M7CR(5) molecule (SEQ ID NO:160) includes the BB2121 CAR molecule (SEQ ID NO:158), P2A (SEQ ID NO:3), SP (SEQ ID NO: 2) and tCD34-M7CR(5) (SEQ ID NO: 194).
  • the BB2121-tCD34-M7CR(7) molecule includes the BB2121 CAR molecule (SEQ ID NO:158), P2A (SEQ ID NO:3), SP (SEQ ID NO: 2) and tCD34-M7CR(7) (SEQ ID NO: 195).
  • the BB2121-tCD34-M7CR(8) molecule includes the BB2121 CAR molecule (SEQ ID NO:158), P2A (SEQ ID NO:3), SP (SEQ ID NO: 2) and tCD34-M7CR(8) (SEQ ID NO: 196).
  • the BB2121-tCD34-M7CR (18) molecule includes the BB2121 CAR molecule (SEQ ID NO: 158), P2A (SEQ ID NO: 3), SP (SEQ ID NO: 2) and tCD34-M7CR(18) (SEQ ID NO: 197).
  • all M7CR molecules contain GM-CSFR ⁇ -SP (SEQ ID NO: 2), tCD19 (SEQ ID NO: 17) or tCD34 (SEQ ID NO:) from N-terminus to C-terminus. 189) and different IL7R mutants.
  • the above-mentioned synthesized DNA fragment was inserted into the downstream of the EF1 ⁇ promoter of the pCKW lentiviral expression vector (Genewise Company), and the EGFR sequence in the original vector was replaced to obtain the corresponding expression plasmid (synthesized by Genewise Company).
  • the lentivirus preparation method is the same as Example 4.2, and the CAR-T preparation method is the same as Example 4.3.
  • Figure 32 shows the expansion kinetics of CAR-T cells derived from donor 15 from day 1 to day 9. The overall expansion of each CAR-T is close, and the expansion fold when harvested on day 9 is approximately 90-120 times.
  • Figure 40A shows the expansion kinetics of CAR-T cells derived from donors 13, 16, and 17 from day 1 to day 9.
  • Figure 40B shows the expansion fold at harvest on day 9. The results show that, except for M7CR(5), Other M7CR modifications promote CAR-T cell expansion in vitro.
  • Example 20.3. CAR expression detection and CAR-T cell phenotype detection
  • FIGS. 33A and 33B show the CAR expression levels of donor 15-derived CAR-T cells. CAR molecules can be expressed in all CAR-T cells.
  • the proportion of CAR+ cells in the H9.2.1 group is approximately 53.5%, while H9.2.1-tCD19-M7CR(CPT), H9.2.1-tCD19-M7CR(5), H9
  • the proportions of CAR+ cells in the .2.1-tCD19-M7CR(7), H9.2.1-tCD19-M7CR(8), and H9.2.1-tCD19-M7CR(18) groups were: 26.6%, 24.9%, 29%, 28.7%, 30.8%.
  • the proportion of CAR+ cells in BB2121 is approximately 83.7%, while BB2121-tCD19-M7CR(CPT), BB2121-tCD19-M7CR(5), BB2121-tCD19-M7CR(7), BB2121-tCD19-M7CR(8), BB2121- The proportions of CAR+ cells in tCD19-M7CR(18) were: 79.4%, 80.4%, 82.5%, 82.2%, and 85.7% respectively.
  • Figure 33C is a statistical histogram.
  • Figures 41A and 41B show donors 13, 16, The expression levels of CAR and CD19 in CAR-T cells from 17 sources.
  • the positive rate of CAR expression in H9.2.1 and other M7CR-modified CAR-T cells is between 20-40%.
  • the CAR in M7CR-modified CAR-T cells The expression is more consistent with CD19.
  • Figure 34A and Figure 34B show representative flow cytometry diagrams of CD4 and CD8 cell subsets in CAR-T cells derived from H9.2.1 and BB2121 derived from donor 15 and each M7CR, respectively. As shown in Figure 34C, the proportions of CD4 and CD8 cell subsets among each CAR-T cell were close.
  • Figure 42A shows representative flow cytometry diagrams of CD4 and CD8 cell subsets in CAR-T cells derived from donors 13, 16, and 17. As shown in Figure 42B, the proportions of CD4 and CD8 cell subsets among each CAR-T cell were close.
  • Figure 35A and Figure 35B respectively show representative flow cytometry diagrams of CD45RA and CCR7 expression in H9.2.1 and BB2121 derived from donor 15 and each M7CR modified CAR-T cell.
  • H9.2.1, H9.2.1-tCD19-M7CR(CPT), H9.2.1-tCD19-M7CR(5), H9.2.1-tCD19-M7CR(7), H9.2.1-tCD19-M7CR (8) and the proportions of TN/TSCM cells in H9.2.1-tCD19-M7CR (18) were: 57.3%, 72.2%, 68.7%, 68.8%, 68.3%, and 71.4% respectively.
  • the proportions of TN/TSCM cells in CAR-T cells are: 78.3%, 84.3%, 81.8%, 82.9%, 82.5%, 85.5% respectively.
  • M7CR-modified CAR-T cells maintained a higher proportion of CD45RA + CCR7 + TN/TSCM memory cells.
  • Figure 35C shows a statistical diagram of the proportion of CD45RA+CCR7+ cells derived from H9.2.1 and BB2121 from donor 15 and each M7CR modified CAR-T cell.
  • Figure 43A shows representative flow cytometry diagrams of CD45RA and CCR7 expression in H9.2.1 and each M7CR modified CAR-T cells derived from donors 13, 16, and 17.
  • the proportions of CD45RA + CCR7 + TN/TSCM memory cells in CAR-T cells are approximately: 80.0%, 85.7%, 83.7%, 86.7%, 81.6%, 83.4 respectively %.
  • each M7CR-modified CAR-T cell maintained a higher proportion of CD45RA + CCR7 + TN/TSCM memory cells.
  • Figure 36A and Figure 36B respectively show the expression of intracellular phosphorylated STAT5 (pSTAT5) in CAR-T cells on the 5th day of preparation and after cryopreservation and recovery, compared with NT or H9.2.1 CAR-T cells, BB2121 CAR- Comparing T cells, each M7CR-modified CAR-T cell had a higher pSTAT5 expression level, indicating that M7CR modification activated STAT5 signaling in CAR-T cells.
  • the M7CR order is: M7CR(8) ⁇ M7CR(7)>M7CR(5)>M7CR(18).
  • Example 21 Activation experiment of M7CR-modified CAR-T cells with different signal intensities
  • H9.2.1 CAR-T cells and each M7CR-modified CAR-T cell were co-incubated with PANC1 or NUGC-4 tumor cells when E:T was 1:1. After 24 h, 100 ⁇ L of supernatant was collected for cytokine detection. The cells were harvested and stained for CD25 and CD69. See Example 13 for cell staining methods and procedures.
  • Figure 37A shows representative flow cytometry diagrams of CD25 and CD69 expression after H9.2.1 CAR-T cells derived from donor 15 and each M7CR-modified CAR-T cell were cultured with NUGC-4 tumor cells for 24 hours.
  • Figure 37B shows the statistical results. Compared with NT, H9.2.1 CAR-T cells were significantly activated after incubation with NUGC-4 for 24 hours. Compared with H9.2.1 CAR-T cells, the proportion of CD25+CD69 + cells in each M7CR-modified CAR-T cell increased, and the intensity of the increase was consistent with the above-mentioned M7CR-activated STAT5 signal intensity, indicating that M7CR promotes CAR-T by activating STAT5 signal Activation of cells following antigen stimulation.
  • Figure 44A shows the statistical chart of the proportion of CD25 + CD69 + cells in CAR + or CAR - cells after H9.2.1 CAR-T cells derived from donors 13, 16, and 17 and each M7CR-modified CAR-T cell were cultured with PANC1 tumor cells for 24 hours. .
  • the proportion of CD25+CD69 + cells in each M7CR-modified CAR + cell increased, while the proportion of CD25+CD69 + cells in CAR- cells did not increase significantly, indicating that M7CR expression increased the low antigen CAR-T cell activation after stimulation of expressing target cells.
  • Figure 44B shows the proportion of CD25 + CD69 + cells in CAR + or CAR - cells after H9.2.1 CAR-T cells derived from donors 13, 16, and 17 and each M7CR-modified CAR-T cell were cultured with NUGC-4 tumor cells for 24 hours. summary graph. Compared with H9.2.1 CAR-T cells, except for M7CR(5), the proportion of CD25+CD69 + cells in each M7CR-modified CAR + cell increased; while the proportion of CD25+CD69 + cells in CAR - cells did not increase significantly, indicating that M7CR expression improves CAR-T cell activation after stimulation of medium and high antigen-expressing target cells.
  • Example 22 M7CR modified CAR-T killing activity with different signal intensities
  • Cytotoxicity % (luminescence of NT group – luminescence of test group)/luminescence of NT group
  • Example 21 The cell culture supernatant harvested in Example 21 was used to detect cytokines using BD TM Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit II. The experimental procedures are shown in Example 8.
  • CBA Cytometric Bead Array
  • Figure 39 shows the levels of IL-2, IFN- ⁇ and TNF ⁇ cytokines in the supernatant after tumor cells activated CAR-T cells for 24 hours.
  • the results showed that compared with NT, the concentrations of IL-2, IFN- ⁇ , and TNF- ⁇ in the supernatant of CAR-T cells in each group increased significantly after incubation with NUGC-4 for 24 hours.
  • M7CR-modified CAR-T cells secreted IL-2, IFN- ⁇ , and TNF- ⁇ cytokines significantly increased, indicating that M7CR modification promotes CAR-T cells to secrete effector cytokines.
  • Example 24 In vivo efficacy experiment of H9.2.1M7CR-CAR-T with different signal intensities
  • mice were selected, and NUGC-4-Gluc cells (1 ⁇ 10 6 NUGC-4-Gluc cells per mouse) were injected intraperitoneally on Day-5 to create a model.
  • the mice were modeled through a small animal in vivo imaging system. The model condition was tested, and the animals were divided into groups (3 animals in each group) when the tumor burden was 1 ⁇ 10 9 p/s on Day 0.
  • Each mouse was then given 5 ⁇ 10 5 CAR-T cells (from donor 15) via intraperitoneal injection, and control mice were given NT cells (dose related to maximum total T cells The number of cells is the same). Mice burden, and the number of CAR-T cells in peripheral blood, were imaged weekly.
  • Example 25 In vivo efficacy experiment of BB2121M7CR-CAR-T
  • mice were selected, and H929 myeloma cells (2 ⁇ 10 6 H929 myeloma cells per mouse) were subcutaneously injected into the mice on Day-9 for modeling.
  • the tumor volume was 100-200mm 3 , and the animals were subjected to Randomly group (5 animals per group).
  • Each mouse was then given 1 ⁇ 10 6 BB2121 CAR-T cells or M7CR(8)-modified CAR-T cells (from donor 17) via intravenous injection.
  • the tumor volume of mice was measured weekly, and the number of CAR-T cells in peripheral blood was detected.
  • a viral expression plasmid was constructed for expressing the chimeric receptor TGF ⁇ RII-containing TGF ⁇ RII extracellular domain (ECD) (SEQ ID NO: 198) and IL7R mutant (also called IL7Rm or M7R in the text). M7CR.
  • IL7Rm7 SEQ ID NO: 33 was selected to construct TGF ⁇ RII-M7CR(7) (SEQ ID NO: 200).
  • tCD19-M7CR SEQ ID NO:191
  • dominant negative TGF ⁇ RII mutant also called dnTGF ⁇ RII in the article
  • SEQ ID NO:199 TGF ⁇ RII ECD
  • CD28 CSD SEQ ID NO:143
  • TGF ⁇ RII-CD28 SEQ ID NO:201
  • TGF ⁇ RII ECD SEQ ID NO:198
  • 4-1BB CSD SEQ ID NO:11
  • chimeric receptors such as TGF ⁇ RII-M7CR(7), dnTGF ⁇ RII, TGF ⁇ RII-CD28 and TGF ⁇ RII-41BB is connected to the C-terminus of H9.2.1 or H9.2.1-28 CAR polypeptide through P2A to form chimeric receptors such as TGF ⁇ R-M7CR. receptor-modified CAR.
  • the sequence of the constructed H9.2.1 CAR is shown in SEQ ID NO: 100
  • the sequence of the constructed H9.2.1-TGF ⁇ RII-M7CR(7) is shown in SEQ ID NO: 164
  • the sequence of dnTGF ⁇ RII is shown in SEQ ID NO: 165
  • the sequence of the constructed H9.2.1-TGF ⁇ RII-CD28 is shown in SEQ ID NO: 166
  • the sequence of the constructed H9.2.1-28 CAR is shown in SEQ ID NO: 142
  • the sequence of the constructed H9.2.1-28-tCD19-M7CR(7) is shown in SEQ ID NO: 167
  • the sequence of the constructed H9.2.1-28-TGF ⁇ RII-M7CR(7) is shown in SEQ ID NO. : 168
  • the constructed sequence of H9.2.1-28-dnTGF ⁇ RII is shown in SEQ ID NO: 169
  • the constructed sequence of H9.2.1-28-TGF ⁇ RII-BB is shown in SEQ
  • the DNA sequences are H9.2.1 CAR (SEQ ID NO: 100), H9.2.1-tCD19-M7CR(7)(SEQ ID NO:156), H9.2.1-dnTGF ⁇ RII-M7CR(7)(SEQ ID NO:164), H9.2.1-dnTGF ⁇ RII(SEQ ID NO:165), H9 .2.1-dnTGF ⁇ RII-CD28 (SEQ ID NO:166), H9.2.1-28-CAR (SEQ ID NO:142), H9.2.1-28-tCD19-M7CR(7) (SEQ ID NO:167), H9 .2.1-28-dnTGF ⁇ RII-M7CR(7) (SEQ ID NO:168), H9.2.1-28-dnTGF ⁇ RII) (SEQ ID NO:169), H9.2.1-28-dnTGF ⁇ RII-BB (SEQ ID NO:170 )
  • the H9.2.1-tCD19-M7CR(7) molecule includes the H9.2.1 CAR molecule (SEQ ID NO:100), P2A (SEQ ID NO:3) from N-terminus to C-terminus , SP (SEQ ID NO: 2) and tCD19-M7CR (7) (SEQ ID NO: 191).
  • the H9.2.1-TGF ⁇ RII-M7CR(7) molecule contains the H9.2.1 CAR molecule (SEQ ID NO:100), "RAKR" sequence, P2A (SEQ ID NO: 3) and TGF ⁇ RII-M7CR (7) (SEQ ID NO: 200).
  • the H9.2.1-dnTGF ⁇ RII molecule (SEQ ID NO: 165) includes the H9.2.1 CAR molecule (SEQ ID NO: 100), the "RAKR” sequence, and P2A (SEQ ID NO: 3) from the N-terminus to the C-terminus. and dnTGF ⁇ RII (SEQ ID NO:199).
  • the H9.2.1-TGF ⁇ RII-28 molecule (SEQ ID NO:166) includes the H9.2.1 CAR molecule (SEQ ID NO:100), "RAKR” sequence, P2A (SEQ ID NO: 3) and TGF ⁇ RII-CD28 (SEQ ID NO: 201).
  • the H9.2.1-28-tCD19-M7CR(7) molecule includes the H9.2.1-28CAR molecule (SEQ ID NO:142), "RAKR" sequence, P2A (SEQ ID NO: 3), SP (SEQ ID NO: 2), and tCD19-M7CR(7) (SEQ ID NO: 191).
  • the H9.2.1-28-TGF ⁇ RII-M7CR(7) molecule includes the H9.2.1-28 CAR molecule (SEQ ID NO:142) and the "RAKR" sequence from N-terminus to C-terminus , P2A (SEQ ID NO: 3) and TGF ⁇ RII-M7CR (7) (SEQ ID NO: 200).
  • the H9.2.1-28-dnTGF ⁇ RII molecule includes the H9.2.1 CAR molecule (SEQ ID NO:142), "RAKR” sequence, P2A (SEQ ID NO: 3) and dnTGF ⁇ RII (SEQ ID NO: 199).
  • the H9.2.1-28-TGF ⁇ RII-BB molecule includes the H9.2.1 CAR molecule (SEQ ID NO:142), "RAKR” sequence, P2A (SEQ ID NO: 3) and TGF ⁇ RII-BB (SEQ ID NO: 202).
  • the above-mentioned synthesized DNA fragment was inserted into the downstream of the EF1 ⁇ promoter of the pCKW lentiviral expression vector (Genewise Company), and the EGFR sequence in the original vector was replaced to obtain the corresponding expression plasmid (synthesized by Genewise Company).
  • the lentivirus preparation method is the same as Example 4.2, and the CAR-T preparation method is the same as Example 4.3.
  • Figure 48 shows the expansion kinetics of CAR-T cells derived from donors 5, 10, and 18. Overall, the expansion fold of each CAR-T cell is between 15 and 60. H9.2.1-dnTGF ⁇ RII and H9 derived from donor 18 .2.1-28-dnTGF ⁇ RII amplification is low.
  • Example 27.3 Expression detection of CAR and TGF ⁇ RII ECD and CAR-T cell phenotype detection
  • Figure 49A shows a representative flow scatter plot of CAR and TGF ⁇ RII ECD expression in CAR-T cells prepared on day 7. CAR expression can be detected in all CAR-T cells.
  • Figure 49B shows that the CAR positivity rate is approximately 20-80%. between.
  • H9.2.1-TGF ⁇ RII-M7CR H9.2.1-dnTGF ⁇ RII, H9.2.1-TGF ⁇ RII-CD28, H9.2.1-28-TGF ⁇ RII-M7CR
  • TGF ⁇ RII ECD expression can be detected simultaneously in H9.2.1-28-dnTGF ⁇ RII and H9.2.1-28-TGF ⁇ RII-BB CAR-T cells.
  • TGF ⁇ RII-M7CR-modified CAR-T cells the consistency between ECD and CAR expression in TGF ⁇ RII-M7CR-modified CAR-T cells is high. , followed by dnTGF ⁇ RII, TGF ⁇ RII-CD28, and TGF ⁇ RII-BB expression was poor.
  • Figure 50A and Figure 50B show representative flow scatter plots and statistical diagrams of CD4 and CD8 subpopulations in CAR-T cells. Compared with H9.2.1 or H9.2.1-28 CAR-T cells, expression of TGF ⁇ RII-M7CR slightly increased the proportion of CD4 T cells.
  • HUP-T4 with medium and high expression levels of Claudin18.2 was used as the target cell, and an in vitro low-efficiency target ratio long-term killing experiment was carried out to study the killing effect of TGF ⁇ RII-M7CR modified CAR-T on target cells in vitro.
  • the xCELLigence RTCA MP instrument (Agilent company) was used to dynamically detect the killing of target cells by CAR-T cells in real time. Add 50 ⁇ L of culture medium to the E-Plates plate. After the instrument reads the baseline value, add 50 ⁇ L of tumor target cells, and then place them in the machine to dynamically monitor the cell growth. Resuscitate the NT cells and CAR-T cells (CAR-T cells derived from donors 5 and 10) prepared in Example 28.3 and place them in a 37°C cell culture incubator overnight. The next day, add CAR-T into the E-Plates wells of the corresponding group at an E:T ratio of 1:50.

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Abstract

The present invention relates to a constitutive chimeric cytokine receptor, comprising an extracellular domain and a constitutively activated IL-7R mutant. The extracellular domain consists of an effector molecule capable of remodeling the tumor microenvironment, and the constitutively activated IL-7R mutant comprises an IL-7R mutant transmembrane domain and an IL-7R intracellular domain. The present invention also relates to a CAR polypeptide or a TCR polypeptide modified by the constitutive chimeric cytokine receptor, an immune effector cell engineered to express the CAR polypeptide or the TCR polypeptide modified by the constitutive chimeric cytokine receptor, and a method for preparing the immune effector cell. The immune effector cell expressing the CAR polypeptide or the TCR polypeptide modified by the constitutive chimeric cytokine receptor of the present invention can be used for treating a tumor in a subject.

Description

组成型嵌合细胞因子受体及表达其的免疫细胞及应用Constitutive chimeric cytokine receptors and immune cells expressing them and their applications 技术领域Technical field
本发明总体上涉及基因工程和细胞免疫学领域，具体地，本发明涉及用于增强免疫细胞扩增及效应功能的组成型嵌合细胞因子受体，其包含胞外结构域和组成型激活的IL-7R突变体，所述胞外结构域由具有重塑肿瘤微环境的效应分子组成，所述组成型激活的IL-7R突变体包含IL-7R突变跨膜域和IL-7R胞内结构域，免疫细胞(例如T细胞)通过表达组成型嵌合细胞因子受体，从而具有不依赖外源细胞因子激活的组成型IL-7R自激活信号以及胞外结构域上的效应分子功效，用于肿瘤免疫治疗。本发明还涉及所述组成型嵌合细胞因子受体和嵌合抗原受体或T细胞受体的组合及其用途。The present invention relates generally to the fields of genetic engineering and cellular immunology. In particular, the present invention relates to constitutively chimeric cytokine receptors for enhancing immune cell expansion and effector function, which comprise an extracellular domain and a constitutively activated IL-7R mutant, the extracellular domain is composed of effector molecules that reshape the tumor microenvironment, and the constitutively activated IL-7R mutant includes the IL-7R mutant transmembrane domain and the IL-7R intracellular structure Domain, immune cells (such as T cells) express constitutive chimeric cytokine receptors, thereby having constitutive IL-7R self-activating signals independent of exogenous cytokine activation and effector molecule efficacy on the extracellular domain. in tumor immunotherapy. The invention also relates to the combination of said constitutive chimeric cytokine receptor and a chimeric antigen receptor or T cell receptor and their use.
背景技术Background technique
近年来肿瘤免疫治疗领域取得巨大进展，已成为晚期肿瘤临床治疗的基石，其中，以嵌合抗原受体T细胞(CAR-T)为代表的过继细胞免疫疗法(ACT)由于在治疗复发难治性血液系统肿瘤中表现出的前所未有的临床治疗效果而备受瞩目。CAR-T和T细胞受体(TCR)基因修饰的T细胞(TCR-T)都是基因修饰的细胞治疗产品：通过收集肿瘤患者外周血T细胞并激活，采用病毒或非病毒载体介导的基因修饰，使其携带能够特异识别肿瘤细胞抗原的CAR或者TCR，从而赋予T细胞肿瘤特异性识别及杀伤功能。CAR-T细胞已经在治疗复发难治性血液系统肿瘤中展现出前所未有的临床治疗效果，2017年FDA批准首个CAR-T细胞产品用于临床治疗，截止目前FDA已经批准6个CAR-T细胞产品，用于治疗复发难治性B细胞白血病、淋巴瘤及骨髓瘤等。多个TCR-T细胞治疗产品也处于关键注册临床阶段。此外，还有多种类型的免疫细胞治疗处于临床试验中，包括CAR修饰NK细胞(CAR-NK)、基因修饰肿瘤浸润T细胞(TIL)、基因修饰γδT细胞、iNKT细胞、双阴性T细胞(DNT)以及诱导多能干细胞来源(iPSC)的免疫细胞(iNK、iT)等。In recent years, great progress has been made in the field of tumor immunotherapy, and it has become the cornerstone of clinical treatment of advanced tumors. Among them, adoptive cellular immunotherapy (ACT), represented by chimeric antigen receptor T cells (CAR-T), is very effective in the treatment of relapsed and refractory tumors. It has attracted much attention for its unprecedented clinical therapeutic effects in sexual hematological tumors. CAR-T and T cell receptor (TCR) gene-modified T cells (TCR-T) are both genetically modified cell therapy products: by collecting and activating T cells from the peripheral blood of tumor patients, using viral or non-viral vectors to mediated Gene modification allows it to carry a CAR or TCR that can specifically recognize tumor cell antigens, thus endowing T cells with tumor-specific recognition and killing functions. CAR-T cells have demonstrated unprecedented clinical therapeutic effects in the treatment of relapsed and refractory hematological tumors. In 2017, the FDA approved the first CAR-T cell product for clinical treatment. So far, the FDA has approved 6 CAR-T cells. Products for the treatment of relapsed and refractory B-cell leukemia, lymphoma and myeloma. Multiple TCR-T cell therapy products are also in the critical clinical stage of registration. In addition, there are many types of immune cell therapies in clinical trials, including CAR-modified NK cells (CAR-NK), gene-modified tumor-infiltrating T cells (TIL), gene-modified γδ T cells, iNKT cells, double-negative T cells ( DNT) and immune cells (iNK, iT) derived from induced pluripotent stem cells (iPSC), etc.
大量研究探索CAR-T等基因修饰免疫细胞在实体瘤中的应用，但截至目前总体临床疗效甚微。一项统计42项实体瘤CAR-T临床试验的荟萃研究发现，在375例接受CAR-T细胞治疗患者中，客观应答率(ORR)仅13.9％，ORR及疗效持久性远低于血液系统肿瘤。免疫细胞治疗实体瘤效果不佳主要有以下原因：肿瘤没有分泌匹配的趋化因子，基因修饰免疫细胞不能有效进入肿瘤组织部位发挥效应；即使这些基因修饰免疫细胞能够浸润至肿瘤组织中，诸多因素使其很快处于功能失活状态，限制其体内扩增及存续，这些因素包括但不限于：由氧化应激、营养缺失、缺氧、酸性pH等构成的“不友好”肿瘤微环境(TME)；肿瘤细胞产生大量免疫抑制性细胞因子；肿瘤中存在大量调节性T细胞(Treg)、髓源性抑制细胞(MDSC)及肿瘤相关巨噬细胞(TAM)等抑制性免疫细胞；T细胞内源性负反馈调节机制，例如PD-1等负性免疫调节受体表达上调导致功能“耗竭”。实体瘤细胞抗原表达高度异质性，在免疫压力下容易产生抗原缺失突变体，导致免疫逃逸。因此，选择合适的治疗靶点、赋予基因修饰免疫细胞在瘤内持续存续、扩增并能够改造“不友好”的肿瘤抑制性免疫微环境的能力，对于提高实体瘤治疗疗效至关重要。A large number of studies have explored the application of genetically modified immune cells such as CAR-T in solid tumors, but so far the overall clinical efficacy is minimal. A meta-study that counted 42 CAR-T clinical trials on solid tumors found that among 375 patients who received CAR-T cell therapy, the objective response rate (ORR) was only 13.9%, and the ORR and durability of efficacy were much lower than those for hematological tumors. . The main reasons for the poor effect of immune cell therapy on solid tumors are as follows: the tumor does not secrete matching chemokines, and the genetically modified immune cells cannot effectively enter the tumor tissue to exert their effects; even if these genetically modified immune cells can infiltrate into the tumor tissue, many factors It quickly becomes functionally inactivated, limiting its expansion and survival in the body. These factors include but are not limited to: "unfriendly" tumor microenvironment (TME) composed of oxidative stress, nutritional deficiency, hypoxia, acidic pH, etc. ); tumor cells produce a large number of immunosuppressive cytokines; there are a large number of suppressive immune cells such as regulatory T cells (Treg), myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) in tumors; within T cells Source negative feedback regulatory mechanisms, such as the upregulation of expression of negative immune regulatory receptors such as PD-1, lead to functional "exhaustion". The antigen expression of solid tumor cells is highly heterogeneous, and antigen deletion mutants are prone to occur under immune pressure, leading to immune escape. Therefore, selecting appropriate therapeutic targets and endowing genetically modified immune cells with the ability to persist, expand, and transform the "unfriendly" tumor-suppressive immune microenvironment in tumors are crucial to improving the therapeutic efficacy of solid tumors.
生理情况下，初始T细胞获得最佳激活需要3种信号，由TCR提供的第一信号、由CD28、 41BB等共刺激分子提供的第二信号、以及由细胞因子和其受体结合提供的第三信号，其中所述第三信号是初始T细胞获得最佳增殖、分化为效应细胞并发展形成长效记忆性T细胞所必需。Under physiological conditions, optimal activation of initial T cells requires three signals. The first signal provided by TCR, the first signal provided by CD28, The second signal provided by co-stimulatory molecules such as 41BB, and the third signal provided by the binding of cytokines and their receptors, where the third signal is for initial T cells to obtain optimal proliferation, differentiate into effector cells, and develop into long-acting Required for memory T cells.
基因修饰免疫细胞(例如，CAR-T细胞)虽然通过CAR分子能够获得第一和第二信号，但缺失第三信号，从而影响了体内CAR-T的扩增、存续和功能。有报导通过系统性给予外源细胞因子能够促进动物体内CAR-T、TCR-T细胞的体内扩增和功能，但系统性给予细胞因子产生了严重的毒副作用，通过转基因形式使基因修饰细胞自分泌细胞因子也产生类似毒性。因此，目前很多研究积极探索其他策略，包括膜表面表达细胞因子、表达细胞因子转换受体(CSR)或组成型激活的细胞因子受体或其片段。例如，Thomas Shum等(WO2018038945A1)设计了表达T淋巴瘤中天然发现的组成型激活的IL-7R(即IL-7Rα)突变体(C7R)的CAR-T细胞，所述C7R缺失IL-7R天然胞外结构域，但由于IL-7R跨膜区发生了半胱氨酸或脯氨酸突变，形成组成型二聚体，在不依赖胞外结构域与配体结合情况下，激活JAK1激酶，进而激活下游STAT5等转录效应因子，调控下游靶基因表达，最终促进和维持T增殖及存活。C7R修饰的CAR-T细胞相比未修饰的CAR-T细胞，能够在反复杀伤肿瘤细胞同时减少功能耗竭，体内具有更好的增殖、存续及抗肿瘤功能。Although genetically modified immune cells (for example, CAR-T cells) can obtain the first and second signals through CAR molecules, they lack the third signal, thus affecting the expansion, survival, and function of CAR-T in the body. It has been reported that the systemic administration of exogenous cytokines can promote the in vivo expansion and function of CAR-T and TCR-T cells in animals. However, systemic administration of cytokines has produced serious toxic side effects. Gene-modified cells can be automatically modified through transgenes. Secreted cytokines also produce similar toxicity. Therefore, many studies are currently actively exploring other strategies, including expressing cytokines on the membrane surface, expressing cytokine switching receptors (CSR), or constitutively activated cytokine receptors or their fragments. For example, Thomas Shum et al. (WO2018038945A1) designed CAR-T cells expressing a constitutively activated IL-7R (i.e., IL-7Rα) mutant (C7R) found naturally in T lymphomas that lacks the native IL-7R. Extracellular domain, but due to cysteine or proline mutations in the transmembrane region of IL-7R, a constitutive dimer is formed, which activates JAK1 kinase without relying on the extracellular domain and ligand binding. It then activates downstream STAT5 and other transcriptional effectors, regulates the expression of downstream target genes, and ultimately promotes and maintains T proliferation and survival. Compared with unmodified CAR-T cells, C7R-modified CAR-T cells can repeatedly kill tumor cells while reducing functional exhaustion, and have better proliferation, survival and anti-tumor functions in the body.
现有技术中的C7R基因修饰CAR-T细胞虽然赋予了CAR-T细胞提高的体内外扩增、存续能力，但缺乏主动改造“不友好”的肿瘤抑制性免疫微环境的特性，在面临具有重度免疫抑制性TME特征的实体瘤时，这些CAR-T仍然受制于抑制性TME，阻碍其发挥功能。Although the C7R gene-modified CAR-T cells in the existing technology endow CAR-T cells with improved in vitro and in vivo expansion and survival capabilities, they lack the ability to actively transform the "unfriendly" tumor-suppressive immune microenvironment. In solid tumors characterized by severe immunosuppressive TME, these CAR-Ts are still restricted by the inhibitory TME, preventing them from functioning.
发明概述Summary of the invention
本发明人通过研究，开发了一组重组多肽，为组成型嵌合细胞因子受体，其包含胞外结构域和组成型激活的IL-7R突变体，所述胞外结构域由具有重塑肿瘤微环境的效应分子组成。本发明的组成型嵌合细胞因子受体赋予了免疫细胞利用组成型激活的IL-7R突变体持续激活STAT5信号，促进和维持免疫细胞的增殖及存活，还赋予了免疫细胞获得了新的胞外效应分子功效，使免疫细胞具备主动塑造“不友好”TME的能力，通过重构TME使“冷”肿瘤变为“热”肿瘤，免疫细胞处于更“友好”的TME中，将更加有利于发挥抗肿瘤效应。进一步地，将本发明的组成型嵌合细胞因子受体和基因修饰免疫细胞(例如，CAR-T细胞)组合使用，通过激发或增强体内内源抗肿瘤效应机制，有望使组成型嵌合细胞因子受体和基因修饰免疫细胞(例如，CAR-T细胞)产生协同抗肿瘤效果，所述基因修饰免疫细胞包括CAR-T细胞、TCR-T、CAR-NK、基因修饰TIL、γδT细胞、iNKT细胞、DNT以及iPSC来源的iT及iNK细胞等。Through research, the inventors have developed a set of recombinant polypeptides, which are constitutively chimeric cytokine receptors, including extracellular domains and constitutively activated IL-7R mutants. The extracellular domains are composed of remodeling Effector molecular composition of the tumor microenvironment. The constitutive chimeric cytokine receptor of the present invention enables immune cells to utilize constitutively activated IL-7R mutants to continuously activate STAT5 signals, promote and maintain the proliferation and survival of immune cells, and also enables immune cells to acquire new cellular functions. The effect of external effector molecules gives immune cells the ability to actively shape the "unfriendly" TME. By reconstructing the TME, "cold" tumors become "hot" tumors. The immune cells are in a more "friendly" TME, which will be more beneficial. exert anti-tumor effects. Furthermore, by combining the constitutive chimeric cytokine receptor of the present invention with genetically modified immune cells (for example, CAR-T cells), it is expected that the constitutive chimeric cells will be able to activate or enhance the endogenous anti-tumor effect mechanism in the body. Factor receptors and genetically modified immune cells (for example, CAR-T cells), including CAR-T cells, TCR-T, CAR-NK, genetically modified TIL, γδT cells, and iNKT, produce synergistic anti-tumor effects. cells, DNT, iPSC-derived iT and iNK cells, etc.
在第一方面，本发明提供了组成型嵌合细胞因子受体，其包含胞外结构域和组成型激活的IL-7R突变体。所述组成型激活的IL-7R突变体能够持续激活STAT5信号、维持免疫效应细胞(例如，T细胞)非外源细胞因子依赖性存活，所述胞外结构域具有重塑肿瘤微环境及激发机体内源性抗肿瘤免疫应答的效应功能。In a first aspect, the present invention provides a constitutively chimeric cytokine receptor comprising an extracellular domain and a constitutively activated IL-7R mutant. The constitutively activated IL-7R mutant can continuously activate STAT5 signaling and maintain immune effector cells (e.g., T cells) independent of exogenous cytokines. The extracellular domain has the ability to reshape the tumor microenvironment and stimulate Effector function of the body's endogenous anti-tumor immune response.
在一些实施方案中，本发明首先利用外源细胞因子依赖的BaF3细胞系在体外比较了27种不同的组成型激活的IL-7R突变体(本文中也称为IL7Rm或M7R)，所述M7R由携带不同突变的IL7R跨膜区(IL7R-mutant(TM))(序列表中SEQ ID NO:20-SEQ ID NO:46序列的加粗部分)和野生型IL7R(IL7R-WT)胞内段(IL7R-wt(ICD)，SEQ ID NO:19)组成。 在一些具体的实施方案中，将胞外结构域tCD19(SEQ ID NO:17)和所述27种不同的M7R组合构建获得组成型嵌合细胞因子受体，通过检测细胞表面tCD19阳性表达来鉴定M7R表达，所获得的组成型嵌合细胞因子受体也称为tCD19-M7CR。In some embodiments, the present invention first compared 27 different constitutively activated IL-7R mutants (also referred to herein as IL7Rm or M7R) in vitro using the exogenous cytokine-dependent BaF3 cell line. It consists of the IL7R transmembrane region (IL7R-mutant(TM)) carrying different mutations (the bold part of the SEQ ID NO:20-SEQ ID NO:46 sequence in the sequence listing) and the intracellular segment of wild-type IL7R (IL7R-WT). (IL7R-wt(ICD), SEQ ID NO:19). In some specific embodiments, the extracellular domain tCD19 (SEQ ID NO: 17) and the 27 different M7Rs are combined to construct a constitutive chimeric cytokine receptor, which is identified by detecting the positive expression of tCD19 on the cell surface. M7R expression, the resulting constitutive chimeric cytokine receptor is also called tCD19-M7CR.
所述27种M7R体外维持BaF3细胞非外源细胞因子依赖的存活效应的实验结果表明，转导并稳定表达其中19个M7R序列能够维持BaF3细胞非外源细胞因子依赖的存活，M7R基因能够促进BaF3细胞以非外源细胞因子依赖方式增殖。并且随着培养时间延长，存活的BaF3细胞均是M7R表达阳性细胞，表明只有表达了M7R的BaF3细胞能够在不加入外源细胞因子的情况下存活。采用胞内流式细胞术染色分析IL-7R下游信号分子发现，这19个M7R分子(IL7Rm1.1，IL7Rm1.3，IL7Rm3.1，IL7Rm4-IL7Rm19)激活并维持BaF3细胞内STAT5磷酸化，水平与加入外源细胞因子相当，表明这19个M7R通过组成型激活STAT5信号通路，促进及维持BaF3细胞非外源细胞因子依赖的存续。The experimental results of the 27 kinds of M7R in maintaining the non-exogenous cytokine-dependent survival effect of BaF3 cells in vitro showed that transducing and stably expressing 19 M7R sequences can maintain the non-exogenous cytokine-dependent survival of BaF3 cells, and the M7R gene can promote BaF3 cells proliferate in an exogenous cytokine-independent manner. And as the culture time increases, the surviving BaF3 cells are all M7R-positive cells, indicating that only BaF3 cells expressing M7R can survive without the addition of exogenous cytokines. Intracellular flow cytometry staining was used to analyze IL-7R downstream signaling molecules and found that these 19 M7R molecules (IL7Rm1.1, IL7Rm1.3, IL7Rm3.1, IL7Rm4-IL7Rm19) activate and maintain STAT5 phosphorylation in BaF3 cells, and the levels It is equivalent to the addition of exogenous cytokines, indicating that these 19 M7Rs promote and maintain the non-exogenous cytokine-dependent survival of BaF3 cells by constitutively activating the STAT5 signaling pathway.
在另一些实施方案中，在原代T细胞中转导并稳定表达上述19个含不同M7R分子的tCD19-M7CR，发现表达所述M7R分子激活了T细胞内STAT5信号，相比较未转导的或转导IL7R-WT的T细胞，表达M7R分子的T细胞在体外具有更好的存续能力，特别地，IL7Rm4、IL7Rm5、IL7Rm7、IL7Rm8所示的M7R具有非常显著的促生存效应。在此基础上，本发明设计并构建了以细胞因子、免疫效应分子、抑制性分子拮抗剂、或靶向NK细胞激活性受体的效应分子作为所述组成型嵌合细胞因子受体的胞外结构域，将所述胞外结构域与M7R融合而形成本发明的组成型嵌合细胞因子受体(本文中也称为M7CR)。在一些实施方案中，所述M7CR胞外结构域可以是IL-12(IL-12p40或IL-12p70)、IL15(IL-15或IL-15FP，所述IL-15FP是指IL-15和IL-15Rα(选自IL-15Rα或IL-15Rα(Sushi))的融合蛋白，包括IL-15/IL-15Rα和IL-15Rα/IL-15两种形式的融合蛋白)、IL-21、IL-18、IL-9、IL-23、IL-36γ、IFNα2b等细胞因子，当免疫细胞(例如T细胞)表达所述包含细胞因子的M7CR基因时，具有增强的免疫效应功能及抗肿瘤效应；在一些实施方案中，所述M7CR胞外结构域也可以是4-1BB靶向分子部分(例如，4-1BB配体(4-1BBL)、抗4-1BB抗体(α4-1BB))、CD40靶向分子部分(例如，CD40配体(CD40L)、抗CD40抗体(αCD40))、CD83靶向分子部分(例如，抗CD83抗体(αCD83))、FLT3靶向分子部分(例如，FLT3配体(FTL3L)、抗FLT3抗体(αFLT3))、GITR、ICOS、CD2、ICAM-1等免疫效应分子，这些免疫效应分子通过与体内的抗原呈递细胞(APC)例如树突状细胞(DC)或巨噬细胞表面的相关受体或配体相互作用，活化APC，从而激发内源性抗肿瘤免疫应答，进而与免疫细胞(例如T细胞)产生协同抗肿瘤功效；在一些实施方案中，所述M7CR胞外结构域也可以是抗PD-L1抗体、抗CD47分子、抗IL-4分子、TGFβ结合分子(例如，抗TGFβ1分子、TGFβRII)、抗PD-1分子、抗CTLA-4分子、抗LAG-3分子、抗TIGIT分子、抗CD73分子等针对抑制性免疫受体或因子的抗体部分，通过拮抗抑制性免疫受体或因子的免疫抑制效应，达到增强抗肿瘤免疫应答目的，进而与免疫细胞(例如T细胞)产生协同抗肿瘤功效；在一些实施方案中，所述M7CR胞外结构域也可以是靶向NKG2C、NKG2D、NKp30、NKp44、NKp46等NK细胞表面表达的激活性受体的分子部分，例如，抗NKG2C、抗NKG2D、抗NKp30、抗NKp44、抗NKp46等，通过激活内源性NK细胞，取得增强抗肿瘤免疫效应的目的，进而与免疫细胞(例如T细胞)产生协同抗肿瘤功效。In other embodiments, the above 19 tCD19-M7CR containing different M7R molecules were transduced and stably expressed in primary T cells, and it was found that expression of the M7R molecules activated STAT5 signaling in T cells, compared with untransduced or T cells transduced with IL7R-WT and expressing M7R molecules have better survival ability in vitro. In particular, M7R represented by IL7Rm4, IL7Rm5, IL7Rm7, and IL7Rm8 has a very significant pro-survival effect. On this basis, the present invention designs and constructs cells using cytokines, immune effector molecules, inhibitory molecule antagonists, or effector molecules targeting NK cell activating receptors as the constitutive chimeric cytokine receptors. The extracellular domain is fused to M7R to form the constitutive chimeric cytokine receptor of the present invention (also referred to as M7CR herein). In some embodiments, the M7CR extracellular domain can be IL-12 (IL-12p40 or IL-12p70), IL15 (IL-15 or IL-15FP, the IL-15FP refers to IL-15 and IL -15Rα (fusion protein selected from IL-15Rα or IL-15Rα (Sushi)), including IL-15/IL-15Rα and IL-15Rα/IL-15 fusion proteins), IL-21, IL- 18. Cytokines such as IL-9, IL-23, IL-36γ, IFNα2b, etc., when immune cells (such as T cells) express the M7CR gene containing cytokines, have enhanced immune effector functions and anti-tumor effects; in In some embodiments, the M7CR extracellular domain can also be a 4-1BB targeting molecule moiety (e.g., 4-1BB ligand (4-1BBL), anti-4-1BB antibody (α4-1BB)), CD40 target To molecule moieties (e.g., CD40 ligand (CD40L), anti-CD40 antibody (αCD40)), CD83-targeting molecule moieties (e.g., anti-CD83 antibody (αCD83)), FLT3-targeting molecule moieties (e.g., FLT3 ligand (FTL3L) ), anti-FLT3 antibody (αFLT3)), GITR, ICOS, CD2, ICAM-1 and other immune effector molecules. These immune effector molecules interact with antigen-presenting cells (APC) in the body such as dendritic cells (DC) or macrophages. Related receptors or ligands on the surface interact with each other to activate APC, thereby stimulating endogenous anti-tumor immune responses, thereby producing synergistic anti-tumor effects with immune cells (such as T cells); in some embodiments, the M7CR extracellular The domain may also be an anti-PD-L1 antibody, an anti-CD47 molecule, an anti-IL-4 molecule, a TGFβ binding molecule (e.g., anti-TGFβ1 molecule, TGFβRII), an anti-PD-1 molecule, an anti-CTLA-4 molecule, an anti-LAG-3 Molecules, anti-TIGIT molecules, anti-CD73 molecules and other antibody parts directed against inhibitory immune receptors or factors achieve the purpose of enhancing the anti-tumor immune response by antagonizing the immunosuppressive effects of inhibitory immune receptors or factors, and then interact with immune cells (such as T cells) to produce synergistic anti-tumor effects; in some embodiments, the M7CR extracellular domain can also be a molecular part targeting activating receptors expressed on the surface of NK cells such as NKG2C, NKG2D, NKp30, NKp44, NKp46, etc. For example, anti-NKG2C, anti-NKG2D, anti-NKp30, anti-NKp44, anti-NKp46, etc., achieve the purpose of enhancing anti-tumor immune effects by activating endogenous NK cells, and then produce synergistic anti-tumor effects with immune cells (such as T cells).
在第二方面，本发明提供了M7CR修饰的CAR或TCR。本发明的新型M7CR“武装” 的肿瘤靶向T细胞(例如，表达M7CR的CAR-T细胞)获得了初始T细胞最佳激活所需要的3种信号，从而产生更好的T细胞激活、增殖、生存及免疫效应功能，同时，M7CR分子胞外结构域的效应分子通过激活机体内源T细胞、活化APC、拮抗免疫抑制性受体或激活机体NK等固有免疫细胞等机制主动对“不友好”TME进行重塑，促进内源性抗肿瘤效应机制，最终产生协同抗肿瘤免疫效应。In a second aspect, the invention provides M7CR modified CAR or TCR. The invention’s new M7CR “arms” Tumor-targeted T cells (for example, M7CR-expressing CAR-T cells) acquire the three signals required for optimal activation of initial T cells, resulting in better T cell activation, proliferation, survival and immune effector functions, while , the effector molecules in the extracellular domain of the M7CR molecule actively remodel the "unfriendly" TME through mechanisms such as activating the body's endogenous T cells, activating APC, antagonizing immunosuppressive receptors, or activating the body's innate immune cells such as NK, promoting endogenous The original anti-tumor effect mechanism ultimately produces a synergistic anti-tumor immune effect.
在一些实施方案中，本发明制备了同时表达CAR和本发明的M7CR(例如，所述M7CR的胞外结构域(ECD)为tCD19、IL-12(p40或p70)、IL15FP(包括IL-15/IL-15Rα和IL-15Rα/IL-15两种形式的融合蛋白，其中IL-15Rα选自IL-15Rα或IL-15Rα(Sushi))、IL-21、4-1BBL、CD40L、抗PD-L1纳米抗体(PD-L1VHH)，M7R采用IL7Rm8)的病毒载体，并且在体外制备了M7CR修饰的直接靶向肿瘤抗原的传统CAR-T细胞(例如靶向claudin18.2的H9.1.2 CAR)或M7CR修饰的通过P329G突变抗体介导靶向肿瘤的“模块化”PG CAR-T细胞(例如8B CAR)，并对它们的体内外功能进行了评价。In some embodiments, the present invention prepares a method for simultaneously expressing the CAR and the M7CR of the present invention (for example, the extracellular domain (ECD) of the M7CR is tCD19, IL-12 (p40 or p70), IL15FP (including IL-15 / IL-15Rα and IL-15Rα/IL-15 two forms of fusion protein, in which IL-15Rα is selected from IL-15Rα or IL-15Rα (Sushi)), IL-21, 4-1BBL, CD40L, anti-PD- L1 nanobody (PD-L1 VHH ), M7R uses the viral vector of IL7Rm8), and M7CR-modified traditional CAR-T cells that directly target tumor antigens (such as H9.1.2 CAR targeting claudin18.2) are prepared in vitro. Or M7CR-modified "modular" PG CAR-T cells (such as 8B CAR) that target tumors mediated by P329G mutated antibodies, and their functions in vitro and in vivo were evaluated.
通过P2A自裂解肽构建了同时表达CAR和M7CR的双顺反子病毒载体，这些病毒转导的T细胞同时表达CAR和本发明所述的M7CR，且CAR和本发明所述的M7CR表达具有相关性。A bicistronic virus vector that simultaneously expresses CAR and M7CR is constructed through P2A self-cleaving peptide. T cells transduced by these viruses express CAR and M7CR of the present invention at the same time, and the expression of CAR and M7CR of the present invention has a correlation. sex.
细胞表型研究显示，M7R修饰的CAR-T细胞CD4/CD8细胞亚群比例未有显著变化，M7CR修饰CAR-T细胞根据作为M7CR的胞外结构域(ECD)的不同，其对T细胞亚群影响效应不一。例如，IL-12-M7CR修饰的CAR-T细胞维持了更高比例的CD4细胞亚群。Cell phenotype studies show that the CD4/CD8 cell subset ratio of M7R-modified CAR-T cells does not change significantly. M7CR-modified CAR-T cells have different effects on T cell subpopulations based on the extracellular domain (ECD) of M7CR. Group influence has varying effects. For example, IL-12-M7CR-modified CAR-T cells maintained a higher proportion of CD4 cell subsets.
细胞表型研究显示，单独M7R(例如tCD19-M7CR，胞外区域为tCD19，用于考察M7R的作用)修饰的CAR-T细胞维持了高比例Tscm/Tcm等记忆性细胞亚群，与作为阳性对照的C7R(tCD19-M7CR(CPT)，M7R序列来自C7R)效应相当；M7CR修饰的CAR-T细胞，根据作为M7CR的胞外结构域(ECD)的不同，其对T细胞分化效应不一。例如，IL-15-M7CR修饰CAR-T细胞具有更好的记忆表型，而IL-12-M7CR修饰促进了CAR-T细胞分化。Cell phenotype studies have shown that CAR-T cells modified by M7R alone (such as tCD19-M7CR, the extracellular domain is tCD19, used to examine the role of M7R) maintain a high proportion of memory cell subpopulations such as Tscm/Tcm, which is consistent with positive The control C7R (tCD19-M7CR (CPT), M7R sequence comes from C7R) has equivalent effects; M7CR-modified CAR-T cells have different effects on T cell differentiation depending on the extracellular domain (ECD) of M7CR. For example, IL-15-M7CR modified CAR-T cells have a better memory phenotype, while IL-12-M7CR modification promotes CAR-T cell differentiation.
在一些实施方案中，本发明对IL-15-M7CR(所述M7CR胞外ECD结构域为IL15FP)修饰的靶向claudin18.2的H9.2.1 CAR-T细胞表型进行了更详细研究，结果表明，单独IL-15(IL-15-M7CRin，失活了M7R信号)或M7R(tCD19-M7CR)效应显著促进了Tscm记忆性细胞亚群维持，但IL-15-M7CR具有更强的促Tscm记忆性细胞维持效应，提示膜表面表达IL-15和M7R信号产生协同效应。In some embodiments, the present invention conducted a more detailed study on the phenotype of H9.2.1 CAR-T cells modified by IL-15-M7CR (the M7CR extracellular ECD domain is IL15FP) targeting claudin18.2, and the results It shows that the effect of IL-15 (IL-15-M7CRin, inactivating M7R signaling) or M7R (tCD19-M7CR) alone significantly promotes the maintenance of Tscm memory cell subsets, but IL-15-M7CR has a stronger pro-Tscm effect. Memory cell maintenance effect, suggesting a synergistic effect of IL-15 and M7R signals expressed on the membrane surface.
在一些实施方案中，本发明通过体外杀伤实验研究了M7CR修饰的传统CAR-T细胞(例如，靶向claudin18.2的H9.1.2 CAR-T细胞、H9.2.1 CAR-T细胞、H9.2.1-28-L CAR-T细胞)或PG CAR-T细胞(例如靶向claudin18.2的HuR968B CAR-T细胞)对抗原表达阳性的肿瘤细胞的杀伤作用，结果表明，单独M7R(例如tCD19-M7CR，胞外区域为tCD19，用于考察M7R的作用)修饰的CAR-T细胞的杀伤能力显著高于未修饰的CAR-T细胞。在M7R基础上融合胞外效应分子形成M7CR，M7CR修饰CAR-T细胞后能够进一步增加CAR-T细胞体外杀伤功能。例如，4-1BBL-M7CR、抗PD-L1VHH-M7CR、IL-12-M7CR、IL-15-M7CR修饰的传统CAR-T细胞的杀伤能力显著高于未修饰或单独M7R修饰的CAR-T细胞。IL-12-M7CR、IL-15-M7CR修饰的PG CAR-T细胞杀伤效能增强，尤其在针对抗原低表达的肿瘤细胞(比如SNU-601low)情况下更显著。说明胞外效应分子与M7R在促进CAR-T细胞的杀伤作用上具有联合效应。 In some embodiments, the present invention studies M7CR-modified traditional CAR-T cells (e.g., H9.1.2 CAR-T cells, H9.2.1 CAR-T cells, H9.2.1 targeting claudin18.2) through in vitro killing experiments. -28-L CAR-T cells) or PG CAR-T cells (such as HuR968B CAR-T cells targeting claudin18.2) on the killing effect of antigen-expressing tumor cells, the results show that M7R alone (such as tCD19-M7CR , the extracellular region is tCD19, used to examine the role of M7R). The killing ability of modified CAR-T cells is significantly higher than that of unmodified CAR-T cells. On the basis of M7R, extracellular effector molecules are fused to form M7CR. After M7CR modifies CAR-T cells, it can further increase the in vitro killing function of CAR-T cells. For example, the killing capacity of traditional CAR-T cells modified with 4-1BBL-M7CR, anti-PD-L1 VHH -M7CR, IL-12-M7CR, and IL-15-M7CR is significantly higher than that of unmodified or M7R-modified CAR-T cells alone. cell. The killing efficacy of IL-12-M7CR and IL-15-M7CR modified PG CAR-T cells is enhanced, especially when targeting tumor cells with low antigen expression (such as SNU-601 low ). This shows that extracellular effector molecules and M7R have a combined effect in promoting the killing effect of CAR-T cells.
在另一些实施方案中，本发明通过体外反复刺激实验研究了M7CR修饰的PG CAR-T细胞在肿瘤细胞反复刺激情况下的体外增殖能力，结果表明，单独M7R(例如tCD19-M7CR，胞外区域为tCD19，用于考察M7R的作用)修饰PG CAR-T细胞在肿瘤细胞反复刺激情况下具有更好的持续增殖能力。在M7R基础上融合胞外效应分子形成的M7CR，能够进一步增加PG CAR-T细胞体外持续增殖能力，IL-12-M7CR、IL-15-M7CR等M7CR修饰的PG CAR-T细胞具有更强的体外持续增殖能力。进一步地，表型研究发现，在反复肿瘤细胞刺激下，IL-12-M7CR修饰的CAR-T细胞具有更多的CD4+T细胞。细胞因子检测结果表明，IL-12-M7CR修饰的CAR-T细胞IFN-γ和TNF释放水平显著升高。In other embodiments, the present invention studied the in vitro proliferation ability of M7CR-modified PG CAR-T cells under repeated stimulation of tumor cells through repeated in vitro stimulation experiments. The results showed that M7R alone (such as tCD19-M7CR, extracellular region (tCD19, used to examine the role of M7R) modified PG CAR-T cells have better sustained proliferation ability under repeated stimulation of tumor cells. The M7CR formed by fusing extracellular effector molecules on the basis of M7R can further increase the sustained proliferation ability of PG CAR-T cells in vitro. M7CR-modified PG CAR-T cells such as IL-12-M7CR and IL-15-M7CR have stronger Sustained proliferation ability in vitro. Further, phenotypic studies found that IL-12-M7CR-modified CAR-T cells had more CD4 + T cells under repeated tumor cell stimulation. Cytokine detection results showed that the release levels of IFN-γ and TNF from IL-12-M7CR-modified CAR-T cells were significantly increased.
在另一些实施方案中，本发明采用表达不同水平Claudin18.2抗原的肿瘤细胞作为靶细胞，对M7R(tCD19-M7CR)、IL-12-M7CR、IL15-M7CR等M7CR修饰的传统CAR-T细胞(例如，靶向claudin18.2的H9.2.1 CAR-T细胞、H9.2.1-28-L CAR-T细胞)体外杀伤功能进行研究，结果表明，单独M7R(例如tCD19-M7CR，胞外区域为tCD19，用于考察M7R的作用)修饰的CAR-T细胞体外杀伤功能增强，M7CR修饰(例如IL-12-M7CR、IL15-M7CR修饰)增强效应更显著，具有显著增强CAR-T细胞对不同抗原表达水平的靶细胞的杀伤效应，在自2个供者制备的CAR-T细胞中观察到类似效应。体外反复杀伤实验研究结果表明，M7R、IL-12-M7CR、IL15-M7CR等M7CR修饰的CAR-T细胞维持了更好的体外持续杀伤功能，在自2个供者制备的CAR-T细胞中采用2个不同效靶比观察到类似效应。In other embodiments, the present invention uses tumor cells expressing different levels of Claudin18.2 antigen as target cells to target traditional CAR-T cells modified by M7CR such as M7R (tCD19-M7CR), IL-12-M7CR, IL15-M7CR, etc. (For example, H9.2.1 CAR-T cells and H9.2.1-28-L CAR-T cells targeting claudin18.2) were studied on the killing function in vitro. The results showed that M7R alone (such as tCD19-M7CR, the extracellular region is tCD19, used to examine the role of M7R) modified CAR-T cells have enhanced killing function in vitro, M7CR modification (such as IL-12-M7CR, IL15-M7CR modification) has a more significant enhancing effect, and can significantly enhance the response of CAR-T cells to different antigens Expression level of target cell killing effect, similar effects were observed in CAR-T cells prepared from 2 donors. The results of repeated in vitro killing experiments showed that M7CR-modified CAR-T cells such as M7R, IL-12-M7CR, IL15-M7CR, etc. maintained better in vitro sustained killing function. In CAR-T cells prepared from two donors, Similar effects were observed using 2 different efficacy-to-target ratios.
在一些实施方案中，本发明通过对M7CR修饰PG CAR-T细胞的小鼠体内抗肿瘤效应进行研究，结果表明，M7CR修饰的CAR-T细胞在体内具有更强的抗肿瘤作用，且M7CR修饰的CAR-T细胞的增殖能力也强于未修饰的CAR-T细胞。In some embodiments, the present invention studies the anti-tumor effect of M7CR-modified PG CAR-T cells in mice. The results show that M7CR-modified CAR-T cells have stronger anti-tumor effects in vivo, and M7CR-modified CAR-T cells have stronger anti-tumor effects in vivo. The proliferation ability of CAR-T cells is also stronger than that of unmodified CAR-T cells.
在另一些实施方案中，本发明通过对M7CR修饰传统CAR-T细胞(例如，靶向claudin18.2的H9.2.1 CAR-T细胞、H9.2.1-28-L CAR-T细胞)的小鼠体内抗肿瘤效应进行研究，结果表明，M7CR修饰的CAR-T细胞在体内具有更强的抗肿瘤作用，且M7CR修饰的CAR-T细胞的增殖能力也强于未修饰的CAR-T细胞。In other embodiments, the present invention uses M7CR-modified traditional CAR-T cells (e.g., H9.2.1 CAR-T cells, H9.2.1-28-L CAR-T cells targeting claudin18.2) in mice. The anti-tumor effect in vivo was studied, and the results showed that M7CR-modified CAR-T cells had stronger anti-tumor effects in vivo, and the proliferation ability of M7CR-modified CAR-T cells was also stronger than that of unmodified CAR-T cells.
在第三方面，本发明提供了编码本发明的M7CR或编码本发明的M7CR修饰的CAR或TCR的核酸分子、包含编码本发明的M7CR或编码本发明的M7CR修饰的CAR或TCR的核酸分子的载体，和包含本发明的组成型嵌合细胞因子受体M7CR或M7CR修饰的CAR多肽、本发明的核酸分子、或本发明的载体的细胞，优选地，所述细胞是自体T细胞或同种异体T细胞。In a third aspect, the invention provides nucleic acid molecules encoding the M7CR of the invention or encoding the M7CR-modified CAR or TCR of the invention, including nucleic acid molecules encoding the M7CR of the invention or encoding the M7CR-modified CAR or TCR of the invention. Vector, and cells comprising the constitutive chimeric cytokine receptor M7CR or M7CR modified CAR polypeptide of the present invention, the nucleic acid molecule of the present invention, or the vector of the present invention. Preferably, the cells are autologous T cells or allogeneic Allogeneic T cells.
在第四方面，本发明提供了一种产生细胞、例如免疫效应细胞的方法，所述方法包括将编码本发明的M7CR或编码本发明所述M7CR修饰的CAR或TCR的核酸分子(例如，RNA分子，例如mRNA分子)，或包含编码本发明的M7CR或编码本文所述M7CR修饰的CAR或TCR的核酸分子的载体引入(例如转导)免疫效应细胞。In a fourth aspect, the invention provides a method of producing cells, such as immune effector cells, the method comprising converting a nucleic acid molecule (eg, RNA) encoding the M7CR of the invention or encoding the M7CR-modified CAR or TCR of the invention into molecule, such as an mRNA molecule), or a vector comprising a nucleic acid molecule encoding an M7CR of the invention or encoding an M7CR-modified CAR or TCR described herein introduces (eg, transduces) immune effector cells.
在一些实施方案中，所述免疫效应细胞是T细胞、NK细胞，例如，所述T细胞是自体T细胞或同种异体T细胞，例如，所述免疫效应细胞是自人PBMC分离T细胞、NK细胞后制备的。In some embodiments, the immune effector cells are T cells, NK cells, for example, the T cells are autologous T cells or allogeneic T cells, for example, the immune effector cells are T cells isolated from human PBMC, Prepared after NK cells.
在第五方面，本发明提供了药物组合物，其包含选自表达本发明的组成型嵌合细胞因子受体或组成型嵌合细胞因子受体修饰的CAR多肽的免疫效应细胞(例如，T细胞、NK细胞)、编码本发明的组成型嵌合细胞因子受体或组成型嵌合细胞因子受体修饰的CAR多肽的核酸分子、本发明的载体、和它们的任意组合；和任选地可药用辅料。 In a fifth aspect, the invention provides a pharmaceutical composition comprising an immune effector cell (e.g., T cells, NK cells), nucleic acid molecules encoding the constitutive chimeric cytokine receptors or constitutively chimeric cytokine receptor-modified CAR polypeptides of the invention, the vectors of the invention, and any combination thereof; and optionally Medicinal excipients.
在一些实施方案中，当表达本发明的组成型嵌合细胞因子受体修饰的CAR多肽是分子开关调控型CAR多肽时，本发明的药物组合物还包含分子开关，例如，分子开关抗体。In some embodiments, when the constitutive chimeric cytokine receptor-modified CAR polypeptide expressing the invention is a molecular switch-regulated CAR polypeptide, the pharmaceutical composition of the invention further includes a molecular switch, for example, a molecular switch antibody.
在第六方面，本发明涉及第五方面所述的药物组合物的用途，用于在受试者中治疗肿瘤，包括向受试者施用治疗有效量的第五方面所述的药物组合物。In a sixth aspect, the present invention relates to the use of the pharmaceutical composition of the fifth aspect for treating tumors in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of the fifth aspect.
在第七方面，本发明涉及第五方面所述的药物组合物在制备治疗癌症药物中的应用。In a seventh aspect, the present invention relates to the use of the pharmaceutical composition described in the fifth aspect in the preparation of drugs for treating cancer.
在第八方面，本发明提供了一种治疗肿瘤的方法，所述方法包括向受试者施用治疗有效量的第五方面所述的药物组合物。In an eighth aspect, the present invention provides a method for treating tumors, the method comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition according to the fifth aspect.
附图简述Brief description of the drawings
结合以下附图一起阅读时，将更好地理解以下详细描述的本发明的优选实施方案。出于说明本发明的目的，图中显示了目前优选的实施方案。然而，应当理解本发明不限于图中所示实施方案的精确安排和手段。The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the following drawings. For the purpose of illustrating the invention, there is shown in the drawing a presently preferred embodiment. It is to be understood, however, that this invention is not limited to the precise arrangements and instrumentalities illustrated in the drawings.
图1显示了本发明的M7CR和/或CAR转导T细胞后，表达本发明的M7CR和/或CAR的T细胞的作用机制，在所述M7CR中，分别以具有重塑肿瘤微环境的效应分子(例如，细胞因子、免疫效应分子或抑制性分子拮抗剂)作为胞外结构域(ECD)与M7R融合。Figure 1 shows the mechanism of action of T cells expressing the M7CR and/or CAR of the present invention after the M7CR and/or CAR of the present invention transduces T cells. In the M7CR, they respectively have the effect of reshaping the tumor microenvironment. Molecules (eg, cytokines, immune effector molecules, or inhibitory molecule antagonists) are fused to the M7R as an extracellular domain (ECD).
图2A显示了野生型IL7R受体的结构和工程化的突变IL7R受体的结构。Figure 2A shows the structure of the wild-type IL7R receptor and the structure of the engineered mutant IL7R receptor.
图2B显示了在构建的病毒表达质粒中，IL7R-tCD19构建体、IL7R-WT构建体、Figure 2B shows that among the constructed viral expression plasmids, IL7R-tCD19 construct, IL7R-WT construct,
IL7Rm-tCD19构建体的结构示意图。图中，IL7Rm是指由不同突变的IL7R跨膜区(TM区)和IL7R野生型胞内区域(ICD)组成的部分。Structural representation of the IL7Rm-tCD19 construct. In the figure, IL7Rm refers to the part composed of different mutated IL7R transmembrane regions (TM regions) and IL7R wild-type intracellular region (ICD).
图3A显示了27个包含不同突变的M7R序列的tCD19-M7CR基因的慢病毒侵染BaF3细胞后第4天，用流式细胞术检测BaF3细胞表面tCD19表达的结果。图中，BaF3表示未经慢病毒侵染的BaF3细胞；IL7R-WT表示用包含野生型IL7R基因的慢病毒侵染BaF3细胞；IL7R-tCD19表示用包含tCD19、野生型IL7R跨膜区和胞内区的基因的慢病毒侵染BaF3细胞；IL7Rm-tCD19表示用包含tCD19、不同突变的IL7R跨膜区和IL7R野生型胞内区的基因的慢病毒侵染BaF3细胞。Figure 3A shows the results of using flow cytometry to detect the expression of tCD19 on the surface of BaF3 cells on day 4 after infection of BaF3 cells with 27 tCD19-M7CR genes containing different mutated M7R sequences. In the figure, BaF3 represents BaF3 cells without lentivirus infection; IL7R-WT represents BaF3 cells infected with lentivirus containing wild-type IL7R gene; IL7R-tCD19 represents BaF3 cells containing tCD19, wild-type IL7R transmembrane region and intracellular BaF3 cells were infected with lentivirus containing genes from the region; IL7Rm-tCD19 means BaF3 cells were infected with lentivirus containing genes from tCD19, different mutated IL7R transmembrane regions and IL7R wild-type intracellular region.
图3B显示了包含不同突变M7R序列的tCD19-M7CR基因的慢病毒侵染BaF3细胞后，不加入外源mIL-3培养各慢病毒侵染的BaF3细胞，能够提供持续激活的IL7R信号和促进BaF3细胞生长的不同突变M7R序列的结果图。图中各图示的含义同图3A。Figure 3B shows that after the lentivirus containing the tCD19-M7CR gene of different mutated M7R sequences infected BaF3 cells, culturing the BaF3 cells infected with each lentivirus without adding exogenous mIL-3 can provide sustained activation of IL7R signals and promote BaF3 Plot of results of cell growth with different mutated M7R sequences. The meanings of each icon in the figure are the same as in Figure 3A.
图3C显示了用包含不同突变M7R序列的tCD19-M7CR基因的慢病毒侵染BaF3细胞，从第3天开始不加入外源mIL-3，在第3天和第11天时检测表达CD19+的BaF3细胞百分数的结果，其中表达CD19+的BaF3细胞百分数越高，则存活的BaF3细胞越多。Figure 3C shows BaF3 cells were infected with lentiviruses containing tCD19-M7CR genes with different mutated M7R sequences, exogenous mIL-3 was not added starting from day 3, and BaF3 expressing CD19 + was detected on days 3 and 11. The result of cell percentage, the higher the percentage of BaF3 cells expressing CD19 + , the more BaF3 cells survive.
图3D显示了用包含不同突变M7R序列的tCD19-M7CR基因的慢病毒侵染BaF3细胞后，不加入外源mIL-3培养各慢病毒侵染的BaF3细胞，对存活的BaF3细胞进行细胞计数的结果。图中，“Parental BaF3 with IL3”表示未经病毒侵染的BaF3细胞用含有IL3的培养基培养，“parental BaF3 w/o IL3”表示未经病毒侵染的BaF3细胞用不含有IL3的培养基培养。Figure 3D shows that after infecting BaF3 cells with lentiviruses containing the tCD19-M7CR gene of different mutated M7R sequences, the BaF3 cells infected with each lentivirus were cultured without adding exogenous mIL-3, and the surviving BaF3 cells were counted. result. In the figure, "Parental BaF3 with IL3" means that BaF3 cells without virus infection are cultured with medium containing IL3, "parental BaF3 w/o IL3" means that BaF3 cells without virus infection are cultured with medium without IL3 nourish.
图4显示了用包含不同突变M7R序列的tCD19-M7CR基因的慢病毒侵染BaF3细胞后，用抗pSTAT5抗体染色BaF3细胞，检测细胞中STAT5的基础磷酸化水平的结果。图中，ISO表示用抗STAT5的同型对照抗体进行染色，+IL3表示对未经慢病毒侵染的BaF3细胞，加入IL3刺激细胞STAT5激活，作为阳性对照；“without IL3”表示不用IL3刺激未经慢病毒侵 染的BaF3细胞。Figure 4 shows the results of infecting BaF3 cells with lentiviruses containing tCD19-M7CR genes containing different mutated M7R sequences, and then staining BaF3 cells with anti-pSTAT5 antibodies to detect the basal phosphorylation level of STAT5 in the cells. In the figure, ISO means staining with an anti-STAT5 isotype control antibody, +IL3 means adding IL3 to BaF3 cells that have not been infected with lentivirus to stimulate the activation of STAT5 in the cells, as a positive control; "without IL3" means not using IL3 to stimulate the cells without lentivirus. Lentivirus invasion stained BaF3 cells.
图5显示了用包含不同突变M7R序列的tCD19-M7CR基因的慢病毒侵染T细胞后48小时，用流式细胞术检测T细胞表面tCD19表达的结果。图中，UNT表示未经慢病毒侵染的T细胞；IL7R-WT表示用包含野生型IL7R基因的慢病毒侵染T细胞；IL7R-tCD19表示用包含tCD19、野生型IL7R跨膜区和胞内区的基因的慢病毒侵染T细胞；IL7Rm-tCD19表示用包含tCD19、不同突变的IL7R跨膜区和IL7R野生型胞内区的基因的慢病毒侵染T细胞。Figure 5 shows the results of using flow cytometry to detect the expression of tCD19 on the surface of T cells 48 hours after infecting T cells with lentivirus containing the tCD19-M7CR gene of different mutated M7R sequences. In the figure, UNT represents T cells without lentivirus infection; IL7R-WT represents T cells infected with lentivirus containing wild-type IL7R gene; IL7R-tCD19 represents T cells containing tCD19, wild-type IL7R transmembrane region and intracellular IL7Rm-tCD19 indicates that T cells are infected with lentivirus containing genes from tCD19, different mutated IL7R transmembrane regions and IL7R wild-type intracellular region.
图6显示了用包含不同突变M7R序列的tCD19-M7CR基因的慢病毒侵染T细胞后，不加入外源IL-2刺激情况下，用抗pSTAT5抗体染色T细胞，检测细胞中STAT5的基础磷酸化水平的结果。图中，UNT表示未经慢病毒侵染的T细胞；IL7Rm-tCD19表示用包含tCD19、不同突变的IL7R跨膜区和IL7R野生型胞内区的基因的慢病毒侵染T细胞。Figure 6 shows that after infecting T cells with lentivirus containing the tCD19-M7CR gene of different mutated M7R sequences, without adding exogenous IL-2 stimulation, the T cells were stained with anti-pSTAT5 antibodies to detect the basal phosphate of STAT5 in the cells. level results. In the figure, UNT represents T cells without lentivirus infection; IL7Rm-tCD19 represents T cells infected with lentivirus containing genes of tCD19, different mutations of IL7R transmembrane region and IL7R wild-type intracellular region.
图7A显示了用包含不同突变M7R序列的tCD19-M7CR基因的慢病毒侵染T细胞后，不加入外源IL-2刺激情况下，随着时间的推移，计数表达tCD19-M7CR的T细胞数的结果。Figure 7A shows that after infecting T cells with lentiviruses containing tCD19-M7CR genes containing different mutated M7R sequences, without adding exogenous IL-2 stimulation, the number of T cells expressing tCD19-M7CR was counted over time. the result of.
图7B显示了用包含不同突变M7R序列的tCD19-M7CR基因的慢病毒侵染T细胞后，不加入外源IL-2刺激情况下，随着时间的推移，表达tCD19-M7CR的T细胞数的变化倍数。Figure 7B shows the number of T cells expressing tCD19-M7CR over time after infecting T cells with lentivirus containing the tCD19-M7CR gene of different mutated M7R sequences without adding exogenous IL-2 stimulation. Change multiple.
图8显示了M7CR修饰的CAR的结构，其中，M7CR包含胞外结构域ECD和IL7Rm。所述M7CR的N端通过P2A与不同CAR多肽的C端连接，从而构成M7CR修饰的CAR。Figure 8 shows the structure of M7CR-modified CAR, where M7CR contains extracellular domains ECD and IL7Rm. The N-terminus of the M7CR is connected to the C-terminus of different CAR polypeptides through P2A, thereby forming an M7CR-modified CAR.
图9A显示了用包含不同M7CR修饰的H9.1.2 CAR基因的慢病毒侵染T细胞后第9天时CAR及M7CR的表达水平，图9B显示了CD4和CD8阳性细胞比例。图中，“UNT”表示未经慢病毒侵染的T细胞；“H9.1.2”表示H9.1.2 CAR-T细胞，其余为tCD19-M7CR、tCD19-M7CR(CPT)、IL-15/IL-15Rα-M7CR(图中标记为IL-15-M7CR)、IL-12-P70-M7CR(图中标记为IL-12-M7CR)、IL-21-M7CR、CD40L-M7CR、4-1BBL-M7CR、抗PD-L1VHH-M7CR修饰的H9.1.2 CAR-T细胞。Figure 9A shows the expression levels of CAR and M7CR on day 9 after infecting T cells with lentivirus containing H9.1.2 CAR genes containing different M7CR modifications. Figure 9B shows the proportion of CD4 and CD8 positive cells. In the figure, “UNT” represents T cells without lentivirus infection; “H9.1.2” represents H9.1.2 CAR-T cells, and the others are tCD19-M7CR, tCD19-M7CR(CPT), IL-15/IL- 15Rα-M7CR (marked as IL-15-M7CR in the figure), IL-12-P70-M7CR (marked as IL-12-M7CR in the figure), IL-21-M7CR, CD40L-M7CR, 4-1BBL-M7CR, Anti-PD-L1VHH-M7CR modified H9.1.2 CAR-T cells.
图9C显示了用包含不同M7CR修饰的HuR968B CAR基因的慢病毒侵染T细胞后第9天时CAR及M7CR的表达水平，图9D显示了CD4和CD8阳性细胞比例。图中，“UNT”表示未经慢病毒侵染的T细胞；“8B”表示HuR968B CAR-T细胞，其余为tCD19-M7CR、tCD19-M7CR(CPT)、IL-15/IL-15Rα-M7CR(图中标记为IL-15-M7CR)、IL-12-P70-M7CR(图中标记为IL-12-M7CR)、IL-21-M7CR、CD40L-M7CR、4-1BBL-M7CR、抗PD-L1VHH-M7CR修饰的HuR968B CAR-T细胞。Figure 9C shows the expression levels of CAR and M7CR on day 9 after infecting T cells with lentivirus containing HuR968B CAR genes containing different M7CR modifications. Figure 9D shows the proportion of CD4 and CD8 positive cells. In the figure, “UNT” represents T cells without lentivirus infection; “8B” represents HuR968B CAR-T cells, and the others are tCD19-M7CR, tCD19-M7CR(CPT), IL-15/IL-15Rα-M7CR( IL-15-M7CR in the figure), IL-12-P70-M7CR (IL-12-M7CR in the figure), IL-21-M7CR, CD40L-M7CR, 4-1BBL-M7CR, anti-PD-L1 VHH -M7CR modified HuR968B CAR-T cells.
图9E显示了未修饰的H9.1.2 CAR-T样品和M7CR修饰的H9.1.2 CAR-T样品中总T细胞，CD4+和CD8+T的表型通过流式细胞术检测的结果。图中，“UNT”表示未经慢病毒侵染的T细胞；“H9.1.2”表示H9.1.2 CAR-T细胞，其余为M7CR修饰的H9.1.2 CAR-T细胞(图中标记同图9A)。Figure 9E shows the results of flow cytometric detection of the phenotypes of total T cells, CD4 + and CD8 + T in unmodified H9.1.2 CAR-T samples and M7CR-modified H9.1.2 CAR-T samples. In the figure, “UNT” indicates T cells without lentivirus infection; “H9.1.2” indicates H9.1.2 CAR-T cells, and the rest are M7CR-modified H9.1.2 CAR-T cells (the markings in the figure are the same as in Figure 9A ).
图9F显示了未修饰的HuR968B CAR-T样品和M7CR修饰的HuR968B CAR-T样品中总T细胞，CD4+和CD8+T的表型通过流式细胞术检测的结果。图中，“UNT”表示未经慢病毒侵染的T细胞；“8B”表示HuR968B CAR-T细胞，其余为M7CR修饰的HuR968B CAR-T细胞(图中标记同图9C)。Figure 9F shows the results of flow cytometric detection of the phenotypes of total T cells, CD4 + and CD8 + T in unmodified HuR968B CAR-T samples and M7CR-modified HuR968B CAR-T samples. In the figure, “UNT” indicates T cells without lentivirus infection; “8B” indicates HuR968B CAR-T cells, and the rest are M7CR-modified HuR968B CAR-T cells (the markings in the figure are the same as in Figure 9C).
图9G和图9H显示了未修饰的H9.2.1-218 CAR-T样品和M7CR修饰的H9.2.1-218 CAR-T样品中总T细胞，CD4+和CD8+T的表型通过流式细胞术检测的结果。图9G显示了来自供者15的PBMC制备的CAR T细胞，图9H显示了来自供者17的PBMC制备的CAR T细胞。 其中，H9.2.1-tCD19-M7CR表示tCD19-M7CR修饰的H9.2.1-218 CAR-T细胞；H9.2.1-IL-15-M7CR表示IL-15-M7CR修饰的H9.2.1-218 CAR-T细胞；H9.2.1in-IL15-M7CR表示H9.2.1-IL-15-M7CR细胞中CAR结构功能缺失；H9.2.1-IL15-M7CRin表示H9.2.1-IL-15-M7CR细胞中M7CR胞内结构(M7R)功能缺失；H9.2.1-sIL15表示H9.2.1-218 CAR-T细胞和可溶性IL15的组合；H9.2.1-IL-12-M7CR表示IL-12-p70-M7CR修饰的H9.2.1 CAR-T细胞(该H9.2.1 CAR序列如SEQ ID NO:100所示)；H9.2.1-28-IL-15-M7CR表示IL-15-M7CR修饰的、且共刺激结构域为CD28的H9.2.1-28 CAR-T细胞，8E5表示来自CARsgen公司CT041产品(该CAR序列如SEQ ID NO:188所示)。Figure 9G and Figure 9H show the phenotypes of total T cells, CD4 + and CD8 + T in unmodified H9.2.1-218 CAR-T samples and M7CR-modified H9.2.1-218 CAR-T samples by flow cytometry The results of technical testing. Figure 9G shows CAR T cells prepared from PBMC of donor 15, and Figure 9H shows CAR T cells prepared from PBMC of donor 17. Among them, H9.2.1-tCD19-M7CR represents tCD19-M7CR modified H9.2.1-218 CAR-T cells; H9.2.1-IL-15-M7CR represents IL-15-M7CR modified H9.2.1-218 CAR-T cells. cells; H9.2.1in-IL15-M7CR indicates the loss of CAR structure and function in H9.2.1-IL-15-M7CR cells; H9.2.1-IL15-M7CRin indicates the intracellular structure of M7CR in H9.2.1-IL-15-M7CR cells (M7R) loss of function; H9.2.1-sIL15 represents the combination of H9.2.1-218 CAR-T cells and soluble IL15; H9.2.1-IL-12-M7CR represents IL-12-p70-M7CR modified H9.2.1 CAR -T cells (the H9.2.1 CAR sequence is shown in SEQ ID NO: 100); H9.2.1-28-IL-15-M7CR represents H9 modified by IL-15-M7CR and whose costimulatory domain is CD28. 2.1-28 CAR-T cells, 8E5 represents the CT041 product from CARsgen Company (the CAR sequence is shown in SEQ ID NO: 188).
图9I和图9J分别显示了自供者15和17的PBMC制备的CAR-T细胞中，M7R能够持续提供激活信号，激活下游信号通路。图中，H9.2.1-tCD19-M7CR表示tCD19-M7CR修饰的H9.2.1 CAR-T细胞；H9.2.1-IL-12-M7CR表示IL-12-p70-M7CR修饰的H9.2.1 CAR-T细胞；H9.2.1in-IL12-M7CR表示H9.2.1-IL-12-M7CR细胞中CAR结构功能缺失；H9.2.1-IL12-M7CRin表示H9.2.1-IL-12-M7CR细胞中M7CR胞内结构(M7R)功能缺失；H9.2.1-sIL12表示H9.2.1 CAR-T细胞和可溶性IL12的组合。Figure 9I and Figure 9J show that in CAR-T cells prepared from PBMC of donors 15 and 17, respectively, M7R can continuously provide activation signals and activate downstream signaling pathways. In the figure, H9.2.1-tCD19-M7CR represents tCD19-M7CR modified H9.2.1 CAR-T cells; H9.2.1-IL-12-M7CR represents IL-12-p70-M7CR modified H9.2.1 CAR-T cells. ; H9.2.1in-IL12-M7CR indicates the loss of CAR structure and function in H9.2.1-IL-12-M7CR cells; H9.2.1-IL12-M7CRin indicates the intracellular structure of M7CR in H9.2.1-IL-12-M7CR cells ( M7R) loss of function; H9.2.1-sIL12 represents a combination of H9.2.1 CAR-T cells and soluble IL12.
图10A显示了通过Qufikit检测DANG-G18.2，SNU-601high，SNU-601low细胞表面CLDN18.2的分子数量。峰状图中，深色部分表示ISO，浅色部分表示阳性细胞。Figure 10A shows the detection of the number of molecules of CLDN18.2 on the cell surface of DANG-G18.2, SNU-601 high , and SNU-601 low by Qufikit. In the peak diagram, the dark part represents ISO, and the light part represents positive cells.
图10B显示了作为靶细胞的DANG18.2细胞、NUGC-4细胞、SNU-620细胞、PANC-1细胞、SNU-601细胞、Hup-T4细胞中CLDN18.2的表达量，图中ISO为同型抗体对照，K562为CLDN18.2阴性对照细胞。Figure 10B shows the expression level of CLDN18.2 in DANG18.2 cells, NUGC-4 cells, SNU-620 cells, PANC-1 cells, SNU-601 cells, and Hup-T4 cells as target cells. ISO in the figure represents the isotype. Antibody control, K562 is CLDN18.2 negative control cells.
图11A、图11B和图11C显示了分别用未修饰的H9.1.2 CAR-T细胞或不同M7CR修饰的H9.1.2 CAR-T细胞与肿瘤靶细胞DAN-G18.2共孵育，在E:T分别为1:1、1:3、1:10时，各CAR-T细胞对靶细胞的杀伤作用。图中，“PC”表示阳性对照(Positive control,用裂解液对靶细胞进行处理，使得所有靶细胞裂解)；“NT”表示未经慢病毒侵染的T细胞；“Tumor cell only”表示DAN-G18.2细胞系；“H9.1.2”表示H9.1.2 CAR-T细胞，其余为M7CR修饰的H9.1.2 CAR-T细胞(图中标记同图9A)。Figure 11A, Figure 11B and Figure 11C show that unmodified H9.1.2 CAR-T cells or H9.1.2 CAR-T cells modified with different M7CR were co-incubated with tumor target cells DAN-G18.2, respectively, in E:T The killing effect of each CAR-T cell on target cells when the ratio is 1:1, 1:3, and 1:10 respectively. In the figure, “PC” represents positive control (Positive control, target cells are treated with lysis solution to lyse all target cells); “NT” represents T cells without lentivirus infection; “Tumor cell only” represents DAN -G18.2 cell line; “H9.1.2” indicates H9.1.2 CAR-T cells, and the rest are M7CR-modified H9.1.2 CAR-T cells (the markings in the figure are the same as in Figure 9A).
图11D显示了分别用未修饰的HuR968B CAR-T细胞或不同M7CR修饰的HuR968B CAR-T细胞以及含P329G突变A6抗体(2nM)与靶细胞SUN-601high或SUN-601low共孵育，在E:T为1:1时，各CAR-T细胞对靶细胞的杀伤作用。图中，“PC”表示阳性对照(Positive control,用裂解液对靶细胞进行处理，使得所有靶细胞裂解)；“NT”表示未经慢病毒侵染的T细胞；“8B”表示未修饰的HuR968B CAR-T细胞，其余为M7CR修饰的HuR968B CAR-T细胞(图中标记同图9C)。Figure 11D shows that unmodified HuR968B CAR-T cells or HuR968B CAR-T cells modified with different M7CRs and P329G mutation-containing A6 antibodies (2nM) were co-incubated with target cells SUN-601 high or SUN-601 low , respectively. When :T is 1:1, the killing effect of each CAR-T cell on target cells. In the figure, “PC” represents positive control (Positive control, target cells are treated with lysis solution to lyse all target cells); “NT” represents T cells that have not been infected by lentivirus; “8B” represents unmodified HuR968B CAR-T cells, and the rest are M7CR-modified HuR968B CAR-T cells (the labels in the figure are the same as in Figure 9C).
图12A、图12C和图12E显示了分别用未修饰的HuR968B CAR-T细胞或不同M7CR修饰的HuR968B CAR-T细胞以及含P329G突变A6抗体(2nM)与靶细胞SUN-601high共孵育，在E:T为2:1时，使用靶细胞SUN-601high反复刺激多个轮次，计数CAR-T细胞数和CAR-T细胞增殖倍数的结果。图中，“8B”表示未修饰的HuR968B CAR-T细胞，其余为M7CR修饰的HuR968B CAR-T细胞(图中标记含义同图9C)。Figure 12A, Figure 12C and Figure 12E show that unmodified HuR968B CAR-T cells or HuR968B CAR-T cells modified with different M7CR and P329G mutation-containing A6 antibody (2nM) were co-incubated with the target cell SUN-601 high , respectively. When E:T is 2:1, use target cells SUN-601 high to repeatedly stimulate for multiple rounds, and count the results of CAR-T cell number and CAR-T cell proliferation fold. In the figure, “8B” represents unmodified HuR968B CAR-T cells, and the rest are M7CR-modified HuR968B CAR-T cells (the meaning of the marks in the figure is the same as in Figure 9C).
图12B、图12D和图12F显示了分别用未修饰的HuR968B CAR-T细胞或不同M7CR修饰的HuR968B CAR-T细胞以及含P329G突变A6抗体(2nM)与靶细胞SUN-601high共孵育，在E:T为2:1时，使用靶细胞SUN-601high反复刺激多个轮次时，HuR968B CAR-T细胞中CAR+ 细胞的比例和CAR+细胞百分比的变化倍数。图中，“8B”表示未修饰的HuR968B CAR-T细胞，其余为M7CR修饰的HuR968B CAR-T细胞(图中标记含义同图9C)。Figure 12B, Figure 12D and Figure 12F show that unmodified HuR968B CAR-T cells or HuR968B CAR-T cells modified with different M7CR and P329G mutant A6 antibody (2nM) were co-incubated with the target cell SUN-601 high , respectively. When E:T is 2:1, when the target cell SUN-601 high is repeatedly stimulated for multiple rounds, CAR + in HuR968B CAR-T cells Fold changes in the proportion of cells and the percentage of CAR + cells. In the figure, “8B” represents unmodified HuR968B CAR-T cells, and the rest are M7CR-modified HuR968B CAR-T cells (the meaning of the marks in the figure is the same as in Figure 9C).
图13A显示了对图12A-图12F中经第一轮刺激和第三轮刺激后各组中CD4+和CD8+T细胞数的代表性流式细胞术测定结果。Figure 13A shows representative flow cytometry results of CD4 + and CD8 + T cell numbers in each group after the first and third rounds of stimulation in Figures 12A-12F.
图13B显示了对图13A各组中CD4+和CD8+T细胞的比例的统计结果。Figure 13B shows the statistical results of the proportion of CD4 + and CD8 + T cells in each group of Figure 13A.
图13C显示了分别用未修饰的HuR968B CAR-T细胞或不同M7CR修饰的HuR968B CAR-T细胞以及含P329G突变A6抗体(2nM)与靶细胞SUN-601high共孵育，在E:T为2:1时，使用靶细胞SUN-601high反复刺激多个轮次，使用BDTM Cytometric Bead Array(CBA)Human Th1/Th2 Cytokine Kit II对培养上清液中的细胞因子进行检测的结果，图中，“8B”表示HuR968B CAR-T细胞，其余为M7CR修饰的HuR968B CAR-T细胞(图中标记含义同图9C)。Figure 13C shows that unmodified HuR968B CAR-T cells or HuR968B CAR-T cells modified with different M7CRs and P329G mutation-containing A6 antibodies (2nM) were co-incubated with the target cell SUN-601 high , and the E:T was 2: At 1 hour, the target cells SUN-601 high were repeatedly stimulated for multiple rounds, and the BD TM Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit II was used to detect the cytokines in the culture supernatant. In the figure, “8B” indicates HuR968B CAR-T cells, and the rest are M7CR-modified HuR968B CAR-T cells (the meaning of the marks in the figure is the same as in Figure 9C).
图14A-14D显示了在CLDN18.2表达水平不同的靶细胞中，CAR-T细胞的杀伤作用随CLDN18.2的表达水平升高而增强。图中，H9.2.1-tCD19-M7CR表示tCD19-M7CR修饰的H9.2.1 CAR-T细胞；H9.2.1-IL-12-M7CR表示IL-12-M7CR修饰的H9.2.1 CAR-T细胞；H9.2.1in-IL12-M7CR表示H9.2.1-IL-12-M7CR细胞中CAR结构功能缺失；H9.2.1-IL-12-M7CRin表示H9.2.1-IL-12-M7CR细胞中M7CR胞内结构(M7R)功能缺失；H9.2.1-sIL12表示H9.2.1 CAR-T细胞和分泌可溶性IL12的组合。Figures 14A-14D show that in target cells with different expression levels of CLDN18.2, the killing effect of CAR-T cells increases as the expression level of CLDN18.2 increases. In the figure, H9.2.1-tCD19-M7CR represents tCD19-M7CR modified H9.2.1 CAR-T cells; H9.2.1-IL-12-M7CR represents IL-12-M7CR modified H9.2.1 CAR-T cells; H9 .2.1in-IL12-M7CR indicates the loss of CAR structure and function in H9.2.1-IL-12-M7CR cells; H9.2.1-IL-12-M7CRin indicates the intracellular structure of M7CR in H9.2.1-IL-12-M7CR cells ( M7R) loss of function; H9.2.1-sIL12 represents a combination of H9.2.1 CAR-T cells and secretes soluble IL12.
图14E显示了分别用未修饰的H9.2.1 CAR-T细胞或IL-12-M7CR修饰的CAR-T细胞与靶细胞Hup-T4共孵育，在E:T为1:1和1:5时，使用靶细胞Hup-T4反复刺激三轮，各CAR-T细胞对靶细胞的杀伤作用。经持续三轮杀伤实验后，IL-12-M7CR修饰的CAR-T细胞对靶细胞仍然具有较好的杀伤作用，而未经修饰的H9.2.1 CAR-T细胞或单独M7R修饰的H9.2.1 CAR-T细胞在多轮杀伤中，随轮数增加杀伤效果逐渐减弱。图中，H9.2.1表示H9.2.1 CAR-T细胞；H9.2.1-tCD19-M7CR表示tCD19-M7CR修饰的H9.2.1 CAR-T细胞；H9.2.1-IL-12-M7CR表示IL-12-M7CR修饰的H9.2.1 CAR-T细胞。Figure 14E shows the co-incubation of unmodified H9.2.1 CAR-T cells or IL-12-M7CR-modified CAR-T cells with the target cell Hup-T4 at E:T of 1:1 and 1:5, respectively. , using the target cell Hup-T4 to repeatedly stimulate three rounds, and the killing effect of each CAR-T cell on the target cell. After three rounds of continuous killing experiments, IL-12-M7CR modified CAR-T cells still had a better killing effect on target cells, while unmodified H9.2.1 CAR-T cells or M7R-modified H9.2.1 alone In multiple rounds of killing, the killing effect of CAR-T cells gradually weakens as the number of rounds increases. In the figure, H9.2.1 represents H9.2.1 CAR-T cells; H9.2.1-tCD19-M7CR represents tCD19-M7CR modified H9.2.1 CAR-T cells; H9.2.1-IL-12-M7CR represents IL-12- M7CR modified H9.2.1 CAR-T cells.
图15显示了在腹腔注射表达荧光素酶NUGC-4细胞构建胃癌腹腔转移模型中，通过IVIS成像系统检测小鼠肿瘤负荷变化的情况。Figure 15 shows the changes in tumor burden in mice detected by the IVIS imaging system in a gastric cancer abdominal metastasis model constructed by intraperitoneal injection of luciferase-expressing NUGC-4 cells.
图16显示了表达组成型嵌合细胞因子受体的PG CAR-T细胞在小鼠体内的抗肿瘤作用。对于荷瘤小鼠，在施用CAR-T细胞和A6抗体后，随时间增加，IL-12-M7CR修饰的PG CAR-T细胞(图中标记为8B-IL12-M7CR CAR-T)和tCD19-M7CR修饰的PG CAR-T细胞(图中标记为8B-M7R CAR-T)在体内具有更好的抗肿瘤作用。Figure 16 shows the anti-tumor effect of PG CAR-T cells expressing constitutive chimeric cytokine receptors in mice. For tumor-bearing mice, after administration of CAR-T cells and A6 antibodies, IL-12-M7CR-modified PG CAR-T cells (labeled 8B-IL12-M7CR CAR-T in the figure) and tCD19- M7CR-modified PG CAR-T cells (labeled 8B-M7R CAR-T in the figure) have better anti-tumor effects in vivo.
图17显示了表达组成型嵌合细胞因子受体的PG CAR-T细胞在小鼠体内的扩增水平。对于荷瘤小鼠，在施用PG CAR-T细胞和A6抗体后第7天至第28天IL-12-M7CR修饰的PG CAR-T细胞(图中标记为8B-IL12-M7CR CAR-T)和tCD19-M7CR修饰的PG CAR-T细胞(图中标记为8B-M7R CAR-T)具有较高的扩增水平。Figure 17 shows the expansion levels of PG CAR-T cells expressing constitutively chimeric cytokine receptors in mice. For tumor-bearing mice, IL-12-M7CR modified PG CAR-T cells (labeled 8B-IL12-M7CR CAR-T in the figure) from day 7 to day 28 after administration of PG CAR-T cells and A6 antibody and tCD19-M7CR modified PG CAR-T cells (labeled 8B-M7R CAR-T in the figure) have higher expansion levels.
图18显示了表达组成型嵌合细胞因子受体的H9.2.1 CAR-T细胞在小鼠体内的抗肿瘤作用。在施用CAR-T细胞两周后，IL-12-M7CR修饰的CAR T细胞(图中标记为IL12-M7CR-H9.2.1 CAR-T)具有最强的抗肿瘤作用，其次为tCD19-M7CR修饰的CAR-T细胞(图中标记为M7R-H9.2.1-CAR-T)，未修饰的H9.2.1 CAR T细胞在体内抗肿瘤作用最弱。Figure 18 shows the anti-tumor effect of H9.2.1 CAR-T cells expressing constitutive chimeric cytokine receptors in mice. Two weeks after the administration of CAR-T cells, IL-12-M7CR-modified CAR T cells (labeled IL12-M7CR-H9.2.1 CAR-T in the figure) had the strongest anti-tumor effect, followed by tCD19-M7CR-modified CAR-T cells (labeled M7R-H9.2.1-CAR-T in the figure), unmodified H9.2.1 CAR T cells have the weakest anti-tumor effect in vivo.
图19显示了表达组成型嵌合细胞因子受体的传统CAR-T细胞在小鼠体内的抗肿瘤作用。 对于荷瘤小鼠，在施用CAR-T细胞后，随时间增加，IL-12-M7CR修饰的CAR-T细胞(图中标记为IL12-M7CR-H9.2.1 CAR-T)和tCD19-M7CR修饰的CAR-T细胞(图中标记为M7R-H9.2.1 CAR-T)在体内具有更好的抗肿瘤作用。Figure 19 shows the anti-tumor effect of traditional CAR-T cells expressing constitutive chimeric cytokine receptors in mice. For tumor-bearing mice, IL-12-M7CR-modified CAR-T cells (labeled IL12-M7CR-H9.2.1 CAR-T in the figure) and tCD19-M7CR-modified increased over time after administration of CAR-T cells. CAR-T cells (labeled M7R-H9.2.1 CAR-T in the figure) have better anti-tumor effects in vivo.
图20显示了表达组成型嵌合细胞因子受体的传统CAR-T细胞在小鼠体内的扩增水平。对于荷瘤小鼠，在施用CAR-T细胞后第7天至第28天IL-12-M7CR修饰的CAR-T细胞(图中标记为IL12-M7CR-H9.2.1 CAR-T)和tCD19-M7CR修饰的CAR-T细胞(图中标记为M7R-H9.2.1 CAR-T)具有较高的扩增水平。Figure 20 shows the expansion levels of conventional CAR-T cells expressing constitutive chimeric cytokine receptors in mice. For tumor-bearing mice, IL-12-M7CR-modified CAR-T cells (labeled IL12-M7CR-H9.2.1 CAR-T in the figure) and tCD19- M7CR-modified CAR-T cells (labeled M7R-H9.2.1 CAR-T in the figure) have higher amplification levels.
图21显示IL-15-M7CR修饰的CAR结构，其中，M7CR包含IL-15ECD和IL7Rm8。所述M7CR的N端通过P2A与CAR多肽的C端连接，从而构成M7CR修饰的CAR。Figure 21 shows the structure of IL-15-M7CR modified CAR, where M7CR contains IL-15ECD and IL7Rm8. The N-terminus of the M7CR is connected to the C-terminus of the CAR polypeptide through P2A, thereby forming an M7CR-modified CAR.
图22显示CAR-T制备第9天的扩增倍数统计柱状图。“H9.1.2”表示H9.1.2 CAR-T细胞，H9.2.1in-IL15-M7CR表示H9.2.1-IL-15-M7CR细胞中CAR结构功能缺失；H9.2.1-IL15-M7CRin表示H9.2.1-IL-15-M7CR细胞中M7CR胞内结构功能缺失；H9.2.1-sIL15表示表达分泌型sIL-15的H9.2.1 CAR-T，8E5表示来自CARsgen公司CT041产品。Figure 22 shows a statistical histogram of amplification folds on day 9 of CAR-T preparation. “H9.1.2” indicates H9.1.2 CAR-T cells, H9.2.1in-IL15-M7CR indicates the loss of CAR structure and function in H9.2.1-IL-15-M7CR cells; H9.2.1-IL15-M7CRin indicates H9.2.1 -IL-15-M7CR cells have a loss of intracellular structure and function of M7CR; H9.2.1-sIL15 represents the H9.2.1 CAR-T expressing secreted sIL-15, and 8E5 represents the CT041 product from CARsgen.
图23A显示制备的CAR-T细胞CAR或M7CR表达。图23A代表性流式散点图，图23B为统计柱状图。Figure 23A shows CAR or M7CR expression in prepared CAR-T cells. Figure 23A is a representative flow scatter plot, and Figure 23B is a statistical histogram.
图23C、图23D显示CD4和CD8比例，图23C代表性流式散点图，图23D为统计柱状图。“NT”表示未经慢病毒侵染的T细胞。Figure 23C and Figure 23D show the ratio of CD4 and CD8, Figure 23C is a representative flow scatter plot, and Figure 23D is a statistical histogram. “NT” indicates T cells without lentivirus infection.
图23E、图23F显示制备第7天和第9天各CAR-T细胞分化表型，图23E为代表性流式散点图，图23F为统计柱状图。Figure 23E and Figure 23F show the differentiation phenotype of each CAR-T cell on the 7th and 9th days of preparation. Figure 23E is a representative flow cytometry scatter plot, and Figure 23F is a statistical histogram.
图23G和图23H显示在制备第9天各CAR-T细胞胞内磷酸化STAT5表达，图23G为代表性流式细胞图，图23H为统计柱状图。Figure 23G and Figure 23H show the expression of intracellular phosphorylated STAT5 in each CAR-T cell on the 9th day of preparation. Figure 23G is a representative flow cytometry diagram, and Figure 23H is a statistical histogram.
图24A和图24C显示各CAR-T细胞和PANC1靶细胞培养后CD25、CD69表达结果，图24A为代表性流式散点图，图24C为CD25+CD69+细胞统计柱状图。图24B和图24D显示各CAR-T细胞和Hup-T4靶细胞培养后CD25、CD69表达结果，图24B为代表性流式散点图，图24D为CD25+CD69+细胞统计柱状图。Figure 24A and Figure 24C show the CD25 and CD69 expression results after culture of each CAR-T cell and PANC1 target cell. Figure 24A is a representative flow cytometry scatter plot, and Figure 24C is a statistical histogram of CD25 + CD69 + cells. Figure 24B and Figure 24D show the CD25 and CD69 expression results after culture of each CAR-T cell and Hup-T4 target cell. Figure 24B is a representative flow cytometry scatter plot, and Figure 24D is a statistical histogram of CD25 + CD69 + cells.
图25A显示各CAR-T细胞和PANC1靶细胞培养24h后上清中IL-2、IFN-γ和TNFα浓度。图25B显示各CAR-T细胞和Hup-T4靶细胞培养24h后上清中IL-2、IFN-γ和TNFα浓度。Figure 25A shows the concentrations of IL-2, IFN-γ and TNFα in the supernatant of each CAR-T cell and PANC1 target cell after culture for 24 hours. Figure 25B shows the concentrations of IL-2, IFN-γ and TNFα in the supernatant of each CAR-T cell and Hup-T4 target cell after culture for 24 hours.
图26A显示来自2个供者不同CAR-T细胞和PANC-1细胞共培养5天后，CAR+和CAR-细胞增殖的代表性流式细胞图。Baseline为CAR-T细胞标记后直接检测结果。图26B显示来自2个供者不同CAR-T细胞和SUN620细胞共培养5天后，CAR+和CAR-细胞增殖的代表性流式细胞图。Baseline为CAR-T细胞标记后直接检测结果。Figure 26A shows representative flow cytometry plots of CAR + and CAR- cell proliferation after 5 days of co-culture with different CAR-T cells and PANC-1 cells from 2 donors. Baseline is the direct detection result after CAR-T cell labeling. Figure 26B shows representative flow cytometry plots of CAR + and CAR- cell proliferation after co-culture of different CAR-T cells and SUN620 cells from 2 donors for 5 days. Baseline is the direct detection result after CAR-T cell labeling.
图27A显示腹腔NUGC‐4模型中3个不同剂量H9.2.1-IL-15-M7CR CAR‐T细胞或H9.2.1-CD28-IL-15-M7CR CAR-T细胞的体内抗肿瘤效应，小鼠腹腔肿瘤负荷直接通过体内成像监测。图27B显示肿瘤负荷统计图。“NT”表示未经慢病毒侵染的T细胞。图27C显示治疗小鼠的体重变化情况。图27D和图27E分别显示小鼠体内总T细胞及CAR‐T细胞随时间扩增情况，以每100μl小鼠外周血中细胞数量表示。Figure 27A shows the in vivo anti-tumor effects of 3 different doses of H9.2.1-IL-15-M7CR CAR-T cells or H9.2.1-CD28-IL-15-M7CR CAR-T cells in the intraperitoneal NUGC-4 model, mice Abdominal tumor burden is monitored directly by in vivo imaging. Figure 27B shows a statistical graph of tumor burden. “NT” indicates T cells without lentivirus infection. Figure 27C shows changes in body weight of treated mice. Figure 27D and Figure 27E respectively show the expansion of total T cells and CAR-T cells in mice over time, expressed as the number of cells per 100 μl of mouse peripheral blood.
图28显示细胞因子-M7CR修饰的CAR结构，将不同细胞因子作为胞外结构域(ECD)和M7Rm8(SEQ ID NO：34)直接连接构建组成型嵌合细胞因子受体M7CRm8；然后通过P2A 将M7CRm8的N端与H9.2.1 CAR多肽的C端连接，从而构成细胞因子-M7CR修饰的H9.2.1 CAR。tCD19-M7CR作为对照分子。Figure 28 shows the cytokine-M7CR modified CAR structure. Different cytokines are directly connected as the extracellular domain (ECD) and M7Rm8 (SEQ ID NO: 34) to construct a constitutive chimeric cytokine receptor M7CRm8; then through P2A The N-terminus of M7CRm8 is connected to the C-terminus of the H9.2.1 CAR polypeptide to form a cytokine-M7CR modified H9.2.1 CAR. tCD19-M7CR served as a control molecule.
图29A显示供者16来源的各种CAR-T第1天到第9天随时间的扩增曲线图。“H9.1.2”表示H9.1.2 CAR-T细胞，“H9.1.2-218”表示H9.1.2-218 CAR-T细胞，其余为tCD19-M7CR和不同细胞因子-M7CR修饰的H9.1.2 CAR-T细胞。图29B显示供者6，11，17来源的各种CAR-T第1天到第9天随时间的扩增曲线图。图29C显示供者6，11，17来源的各种CAR-T制备第9天的扩增倍数统计柱状图。Figure 29A shows the amplification curves of various CAR-T derived from donor 16 over time from day 1 to day 9. “H9.1.2” represents H9.1.2 CAR-T cells, “H9.1.2-218” represents H9.1.2-218 CAR-T cells, and the rest are H9.1.2 CAR- modified by tCD19-M7CR and different cytokines-M7CR. T cells. Figure 29B shows the amplification curves of various CAR-T derived from donors 6, 11, and 17 over time from day 1 to day 9. Figure 29C shows a statistical histogram of amplification folds on day 9 of preparation of various CAR-Ts derived from donors 6, 11, and 17.
图30A和图30B分别显示供者16来源CAR-T后第7和第9天时CAR和/或作为M7CR的ECD表达的代表性流式散点图和统计图。图30C和图30D分别显示供者6，11，17来源的CAR-T细胞第9天时CAR表达的代表性流式散点图和统计图。“NT”表示未经慢病毒侵染的T细胞。Figures 30A and 30B show representative flow scatter plots and statistical plots of CAR and/or ECD expression as M7CR at days 7 and 9, respectively, after donor 16-derived CAR-T. Figure 30C and Figure 30D show representative flow scatter plots and statistical graphs of CAR expression on day 9 of CAR-T cells derived from donors 6, 11, and 17, respectively. “NT” indicates T cells without lentivirus infection.
图30E显示供者16来源CAR-T细胞制备第7天和第9天时CD4和CD8亚群比例的代表性流式散点图。图30F和图30G分别显示供者6，11，17来源CAR-T细胞制备第9天时CD4和CD8亚群比例的代表性流式散点图和统计图。Figure 30E shows representative flow scatter plots of CD4 and CD8 subpopulation ratios on days 7 and 9 of donor 16-derived CAR-T cell preparation. Figure 30F and Figure 30G respectively show representative flow scatter plots and statistical diagrams of the proportions of CD4 and CD8 subpopulations on day 9 of preparation of CAR-T cells derived from donors 6, 11, and 17.
图30H和图30I分别显示制备第7天和第9天供者16来源CAR-T细胞分化表型的代表性流式散点图和CD45RA+CCR7+细胞比例统计图。CD45RA+CCR7+代表初始T细胞或干性记忆性T细胞(TN/TSCM)、CD45RA-CCR7+代表中心记忆性T细胞(TCM)、CD45RA-CCR7-代表效应记忆性T细胞(TEM)、CD45RA+CCR7-代表效应T细胞(Teff)亚群。Figure 30H and Figure 30I show representative flow cytometry scatter plots and CD45RA + CCR7 + cell proportion statistical diagrams of the differentiation phenotype of donor 16-derived CAR-T cells on days 7 and 9 of preparation, respectively. CD45RA + CCR7 + represents naive T cells or stem memory T cells (TN/TSCM), CD45RA-CCR7 + represents central memory T cells (TCM), CD45RA - CCR7 - represents effector memory T cells (TEM), CD45RA + CCR7 - represents the effector T cell (Teff) subset.
图30J和图30K分别显示制备第9天供者6，11，17来源CAR-T细胞分化表型的代表性流式散点图和统计图。Figure 30J and Figure 30K respectively show representative flow scatter plots and statistical diagrams of the differentiation phenotypes of CAR-T cells derived from donors 6, 11, and 17 on day 9 of preparation.
图30L和图30M分别显示制备第9天供者6，11，17来源CAR-T细胞CD45RA表达代表性流式细胞图和统计图(以平均荧光强度(MFI)表示)。Figure 30L and Figure 30M respectively show representative flow cytometry and statistical diagrams (expressed as mean fluorescence intensity (MFI)) of CD45RA expression in CAR-T cells derived from donors 6, 11, and 17 on day 9 of preparation.
图30N和图30O分别显示制备第9天供者6，11，17来源CAR-T细胞CCR7表达代表性流式细胞图和统计图(以MFI表示)。图31显示用不同信号强度的嵌合受体M7CR修饰的CAR结构。通过P2A将不同信号强度tCD19-M7CR的N端与H9.2.1 CAR多肽的C端连接，从而构成tCD19-M7CR修饰的H9.2.1 CAR。通过P2A将不同信号强度tCD34-M7CR的N端与BB2121 CAR多肽的C端连接，从而构成tCD34-M7CR修饰的BB2121 CAR。tCD19-M7CR(CPT)和tCD34-M7CR(CPT)为阳性对照。Figure 30N and Figure 30O respectively show representative flow cytometry and statistical diagrams (expressed as MFI) of CCR7 expression in CAR-T cells derived from donors 6, 11, and 17 on day 9 of preparation. Figure 31 shows CAR structures modified with chimeric receptor M7CR of different signal intensities. The N-terminus of tCD19-M7CR with different signal strengths is connected to the C-terminus of the H9.2.1 CAR polypeptide through P2A, thereby forming a tCD19-M7CR modified H9.2.1 CAR. The N-terminus of tCD34-M7CR with different signal strengths is connected to the C-terminus of BB2121 CAR polypeptide through P2A to form a tCD34-M7CR modified BB2121 CAR. tCD19-M7CR(CPT) and tCD34-M7CR(CPT) are positive controls.
图32显示供者15来源的H9.2.1和BB2121及其不同信号强度M7CR修饰的CAR-T细胞第1天到第9天的扩增动力学。图中“CAR alone”为H9.2.1 CAR-T细胞或BB2121 CAR-T细胞，其他为M7CR修饰的CAR-T细胞。Figure 32 shows the expansion kinetics of H9.2.1 and BB2121 derived from donor 15 and M7CR-modified CAR-T cells with different signal intensities from day 1 to day 9. “CAR alone” in the picture refers to H9.2.1 CAR-T cells or BB2121 CAR-T cells, and the others are M7CR-modified CAR-T cells.
图33A和图33B分别显示供者15来源的H9.2.1和BB2121及其不同信号强度M7CR修饰的CAR-T细胞CAR表达水平的代表性流式细胞图，图33C显示CAR表达阳性率统计图。Figures 33A and 33B respectively show representative flow cytometry diagrams of CAR expression levels of H9.2.1 and BB2121 derived from donor 15 and M7CR-modified CAR-T cells with different signal intensities. Figure 33C shows a statistical diagram of the positive rate of CAR expression.
图34A和图34B分别显示供者15来源的H9.2.1和BB2121及其不同信号强度M7CR修饰的CAR-T细胞中CD4和CD8细胞亚群代表性流式细胞图。图34C显示CD4和CD8细胞亚群比例统计图。Figure 34A and Figure 34B respectively show representative flow cytometry diagrams of CD4 and CD8 cell subsets in H9.2.1 and BB2121 derived from donor 15 and their M7CR-modified CAR-T cells with different signal intensities. Figure 34C shows a statistical graph of CD4 and CD8 cell subset proportions.
图35A和图35B分别显示供者15来源的H9.2.1和BB2121及其不同信号强度M7CR修饰的CAR-T细胞分化表型表达的代表性流式散点图。图35C显示CD45RA+CCR7+细胞比例统计图。 Figures 35A and 35B show representative flow scatter plots of the differentiation phenotype expression of H9.2.1 and BB2121 derived from donor 15 and their M7CR-modified CAR-T cells with different signal intensities, respectively. Figure 35C shows a statistical graph of the proportion of CD45RA+CCR7+ cells.
图36A和图36B分别显示供者15来源的制备第5天的CAR-T细胞和冻存复苏后CAR-T细胞胞内磷酸化STAT5(pSTAT5)表达的代表性统计图。图中“CAR alone”为H9.2.1 CAR-T细胞或BB2121 CAR-T细胞，其他为M7CR修饰的H9.2.1或BB2121 CAR-T细胞。Figure 36A and Figure 36B respectively show representative statistical diagrams of the expression of intracellular phosphorylated STAT5 (pSTAT5) in CAR-T cells derived from donor 15 on the 5th day of preparation and in CAR-T cells after cryopreservation and recovery. “CAR alone” in the picture refers to H9.2.1 CAR-T cells or BB2121 CAR-T cells, and the others are M7CR-modified H9.2.1 or BB2121 CAR-T cells.
图37A显示供者15来源的H9.2.1及其不同信号强度M7CR修饰的CAR-T细胞和NUGC-4靶细胞培养后CD25、CD69表达代表性流式散点图，图37B显示CD25+CD69+细胞比例统计图。“NT”表示未经慢病毒侵染的T细胞，“CAR alone”为H9.2.1 CAR-T细胞，其他为M7CR修饰的H9.2.1 CAR-T细胞，8E5为对照CAR-T细胞。Figure 37A shows a representative flow scatter diagram of CD25 and CD69 expression after culture of H9.2.1 derived from donor 15 and M7CR-modified CAR-T cells with different signal intensities and NUGC-4 target cells. Figure 37B shows CD25+CD69+ cells. Proportion chart. “NT” represents T cells without lentivirus infection, “CAR alone” represents H9.2.1 CAR-T cells, others represent M7CR-modified H9.2.1 CAR-T cells, and 8E5 represents control CAR-T cells.
图38显示供者15来源的H9.2.1及其不同信号强度M7CR修饰的CAR-T细胞杀伤NUGC-4靶细胞的统计柱状图。Figure 38 shows a statistical histogram showing the killing of NUGC-4 target cells by H9.2.1 derived from donor 15 and its M7CR-modified CAR-T cells with different signal intensities.
图39显示供者15来源的H9.2.1及其不同信号强度M7CR修饰的CAR-T细胞和NUGC-4靶细胞共培养24h后上清中IL-2、IFN-γ和TNFα浓度。Figure 39 shows the concentrations of IL-2, IFN-γ and TNFα in the supernatant of H9.2.1 derived from donor 15 and M7CR-modified CAR-T cells with different signal intensities and NUGC-4 target cells co-cultured for 24 hours.
图40A显示供者13、16、17来源的H9.2.1及其不同信号强度M7CR修饰的CAR-T细胞第1天到第9天的扩增动力学，图40B显示第9天收获时的CAR-T细胞扩增倍数。“NT”表示未经慢病毒侵染的T细胞，“CAR alone”为H9.2.1 CAR-T细胞，8E5为对照CAR-T细胞。Figure 40A shows the expansion kinetics of H9.2.1 and M7CR-modified CAR-T cells derived from donors 13, 16, and 17 with different signal intensities from day 1 to day 9, and Figure 40B shows the CAR at harvest on day 9 -T cell expansion fold. “NT” represents T cells without lentivirus infection, “CAR alone” represents H9.2.1 CAR-T cells, and 8E5 represents control CAR-T cells.
图41A显示供者13、16、17来源的H9.2.1及其不同信号强度M7CR修饰的CAR-T细胞CAR和tCD19表达的代表性流式细胞图，图41B显示CAR表达阳性率统计图。Figure 41A shows representative flow cytometry diagrams of CAR and tCD19 expression in H9.2.1 and M7CR-modified CAR-T cells with different signal intensities derived from donors 13, 16, and 17, and Figure 41B shows a statistical diagram of the positive rate of CAR expression.
图42A显示供者13、16、17来源的H9.2.1及其不同信号强度M7CR修饰的CAR-T细胞中CD4和CD8细胞亚群的代表性流式散点图。图42B显示CD4和CD8细胞亚群比例统计图。Figure 42A shows representative flow scatter plots of CD4 and CD8 cell subsets in H9.2.1 and M7CR-modified CAR-T cells derived from donors 13, 16, and 17 with different signal intensities. Figure 42B shows a statistical graph of CD4 and CD8 cell subset proportions.
图43A显示供者13、16、17来源的H9.2.1及其不同信号强度M7CR修饰的CAR-T细胞分化表型表达的代表性流式散点图。图43B显示CD45RA+CCR7+细胞比例统计图。图43C和图43D分别显示供者13、16、17来源的H9.2.1及其不同信号强度M7CR修饰的CAR-T细胞CD45RA表达的代表性流式细胞图和统计图(以MFI表示)。如图43E和图43F分别显示供者13、16、17来源H9.2.1及其不同信号强度M7CR修饰CAR-T细胞CCR7表达代表性流式细胞图和统计图(以MFI表示)。Figure 43A shows a representative flow scatter diagram of the differentiation phenotype expression of H9.2.1 derived from donors 13, 16, and 17 and its M7CR-modified CAR-T cells with different signal intensities. Figure 43B shows a statistical graph of CD45RA + CCR7 + cell proportions. Figure 43C and Figure 43D respectively show representative flow cytometry and statistical diagrams (expressed as MFI) of CD45RA expression in H9.2.1 derived from donors 13, 16, and 17 and their M7CR-modified CAR-T cells with different signal intensities. Figure 43E and Figure 43F respectively show representative flow cytometry and statistical diagrams (expressed as MFI) of CCR7 expression in H9.2.1 derived from donors 13, 16, and 17 and their different signal intensity M7CR-modified CAR-T cells.
如图44A和图44B分别显示供者13、16、17来源的H9.2.1及其不同信号强度M7CR修饰的CAR-T细胞与PANC1或NUGC-4肿瘤细胞培养24h后CAR+和CAR-细胞中CD25+CD69+细胞比例统计图。As shown in Figure 44A and Figure 44B, respectively, H9.2.1 derived from donors 13, 16, and 17 and their different signal intensity M7CR-modified CAR-T cells were cultured with PANC1 or NUGC-4 tumor cells for 24 hours in CAR + and CAR - cells. CD25 + CD69 + cell proportion statistical graph.
图45A显示供者15来源的H9.2.1及其不同信号强度M7CR修饰CAR-T细胞在NUGC4腹腔肿瘤模型中的抗肿瘤效应，通过每周成像监测小鼠腹腔肿瘤生长。“NT”表示未经慢病毒侵染的T细胞。图45B显示治疗小鼠的肿瘤负荷统计图。图45C显示治疗小鼠的体重变化情况。图45D和图45E分别表示小鼠体内总T细胞及CAR‐T细胞随时间扩增情况，以每100μl小鼠外周血中细胞数量表示。Figure 45A shows the anti-tumor effect of donor 15-derived H9.2.1 and its different signal intensity M7CR modified CAR-T cells in the NUGC4 abdominal tumor model, and mouse abdominal tumor growth was monitored by weekly imaging. “NT” indicates T cells without lentivirus infection. Figure 45B shows a statistical graph of tumor burden in treated mice. Figure 45C shows changes in body weight of treated mice. Figure 45D and Figure 45E respectively show the expansion of total T cells and CAR-T cells in mice over time, expressed as the number of cells per 100 μl of mouse peripheral blood.
图46A显示供者17来源的BB2121及其M7CR(8)修饰的CAR-T细胞在皮下H929肿瘤模型中抗肿瘤效应。“CAR alone”为施用BB2121 CAR-T细胞组。图46B显示治疗小鼠的体重变化情况。Figure 46A shows the anti-tumor effects of donor 17-derived BB2121 and its M7CR(8) modified CAR-T cells in the subcutaneous H929 tumor model. “CAR alone” is the group administered BB2121 CAR-T cells. Figure 46B shows changes in body weight of treated mice.
图47显示TGFβRII-M7CR CAR结构图。通过P2A将TGFβRII-M7CR、dnTGFβRII、TGFβRII-CD28和TGFβRII-41BB等嵌合受体N端与H9.2.1或H9.2.1-28 CAR多肽的C端连 接，从而构成TGFβR-M7CR等嵌合受体修饰的CAR。Figure 47 shows the structure diagram of TGFβRII-M7CR CAR. The N-terminus of chimeric receptors such as TGFβRII-M7CR, dnTGFβRII, TGFβRII-CD28 and TGFβRII-41BB is connected to the C-terminus of H9.2.1 or H9.2.1-28 CAR polypeptide through P2A are connected to form a CAR modified by chimeric receptors such as TGFβR-M7CR.
图48显示供者5，10，18来源的H9.2.1及其dnTGFβRII、TGFβRII-CD28、TGFβRII-M7CR修饰的CAR-T细胞、H9.2.1-28及其dnTGFβRII、TGFβRII-BB、TGFβRII-M7CR修饰的CAR-T细胞随时间扩增动力学。Figure 48 shows CAR-T cells derived from H9.2.1 and its dnTGFβRII, TGFβRII-CD28, and TGFβRII-M7CR modifications derived from donors 5, 10, and 18, and H9.2.1-28 and its dnTGFβRII, TGFβRII-BB, and TGFβRII-M7CR modifications. Kinetics of CAR-T cell expansion over time.
图49A显示图48中的各CAR-T细胞中CAR和作为ECD的TGFβRII表达的代表性流式散点图。图49B显示图48中的各CAR-T细胞CAR阳性率统计柱状图。Figure 49A shows a representative flow scatter plot of CAR and TGFβRII expression as ECD in each CAR-T cell in Figure 48. Figure 49B shows a statistical histogram of the CAR positivity rate of each CAR-T cell in Figure 48.
图50A和50B分别显示图48中的各CAR‐T细胞中CD4和CD8亚群的代表性流式散点图和CD4、CD8亚群比例统计图。Figures 50A and 50B respectively show the representative flow scatter plots and CD4 and CD8 subpopulation ratio statistical diagrams of CD4 and CD8 subpopulations in each CAR-T cell in Figure 48.
图51A显示供者5来源的H9.2.1及其dnTGFβRII、TGFβRII-CD28、TGFβRII-M7CR修饰的CAR-T细胞在低效靶比(E:T＝1:50)、加入外源TGFβ1情况下对于Hup-T4细胞的实时动态杀伤效应。“NT”表示未经慢病毒侵染的T细胞。图51B显示供者5来源的H9.2.1-28及其dnTGFβRII、TGFβRII-BB、TGFβRII-M7CR修饰的CAR-T细胞在低效靶比(E:T＝1:50)、加入外源TGFβ1情况下对于Hup-T4细胞实时动态杀伤效应。图51C显示供者10来源的H9.2.1及其dnTGFβRII、TGFβRII-CD28、TGFβRII-M7CR修饰的CAR-T细胞在低效靶比(E:T＝1:50)、加入外源TGFβ1情况下对于Hup-T4细胞的实时动态杀伤效应。图51D显示供者10来源的H9.2.1-28及其dnTGFβRII、TGFβRII-BB、TGFβRII-M7CR修饰的CAR-T细胞在低效靶比(E:T＝1:50)、加入外源TGFβ1情况下对于Hup-T4细胞实时动态杀伤效应。Figure 51A shows that H9.2.1 derived from donor 5 and its dnTGFβRII, TGFβRII-CD28, and TGFβRII-M7CR-modified CAR-T cells have a low efficiency target ratio (E:T=1:50) and the addition of exogenous TGFβ1. Real-time dynamic killing effect of Hup-T4 cells. “NT” indicates T cells without lentivirus infection. Figure 51B shows H9.2.1-28 derived from donor 5 and its dnTGFβRII, TGFβRII-BB, and TGFβRII-M7CR-modified CAR-T cells at a low-efficiency target ratio (E:T=1:50) and the addition of exogenous TGFβ1 Real-time dynamic killing effect on Hup-T4 cells. Figure 51C shows that H9.2.1 derived from donor 10 and its dnTGFβRII, TGFβRII-CD28, and TGFβRII-M7CR-modified CAR-T cells have a low efficiency target ratio (E:T=1:50) and the addition of exogenous TGFβ1. Real-time dynamic killing effect of Hup-T4 cells. Figure 51D shows H9.2.1-28 derived from donor 10 and its dnTGFβRII, TGFβRII-BB, and TGFβRII-M7CR modified CAR-T cells at a low-efficiency target ratio (E:T=1:50) and the addition of exogenous TGFβ1 Real-time dynamic killing effect on Hup-T4 cells.
发明详述Detailed description of the invention
除非另外限定，否则本文中所用的全部技术与科学术语具有如本发明所属领域的普通技术人员通常理解的相同含义。本文所提及的全部出版物、专利申请、专利和其他参考文献通过引用的方式完整地并入。此外，本文中所述的材料、方法和例子仅是说明性的并且不意在是限制性的。本发明的其他特征、目的和优点将从本说明书及附图并且从后附的权利要求书中显而易见。Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Furthermore, the materials, methods, and examples described herein are illustrative only and not intended to be limiting. Other features, objects and advantages of the invention will be apparent from the description and drawings, and from the appended claims.
I.定义I.Definition
为了解释本说明书，将使用以下定义，并且只要适当，以单数形式使用的术语也可以包括复数，并且反之亦然。要理解，本文所用的术语仅是为了描述具体的实施方案，并且不意欲是限制性的。For the purpose of interpreting this specification, the following definitions will be used and, wherever appropriate, terms used in the singular may also include the plural and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。The term "about" when used in conjunction with a numerical value is intended to encompass a range of numerical values having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.
如本文所用，术语“和/或”意指可选项中的任一项或可选项的两项或多项。As used herein, the term "and/or" means any one of the options or two or more of the options.
在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”某个具体序列的结构域时，也旨在涵盖由该具体序列组成的结构域。When the term "comprises" or "includes" is used herein, it also encompasses a combination of the stated elements, integers, or steps unless otherwise indicated. For example, when reference is made to a domain that "comprises" a particular sequence, it is also intended to encompass a domain that consists of that particular sequence.
“组成型激活的IL-7R突变体”是指在野生型IL-7受体α链(IL7Rα)跨膜区发生突变而产生的突变IL-7R，其能够在不依赖野生型IL7Rα的配体结合的情况下，发生二聚化并激活下游STAT5信号通路。"Constitutively active IL-7R mutant" refers to a mutant IL-7R produced by mutations in the transmembrane region of the wild-type IL-7 receptor alpha chain (IL7Rα), which can activate ligands independent of wild-type IL7Rα. Upon binding, dimerization occurs and the downstream STAT5 signaling pathway is activated.
术语“自体的”指这样的任何物质，所述物质从稍后将向个体再次引入所述物质的相同个体衍生。 The term "autologous" refers to any substance derived from the same individual to whom the substance is later reintroduced.
术语“同种异体的”指这样的任何物质，所述物质从与引入所述物质的个体相同的物种的不同动物衍生。当一个或多个基因座处的基因不相同时，两位或更多位个体据称彼此是同种异体的。在一些方面，来自相同物种的个体的同种异体物质可以在遗传上足够地不相似以发生抗原性相互作用。The term "allogeneic" refers to any substance derived from a different animal of the same species as the individual into which the substance is introduced. Two or more individuals are said to be allogeneic to each other when the genes at one or more loci are not identical. In some aspects, allogeneic agents from individuals of the same species can be genetically dissimilar enough for antigenic interaction to occur.
术语“异种的”指从不同物种的动物衍生的移植物。The term "xenogeneic" refers to a graft derived from an animal of a different species.
如本文所用的术语“单采血液成分术”指本领域认可的体外方法，借助所述方法，供体或患者的血液从供体或患者取出并且穿过这样的装置，所述装置分离选择的特定组分并将剩余部分返回供体或患者的循环，例如，通过再输血。因此，在“单采样品”的语境中，指使用单采血液成分术获得的样品。The term "apheresis" as used herein refers to an art-recognized extracorporeal method by which blood from a donor or patient is removed from the donor or patient and passed through a device that separates selected specific components and return the remainder to the donor or patient's circulation, for example, by retransfusion. Therefore, in the context of "single sample", it refers to a sample obtained using apheresis.
术语“免疫效应细胞”指参与免疫应答，例如参与促进免疫效应反应的细胞。免疫效应细胞的例子包括T细胞，例如，α/βT细胞和γ/δT细胞、B细胞、天然杀伤(NK)细胞、天然杀伤T(NKT)细胞、肥大细胞、和髓细胞衍生的吞噬细胞。The term "immune effector cells" refers to cells involved in an immune response, such as in promoting an immune effector response. Examples of immune effector cells include T cells, eg, alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.
“免疫效应功能”、“免疫效应应答”或“免疫效应反应”指例如免疫效应细胞的增强或促进免疫攻击靶细胞的功能或应答。例如，免疫效应功能或应答指促进杀伤靶细胞或抑制靶细胞生长或增殖的T细胞或NK细胞特性。在T细胞的情况下，初级刺激和共刺激是免疫效应功能或应答的例子。"Immune effector function", "immune effector response" or "immune effector response" refers to, for example, a function or response of an immune effector cell that enhances or promotes an immune attack on a target cell. For example, immune effector functions or responses refer to properties of T cells or NK cells that promote killing of target cells or inhibit the growth or proliferation of target cells. In the case of T cells, primary stimulation and costimulation are examples of immune effector functions or responses.
术语“效应功能”指细胞的特化功能。T细胞的效应功能例如可以是溶细胞活性或辅助活性，包括分泌细胞因子。The term "effector function" refers to a specialized function of a cell. The effector function of T cells may be, for example, cytolytic activity or auxiliary activity, including secretion of cytokines.
术语“T细胞激活”是指T淋巴细胞，特别是细胞毒性T淋巴细胞的一种或多种细胞应答，选自：增殖、分化、细胞因子分泌、细胞毒性效应分子释放、细胞毒活性和活化标志物的表达。本发明的嵌合抗原受体能够诱导T细胞激活。用于测量T细胞激活的合适测定法在实施例中描述，并是本领域中已知的。The term "T cell activation" refers to one or more cellular responses of T lymphocytes, in particular cytotoxic T lymphocytes, selected from: proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity and activation Expression of markers. The chimeric antigen receptor of the present invention can induce T cell activation. Suitable assays for measuring T cell activation are described in the Examples and are known in the art.
术语“慢病毒”指逆转录病毒科(Retroviridae)的一个属。慢病毒在逆转录病毒当中的独特之处在于能够感染非分裂性细胞；它们可以递送显著量的遗传信息至宿主细胞，从而它们是基因递送载体的最高效方法之一。HIV、SIV和FIV均是慢病毒的例子。The term "lentivirus" refers to a genus of the family Retroviridae. Lentiviruses are unique among retroviruses in their ability to infect non-dividing cells; they can deliver significant amounts of genetic information to host cells, making them one of the most efficient methods of gene delivery vectors. HIV, SIV and FIV are examples of lentiviruses.
术语“慢病毒载体”指从慢病毒基因组的至少一部分衍生的载体，尤其包括如Milone等人,Mol.Ther.17(8):1453–1464(2009)中提供的自我失活慢病毒载体。可以在临床使用的慢病毒载体的其他例子例如包括但不限于来自Oxford BioMedica的基因递送技术、来自Lentigen的LENTIMAXTM载体系统等。非临床类型的慢病毒载体也是可获得的并且是本领域技术人员已知的。The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome, including in particular self-inactivating lentiviral vectors as provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors that may be used clinically include, but are not limited to, lentiviral vectors from Oxford BioMedica Gene delivery technology, LENTIMAX TM vector system from Lentigen, etc. Non-clinical types of lentiviral vectors are also available and known to those skilled in the art.
术语“肿瘤”和“癌症”在本文中互换地使用，涵盖实体瘤和液体肿瘤。The terms "tumor" and "cancer" are used interchangeably herein to encompass both solid and liquid tumors.
术语“癌症”和“癌性”是指哺乳动物中细胞生长不受调节的生理疾患。The terms "cancer" and "cancerous" refer to a physiological disorder in mammals in which cell growth is unregulated.
术语“肿瘤”指所有赘生性(neoplastic)细胞生长和增殖，无论是恶性的还是良性的，及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”、“癌性”和“肿瘤”在本文中提到时并不互相排斥。The term "neoplastic" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer", "cancerous" and "neoplasm" when used herein are not mutually exclusive.
术语“Claudins”是一类存在于上皮和内皮紧密连接中的整合素膜蛋白，是紧密连接的重要组成部分，1998年由Shoichiro Tsukita等人发现。该家族有24个成员。人类Claudin 18基因有两个可供选择的1号外显子，因而产生Claudin 18.1(本文中也称为“CLDN18.1”)和Claudin 18.2(本文中也称为“CLDN18.2”)两种蛋白亚型，两者在第1个胞外结构域约50个氨基酸的 序列上只有7个氨基酸残基的差异。The term "Claudins" is a type of integrin membrane protein that exists in epithelial and endothelial tight junctions and is an important component of tight junctions. It was discovered by Shoichiro Tsukita et al. in 1998. The family has 24 members. The human Claudin 18 gene has two alternative exons 1, resulting in two proteins, Claudin 18.1 (also referred to as "CLDN18.1" in this article) and Claudin 18.2 (also referred to as "CLDN18.2" in this article) Isoforms, both of which have about 50 amino acids in the first extracellular domain There are only 7 amino acid residue differences in sequence.
Claudin 18.2在癌组织和正常组织的表达上存在显著差异性，这可能源于Claudin 18.2启动子区域CREB结合位点在正常组织中CpG高度甲基化，而在细胞癌变过程中CpG甲基化水平降低，进而CREB参与激活Claudin18.2的转录。There is a significant difference in the expression of Claudin 18.2 in cancer tissues and normal tissues. This may be due to the fact that the CREB binding site in the promoter region of Claudin 18.2 is highly methylated in CpG in normal tissues, while the level of CpG methylation in the process of cell canceration decrease, and then CREB participates in activating the transcription of Claudin18.2.
“肿瘤免疫逃逸”指肿瘤逃避免疫识别和清除的过程。如此，作为治疗概念，肿瘤免疫在此类逃避减弱时得到“治疗”，并且肿瘤被免疫系统识别并攻击。肿瘤识别的例子包括肿瘤结合，肿瘤收缩和肿瘤清除。"Tumor immune escape" refers to the process by which tumors escape immune recognition and clearance. Thus, as a therapeutic concept, tumor immunity is "cured" when such evasion is attenuated, and tumors are recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.
如本文所用，术语“结合”或“特异性结合”意指结合作用对抗原是选择性的并且可以与不想要的或非特异的相互作用区别。抗体与特定抗原结合的能力可以通过酶联免疫吸附测定法(ELISA)、SPR或生物膜层干涉技术或本领域已知的其他常规结合测定法测定。As used herein, the term "binding" or "specific binding" means that the binding is selective for the antigen and can be distinguished from undesired or non-specific interactions. The ability of an antibody to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA), SPR or biofilm layer interference techniques, or other conventional binding assays known in the art.
术语“刺激”指由刺激分子(例如，TCR/CD3复合体)与其相应配体的结合所诱导的初次应答，所述初次应答因而介导信号转导事件，例如但不限于借助TCR/CD3复合体的信号转导。刺激可以介导某些分子改变的表达，如下调TGF-β和/或细胞骨架结构的再组织等。The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., the TCR/CD3 complex) to its corresponding ligand, which primary response thereby mediates a signaling event, such as, but not limited to, via the TCR/CD3 complex body signal transduction. Stimulation can mediate the expression of certain molecular changes, such as down-regulation of TGF-β and/or reorganization of cytoskeletal structure.
术语“刺激分子”指由提供初级胞质信号传导序列的T细胞表达的分子，所述的初级胞质信号传导序列在T细胞信号传导途径的至少某个方面以刺激性方式调节TCR复合体的初级活化。在一个实施方案中，初级信号例如通过TCR/CD3复合体与载有肽的MHC分子的结合引发并且导致介导T细胞反应，包括但不限于增殖、活化、分化等。The term "stimulatory molecule" refers to a molecule expressed by a T cell that provides a primary cytoplasmic signaling sequence that modulates the TCR complex in a stimulatory manner in at least some aspect of the T cell signaling pathway. Primary activation. In one embodiment, the primary signal is initiated, for example, by binding of a TCR/CD3 complex to a peptide-loaded MHC molecule and results in the mediation of a T cell response, including but not limited to proliferation, activation, differentiation, and the like.
术语“CD3ζ”定义为GenBan登录号BAG36664.1提供的蛋白质或其等同物，并且“CD3ζ刺激信号结构域”定义为来自CD3ζ链胞质结构域的氨基酸残基，所述氨基酸残基足以在功能上传播T细胞活化必需的初始信号。在一个实施方案中，CD3ζ的胞质结构域包含GenBank登录号BAG36664.1的残基52至残基164或作为其功能直向同源物的来自非人类物种(例如，小鼠、啮齿类、猴、猿等)的等同残基。在一个实施方案中，“CD3ζ刺激信号结构域”是在SEQ ID NO:12中提供的序列或其变体。The term "CD3ζ" is defined as the protein provided by GenBan accession number BAG36664.1 or its equivalent, and "CD3ζ stimulatory signaling domain" is defined as the amino acid residues from the cytoplasmic domain of the CD3ζ chain that are sufficient to functionally propagates the initial signal necessary for T cell activation. In one embodiment, the cytoplasmic domain of CD3ζ comprises residues 52 to 164 of GenBank accession number BAG36664.1 or as a functional ortholog thereof from a non-human species (e.g., mouse, rodent, equivalent residues of monkeys, apes, etc.). In one embodiment, the "CD3ζ stimulating signal domain" is the sequence provided in SEQ ID NO: 12 or a variant thereof.
术语“共刺激分子”是指细胞上的与共刺激配体特异性结合从而介导细胞的共刺激反应(例如但不限于增殖)的相应结合配偶体。共刺激分子是除抗原受体或其配体之外的有助于有效免疫应答的细胞表面分子。共刺激分子包括但不限于MHC I类分子、TNF受体蛋白、免疫球蛋白样蛋白、细胞因子受体、整联蛋白、信号传导淋巴细胞活化分子(SLAM蛋白)、激活NK细胞受体、OX40、CD40、GITR、4-1BB(即CD137)、CD27和CD28。在一些实施方案中，“共刺激分子”是CD28、4-1BB(即CD137)。共刺激信号结构域是指共刺激分子的胞内部分。The term "costimulatory molecule" refers to a corresponding binding partner on a cell that specifically binds to a costimulatory ligand thereby mediating a costimulatory response (such as, but not limited to, proliferation) of the cell. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an effective immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activated NK cell receptors, OX40 , CD40, GITR, 4-1BB (ie CD137), CD27 and CD28. In some embodiments, the "costimulatory molecule" is CD28, 4-1BB (ie, CD137). The costimulatory signaling domain refers to the intracellular part of the costimulatory molecule.
术语“4-1BB”指TNFR超家族成员，所述成员具有作为GenBank登录号AAA62478.2提供的氨基酸序列或来自非人类物种(例如，小鼠、啮齿类、猴、猿等)的等同残基；并且“4-1BB共刺激信号结构域”定义为GenBank登录号AAA62478.2的氨基酸残基214-255或来自非人类物种(例如，小鼠、啮齿类、猴、猿等)的等同残基。在一个实施方案中，“4-1BB共刺激结构域”是作为SEQ ID NO:11提供的序列或来自非人类物种(例如，小鼠、啮齿类、猴、猿等)的等同残基。The term "4-1BB" refers to a TNFR superfamily member having the amino acid sequence provided as GenBank accession number AAA62478.2 or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.) ; and "4-1BB costimulatory signaling domain" is defined as amino acid residues 214-255 of GenBank accession number AAA62478.2 or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.) . In one embodiment, the "4-1BB costimulatory domain" is the sequence provided as SEQ ID NO: 11 or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.).
术语“信号传导途径”指在从细胞一个部分传播信号至细胞的另一个部分中发挥作用的多种信号传导分子之间的生物化学关系。The term "signaling pathway" refers to the biochemical relationships between various signaling molecules that play a role in propagating signals from one part of a cell to another part of the cell.
在谈及组成型嵌合细胞因子受体的胞外结构域时，所述胞外结构域可以是细胞因子，且所述“细胞因子”是由一种细胞群释放，作为细胞间介质作用于另一细胞的蛋白质的通称。 此类细胞因子的例子有淋巴因子、单核因子、白介素(IL)，诸如IL-1，IL-1α，IL-2，IL-3，IL-4，IL-5，IL-6，IL-8，IL-9，IL-11，IL-12，IL15，IL-21、IL-18、IL-9、IL-23、IL-36γ；肿瘤坏死因子，诸如TNF-α或TNF-β；及其它多肽因子，包括干扰素。在一些实施方案中，作为本发明的组成型嵌合细胞因子受体的胞外结构域的“细胞因子”选自任一IL-12(例如，IL-12p40或IL-12p70)、IL15(例如IL-15或IL-15FP，所述IL-15FP是指IL-15和IL-15Rα(选自IL-15Rα或IL-15Rα(Sushi))的融合蛋白，包括IL-15/IL-15Rα和IL-15Rα/IL-15两种形式的融合蛋白))、IL-21、IL-18、IL-9、IL-23、IL-36γ和IFNα2b。When referring to the extracellular domain of a constitutively chimeric cytokine receptor, the extracellular domain may be a cytokine, and the "cytokine" is released by a cell population and acts as an intercellular mediator on A general name for a protein from another cell. Examples of such cytokines are lymphokines, monokines, interleukins (IL), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL- 8. IL-9, IL-11, IL-12, IL15, IL-21, IL-18, IL-9, IL-23, IL-36γ; tumor necrosis factor, such as TNF-α or TNF-β; and Other polypeptide factors, including interferons. In some embodiments, the "cytokine" that is the extracellular domain of the constitutive chimeric cytokine receptor of the invention is selected from any of IL-12 (e.g., IL-12p40 or IL-12p70), IL15 (e.g., IL-15 or IL-15FP, the IL-15FP refers to the fusion protein of IL-15 and IL-15Rα (selected from IL-15Rα or IL-15Rα (Sushi)), including IL-15/IL-15Rα and IL -15Rα/IL-15 two forms of fusion protein)), IL-21, IL-18, IL-9, IL-23, IL-36γ and IFNα2b.
在谈及组成型嵌合细胞因子受体的胞外结构域时，所述胞外结构域可以是免疫效应分子，且所述“免疫效应分子”可以选自：(i)增强抗原呈递(例如，肿瘤抗原呈递)的分子；(ii)增强效应细胞反应的分子(例如，活化和/或动员B细胞和/或T细胞)。所述“免疫效应分子”例如是以下分子或其激动剂：GITR、OX40、ICOS、SLAM(例如，SLAMF7)、HVEM、LIGHT、CD2、CD27、CD28、CDS、ICAM1、LFA-1(CD11a/CD18)、ICOS(CD278)，4-1BB(CD137)、CD30、CD40、BAFFR、CD7、CD160、B7-H3或CD83。在一些实施方案中，作为本发明的组成型嵌合细胞因子受体的胞外结构域的“免疫效应分子”选自任一4-1BB靶向分子部分(例如，4-1BB配体、抗4-1BB抗体)、CD40靶向分子部分(例如，CD40配体、抗CD40抗体)、CD83靶向分子部分(例如，抗CD83抗体)、FLT3配体、GITR、ICOS、CD2和ICAM1。Referring to the extracellular domain of a constitutively chimeric cytokine receptor, the extracellular domain may be an immune effector molecule, and the "immune effector molecule" may be selected from: (i) enhancing antigen presentation (e.g. , tumor antigen presentation); (ii) molecules that enhance effector cell responses (e.g., activate and/or mobilize B cells and/or T cells). The "immune effector molecule" is, for example, the following molecules or their agonists: GITR, OX40, ICOS, SLAM (e.g., SLAMF7), HVEM, LIGHT, CD2, CD27, CD28, CDS, ICAM1, LFA-1 (CD11a/CD18 ), ICOS (CD278), 4-1BB (CD137), CD30, CD40, BAFFR, CD7, CD160, B7-H3 or CD83. In some embodiments, the "immune effector molecule" that is the extracellular domain of the constitutive chimeric cytokine receptor of the invention is selected from any 4-1BB targeting molecule moiety (e.g., 4-1BB ligand, anti- 4-1BB antibody), CD40 targeting molecule moieties (e.g., CD40 ligand, anti-CD40 antibody), CD83 targeting molecule moieties (e.g., anti-CD83 antibody), FLT3 ligand, GITR, ICOS, CD2 and ICAM1.
在谈及组成型嵌合细胞因子受体的胞外结构域时，所述胞外结构域可以是抑制性分子拮抗剂，且所述“抑制性分子拮抗剂”是减少肿瘤免疫抑制的药剂。所述抑制性分子包括但不限于PD-1、PD-L1、CD47、TIM-3、IL-4、TGFβ、LAG-3、VISTA、B7-H4、CTLA-4、CD73或TIGIT。在一些实施方案中，作为本发明的组成型嵌合细胞因子受体的胞外结构域的“抑制性分子拮抗剂”选自任一抗PD-L1分子、抗CD47分子、抗IL-4分子、TGFβ结合分子(例如，抗TGFβ1分子、TGFβRII)、抗PD-1分子、抗CTLA-4分子、抗LAG-3分子、抗TIGIT分子和抗CD73分子。Referring to the extracellular domain of a constitutive chimeric cytokine receptor, the extracellular domain may be an inhibitory molecule antagonist, and the "inhibitory molecule antagonist" is an agent that reduces tumor immunosuppression. Such inhibitory molecules include, but are not limited to, PD-1, PD-L1, CD47, TIM-3, IL-4, TGFβ, LAG-3, VISTA, B7-H4, CTLA-4, CD73 or TIGIT. In some embodiments, the "inhibitory molecule antagonist" of the extracellular domain of the constitutive chimeric cytokine receptor of the invention is selected from the group consisting of any anti-PD-L1 molecule, anti-CD47 molecule, and anti-IL-4 molecule , TGFβ binding molecules (eg, anti-TGFβ1 molecules, TGFβRII), anti-PD-1 molecules, anti-CTLA-4 molecules, anti-LAG-3 molecules, anti-TIGIT molecules, and anti-CD73 molecules.
在谈及组成型嵌合细胞因子受体的胞外结构域时，所述胞外结构域可以是靶向NK细胞激活性受体的效应分子，且所述“靶向NK细胞激活性受体的效应分子”是与NK细胞激活性受体结合后，能够激活NK细胞的一类分子。所述NK细胞激活性受体包括但不限于NK细胞上的NKG2C、NKG2D、NKp30、NKp44和NKp46。在一些实施方案中，作为本发明的组成型嵌合细胞因子受体的胞外结构域的“靶向NK细胞激活性受体的效应分子”选自靶向NK细胞激活性受体NKG2C、NKG2D、NKp30、NKp44和NKp46的分子，例如，抗NKG2C、抗NKG2D、抗NKp30、抗NKp44、抗NKp46，通过激活内源性NK细胞，获得增强的抗肿瘤免疫效应。When referring to the extracellular domain of a constitutively chimeric cytokine receptor, the extracellular domain may be an effector molecule targeting an NK cell activating receptor, and "targeting an NK cell activating receptor" "Effector molecules" are a class of molecules that can activate NK cells after binding to NK cell activating receptors. The NK cell activating receptors include, but are not limited to, NKG2C, NKG2D, NKp30, NKp44 and NKp46 on NK cells. In some embodiments, the "effector molecule targeting NK cell activating receptors" as the extracellular domain of the constitutive chimeric cytokine receptor of the invention is selected from the group consisting of targeting NK cell activating receptors NKG2C, NKG2D , NKp30, NKp44 and NKp46 molecules, such as anti-NKG2C, anti-NKG2D, anti-NKp30, anti-NKp44, and anti-NKp46, obtain enhanced anti-tumor immune effects by activating endogenous NK cells.
术语“抗体”在本文中以最广意义使用，指包含抗原结合位点的蛋白质，涵盖各种结构的天然抗体和人工抗体，包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如，双特异性抗体)、单链抗体、完整抗体和抗体片段。The term "antibody" is used in the broadest sense herein to refer to proteins containing antigen-binding sites, encompassing natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies ( For example, bispecific antibodies), single-chain antibodies, intact antibodies, and antibody fragments.
“抗体片段”或“抗原结合片段”在本文中可互换地使用，指与完整抗体不同的分子，其包含完整抗体的一部分且结合完整抗体所结合的抗原。抗体片段的例子包括但不限于Fab、Fab’、F(ab’)2、Fv、单链Fv、单链Fab、双体抗体(diabody)。"Antibody fragment" or "antigen-binding fragment" are used interchangeably herein to refer to a molecule, distinct from an intact antibody, that contains a portion of an intact antibody and binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single chain Fv, single chain Fab, diabody.
术语“scFv”指一种融合蛋白，其包含至少一个包含轻链可变区的抗体片段和至少一个包含重链可变区的抗体片段，其中轻链可变区和重链可变区任选地借助柔性短多肽接头连续地 连接，并且能够表达为单链多肽，并且其中scFv保留衍生它的完整抗体的特异性。除非另外指出，否则如本文所用，scFv可以具有按任何顺序(例如，相对于多肽的N末端和C末端)的VL可变区和VH可变区，scFv可以包含VL-接头-VH或可以包含VH-接头-VL。The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are optionally Continuously with the help of flexible short peptide linkers ligated and capable of expression as a single-chain polypeptide in which the scFv retains the specificity of the intact antibody from which it was derived. Unless otherwise indicated, as used herein, a scFv may have a VL variable region and a VH variable region in any order (eg, relative to the N-terminus and C-terminus of the polypeptide), the scFv may comprise a VL-linker-VH or may comprise VH-joint-VL.
“互补决定区”或“CDR区”或“CDR”或“高变区”是抗体可变结构域中在序列上高变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基(“抗原接触点”)的区域。CDR主要负责与抗原表位结合。重链和轻链的CDR通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。位于抗体重链可变结构域内的CDR被称作CDR H1、CDR H2和CDR H3，而位于抗体轻链可变结构域内的CDR被称作CDR L1、CDR L2和CDR L3。在一个给定的轻链可变区或重链可变区氨基酸序列中，各CDR的精确氨基酸序列边界可以使用许多公知的抗体CDR指派系统的任一种或其组合确定，所述指派系统包括例如：基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342:877-883，Al-Lazikani等人,“Standard conformations for the canonical structures of immunoglobulins”,Journal of Molecular Biology,273,927-948(1997))，基于抗体序列可变性的Kabat(Kabat等人,Sequences of Proteins of Immunological Interest,第4版,U.S.Department of Health and Human Services,National Institutes of Health(1987)),AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(万维网imgt.cines.fr/)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。A "complementarity determining region" or "CDR region" or "CDR" or "hypervariable region" is an antibody variable domain that is hypervariable in sequence and forms a structurally defined loop (a "hypervariable loop") and/or A region containing antigen contact residues ("antigen contact points"). CDRs are mainly responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are generally referred to as CDR1, CDR2 and CDR3 and are numbered sequentially starting from the N-terminus. The CDRs located within the variable domain of the antibody heavy chain are called CDR H1, CDR H2, and CDR H3, while the CDRs located within the variable domain of the antibody light chain are called CDR L1, CDR L2, and CDR L3. The precise amino acid sequence boundaries of each CDR in a given light chain variable region or heavy chain variable region amino acid sequence can be determined using any one or a combination of many well-known antibody CDR assignment systems, including For example: Chothia based on the three-dimensional structure of antibodies and the topology of CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (World Wide Web imgt.cines.fr/), and North CDR definition based on affinity propagation clustering using a large number of crystal structures .
除非另有说明，否则在本发明中，术语“CDR”或“CDR序列”涵盖以上述任一种方式确定的CDR序列。Unless otherwise stated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above ways.
CDR也可以基于与参考CDR序列(例如本发明示例的CDR之任一)具有相同的Kabat编号位置而确定。在本发明中，当提及抗体可变区和具体CDR序列(包括重链可变区残基)时，是指根据Kabat编号系统的编号位置。CDRs may also be determined based on having the same Kabat number position as a reference CDR sequence (eg, any of the CDRs exemplified herein). In the present invention, when reference is made to antibody variable regions and specific CDR sequences (including heavy chain variable region residues), reference is made to the numbering positions according to the Kabat numbering system.
尽管CDR在抗体与抗体之间是不同的，但是CDR内只有有限数量的氨基酸位置直接参与抗原结合。使用Kabat,Chothia,AbM和Contact方法中的至少两种，可以确定最小重叠区域，从而提供用于抗原结合的“最小结合单位”。最小结合单位可以是CDR的一个子部分。正如本领域技术人员明了，通过抗体的结构和蛋白折叠，可以确定CDR序列其余部分的残基。因此，本发明也考虑本文所给出的任何CDR的变体。例如，在一个CDR的变体中，最小结合单位的氨基酸残基可以保持不变，而根据Kabat或Chothia或AbM定义的其余CDR残基可以被保守氨基酸残基替代。Although CDRs vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM and Contact methods, the minimal overlapping region can be determined, thus providing the "minimum binding unit" for antigen binding. The smallest binding unit may be a subportion of a CDR. As will be apparent to those skilled in the art, the remainder of the CDR sequence can be determined from the structure of the antibody and protein folding. Therefore, variants of any CDR given herein are also contemplated by the present invention. For example, in a variant of a CDR, the amino acid residues of the minimal binding unit can remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia or AbM can be replaced by conserved amino acid residues.
术语“可变区”或“可变结构域”是指参与抗体与抗原结合的抗体重链或轻链的结构域。天然抗体的重链和轻链的可变结构域通常具有相似的结构，其中每个结构域包含四个保守的构架区(FR)和三个互补决定区(CDR)。(参见，例如，Kindt等Kuby Immunology，6th ed.，W.H.Freeman and Co.91页(2007))。单个VH或VL结构域可以足以给予抗原结合特异性。The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies generally have similar structures, with each domain containing four conserved framework regions (FR) and three complementarity determining regions (CDR). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co. p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity.
术语“Fc区”在本文中用于定义免疫球蛋白重链的C端区域，所述区域包含至少一部分的恒定区。该术语包括天然序列Fc区和变体Fc区。在某些实施方案中，人IgG重链Fc区从Cys226或Pro230延伸至重链的羰基端。然而，Fc区的C端赖氨酸(Lys447)可以存在或者可以不存在。除非另外说明，Fc区或恒定区中的氨基酸残基的编号是根据EU编号系统，其也被称为EU索引，如在Kabat等，Sequences of Proteins of Immunological Interest，5th Ed.Public Health Service，National Institutes of Health，Bethesda，MD，1991中所述。术语 “功能变体”表示与由本发明核酸序列编码的多肽具有基本或显著序列同一性或相似性的多肽，所述功能变体保留本发明核酸序列编码的多肽的生物学活性。功能变体可以例如包含在由本发明核酸序列编码的多肽的氨基酸序列中具有至少一个保守的氨基酸置换。The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which region contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In certain embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carbonyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise stated, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, which is also known as the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. the term "Functional variant" refers to a polypeptide having substantial or significant sequence identity or similarity with a polypeptide encoded by a nucleic acid sequence of the invention, which functional variant retains the biological activity of a polypeptide encoded by a nucleic acid sequence of the invention. Functional variants may, for example, comprise at least one conservative amino acid substitution in the amino acid sequence of the polypeptide encoded by the nucleic acid sequence of the invention.
术语“保守的序列修饰”、“保守的序列变化”可互换地使用，指未显著影响或改变含有氨基酸序列的多肽的生物学活性的氨基酸修饰或变化。这类种保守修饰包括氨基酸取代、添加和缺失。可以通过本领域已知的标准技术，如位点定向诱变和PCR介导的诱变向本发明的多肽引入修饰。保守性取代是氨基酸残基由具有相似侧链的氨基酸残基替换的氨基酸取代。已经在本领域中定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸、组氨酸)、酸性侧链(例如，天冬氨酸、谷氨酸)、不带电荷极性侧链(例如，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、蛋氨酸)、β-侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因而，可以将本发明多肽内部的一个或多个氨基酸残基替换为来自相同侧链家族的其他氨基酸残基，并且可以使用本文所述的功能测定法测试改变的多肽的生物学活性。The terms "conservative sequence modification" and "conservative sequence change" are used interchangeably and refer to amino acid modifications or changes that do not significantly affect or alter the biological activity of the polypeptide containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the polypeptides of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative substitution is an amino acid substitution in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include those with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., Glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenyl Alanine, tryptophan, histidine) amino acids. Thus, one or more amino acid residues within a polypeptide of the invention can be replaced with other amino acid residues from the same side chain family, and the altered polypeptide can be tested for biological activity using the functional assays described herein.
“分离的”核酸是指这样的核酸分子，其已经与其天然环境的组分分离。分离的核酸包括包含在通常包含该核酸分子的细胞中的核酸分子，但是该核酸分子存在于染色体外或在不同于其天然染色体位置的染色体位置处。An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. Isolated nucleic acid includes nucleic acid molecules that are contained in cells that normally contain the nucleic acid molecule, but that are present extrachromosomally or at a chromosomal location that is different from its native chromosomal location.
术语“荧光激活细胞分选”或“FACS”是指专门类型的流式细胞术。它提供了根据每个细胞的特定光散射和荧光特征，将生物细胞的异质混合物以每次一个细胞分拣到两个或更多个容器中的方法(FlowMetric.“Sorting Out Fluorescence Activated Cell Sorting”.2017-11-09)。用于进行FACS的仪器是本领域技术人员已知的并且对于公众是可商购获得的。这种仪器的实例包括Becton Dickinson(Foster City，CA)的FACS Star Plus、FACScan和FACSort仪器、来自Coulter Epics Division(Hialeah，FL)的Epics C和来自Cytomation(Colorado Springs，Colorado)的MoFlo。The term "fluorescence-activated cell sorting" or "FACS" refers to a specialized type of flow cytometry. It provides a method to sort heterogeneous mixtures of biological cells into two or more containers one cell at a time based on the specific light scattering and fluorescence characteristics of each cell (FlowMetric. "Sorting Out Fluorescence Activated Cell Sorting ".2017-11-09). Instruments for performing FACS are known to those skilled in the art and are commercially available to the public. Examples of such instruments include the FACS Star Plus, FACScan, and FACSort instruments from Becton Dickinson (Foster City, CA), the Epics C from Coulter Epics Division (Hialeah, FL), and the MoFlo from Cytomation (Colorado Springs, Colorado).
术语“可药用辅料”指与活性物质一起施用的稀释剂、佐剂(例如弗氏佐剂(完全和不完全的))、赋形剂、缓冲剂或稳定剂等。The term "pharmaceutically acceptable excipient" refers to diluents, adjuvants (such as Freund's adjuvant (complete and incomplete)), excipients, buffers or stabilizers, etc., which are administered with the active substance.
用于本文时，“治疗”指减缓、中断、阻滞、缓解、停止、降低、或逆转已存在的症状、病症、病况或疾病的进展或严重性。想要的治疗效果包括但不限于防止疾病出现或复发、减轻症状、减小疾病的任何直接或间接病理学后果、防止转移、降低病情进展速率、改善或缓和疾病状态，以及缓解或改善预后。As used herein, "treating" means slowing, interrupting, retarding, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Desired therapeutic effects include, but are not limited to, preventing the emergence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis.
“治疗有效量”指以需要的剂量并持续需要的时间段，有效实现所需治疗结果的量。治疗有效量可以根据多种因素如疾病状态、个体的年龄、性别和重量而变动。相对于未治疗的对象，“治疗有效量”优选地抑制可度量参数(例如肿瘤生长率、肿瘤体积等)至少约20％、更优选地至少约40％、甚至更优选地至少约50％、60％或70％和仍更优选地至少约80％或90％。可以在预示人肿瘤中的功效的动物模型系统中评价化合物抑制可度量参数(例如，癌症)的能力。A "therapeutically effective amount" means an amount effective to achieve the desired therapeutic result, at the required doses and for the required period of time. The therapeutically effective amount may vary depending on factors such as disease state, age, sex and weight of the individual. A "therapeutically effective amount" preferably inhibits a measurable parameter (eg, tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, relative to an untreated subject. 60% or 70% and still more preferably at least about 80% or 90%. The ability of a compound to inhibit a measurable parameter (eg, cancer) can be evaluated in animal model systems that are predictive of efficacy in human tumors.
II.本发明的组成型嵌合细胞因子受体II. Constitutive chimeric cytokine receptors of the invention
本发明涉及能够持续激活STAT5信号、维持免疫效应细胞(例如，T细胞)外源细胞因子非依赖性存活、且具有重塑肿瘤微环境的效应分子的组成型嵌合细胞因子受体。具体地，本发明的组成型嵌合细胞因子受体包含: The present invention relates to constitutive chimeric cytokine receptors that can continuously activate STAT5 signaling, maintain exogenous cytokine-independent survival of immune effector cells (eg, T cells), and have effector molecules that reshape the tumor microenvironment. Specifically, the constitutive chimeric cytokine receptor of the present invention includes:
(i)胞外结构域，所述胞外结构域由具有重塑肿瘤微环境的效应分子组成；和(i) an extracellular domain consisting of effector molecules that reshape the tumor microenvironment; and
(ii)组成型激活的IL-7R突变体，由携带不同突变的IL7R跨膜区(IL7R-mutant(TM))和IL7Rα胞内段组成。(ii) Constitutively activated IL-7R mutants, consisting of the IL7R transmembrane region (IL7R-mutant(TM)) carrying different mutations and the IL7Rα intracellular segment.
在一些实施方案中，本发明的组成型嵌合细胞因子受体的所述(i)胞外结构域选自细胞因子、免疫效应分子、抑制性分子拮抗剂或靶向NK细胞激活性受体的效应分子。In some embodiments, said (i) extracellular domain of the constitutive chimeric cytokine receptor of the invention is selected from the group consisting of cytokines, immune effector molecules, inhibitory molecule antagonists, or NK cell-targeted activating receptors effector molecules.
当本发明的组成型嵌合细胞因子受体的所述(i)胞外结构域是细胞因子时，所述细胞因子可以是IL-12(IL-12-P40或IL-12-P70)、IL15(IL-15或IL-15FP，所述IL-15FP是指IL-15和IL-15Rα(选自IL-15Rα或IL-15Rα(Sushi))的融合蛋白，包括IL-15/IL-15Rα和IL-15Rα/IL-15两种形式的融合蛋白)、IL-21、IL-18、IL-9、IL-23、IL-36γ、IFNα2b等细胞因子，采用这些细胞因子基因修饰的免疫细胞具有增强的免疫效应功能及抗肿瘤效应。When the (i) extracellular domain of the constitutive chimeric cytokine receptor of the present invention is a cytokine, the cytokine may be IL-12 (IL-12-P40 or IL-12-P70), IL15 (IL-15 or IL-15FP, the IL-15FP refers to the fusion protein of IL-15 and IL-15Rα (selected from IL-15Rα or IL-15Rα (Sushi)), including IL-15/IL-15Rα and IL-15Rα/IL-15 two forms of fusion protein), IL-21, IL-18, IL-9, IL-23, IL-36γ, IFNα2b and other cytokines, using immune cells genetically modified by these cytokines It has enhanced immune effector function and anti-tumor effect.
当本发明的组成型嵌合细胞因子受体的所述(i)胞外结构域是免疫效应分子时，所述免疫效应分子可以是4-1BB靶向分子部分(例如，4-1BB配体(4-1BBL)、抗4-1BB抗体(α4-1BB))、CD40靶向分子部分(例如，CD40配体(CD40L)、抗CD40抗体(αCD40))、CD83靶向分子部分(例如，抗CD83抗体(αCD83))、FLT3配体(FTL3L)、GITR、ICOS、CD2、ICAM1等，这些免疫效应分子通过与体内专职的抗原呈递细胞(APC)例如树突状细胞(DC)表面的相关受体或配体相互作用，活化APC，从而激发内源性抗肿瘤免疫应答。When the (i) extracellular domain of the constitutive chimeric cytokine receptor of the present invention is an immune effector molecule, the immune effector molecule may be a 4-1BB targeting molecule moiety (e.g., 4-1BB ligand (4-1BBL), anti-4-1BB antibody (α4-1BB)), CD40-targeting molecule moiety (e.g., CD40 ligand (CD40L), anti-CD40 antibody (αCD40)), CD83-targeting molecule moiety (e.g., anti- CD83 antibody (αCD83)), FLT3 ligand (FTL3L), GITR, ICOS, CD2, ICAM1, etc., these immune effector molecules are recognized by receptors on the surface of professional antigen-presenting cells (APC) such as dendritic cells (DC) in the body. The body or ligand interacts to activate APC, thereby stimulating endogenous anti-tumor immune response.
当本发明的组成型嵌合细胞因子受体的所述(i)胞外结构域是抑制性分子拮抗剂时，所述抑制性分子拮抗剂可以是抗PD-L1分子、抗CD47分子、抗IL-4分子、TGFβ结合分子(例如，抗TGFβ1分子、TGFβRII)、抗PD-1分子、抗CTLA-4分子、抗LAG-3分子、抗TIGIT分子、抗CD73分子等针对抑制性免疫受体或因子的抗体部分，例如，抗PD-L1VHH，通过拮抗抑制性免疫受体或因子的免疫抑制效应，达到增强抗肿瘤免疫应答目的。When the (i) extracellular domain of the constitutive chimeric cytokine receptor of the present invention is an inhibitory molecule antagonist, the inhibitory molecule antagonist may be an anti-PD-L1 molecule, an anti-CD47 molecule, or an anti-CD47 molecule. IL-4 molecules, TGFβ binding molecules (e.g., anti-TGFβ1 molecules, TGFβRII), anti-PD-1 molecules, anti-CTLA-4 molecules, anti-LAG-3 molecules, anti-TIGIT molecules, anti-CD73 molecules, etc. target inhibitory immune receptors Or the antibody part of the factor, for example, anti-PD-L1 VHH , achieves the purpose of enhancing the anti-tumor immune response by antagonizing the immunosuppressive effect of inhibitory immune receptors or factors.
在一些实施方案中，本发明的组成型嵌合细胞因子受体的所述(i)胞外结构域选自靶向NKG2C、NKG2D、NKp30、NKp44、NKp46等NK细胞表面表达的激活性受体的分子部分，例如，抗NKG2C、抗NKG2D、抗NKp30、抗NKp44、抗NKp46等抗体部分，通过激活内源性NK细胞，取得增强抗肿瘤免疫效应的目的。In some embodiments, the (i) extracellular domain of the constitutive chimeric cytokine receptor of the invention is selected from activating receptors targeting NK cell surface expression such as NKG2C, NKG2D, NKp30, NKp44, NKp46, etc. The molecular parts, such as anti-NKG2C, anti-NKG2D, anti-NKp30, anti-NKp44, anti-NKp46 and other antibody parts, achieve the purpose of enhancing the anti-tumor immune effect by activating endogenous NK cells.
在一些实施方案中，本发明的组成型嵌合细胞因子受体的所述(ii)组成型激活的IL-7R突变体包含任一选自SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:28、SEQ ID NO:30至SEQ ID NO:45所示的氨基酸序列，优选地，所述组成型激活的IL-7R突变体包含任一选自SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:34和SEQ ID NO:44所示的氨基酸序列，最优选地，所述组成型激活的IL-7R突变体包含SEQ ID NO:34所示的氨基酸序列。In some embodiments, the (ii) constitutively activated IL-7R mutant of the constitutively chimeric cytokine receptor of the invention comprises any one selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:28, SEQ ID NO:30 to the amino acid sequence shown in SEQ ID NO:45, preferably, the constitutively activated IL-7R mutant includes any one selected from SEQ ID NO:30, SEQ ID NO :31, the amino acid sequence shown in SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:44, most preferably, the constitutively activated IL-7R mutant includes the amino acid sequence shown in SEQ ID NO:34 Amino acid sequence.
本发明的组成型嵌合细胞因子受体是一种组成型二聚体，能够不依赖IL-7R与其配体的结合且不依赖于与共同γ信号链(γc)组合而激活IL-7R的胞内信号传导，活化JAK1激酶，进而磷酸化下游STAT5等转录效应因子，调控下游靶基因表达，最终促进和维持T增殖及存活。The constitutive chimeric cytokine receptor of the present invention is a constitutive dimer that can activate IL-7R independently of the binding of IL-7R to its ligand and independent of combination with a common γ signal chain (γc). Intracellular signaling activates JAK1 kinase, which in turn phosphorylates downstream STAT5 and other transcriptional effectors, regulates the expression of downstream target genes, and ultimately promotes and maintains T proliferation and survival.
本发明的组成型嵌合细胞因子受体在包含所述(ii)组成型激活的IL-7R突变体基础上，通过包含所述(i)胞外结构域作为效应分子来重塑肿瘤微环境。The constitutive chimeric cytokine receptor of the present invention reshapes the tumor microenvironment by including the (i) extracellular domain as an effector molecule on the basis of (ii) the constitutively activated IL-7R mutant. .
III.本发明的组成型嵌合细胞因子受体与CAR多肽的共表达III. Co-expression of the constitutive chimeric cytokine receptor and CAR polypeptide of the present invention
本发明的组成型嵌合细胞因子受体与嵌合抗原受体(CAR)多肽共表达于T细胞时，所述组成型嵌合细胞因子受体由于包含胞外结构域和组成型激活的IL-7R突变体，其通过所述组 成型激活的IL-7R突变体持续激活STAT5信号，促进和维持免疫细胞的增殖及存活，并通过所述胞外结构域赋予了免疫细胞获得了新的胞外效应分子功效，使其修饰的免疫细胞(例如，CAR-T细胞)具备主动塑造“不友好”肿瘤微环境(TME)的能力，通过重构TME使“冷”肿瘤变为“热”肿瘤，其修饰的免疫细胞(例如，CAR-T细胞)处于更“友好”的TME中，将更加有利于发挥抗肿瘤效应。When the constitutive chimeric cytokine receptor of the present invention and the chimeric antigen receptor (CAR) polypeptide are co-expressed in T cells, the constitutive chimeric cytokine receptor contains an extracellular domain and a constitutively activated IL -7R mutant, which passed the group The activated IL-7R mutant continuously activates STAT5 signaling, promotes and maintains the proliferation and survival of immune cells, and gives immune cells new extracellular effector molecule effects through the extracellular domain, making it a modified immune system. Cells (e.g., CAR-T cells) have the ability to actively shape the "unfriendly" tumor microenvironment (TME), turning "cold" tumors into "hot" tumors by remodeling the TME, and their modified immune cells (e.g., CAR -T cells) in a more "friendly" TME will be more conducive to exerting anti-tumor effects.
在一些实施方案中，所述CAR多肽是传统CAR多肽，其直接靶向一种或多种癌相关抗原。例如，所述癌相关抗原(也称为“肿瘤抗原”)选自以下一种或多种：CD19；CD20；CD22；CD24；CD30；CD123；CD171；CD33表皮生长因子受体变体III(EGFRvIII)；神经节苷脂G2(GD2)；TNF受体家族成员B细胞成熟(BCMA)；前列腺特异性膜抗原(PSMA)；Fms样酪氨酸激酶3(FLT3)；肿瘤相关的糖蛋白72(TAG72)；CD38；CD44v6；癌胚抗原(CEA)；上皮细胞粘附分子(EPCAM)；B7H3(CD276)；KIT(CD117)；白介素13受体亚基α-2(IL-13Ra2或CD213A2)；间皮素；白介素11受体α(IL-11Ra)；前列腺干细胞抗原(PSCA)；蛋白酶丝氨酸21；血管内皮生长因子受体2(VEGFR2)；路易斯(Y)抗原；血小板衍生生长因子受体β(PDGFR-β)；阶段特异性胚胎抗原-4(SSEA-4)；叶酸受体α；受体酪氨酸蛋白激酶ERBB2(Her2/neu)；细胞表面相关的粘蛋白1(MUC1)；表皮生长因子受体(EGFR)；神经细胞粘附分子(NCAM)；前列腺酸性磷酸酶(PAP)；突变的延伸因子2(ELF2M)；肝配蛋白B2；成纤维细胞活化蛋白α(FAP)；胰岛素样生长因子1受体(IGF-I受体)；肝配蛋白A型受体2(EphA2)；岩藻糖基GM1；唾液酸基路易斯粘附分子(sLe)；转谷氨酰胺酶5(TGS5)；高分子量黑素瘤相关抗原(HMWMAA)；邻乙酰基GD2神经节苷脂(OAcGD2)；叶酸受体β；肿瘤血管内皮标记1(TEM1/CD248)；肿瘤血管内皮标记7相关的(TEM7R)；Claudin 6(CLDN6)；CLDN18.2；促甲状腺激素受体(TSHR)；G蛋白偶联受体C类5组，成员D(GPRC5D)；X染色体开放阅读框61(CXORF61)；CD97；CD179a；间变性淋巴瘤激酶(ALK)；聚唾液酸；胎盘特异性1(PLAC1)；globoH葡糖苷神经酰胺的己糖部分(GloboH)；乳腺分化抗原(NY-BR-1)；uroplakin 2(UPK2)；甲型肝炎病毒细胞受体1(HAVCR1)；肾上腺素受体β3(ADRB3)；pannexin 3(PANX3)；G蛋白偶联受体20(GPR20)；淋巴细胞抗原6复合物，基因座K9(LY6K)；嗅觉受体51E2(OR51E2)；TCRγ交替阅读框蛋白(TARP)；肾母细胞瘤蛋白(WT1)；癌/睾丸抗原1(NY-ESO-1)；癌症/睾丸抗原2(LAGE-1A)；黑素瘤相关抗原1(MAGE-A1)；ETS易位变异基因6，位于染色体12p(ETV6-AML)；精子蛋白17(SPA17)；X抗原家族，成员1A(XAGE1)；血管生成素结合细胞表面受体2(Tie2)；黑素瘤癌睾丸抗原-1(MAD-CT-1)；黑素瘤癌睾丸抗原-2(MAD-CT-2)；FOS相关抗原1；肿瘤蛋白质p53(p53)；p53突变体；prostein；存活蛋白；端粒酶；前列腺癌肿瘤抗原-1(PCTA-1或半乳凝素8)，由T细胞识别的黑素瘤抗原1(MelanA或MART1)；大鼠肉瘤(Ras)突变体；人端粒酶逆转录酶(hTERT)；肉瘤易位断点；细胞凋亡的黑素瘤抑制剂(ML-IAP)；ERG(跨膜蛋白酶，丝氨酸2(TMPRSS2)ETS融合基因)；N-乙酰葡糖胺基转移酶V(NA17)；配对盒蛋白Pax-3(PAX3)；雄激素受体；细胞周期蛋白B1；V-myc鸟髓细胞瘤病病毒癌基因神经母细胞瘤衍生的同源物(MYCN)；Ras同源物家族成员C(RhoC)；酪氨酸酶相关蛋白2(TRP-2)；细胞色素P450 1B1(CYP1B1)；CCCTC结合因子(锌指蛋白)样(BORIS或印记位点的调节物的兄弟)，由T细胞识别的鳞状细胞癌抗原3(SART3)；配对盒蛋白Pax-5(PAX5)；顶体蛋白酶原结合蛋白sp32(OY-TES1)；淋巴细胞特异性蛋白酪氨酸激酶(LCK)；A激酶锚定蛋白4(AKAP-4)；滑膜肉瘤，X断点2(SSX2)；高级糖化终产物受体(RAGE-1)； 肾泛素1(RU1)；肾泛素2(RU2)；豆荚蛋白酶；人类乳头瘤病毒E6(HPV E6)；人类乳头瘤病毒E7(HPV E7)；肠羧基酯酶；突变的热休克蛋白70-2(mut hosp 70-2)；CD79a；CD79b；CD72；白细胞相关免疫球蛋白样受体1(LAIR1)；IgA受体的Fc片段(FCAR或CD89)；白细胞免疫球蛋白样受体亚家族A成员2(LILRA2)；CD300分子样家族成员f(CD300LF)；C型凝集素结构域家族12成员A(CLEC12A)；骨髓基质细胞抗原2(BST2)；含有EGF样模块粘蛋白样激素受体样2(EMR2)；淋巴细胞抗原75(LY75)；磷脂酰肌醇蛋白聚糖-3(GPC3)；Fc受体样5(FCRL5)；与免疫球蛋白λ样多肽1(IGLL1)。In some embodiments, the CAR polypeptide is a traditional CAR polypeptide that directly targets one or more cancer-associated antigens. For example, the cancer-associated antigen (also known as "tumor antigen") is selected from one or more of the following: CD19; CD20; CD22; CD24; CD30; CD123; CD171; CD33 epidermal growth factor receptor variant III (EGFRvIII ); ganglioside G2 (GD2); TNF receptor family member B cell maturation (BCMA); prostate-specific membrane antigen (PSMA); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 ( TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin 13 receptor subunit α-2 (IL-13Ra2 or CD213A2); Mesothelin; interleukin 11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); protease serine 21; vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; platelet-derived growth factor receptor beta (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); folate receptor α; receptor tyrosine protein kinase ERBB2 (Her2/neu); cell surface-associated mucin 1 (MUC1); epidermis Growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostatic acid phosphatase (PAP); mutated elongation factor 2 (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin growth factor 1 receptor (IGF-I receptor); ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); transglutaminase 5 ( TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl GD2 ganglioside (OAcGD2); folate receptor beta; tumor endothelial marker 1 (TEM1/CD248); tumor endothelial marker 7 related ( TEM7R); Claudin 6 (CLDN6); CLDN18.2; thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97 ; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexose portion of globoH glucosylceramide (GloboH); breast differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, gene Locus K9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCRγ alternating reading frame protein (TARP); Wilms tumor protein (WT1); Cancer/testis antigen 1 (NY-ESO-1); Cancer/testis antigen 2 (LAGE-1A); melanoma-associated antigen 1 (MAGE-A1); ETS translocation variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1) ; Angiopoietin-binding cell surface receptor 2 (Tie2); Melanoma cancer testis antigen-1 (MAD-CT-1); Melanoma cancer testis antigen-2 (MAD-CT-2); FOS-related antigen 1 ; Tumor protein p53 (p53); p53 mutants; prostein; survivin; telomerase; prostate cancer tumor antigen-1 (PCTA-1 or galectin-8), melanoma antigen 1 recognized by T cells ( MelanA or MART1); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane Protease, serine 2 (TMPRSS2, ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; V-myc bird Myelocytoma viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450 1B1 (CYP1B1 ); CCCTC-binding factor (zinc finger protein)-like (brother of BORIS or regulator of imprinted loci), squamous cell carcinoma antigen 3 (SART3) recognized by T cells; paired box protein Pax-5 (PAX5); IM In vivo protease-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A-kinase-anchored protein 4 (AKAP-4); synovial sarcoma, breakpoint X 2 (SSX2); advanced Receptor for glycation end products (RAGE-1); Renal ubiquitin 1 (RU1); Renal ubiquitin 2 (RU2); Legumin; Human papillomavirus E6 (HPV E6); Human papillomavirus E7 (HPV E7); Intestinal carboxyl esterase; Mutated heat shock protein 70 -2 (mut hosp 70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); mucin-like hormone receptor containing EGF-like module like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1).
在一些实施方案中，由传统CAR多肽直接靶向的癌相关抗原是CLDN18.2，所述传统CAR多肽从N端至C端包含信号肽、癌相关抗原结合结构域、跨膜结构域、共刺激信号传导结构域和主信号传导结构域。In some embodiments, the cancer-associated antigen directly targeted by a traditional CAR polypeptide comprising a signal peptide, a cancer-associated antigen-binding domain, a transmembrane domain, a covalent Stimulatory signaling domain and main signaling domain.
在一些实施方案中，所编码的CAR多肽的癌相关抗原结合结构域包含针对癌相关抗原的抗体、抗体片段、scFv、Fv、Fab、(Fab')2、单结构域抗体(SDAB)、VH或VL结构域，或骆驼科VHH结构域。In some embodiments, the encoded cancer-associated antigen binding domain of the CAR polypeptide comprises an antibody, antibody fragment, scFv, Fv, Fab, (Fab')2, single domain antibody (SDAB), VH Or VL domain, or Camelidae VHH domain.
在一些实施方案中，CAR多肽的跨膜结构域包含选自以下的跨膜结构域：T细胞受体的α、β、或ζ的跨膜结构域、CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、KIRDS2、OX40、CD2、CD27、LFA-1(CD11a、CD18)、ICOS(CD278)、4-1BB(CD137)、GITR、CD40、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、CD160、CD19、IL2Rβ、IL2Rγ、IL7Rα、ITGA1、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、TNFR2、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100(SEMA4D)、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、PAG/Cbp、NKp44、NKp30、NKp46、NKG2D、和/或NKG2C的跨膜结构域。In some embodiments, the transmembrane domain of the CAR polypeptide comprises a transmembrane domain selected from the group consisting of the alpha, beta, or zeta transmembrane domain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1(CD11a, CD18), ICOS(CD278), 4-1BB( CD137), GITR, CD40, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80(KLRF1), CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f , ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), SLAMF6(NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3) , BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, and/or the transmembrane domain of NKG2C.
在某些实施方案中，CAR多肽的跨膜结构域包含CD8跨膜结构域的氨基酸序列，其具有SEQ ID NO:8的一个、两个或三个氨基酸修饰的序列。在一个实施方案中，跨膜结构域包含SEQ ID NO:8的序列。In certain embodiments, the transmembrane domain of the CAR polypeptide comprises the amino acid sequence of the CD8 transmembrane domain having one, two, or three amino acid modifications of SEQ ID NO: 8. In one embodiment, the transmembrane domain comprises the sequence of SEQ ID NO: 8.
在某些实施方案中，癌相关抗原结合结构域由铰链区连接到所述跨膜结构域。在一个实施方案中，铰链区包含CD8铰链的氨基酸序列，例如，SEQ ID NO:7，或与SEQ ID NO:7具有一个、两个或三个氨基酸修饰的序列。In certain embodiments, the cancer-associated antigen binding domain is connected to the transmembrane domain by a hinge region. In one embodiment, the hinge region comprises the amino acid sequence of the CD8 hinge, e.g., SEQ ID NO:7, or a sequence with one, two, or three amino acid modifications to SEQ ID NO:7.
在其他实施方案中，CAR多肽包含胞内信号传导结构域，例如主信号传导结构域(primary signaling domain)和/或共刺激信号传导结构域。在一些实施方案中，胞内信号传导结构域包含主信号传导结构域。在一些实施方案中，胞内信号传导结构域包含共刺激信号传导结构域。在一些实施方案中，胞内信号传导结构域包含主信号传导结构域和共刺激信号传导结构域。In other embodiments, the CAR polypeptide comprises an intracellular signaling domain, such as a primary signaling domain and/or a costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises a primary signaling domain. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the intracellular signaling domain includes a primary signaling domain and a costimulatory signaling domain.
在某些实施方案中，主信号传导结构域包含选自CD3ζ、CD3γ、CD3δ、CD3ε、常见FcRγ(FCER1G)、FcRβ(FcεR1b)、CD79a、CD79b、FcγRIIa、DAP10和DAP12的蛋白质的功能信号传导结构域。In certain embodiments, the primary signaling domain comprises a functional signaling structure of a protein selected from the group consisting of CD3ζ, CD3γ, CD3δ, CD3ε, common FcRγ (FCER1G), FcRβ (FcεR1b), CD79a, CD79b, FcγRIIa, DAP10, and DAP12 area.
在一个实施方案中，CAR多肽的主信号传导结构域包含CD3ζ的功能信号传导结构域。 CD3ζ主信号传导结构域可包含具有SEQ ID NO:12的氨基酸序列的1、2或3个氨基酸修饰。在一些实施方案中，主信号传导结构域包含SEQ ID NO:12的序列。In one embodiment, the primary signaling domain of the CAR polypeptide comprises a functional signaling domain of CD3ζ. The CD3ζ primary signaling domain may comprise 1, 2 or 3 amino acid modifications having the amino acid sequence of SEQ ID NO:12. In some embodiments, the primary signaling domain comprises the sequence of SEQ ID NO: 12.
在一些实施方案中，CAR多肽的胞内信号传导结构域包含主信号传导结构域和共刺激信号传导结构域。在一些实施方案中，共刺激信号传导结构域包含选自如下一种或多种的蛋白质的功能信号传导结构域：CD27、CD28、4-1BB(CD137)、OX40、CD30、CD40、PD1、ICOS，淋巴细胞功能相关抗原-1(LFA-1)、CD2、CD7、LIGHT、NKG2C、B7-H3、特异性结合CD83的配体、CDS、ICAM-1、GITR、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、CD160、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、TNFR2、TRANCE/RANKL、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100(SEMA4D)、CD69、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、NKp44、NKp30、NKp46或NKG2D。In some embodiments, the intracellular signaling domain of a CAR polypeptide comprises a primary signaling domain and a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain comprises a functional signaling domain of a protein selected from one or more of the following: CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD1, ICOS , Lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligand specifically binding to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM(LIGHTR), SLAMF7 , NKp80(KLRF1), CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME( SLAMF8), SELPLG(CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, or NKG2D.
在一些实施方案中，CAR多肽的共刺激信号传导结构域包含具有SEQ ID NO:11的氨基酸序列的1、2、或3个氨基酸修饰。在一些实施方案中，编码的共刺激信号传导结构域包含SEQ ID NO:11的序列。In some embodiments, the costimulatory signaling domain of the CAR polypeptide comprises 1, 2, or 3 amino acid modifications having the amino acid sequence of SEQ ID NO: 11. In some embodiments, the encoded costimulatory signaling domain comprises the sequence of SEQ ID NO: 11.
在一些实施方案中，CAR还包含信号肽序列。在一个实施方案中，信号肽序列包含SEQ ID NO:1的序列。In some embodiments, the CAR further comprises a signal peptide sequence. In one embodiment, the signal peptide sequence comprises the sequence of SEQ ID NO: 1.
在某些实施方案中，CAR多肽的癌相关抗原结合结构域对癌相关抗原具有10-4M到10-8M的结合亲和力KD。In certain embodiments, the cancer-associated antigen binding domain of the CAR polypeptide has a binding affinity K D for the cancer-associated antigen of 10 −4 M to 10 −8 M.
在一些实施方案中，传统CAR多肽包含传统CLDN18.2 CAR多肽。In some embodiments, the conventional CAR polypeptide comprises a conventional CLDN18.2 CAR polypeptide.
在一个实施方案中，传统CLDN18.2 CAR多肽包含：In one embodiment, a traditional CLDN18.2 CAR polypeptide comprises:
(1)特异性结合CLDN18.2分子的H9.1.2抗体scFv序列，其包含重链可变区和轻链可变区，(1) H9.1.2 antibody scFv sequence that specifically binds to CLDN18.2 molecules, which includes a heavy chain variable region and a light chain variable region,
其中：in:
所述重链可变区包含根据Kabat编号的氨基酸序列SYNIH(SEQ ID NO:106)所示的CDR H1、或所述CDR H1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；氨基酸序列YIAPFQGDARYNQKFKG(SEQ ID NO:107)所示的CDR H2、或所述CDR H2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和氨基酸序列LNRGQSLDY(SEQ ID NO:108)所示的CDR H3、或所述CDR H3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；所述轻链可变区包含根据Kabat编号的氨基酸序列KSSQSLFNAGNQRNYLT(SEQ ID NO:109)所示的CDR L1、或所述CDR L1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；氨基酸序列WASTRES(SEQ ID NO:110)所示的CDR L2、或所述CDR L2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和氨基酸序列QNNYIYPLT(SEQ ID NO:111)所示的CDR L3、或所述CDR L3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；The heavy chain variable region includes a CDR H1 represented by the Kabat numbered amino acid sequence SYNIH (SEQ ID NO: 106), or a variant of the CDR H1 with no more than 2 amino acid changes or no more than 1 amino acid change. ; CDR H2 shown in the amino acid sequence YIAPFQGDARYNQKFKG (SEQ ID NO: 107), or a variant of the CDR H2 with no more than 2 amino acid changes or no more than 1 amino acid change; and the amino acid sequence LNRGQSLDY (SEQ ID NO: 108 ), or a variant of the CDR H3 with no more than 2 amino acid changes or no more than 1 amino acid change; the light chain variable region includes the amino acid sequence KSSQSLFNAGNQRNYLT (SEQ ID NO: The CDR L1 shown in 109), or a variant of the CDR L1 with no more than 2 amino acid changes or no more than 1 amino acid change; the CDR L2 shown with the amino acid sequence WASTRES (SEQ ID NO: 110), or the CDR L1 with no more than 2 amino acid changes or no more than 1 amino acid change. Variants of CDR L2 with no more than 2 amino acid changes or no more than 1 amino acid change; and CDR L3 shown in the amino acid sequence QNNYIYPLT (SEQ ID NO: 111), or with no more than 2 amino acid changes of the CDR L3 or Variants with no more than 1 amino acid change;
其中所述氨基酸变化是氨基酸的添加、缺失或取代；wherein said amino acid change is the addition, deletion or substitution of amino acids;
例如，所述重链可变区包含SEQ ID NO:14的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:13 的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；For example, the heavy chain variable region comprises or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 14 Identity sequence, and the light chain variable region comprises SEQ ID NO:13 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;
(2)铰链区，其选自CD8铰链区(SEQ ID NO:7)，或其具有至少80％的序列同一性的铰链区。(2) A hinge region selected from the CD8 hinge region (SEQ ID NO: 7), or a hinge region having at least 80% sequence identity.
(3)跨膜区(TM)，其选自CD8跨膜结构域或其具有1-5个氨基酸修饰的变体，例如，SEQ ID NO:8所示的序列或其具有1-2个氨基酸修饰的变体；(3) Transmembrane region (TM), which is selected from the CD8 transmembrane domain or a variant thereof having 1-5 amino acid modifications, for example, the sequence shown in SEQ ID NO:8 or having 1-2 amino acids Modified variants;
(4)共刺激信号结构域(CSD)，其选自4-1BB共刺激结构域或其具有1-5个氨基酸修饰的变体，例如，SEQ ID NO:11所示的序列或其具有1-2个氨基酸修饰的变体；(4) Costimulatory signal domain (CSD), which is selected from the 4-1BB costimulatory domain or a variant thereof with 1-5 amino acid modifications, for example, the sequence shown in SEQ ID NO: 11 or a variant thereof with 1 -2 amino acid modified variants;
(5)刺激信号结构域(SSD)，为CD3ζ信号传导结构域或其具有1-10个氨基酸修饰的变体，例如，SEQ ID NO:12所示的序列或其具有1-10个、1-5个氨基酸修饰的变体(5) Stimulating signal domain (SSD), which is a CD3ζ signaling domain or a variant thereof with 1-10 amino acid modifications, for example, the sequence shown in SEQ ID NO: 12 or a sequence with 1-10, 1 -5 amino acid modified variants
任选地，所述传统CLDN18.2 CAR多肽还包含位于N端的信号肽序列，例如，SEQ ID NO:1所示的信号肽序列，Optionally, the traditional CLDN18.2 CAR polypeptide also includes a signal peptide sequence located at the N-terminus, for example, the signal peptide sequence shown in SEQ ID NO: 1,
在一个具体实施方案中，传统CLDN18.2 CAR多肽包含，例如本文所述的H9.1.2 CAR(SEQ ID NO:16)。In a specific embodiment, a traditional CLDN18.2 CAR polypeptide includes, for example, the H9.1.2 CAR (SEQ ID NO: 16) described herein.
在一个实施方案中，传统CLDN18.2 CAR多肽包含：In one embodiment, a conventional CLDN18.2 CAR polypeptide comprises:
(1)特异性结合CLDN18.2分子的H9.2.1抗体scFv序列，其包含重链可变区和轻链可变区，其中：(1) H9.2.1 antibody scFv sequence that specifically binds to CLDN18.2 molecules, which contains a heavy chain variable region and a light chain variable region, wherein:
所述重链可变区包含根据Kabat编号的氨基酸序列SYNIH(SEQ ID NO:112)所示的CDR H1、或所述CDR H1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；氨基酸序列YIAPFQGDARYNQKFKG(SEQ ID NO:113)所示的CDR H2、或所述CDR H2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和氨基酸序列LNRGNALDY(SEQ ID NO:114)所示的CDR H3、或所述CDR H3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；所述轻链可变区包含根据Kabat编号的氨基酸序列KSSQSLFQSGNQRNYLT(SEQ ID NO:115)所示的CDR L1、或所述CDR L1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；氨基酸序列WASTRES(SEQ ID NO:116)所示的CDR L2、或所述CDR L2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和氨基酸序列QNNYIYPLT(SEQ ID NO:117)所示的CDR L3、或所述CDR L3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；The heavy chain variable region includes a CDR H1 represented by the Kabat numbered amino acid sequence SYNIH (SEQ ID NO: 112), or a variant of the CDR H1 with no more than 2 amino acid changes or no more than 1 amino acid change. ; CDR H2 shown in the amino acid sequence YIAPFQGDARYNQKFKG (SEQ ID NO: 113), or a variant of the CDR H2 with no more than 2 amino acid changes or no more than 1 amino acid change; and the amino acid sequence LNRGNALDY (SEQ ID NO: 114 ), or a variant of the CDR H3 with no more than 2 amino acid changes or no more than 1 amino acid change; the light chain variable region includes the amino acid sequence KSSQSLFQSGNQRNYLT (SEQ ID NO: The CDR L1 shown in 115), or a variant of the CDR L1 with no more than 2 amino acid changes or no more than 1 amino acid change; the CDR L2 shown with the amino acid sequence WASTRES (SEQ ID NO: 116), or the CDR L2 with no more than 1 amino acid change Variants of CDR L2 with no more than 2 amino acid changes or no more than 1 amino acid change; and CDR L3 shown in the amino acid sequence QNNYIYPLT (SEQ ID NO: 117), or with no more than 2 amino acid changes of the CDR L3 or Variants with no more than 1 amino acid change;
其中所述氨基酸变化是氨基酸的添加、缺失或取代；wherein said amino acid change is the addition, deletion or substitution of amino acids;
例如，所述重链可变区包含SEQ ID NO:99的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:98的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；For example, the heavy chain variable region comprises or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 99 Sequence identity, and the light chain variable region contains or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or Sequences with 99% identity;
(2)铰链区，其选自CD8铰链区(SEQ ID NO：7或SEQ ID NO：146)，或其具有至少80％的序列同一性的铰链区。(2) A hinge region selected from the CD8 hinge region (SEQ ID NO: 7 or SEQ ID NO: 146), or a hinge region having at least 80% sequence identity.
(3)跨膜区(TM)，其选自CD8跨膜结构域或其具有1-5个氨基酸修饰的变体，例如，SEQ ID NO:8或SEQ ID NO:147所示的序列或其具有1-2个氨基酸修饰的变体；(3) Transmembrane region (TM), which is selected from the CD8 transmembrane domain or a variant thereof with 1-5 amino acid modifications, for example, the sequence shown in SEQ ID NO:8 or SEQ ID NO:147 or its Variants with 1-2 amino acid modifications;
(4)共刺激信号结构域(CSD)，其选自4-1BB共刺激结构域或其具有1-5个氨基酸修饰的变体，例如，SEQ ID NO:11所示的序列或其具有1-2个氨基酸修饰的变体；或其选自CD28共刺激结构域或其具有1-5个氨基酸修饰的变体，例如，SEQ ID NO:143所示的序列或其具 有1-2个氨基酸修饰的变体；(4) Costimulatory signal domain (CSD), which is selected from the group consisting of 4-1BB costimulatory domain or a variant thereof with 1-5 amino acid modifications, for example, the sequence shown in SEQ ID NO: 11 or a variant thereof with 1 - A variant with 2 amino acid modifications; or it is selected from the CD28 costimulatory domain or a variant thereof with 1-5 amino acid modifications, for example, the sequence shown in SEQ ID NO: 143 or the sequence thereof Variants with 1-2 amino acid modifications;
(5)刺激信号结构域(SSD)，为CD3ζ信号传导结构域或其具有1-10个氨基酸修饰的变体，例如，SEQ ID NO:12所示的序列或其具有1-10个、1-5个氨基酸修饰的变体(5) Stimulating signal domain (SSD), which is a CD3ζ signaling domain or a variant thereof with 1-10 amino acid modifications, for example, the sequence shown in SEQ ID NO: 12 or a sequence with 1-10, 1 -5 amino acid modified variants
任选地，所述传统CLDN18.2 CAR多肽还包含位于N端的信号肽序列，例如，SEQ ID NO:1所示的信号肽序列，Optionally, the traditional CLDN18.2 CAR polypeptide also includes a signal peptide sequence located at the N-terminus, for example, the signal peptide sequence shown in SEQ ID NO: 1,
在一个具体实施方案中，传统CLDN18.2 CAR多肽包含例如本文所述的H9.2.1 CAR(SEQ ID NO:100)。In a specific embodiment, a traditional CLDN18.2 CAR polypeptide comprises, for example, a H9.2.1 CAR (SEQ ID NO: 100) described herein.
在一个具体实施方案中，传统CLDN18.2 CAR多肽包含例如本文所述的H9.2.1-218 CAR(SEQ ID NO:144)。In a specific embodiment, a conventional CLDN18.2 CAR polypeptide includes, for example, the H9.2.1-218 CAR (SEQ ID NO: 144) described herein.
在一个具体实施方案中，传统CLDN18.2 CAR多肽包含例如本文所述的H9.2.1-28-L CAR(SEQ ID NO:142)。In a specific embodiment, a conventional CLDN18.2 CAR polypeptide comprises, for example, the H9.2.1-28-L CAR (SEQ ID NO: 142) described herein.
在一些实施方案中，所述CAR多肽是分子开关调控型CAR多肽，其不直接靶向一种或多种癌相关抗原，而是通过“分子开关”来靶向一种或多种癌相关抗原。例如，通过将Pro329Gly(抗体Fc段根据EU编号的第329位脯氨酸突变为甘氨酸，简写为P329G)突变抗体作为“分子开关”，构建的这样的CAR分子，其能够特异结合包含P329G突变Fc结构域的抗体而不结合不包含P329G突变Fc结构域的抗体，由此，通过将表达所述CAR的免疫效应细胞(例如，T细胞、NK细胞)与作为“分子开关”的靶向癌相关抗原的P329G突变抗体组合，用于治疗肿瘤。In some embodiments, the CAR polypeptide is a molecular switch-regulated CAR polypeptide, which does not directly target one or more cancer-associated antigens, but targets one or more cancer-associated antigens through a "molecular switch" . For example, by using the Pro329Gly (antibody Fc segment is based on the mutation of proline at position 329 of EU numbering to glycine, abbreviated as P329G) mutant antibody as a "molecular switch", a CAR molecule is constructed that can specifically bind to the Fc containing the P329G mutant Domain-specific antibodies do not bind to antibodies that do not contain the P329G mutated Fc domain, whereby immune effector cells (e.g., T cells, NK cells) expressing the CAR are associated with targeted cancers as a "molecular switch" P329G mutated antibody combination of the antigen for the treatment of tumors.
在一个实施方案中，所述分子开关调控型CLDN18.2 CAR多肽包含：In one embodiment, the molecular switch-regulated CLDN18.2 CAR polypeptide comprises:
(1)人源化抗P329G突变scFv序列，其中所述scFv序列包含能够特异性结合包含P329G突变的抗体Fc结构域，但不能特异性结合未突变的亲本抗体Fc结构域的如下序列：(1) A humanized anti-P329G mutation scFv sequence, wherein the scFv sequence includes the following sequence that is capable of specifically binding to an antibody Fc domain containing a P329G mutation, but is unable to specifically bind to an unmutated parent antibody Fc domain:
(i)重链可变区，其包含根据Kabat编号的(i) A heavy chain variable region comprising a number according to Kabat
(a)氨基酸序列RYWMN(SEQ ID NO:118)所示的重链互补决定区CDR H1、或所述CDR H1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；(a) The heavy chain complementarity determining region CDR H1 shown in the amino acid sequence RYWMN (SEQ ID NO: 118), or a variant of the CDR H1 with no more than 2 amino acid changes or no more than 1 amino acid change;
(b)氨基酸序列EITPDSSTINYAPSLKG(SEQ ID NO:119)所示的CDR H2、或所述CDR H2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和(b) CDR H2 shown in the amino acid sequence EITPDSSTINYAPSLKG (SEQ ID NO: 119), or a variant of the CDR H2 with no more than 2 amino acid changes or no more than 1 amino acid change; and
(c)氨基酸序列PYDYGAWFAS(SEQ ID NO:120)所示的CDR H3、或所述CDR H3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和(c) CDR H3 shown in the amino acid sequence PYDYGAWFAS (SEQ ID NO: 120), or a variant of the CDR H3 with no more than 2 amino acid changes or no more than 1 amino acid change; and
(ii)轻链可变区，其包含根据Kabat编号的(ii) A light chain variable region comprising a number according to Kabat
(d)氨基酸序列RSSTGAVTTSNYAN(SEQ ID NO:121)所示的轻链互补决定区(CDR L)1、或所述CDR L1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；(d) The light chain complementarity determining region (CDR L) 1 shown in the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 121), or a variant of the CDR L1 with no more than 2 amino acid changes or no more than 1 amino acid change;
(e)氨基酸序列GTNKRAP(SEQ ID NO:122)所示的CDR L2、或所述CDR L2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和(e) CDR L2 shown in the amino acid sequence GTNKRAP (SEQ ID NO: 122), or a variant of the CDR L2 with no more than 2 amino acid changes or no more than 1 amino acid change; and
(f)氨基酸序列ALWYSNHWV(SEQ ID NO:123)所示的CDR L3、或所述CDR L3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；(f) CDR L3 shown in the amino acid sequence ALWYSNHWV (SEQ ID NO: 123), or a variant of the CDR L3 with no more than 2 amino acid changes or no more than 1 amino acid change;
其中所述氨基酸变化是氨基酸的添加、缺失或取代wherein the amino acid change is the addition, deletion or substitution of an amino acid
例如，所述重链可变区包含SEQ ID NO:9的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:10的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的 序列；For example, the heavy chain variable region comprises or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 9 Sequence identity, and the light chain variable region comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical sequence;
(2)铰链区，其选自CD8铰链区(SEQ ID NO:7)，或其具有至少80％的序列同一性的铰链区；(2) Hinge region, which is selected from the CD8 hinge region (SEQ ID NO:7), or a hinge region with at least 80% sequence identity;
(3)跨膜区(TM)，其选自CD8跨膜结构域或其具有1-5个氨基酸修饰的变体，例如，SEQ ID NO:8所示的序列或其具有1-2个氨基酸修饰的变体；(3) Transmembrane region (TM), which is selected from the CD8 transmembrane domain or a variant thereof having 1-5 amino acid modifications, for example, the sequence shown in SEQ ID NO:8 or having 1-2 amino acids Modified variants;
(4)共刺激信号结构域(CSD)，其选自4-1BB共刺激结构域或其具有1-5个氨基酸修饰的变体，例如，SEQ ID NO:11所示的序列或其具有1-2个氨基酸修饰的变体；(4) Costimulatory signal domain (CSD), which is selected from the 4-1BB costimulatory domain or a variant thereof with 1-5 amino acid modifications, for example, the sequence shown in SEQ ID NO: 11 or a variant thereof with 1 -2 amino acid modified variants;
(5)刺激信号结构域(SSD)，为CD3ζ信号传导结构域或其具有1-10个氨基酸修饰的变体，例如，SEQ ID NO:12所示的序列或其具有1-10个、1-5个氨基酸修饰的变体。(5) Stimulating signal domain (SSD), which is a CD3ζ signaling domain or a variant thereof with 1-10 amino acid modifications, for example, the sequence shown in SEQ ID NO: 12 or a sequence with 1-10, 1 -5 amino acid modified variants.
任选地，所述分子开关调控型CLDN18.2 CAR多肽还包含位于N端的信号肽序列，例如，SEQ ID NO:1所示的信号肽序列。Optionally, the molecular switch-regulated CLDN18.2 CAR polypeptide also includes a signal peptide sequence located at the N-terminus, for example, the signal peptide sequence shown in SEQ ID NO:1.
在一个具体实施方案中，分子开关调控型CLDN18.2 CAR多肽包含例如本文所述的HuR968B CAR(SEQ ID NO:15)In a specific embodiment, a molecular switch-regulated CLDN18.2 CAR polypeptide comprises, for example, a HuR968B CAR (SEQ ID NO: 15) described herein
在一个实施方案中，所述作为“分子开关”的靶向癌相关抗原的P329G突变抗体包含重链可变区和轻链可变区，其中：所述重链可变区包含根据Kabat编号的氨基酸序列SYVMS(SEQ ID NO:124)所示的CDR H1、或所述CDR H1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；氨基酸序列TISHSGGSTYYADSVKG(SEQ ID NO:125)所示的CDR H2、或所述CDR H2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和氨基酸序列DAPYYDILTGYRY(SEQ ID NO:126)所示的CDR H3、或所述CDR H3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；所述轻链可变区包含根据Kabat编号的氨基酸序列RASQSISSWLA(SEQ ID NO:127)所示的CDR L1、或所述CDR L1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；氨基酸序列KASSLES(SEQ ID NO:128)所示的CDR L2、或所述CDR L2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和氨基酸序列QQYNSYSYT(SEQ ID NO:129)所示的CDR L3、或所述CDR L3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；In one embodiment, the P329G mutant antibody targeting cancer-associated antigen as a "molecular switch" includes a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region includes a heavy chain variable region according to Kabat numbering The CDR H1 shown in the amino acid sequence SYVMS (SEQ ID NO: 124), or a variant of the CDR H1 with no more than 2 amino acid changes or no more than 1 amino acid change; the amino acid sequence TISHSGGSTYYADSVKG (SEQ ID NO: 125) The CDR H2 shown, or a variant of the CDR H2 with no more than 2 amino acid changes or no more than 1 amino acid change; and the CDR H3 shown with the amino acid sequence DAPYYDILTGYRY (SEQ ID NO: 126), or the CDR H3 A variant with no more than 2 amino acid changes or no more than 1 amino acid change; the light chain variable region includes the CDR L1 shown according to the amino acid sequence RASQSISSWLA (SEQ ID NO: 127) numbered by Kabat, or the CDR Variants with no more than 2 amino acid changes or no more than 1 amino acid change in L1; CDR L2 shown in the amino acid sequence KASSLES (SEQ ID NO: 128), or no more than 2 amino acid changes in the CDR L2 or no more than 1 amino acid change in L1; Variants with 1 amino acid change; and CDR L3 shown in the amino acid sequence QQYNSYSYT (SEQ ID NO: 129), or variants of the CDR L3 with no more than 2 amino acid changes or no more than 1 amino acid change;
其中所述氨基酸变化是氨基酸的添加、缺失或取代；wherein said amino acid change is the addition, deletion or substitution of amino acids;
例如，所述重链可变区包含SEQ ID NO:130的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:131的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列。For example, the heavy chain variable region comprises or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 130 Sequence identity, and the light chain variable region contains or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical sequence.
在一些实施方案中，表达分子开关调控型CAR多肽(例如，SEQ ID NO:15所示的HuR968B CAR)的CAR-T细胞与P329G突变的抗CLDN18.2抗体(例如，P329G突变的HB37A6PG Ab，本文中又称A6抗体，)(参见中国申请号202111416497.1)的组合显示出持续杀伤肿瘤细胞的能力，并且维持了CAR分子的特异性，表达分子开关调控型CAR多肽的T细胞在与肿瘤细胞的共培养实验中，只有在P329G突变抗体存在情况下所述表达分子开关调控型CAR多肽的T细胞才能被激活、增殖、分泌效应细胞因子并对表达或过表达CLDN18.2的肿瘤细胞产生杀伤效应。In some embodiments, CAR-T cells expressing a molecular switch-regulated CAR polypeptide (e.g., the HuR968B CAR set forth in SEQ ID NO: 15) are combined with a P329G mutated anti-CLDN18.2 antibody (e.g., a P329G mutated HB37A6PG Ab, The combination of A6 antibody, also called A6 antibody in this article (see Chinese Application No. 202111416497.1), shows the ability to continuously kill tumor cells and maintains the specificity of the CAR molecule. T cells expressing molecular switch-regulated CAR polypeptides interact with tumor cells. In co-culture experiments, only in the presence of the P329G mutant antibody can the T cells expressing the molecular switch-regulated CAR polypeptide be activated, proliferate, secrete effector cytokines, and produce a killing effect on tumor cells expressing or overexpressing CLDN18.2 .
为了在免疫效应细胞上共表达本发明的组成型嵌合细胞因子受体和嵌合抗原受体(CAR)多肽，可以将所述组成型嵌合细胞因子受体构建在一个构建体上，将所述CAR多肽构建在另 一个构建体上，将这两个构建体共同导入免疫效应细胞而表达。In order to co-express the constitutive chimeric cytokine receptor and the chimeric antigen receptor (CAR) polypeptide of the present invention on immune effector cells, the constitutive chimeric cytokine receptor can be constructed on one construct, The CAR polypeptide is constructed in another On one construct, the two constructs are co-introduced into immune effector cells for expression.
备选地，在一个核酸构建体上构建编码组成型嵌合细胞因子受体修饰的CAR多肽的核酸，所述组成型嵌合细胞因子受体修饰的CAR多肽包含位于CAR多肽的N端或者C端的本发明组成型嵌合细胞因子受体，且所述组成型嵌合细胞因子受体与CAR多肽之间具有自切割肽，使得核酸构建体产生包含通过自切割肽连接的本发明的组成型嵌合细胞因子受体和CAR多肽，不需要任何外部切割活性，将核酸构建体产生的多肽切割成单独的组成型嵌合细胞因子受体和单独的CAR多肽。Alternatively, a nucleic acid encoding a constitutively chimeric cytokine receptor-modified CAR polypeptide is constructed on a nucleic acid construct, the constitutively chimeric cytokine receptor-modified CAR polypeptide comprising a protein located at the N-terminus or C of the CAR polypeptide. end of the constitutive chimeric cytokine receptor of the present invention, and there is a self-cleaving peptide between the constitutive chimeric cytokine receptor and the CAR polypeptide, so that the nucleic acid construct generates a constitutive chimeric cytokine receptor of the present invention connected by the self-cleaving peptide. Chimeric cytokine receptors and CAR polypeptides do not require any external cleavage activity to cleave the polypeptide produced by the nucleic acid construct into separate constitutive chimeric cytokine receptors and separate CAR polypeptides.
所述“自切割肽”是指这样的肽，该肽发挥功能使得当产生从N端至C端包含第一多肽、自切割肽和第二多肽的融合多肽时，该融合多肽被切割成独特的且离散的第一多肽和第二多肽，而不需要任何外部切割活性。自切割肽可以是来自口蹄病毒或心脏病毒的2A自切割肽。The "self-cleaving peptide" refers to a peptide that functions such that when a fusion polypeptide is produced that includes a first polypeptide, a self-cleaving peptide, and a second polypeptide from the N-terminus to the C-terminus, the fusion polypeptide is cleaved. into unique and discrete first and second polypeptides without the need for any external cleavage activity. The self-cleaving peptide may be a 2A self-cleaving peptide from foot-and-mouth virus or cardiovirus.
在一些实施方案中，所述自切割肽为SEQ ID NO:3所示的P2A或其具有1-5个氨基酸修饰的变体。In some embodiments, the self-cleaving peptide is P2A shown in SEQ ID NO: 3 or a variant thereof with 1-5 amino acid modifications.
在一些实施方案中，本发明的组成型嵌合细胞因子受体修饰的CAR多肽从N端至C端In some embodiments, the constitutively chimeric cytokine receptor-modified CAR polypeptides of the invention extend from the N-terminus to the C-terminus
含CAR多肽、自切割肽和组成型嵌合细胞因子受体。Contains CAR peptides, self-cleaving peptides and constitutive chimeric cytokine receptors.
IV.编码本发明的组成型嵌合细胞因子受体或者编码本发明的组成型嵌合细胞因子受体修饰的CAR多肽的核酸分子、载体和表达细胞IV. Nucleic acid molecules, vectors and expression cells encoding the constitutive chimeric cytokine receptor of the present invention or encoding the constitutively chimeric cytokine receptor-modified CAR polypeptide of the present invention
本发明提供了编码本发明的组成型嵌合细胞因子受体或者编码本发明的组成型嵌合细胞因子受体修饰的CAR多肽的核酸分子。在一个实施方案中，核酸分子作为DNA构建体提供。The present invention provides nucleic acid molecules encoding the constitutive chimeric cytokine receptor of the present invention or encoding the CAR polypeptide modified by the constitutive chimeric cytokine receptor of the present invention. In one embodiment, the nucleic acid molecules are provided as DNA constructs.
IV.1.编码本发明的组成型嵌合细胞因子受体的DNA构建体IV.1. DNA construct encoding the constitutive chimeric cytokine receptor of the invention
在一些实施方案中，编码本发明的组成型嵌合细胞因子受体的DNA构建体从N端至C端包含编码信号肽的多核苷酸、编码由具有重塑肿瘤微环境的效应分子组成的胞外结构域的多核苷酸、编码IL-7R突变跨膜结构域和IL-7R胞内结构域的多核苷酸。任选地，在编码所述胞外结构域的多核苷酸与编码IL-7R突变跨膜结构域和IL-7R胞内结构域的多核苷酸之间存在编码铰链区的多核苷酸，所述铰链区例如SEQ ID NO:6所示的Flag Tag或其功能变体。In some embodiments, the DNA construct encoding the constitutive chimeric cytokine receptor of the invention includes from the N-terminus to the C-terminus a polynucleotide encoding a signal peptide, encoding a polynucleotide composed of an effector molecule with the ability to remodel the tumor microenvironment. Polynucleotides for the extracellular domain, polynucleotides encoding the IL-7R mutant transmembrane domain and the IL-7R intracellular domain. Optionally, there is a polynucleotide encoding a hinge region between the polynucleotide encoding the extracellular domain and the polynucleotide encoding the IL-7R mutant transmembrane domain and the IL-7R intracellular domain, so The hinge region is, for example, the Flag Tag shown in SEQ ID NO: 6 or a functional variant thereof.
在一些实施方案中，信号肽包含SEQ ID NO:2的序列或其功能变体。In some embodiments, the signal peptide comprises the sequence of SEQ ID NO: 2 or a functional variant thereof.
在一些实施方案中，编码IL-7R突变跨膜结构域和IL-7R胞内结构域的多核苷酸包含编码任一选自SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:28、SEQ ID NO:30至SEQ ID NO:45所示的氨基酸序列的多核苷酸，优选地，包含编码任一选自SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:34和SEQ ID NO:44所示的氨基酸序列的多核苷酸，最优选地，包含编码SEQ ID NO:34所示的氨基酸序列的多核苷酸。In some embodiments, the polynucleotide encoding the IL-7R mutant transmembrane domain and the IL-7R intracellular domain comprises encoding any one selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 28 , SEQ ID NO:30 to the polynucleotide of the amino acid sequence shown in SEQ ID NO:45, preferably, include coding for any one selected from the group consisting of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ The polynucleotide of the amino acid sequence shown in ID NO:34 and SEQ ID NO:44, most preferably, includes a polynucleotide encoding the amino acid sequence shown in SEQ ID NO:34.
在一些实施方案中，由具有重塑肿瘤微环境的效应分子组成的胞外结构域是细胞因子，所述细胞因子可以是例如IL-12(IL-12-P40或IL-12-P70)、IL15(IL-15或IL-15FP，所述IL-15FP是指IL-15和IL-15Rα(选自IL-15Rα或IL-15Rα(Sushi))的融合蛋白，包括IL-15/IL-15Rα和IL-15Rα/IL-15两种形式的融合蛋白)、IL-21、IL-18、IL-9、IL-23、IL-36γ、IFNα2b等细胞因子，免疫细胞(例如T细胞)表达所述包含细胞因子的M7CR基因，具有增强的免疫效应功能及抗肿瘤效应。在一些实施方案中，所述细胞因子是SEQ ID NO：47所示的IL-15或其功能变体；SEQ ID NO：48所示的IL-15FP(IL15/IL15Rα(Sushi)融合蛋白)或其功能变体；SEQ ID NO：140所示的IL-15FP(IL15/IL15Rα融合蛋白)或其功能变体；SEQ ID NO：141所示的IL-15FP(IL15Rα(Sushi)/IL15融合蛋白)或其功能变体；SEQ  ID NO：49所示的IL-12-P70或其功能变体；SEQ ID NO：50所示的IL-12-p40或其功能变体；SEQ ID NO：51所示的IL-21或其功能变体；SEQ ID NO：52所示的IL-9或其功能变体；SEQ ID NO：53所示的IL-18或其功能变体；SEQ ID NO：54所示的IL-23或其功能变体；SEQ ID NO：55所示的IL-36γ或其功能变体；SEQ ID NO：56所示的IFNα2b或其功能变体。In some embodiments, the extracellular domain consisting of effector molecules that remodel the tumor microenvironment is a cytokine, which may be, for example, IL-12 (IL-12-P40 or IL-12-P70), IL15 (IL-15 or IL-15FP, the IL-15FP refers to the fusion protein of IL-15 and IL-15Rα (selected from IL-15Rα or IL-15Rα (Sushi)), including IL-15/IL-15Rα and IL-15Rα/IL-15 two forms of fusion proteins), IL-21, IL-18, IL-9, IL-23, IL-36γ, IFNα2b and other cytokines, which are expressed by immune cells (such as T cells) The M7CR gene containing cytokines has enhanced immune effector function and anti-tumor effect. In some embodiments, the cytokine is IL-15 set forth in SEQ ID NO: 47 or a functional variant thereof; IL-15FP (IL15/IL15Rα (Sushi) fusion protein) set forth in SEQ ID NO: 48; or Its functional variant; IL-15FP (IL15/IL15Rα fusion protein) shown in SEQ ID NO: 140 or its functional variant; IL-15FP (IL15Rα (Sushi)/IL15 fusion protein) shown in SEQ ID NO: 141 or functional variants thereof; SEQ IL-12-P70 or its functional variant shown in ID NO: 49; IL-12-p40 or its functional variant shown in SEQ ID NO: 50; IL-21 or its functional variant shown in SEQ ID NO: 51 Functional variant; IL-9 shown in SEQ ID NO: 52 or a functional variant thereof; IL-18 shown in SEQ ID NO: 53 or a functional variant thereof; IL-23 shown in SEQ ID NO: 54 or Its functional variant; IL-36γ shown in SEQ ID NO: 55 or its functional variant; IFNα2b shown in SEQ ID NO: 56 or its functional variant.
在一些实施方案中，由具有重塑肿瘤微环境的效应分子组成的胞外结构域是免疫效应分子，所述免疫效应分子可以是例如4-1BB靶向分子部分(例如，4-1BB配体(4-1BBL)、抗4-1BB抗体(α4-1BB))、CD40靶向分子部分(例如，CD40配体(CD40L)、抗CD40抗体(αCD40))、CD83靶向分子部分(例如，抗CD83抗体(αCD83))、FLT3配体(FTL3L)、GITR、ICOS、CD2、ICAM1等，这些免疫效应分子通过与体内专职的抗原呈递细胞(APC)例如树突状细胞(DC)表面的相关受体或配体相互作用，活化APC，从而激发内源性抗肿瘤免疫应答，进而与免疫细胞(例如T细胞)产生协同抗肿瘤功效。在一些实施方案中，所述免疫效应分子是SEQ ID NO：57所示的4-1BBL或其功能变体；SEQ ID NO：58所示的CD40L或其功能变体；SEQ ID NO：59所示的FLT3L或其功能变体；SEQ ID NO：60所示的ICOS或其功能变体；SEQ ID NO：61所示的GITR或其功能变体；SEQ ID NO：62所示的ICAM-1或其功能变体；SEQ ID NO：63所示的CD2或其功能变体；SEQ ID NO：64所示的抗4-1BB或其功能变体；SEQ ID NO：65所示的抗CD40或其功能变体；SEQ ID NO：66所示的抗CD83或其功能变体。In some embodiments, the extracellular domain consisting of effector molecules that remodel the tumor microenvironment is an immune effector molecule, which may be, for example, a 4-1BB targeting molecule moiety (e.g., a 4-1BB ligand (4-1BBL), anti-4-1BB antibody (α4-1BB)), CD40-targeting molecule moiety (e.g., CD40 ligand (CD40L), anti-CD40 antibody (αCD40)), CD83-targeting molecule moiety (e.g., anti- CD83 antibody (αCD83)), FLT3 ligand (FTL3L), GITR, ICOS, CD2, ICAM1, etc., these immune effector molecules are recognized by receptors on the surface of professional antigen-presenting cells (APC) such as dendritic cells (DC) in the body. The APC interacts with the body or ligand to activate the APC, thus stimulating the endogenous anti-tumor immune response, thereby producing a synergistic anti-tumor effect with immune cells (such as T cells). In some embodiments, the immune effector molecule is 4-1BBL or a functional variant thereof shown in SEQ ID NO: 57; CD40L or a functional variant thereof shown in SEQ ID NO: 58; SEQ ID NO: 59 FLT3L shown in SEQ ID NO: 60 or its functional variant; ICOS shown in SEQ ID NO: 60 or its functional variant; GITR shown in SEQ ID NO: 61 or its functional variant; ICAM-1 shown in SEQ ID NO: 62 Or its functional variant; CD2 or its functional variant shown in SEQ ID NO: 63; Anti-4-1BB or its functional variant shown in SEQ ID NO: 64; Anti-CD40 or its functional variant shown in SEQ ID NO: 65 Its functional variant; anti-CD83 shown in SEQ ID NO: 66 or its functional variant.
在一些实施方案中，由具有重塑肿瘤微环境的效应分子组成的胞外结构域是抑制性分子拮抗剂，所述抑制性分子拮抗剂可以是例如抗PD-L1分子、抗CD47分子、抗IL-4分子、TGFβ结合分子(例如，抗TGFβ1分子、TGFβRII)、抗PD-1分子、抗CTLA-4分子、抗LAG-3分子、抗TIGIT分子、抗CD73分子等针对抑制性免疫受体或因子的抗体部分，通过拮抗抑制性免疫受体或因子的免疫抑制效应，达到增强抗肿瘤免疫应答目的，进而与免疫细胞(例如T细胞)产生协同抗肿瘤功效。在一些实施方案中，所述抑制性分子拮抗剂是SEQ ID NO：67所示的抗TGFβ分子或其功能变体；SEQ ID NO：198所示的TGFβRII ECD或其功能变体；SEQ ID NO：199所示的TGFβRII或其功能变体；SEQ ID NO：68所示的抗PD-L1VHH或其功能变体；SEQ ID NO：69所示的抗CD47分子或其功能变体；SEQ ID NO：70所示的抗IL-4分子或其功能变体；SEQ ID NO：71所示的抗PD-1分子或其功能变体；SEQ ID NO：72所示的抗CTLA-4分子或其功能变体；SEQ ID NO：73所示的抗LAG-3分子或其功能变体；SEQ ID NO：74所示的抗TIGIT分子或其功能变体；SEQ ID NO：75所示的抗CD73分子或其功能变体。In some embodiments, the extracellular domain consisting of effector molecules that remodel the tumor microenvironment is an inhibitory molecule antagonist, which may be, for example, an anti-PD-L1 molecule, an anti-CD47 molecule, an anti- IL-4 molecules, TGFβ binding molecules (e.g., anti-TGFβ1 molecules, TGFβRII), anti-PD-1 molecules, anti-CTLA-4 molecules, anti-LAG-3 molecules, anti-TIGIT molecules, anti-CD73 molecules, etc. target inhibitory immune receptors Or the antibody part of the factor, by antagonizing the immunosuppressive effect of inhibitory immune receptors or factors, achieves the purpose of enhancing the anti-tumor immune response, and then produces a synergistic anti-tumor effect with immune cells (such as T cells). In some embodiments, the inhibitory molecule antagonist is an anti-TGFβ molecule set forth in SEQ ID NO: 67 or a functional variant thereof; a TGFβRII ECD set forth in SEQ ID NO: 198 or a functional variant thereof; SEQ ID NO : TGFβRII represented by 199 or a functional variant thereof; anti-PD-L1 VHH represented by SEQ ID NO: 68 or a functional variant thereof; anti-CD47 molecule represented by SEQ ID NO: 69 or a functional variant thereof; SEQ ID The anti-IL-4 molecule shown in NO: 70 or its functional variant; the anti-PD-1 molecule shown in SEQ ID NO: 71 or its functional variant; the anti-CTLA-4 molecule shown in SEQ ID NO: 72 or Its functional variant; the anti-LAG-3 molecule shown in SEQ ID NO: 73 or its functional variant; the anti-TIGIT molecule shown in SEQ ID NO: 74 or its functional variant; the anti-LAG-3 molecule shown in SEQ ID NO: 75 CD73 molecules or functional variants thereof.
在一些实施方案中，由具有重塑肿瘤微环境的效应分子组成的胞外结构域是靶向NK细胞激活性受体的效应分子，所述靶向NK细胞激活性受体的效应分子可以是靶向NKG2C、NKG2D、NKp30、NKp44、NKp46等NK细胞表面表达的激活性受体的分子部分，例如，抗NKG2C、抗NKG2D、抗NKp30、抗NKp44、抗NKp46等抗体部分，通过激活内源性NK细胞，取得增强抗肿瘤免疫效应的目的，进而与免疫细胞(例如T细胞)产生协同抗肿瘤功效。在一些实施方案中，所述NK细胞激活分子是SEQ ID NO：76所示的抗NKG2D或其功能变体；SEQ ID NO：77所示的抗NKG2C或其功能变体；SEQ ID NO：78所示的抗NKp30或其功能变体；SEQ ID NO：79所示的抗NKp46或其功能变体。In some embodiments, the extracellular domain consisting of an effector molecule that remodels the tumor microenvironment is an effector molecule targeting an NK cell activating receptor, and the effector molecule targeting an NK cell activating receptor may be Target the molecular parts of activating receptors expressed on the surface of NK cells such as NKG2C, NKG2D, NKp30, NKp44, NKp46, etc., for example, anti-NKG2C, anti-NKG2D, anti-NKp30, anti-NKp44, anti-NKp46 and other antibody parts, by activating endogenous NK cells achieve the purpose of enhancing anti-tumor immune effects and then produce synergistic anti-tumor effects with immune cells (such as T cells). In some embodiments, the NK cell activating molecule is anti-NKG2D or a functional variant thereof set forth in SEQ ID NO: 76; anti-NKG2C or a functional variant thereof set forth in SEQ ID NO: 77; SEQ ID NO: 78 The anti-NKp30 or functional variant thereof as shown; the anti-NKp46 or functional variant thereof as shown in SEQ ID NO:79.
IV.2.编码本发明的组成型嵌合细胞因子受体修饰的CAR多肽的DNA构建体 IV.2. DNA construct encoding the constitutively chimeric cytokine receptor-modified CAR polypeptide of the invention
在一些实施方案中，编码本发明的组成型嵌合细胞因子受体修饰的CAR多肽的DNA构建体从N端至C端包含编码组成型嵌合细胞因子受体的多核苷酸、编码自切割肽的多核苷酸和编码CAR多肽的的多核苷酸。In some embodiments, a DNA construct encoding a constitutively chimeric cytokine receptor-modified CAR polypeptide of the invention includes from the N-terminus to the C-terminus a polynucleotide encoding a constitutively chimeric cytokine receptor, encoding a self-cleaving peptide polynucleotides and polynucleotides encoding CAR polypeptides.
在一些实施方案中，编码本发明的组成型嵌合细胞因子受体修饰的CAR多肽的DNA构建体从N端至C端包含编码CAR多肽的的多核苷酸、编码自切割肽的多核苷酸和编码组成型嵌合细胞因子受体的多核苷酸。In some embodiments, a DNA construct encoding a constitutively chimeric cytokine receptor modified CAR polypeptide of the invention includes from N-terminus to C-terminus a polynucleotide encoding a CAR polypeptide, a polynucleotide encoding a self-cleaving peptide and polynucleotides encoding constitutively chimeric cytokine receptors.
所述编码组成型嵌合细胞因子受体的多核苷酸如上文所述。The polynucleotide encoding a constitutively chimeric cytokine receptor is as described above.
所述编码自切割肽的多核苷酸是例如编码SEQ ID NO:3所示的P2A或其具有1-5个氨基酸修饰的变体的多核苷酸。The polynucleotide encoding a self-cleaving peptide is, for example, a polynucleotide encoding P2A shown in SEQ ID NO: 3 or a variant thereof with 1-5 amino acid modifications.
所述编码CAR多肽的的多核苷酸可以是编码现有技术中已知的任一CAR多肽的多核苷酸。The polynucleotide encoding a CAR polypeptide may be a polynucleotide encoding any CAR polypeptide known in the art.
在一些实施方案中，所述CAR多肽是传统CAR多肽，其直接靶向一种或多种上文所述的癌相关抗原。在一些实施方案中，所述CAR多肽是直接靶向CLDN18.2的传统CAR多肽，其从N端至C端包含信号肽、癌相关抗原结合结构域、跨膜结构域、共刺激信号传导结构域和主信号传导结构域。In some embodiments, the CAR polypeptide is a traditional CAR polypeptide that directly targets one or more of the cancer-associated antigens described above. In some embodiments, the CAR polypeptide is a traditional CAR polypeptide that directly targets CLDN18.2, which includes a signal peptide, a cancer-associated antigen binding domain, a transmembrane domain, and a costimulatory signaling structure from the N-terminus to the C-terminus. domain and the main signaling domain.
在一个具体实施方案中，所述传统CAR多肽从N端至C端包含：SEQ ID NO:1所示的CD8信号肽或其具有1-5个氨基酸修饰的变体；SEQ ID NO:13所示的VL-(G4S)n肽接头-SEQ ID NO:14所示的VH，其中“n”是1至10的整数，例如2至4的整数，例如SEQ ID NO:4、SEQ ID NO:5所示的序列；SEQ ID NO:7所示的CD8铰链区或其具有1-5个氨基酸修饰的变体；SEQ ID NO:8所示的跨膜结构域或其具有1-5个氨基酸修饰的变体；SEQ ID NO:11所示的共刺激信号传导结构域或其具有1-5个氨基酸修饰的变体；SEQ ID NO:12所示的主信号传导结构域或其具有1-5个氨基酸修饰的变体。在具体实施方案中，所述传统CAR多肽是具有SEQ ID NO:16所示氨基酸序列的H9.1.2 CAR。In a specific embodiment, the traditional CAR polypeptide includes from N-terminus to C-terminus: the CD8 signal peptide shown in SEQ ID NO:1 or a variant thereof with 1-5 amino acid modifications; the CD8 signal peptide shown in SEQ ID NO:13 VL-(G 4 S) n peptide linker shown - VH shown in SEQ ID NO: 14, wherein "n" is an integer from 1 to 10, such as an integer from 2 to 4, such as SEQ ID NO: 4, SEQ ID The sequence shown in NO:5; the CD8 hinge region shown in SEQ ID NO:7 or its variant with 1-5 amino acid modifications; the transmembrane domain shown in SEQ ID NO:8 or its variant with 1-5 Variants with amino acid modifications; costimulatory signaling domain shown in SEQ ID NO:11 or variants thereof with 1-5 amino acid modifications; main signaling domain shown in SEQ ID NO:12 or variants thereof with Variants with 1-5 amino acid modifications. In specific embodiments, the conventional CAR polypeptide is a H9.1.2 CAR having the amino acid sequence set forth in SEQ ID NO:16.
在一个具体实施方案中，所述传统CAR多肽从N端至C端包含：SEQ ID NO:1所示的CD8信号肽或其具有1-5个氨基酸修饰的变体；SEQ ID NO:98所示的VL-(G4S)n肽接头-SEQ ID NO:99所示的VH，其中“n”是1至10的整数，例如2至4的整数，例如SEQ ID NO:4、SEQ ID NO:5所示的序列；SEQ ID NO:7所示的CD8铰链区或其具有1-5个氨基酸修饰的变体；SEQ ID NO:8所示的跨膜结构域或其具有1-5个氨基酸修饰的变体；SEQ ID NO:11所示的共刺激信号传导结构域或其具有1-5个氨基酸修饰的变体；SEQ ID NO:12所示的主信号传导结构域或其具有1-5个氨基酸修饰的变体。在具体实施方案中，所述传统CAR多肽是具有SEQ ID NO:100所示氨基酸序列的H9.2.1 CAR。In a specific embodiment, the traditional CAR polypeptide includes from N-terminus to C-terminus: the CD8 signal peptide shown in SEQ ID NO: 1 or a variant thereof with 1-5 amino acid modifications; the CD8 signal peptide shown in SEQ ID NO: 98 VL-(G 4 S) n peptide linker shown - VH shown in SEQ ID NO: 99, wherein "n" is an integer from 1 to 10, such as an integer from 2 to 4, such as SEQ ID NO: 4, SEQ ID The sequence shown in NO:5; the CD8 hinge region shown in SEQ ID NO:7 or its variant with 1-5 amino acid modifications; the transmembrane domain shown in SEQ ID NO:8 or its variant with 1-5 Variants with amino acid modifications; costimulatory signaling domain shown in SEQ ID NO:11 or variants thereof with 1-5 amino acid modifications; main signaling domain shown in SEQ ID NO:12 or variants thereof with Variants with 1-5 amino acid modifications. In a specific embodiment, the conventional CAR polypeptide is a H9.2.1 CAR having the amino acid sequence set forth in SEQ ID NO: 100.
在一个具体实施方案中，所述传统CAR多肽从N端至C端包含：SEQ ID NO:1所示的CD8信号肽或其具有1-5个氨基酸修饰的变体；SEQ ID NO:98所示的VL-接头-SEQ ID NO:99所示的VH，例如，所述接头为SEQ ID NO:145所示的序列；SEQ ID NO:147所示的CD8铰链区或其具有1-5个氨基酸修饰的变体；SEQ ID NO:148所示的跨膜结构域或其具有1-5个氨基酸修饰的变体；SEQ ID NO:11所示的共刺激信号传导结构域或其具有1-5个氨基酸修饰的变体；SEQ ID NO:12所示的主信号传导结构域或其具有1-5个氨基酸修饰的变体。在具体实施方案中，所述传统CAR多肽是具有SEQ ID NO:144所示氨基酸序列的H9.2.1-218 CAR。In a specific embodiment, the traditional CAR polypeptide includes from N-terminus to C-terminus: the CD8 signal peptide shown in SEQ ID NO:1 or a variant thereof with 1-5 amino acid modifications; the CD8 signal peptide shown in SEQ ID NO:98 The VL-linker shown in - the VH shown in SEQ ID NO: 99, for example, the linker is the sequence shown in SEQ ID NO: 145; the CD8 hinge region shown in SEQ ID NO: 147 or it has 1-5 Amino acid modified variants; the transmembrane domain shown in SEQ ID NO:148 or its variants having 1-5 amino acid modifications; the costimulatory signaling domain shown in SEQ ID NO:11 or its variants having 1- Variants with 5 amino acid modifications; the main signaling domain shown in SEQ ID NO: 12 or variants thereof with 1-5 amino acid modifications. In specific embodiments, the traditional CAR polypeptide is a H9.2.1-218 CAR having the amino acid sequence set forth in SEQ ID NO: 144.
在一个具体实施方案中，所述传统CAR多肽从N端至C端包含：SEQ ID NO:1所示的 CD8信号肽或其具有1-5个氨基酸修饰的变体；SEQ ID NO:98所示的VL-接头-SEQ ID NO:99所示的VH，例如，所述接头为SEQ ID NO:145所示的序列；SEQ ID NO:147所示的CD8铰链区或其具有1-5个氨基酸修饰的变体；SEQ ID NO:148所示的跨膜结构域或其具有1-5个氨基酸修饰的变体；SEQ ID NO:143所示的共刺激信号传导结构域或其具有1-5个氨基酸修饰的变体；SEQ ID NO:12所示的主信号传导结构域或其具有1-5个氨基酸修饰的变体。在具体实施方案中，所述传统CAR多肽是具有SEQ ID NO:142所示氨基酸序列的H9.2.1-28-L CAR。In a specific embodiment, the traditional CAR polypeptide includes from N-terminus to C-terminus: as shown in SEQ ID NO:1 CD8 signal peptide or a variant thereof with 1-5 amino acid modifications; VL-linker shown in SEQ ID NO:98-VH shown in SEQ ID NO:99, for example, the linker is shown in SEQ ID NO:145 The sequence shown; the CD8 hinge region shown in SEQ ID NO: 147 or its variant with 1-5 amino acid modifications; the transmembrane domain shown in SEQ ID NO: 148 or its variant with 1-5 amino acid modifications Variant; the costimulatory signaling domain shown in SEQ ID NO:143 or a variant thereof having 1-5 amino acid modifications; the main signaling domain shown in SEQ ID NO:12 or a variant thereof having 1-5 Amino acid modified variants. In a specific embodiment, the conventional CAR polypeptide is a H9.2.1-28-L CAR having the amino acid sequence set forth in SEQ ID NO:142.
在一些实施方案中，所述CAR多肽是分子开关调控型CAR多肽。在一些实施方案中，所述CAR多肽是通过与作为分子开关的P329G突变(所述P329G突变也简称为“PG”)抗癌相关抗原的抗体组合来靶向癌相关抗原，其从N端至C端包含信号肽、抗PG抗体scFv序列、跨膜结构域、共刺激信号传导结构域和主信号传导结构域。在具体实施方案中，所述分子开关调控型CAR多肽从N端至C端包含：SEQ ID NO:1所示的CD8信号肽或其具有1-5个氨基酸修饰的变体；SEQ ID NO:9所示的抗PG抗体VH-(G4S)n肽接头-SEQ ID NO:10所示的抗PG抗体VL，其中“n”是1至10的整数，例如2至4的整数，例如SEQ ID NO:4、SEQ ID NO:5所示的序列；GGGGS铰链；SEQ ID NO:8所示的跨膜结构域或其具有1-5个氨基酸修饰的变体；SEQ ID NO:11所示的共刺激信号传导结构域或其具有1-5个氨基酸修饰的变体；SEQ ID NO:12所示的主信号传导结构域或其具有1-5个氨基酸修饰的变体。在具体实施方案中，所述分子开关调控型CAR多肽是具有SEQ ID NO:15所示氨基酸序列的HuR968B CAR。In some embodiments, the CAR polypeptide is a molecular switch-regulated CAR polypeptide. In some embodiments, the CAR polypeptide targets the cancer-associated antigen by combining it with an antibody against the cancer-associated antigen of the P329G mutation (the P329G mutation is also referred to as "PG") as a molecular switch, from the N-terminus to The C-terminus contains signal peptide, anti-PG antibody scFv sequence, transmembrane domain, costimulatory signaling domain and main signaling domain. In a specific embodiment, the molecular switch-regulated CAR polypeptide includes from the N-terminus to the C-terminus: the CD8 signal peptide shown in SEQ ID NO: 1 or a variant thereof with 1-5 amino acid modifications; SEQ ID NO: Anti-PG antibody VH shown in 9-(G 4 S) n peptide linker-anti-PG antibody VL shown in SEQ ID NO: 10, wherein "n" is an integer from 1 to 10, such as an integer from 2 to 4, such as Sequences shown in SEQ ID NO:4 and SEQ ID NO:5; GGGGS hinge; transmembrane domain shown in SEQ ID NO:8 or variants thereof with 1-5 amino acid modifications; SEQ ID NO:11 The co-stimulatory signaling domain shown in SEQ ID NO: 12 or a variant thereof having 1-5 amino acid modifications; the main signaling domain shown in SEQ ID NO: 12 or a variant thereof having 1-5 amino acid modifications. In a specific embodiment, the molecular switch-regulated CAR polypeptide is a HuR968B CAR having the amino acid sequence shown in SEQ ID NO: 15.
在一些实施方案中，编码本发明的组成型嵌合细胞因子受体修饰的CAR多肽的DNA构建体包含编码任一SEQ ID NO:80-SEQ ID NO:95、SEQ ID NO:101、SEQ ID NO:104、SEQ ID NO:133、SEQ ID NO:136、SEQ ID NO:137、SEQ ID NO:138的氨基酸序列的多核苷酸和其功能变体。In some embodiments, a DNA construct encoding a constitutively chimeric cytokine receptor modified CAR polypeptide of the invention comprises encoding any of SEQ ID NO:80-SEQ ID NO:95, SEQ ID NO:101, SEQ ID Polynucleotides of the amino acid sequences of NO:104, SEQ ID NO:133, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138 and functional variants thereof.
本发明还提供了插入有本发明DNA构建体的载体。通过将编码本发明DNA构建体的核酸有效连接至启动子插入表达载体中，实现DNA构建体上的多核苷酸所编码多肽的表达。载体可以适合在真核生物中复制和整合。常见的克隆载体含有用于调节所需核酸序列的表达的转录和翻译终止子、起始序列和启动子。The invention also provides a vector into which the DNA construct of the invention is inserted. By effectively linking the nucleic acid encoding the DNA construct of the present invention to a promoter and inserting it into an expression vector, the expression of the polypeptide encoded by the polynucleotide on the DNA construct is achieved. The vector may be suitable for replication and integration in eukaryotic organisms. Common cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters for regulating expression of the desired nucleic acid sequence.
已经开发了众多基于病毒的系统用于转移基因至哺乳动物细胞中。例如，逆转录病毒提供了用于基因递送系统的便利平台。可以使用本领域已知的技术，将选择的基因插入载体并且包装在逆转录病毒粒子中。随后可以分离重组病毒并将其在体内或离体递送至受试者的细胞。众多逆转录病毒系统是本领域已知的。在一些实施方案中，使用慢病毒载体。Numerous virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide convenient platforms for gene delivery systems. The selected genes can be inserted into the vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject's cells in vivo or ex vivo. Numerous retroviral systems are known in the art. In some embodiments, lentiviral vectors are used.
衍生自逆转录病毒(如慢病毒)的载体是实现长期基因转移的合适工具，因为它们允许转基因的长期、稳定整合和其在子代细胞中增殖。慢病毒载体具有胜过衍生自癌-逆转录病毒(如鼠白血病病毒)的载体的额外优点，因为它们可以转导非增殖性细胞，如肝细胞。它们还具有额外的低免疫原性优点。逆转录病毒载体也可以例如是γ逆转录病毒载体。γ逆转录病毒载体可以例如包含启动子、包装信号(ψ)、引物结合位点(PBS)、一个或多个(例如，两个)长末端重复序列(LTR)和目的转基因，例如，编码本发明的组成型嵌合细胞因子受体的基因或者编码本发明的组成型嵌合细胞因子受体修饰的CAR多肽的基因。γ逆转录病毒载体可以缺少病毒结构性基因如gag、pol和env。 Vectors derived from retroviruses (e.g., lentiviruses) are suitable tools for long-term gene transfer because they allow long-term, stable integration of the transgene and its propagation in progeny cells. Lentiviral vectors have the additional advantage over vectors derived from onco-retroviruses (such as murine leukemia virus) in that they can transduce non-proliferating cells, such as hepatocytes. They also have the additional advantage of being low immunogenicity. Retroviral vectors may also be, for example, gamma retroviral vectors. A gamma retroviral vector may, for example, comprise a promoter, a packaging signal (ψ), a primer binding site (PBS), one or more (e.g., two) long terminal repeats (LTRs) and a transgene of interest, e.g., the codebook The gene of the constitutive chimeric cytokine receptor of the invention or the gene encoding the CAR polypeptide modified by the constitutive chimeric cytokine receptor of the invention. Gamma retroviral vectors can lack viral structural genes such as gag, pol and env.
能够在哺乳动物T细胞中表达本发明的转基因的启动子的例子是EF1a启动子。天然EF1a启动子驱动延伸因子-1复合体的α亚基表达，所述α亚基负责酶促递送氨酰基tRNA至核糖体。已经在哺乳动物表达质粒中广泛使用了EF1a启动子并且已经显示有效驱动了克隆至慢病毒载体中的转基因的表达。参见，例如，Milone等人,Mol.Ther.17(8):1453–1464(2009)。An example of a promoter capable of expressing the transgene of the invention in mammalian T cells is the EF1a promoter. The native EF1a promoter drives expression of the alpha subunit of the elongation factor-1 complex, which is responsible for enzymatic delivery of aminoacyl tRNA to ribosomes. The EF1a promoter has been widely used in mammalian expression plasmids and has been shown to efficiently drive expression of transgenes cloned into lentiviral vectors. See, eg, Milone et al., Mol. Ther. 17(8):1453–1464 (2009).
启动子的另一个例子是立即早期巨细胞病毒(CMV)启动子序列。这个启动子序列是能够驱动与之有效连接的任何多核苷酸序列高水平表达的组成型强启动子序列。但是，也可以使用其他组成型启动子序列，所述其他组成型启动子序列包括但不限于猴病毒40(SV40)早期启动子、小鼠乳腺瘤病毒(MMTV)、人类免疫缺陷病毒(HIV)长末端重复序列(LTR)启动子、MoMuLV启动子、鸟类白血病病毒启动子、埃巴病毒立即早期启动子、劳斯肉瘤病毒启动子以及人基因启动子，如但不限于肌动蛋白启动子、肌球蛋白启动子、延伸因子-1α启动子、血红蛋白启动子和肌酸激酶启动子。另外，本发明不应当限于使用组成型启动子。还构思了诱导型启动子作为本发明的部分。Another example of a promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a constitutively strong promoter sequence capable of driving high-level expression of any polynucleotide sequence to which it is operably linked. However, other constitutive promoter sequences may also be used, including, but not limited to, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) Long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to the actin promoter , myosin promoter, elongation factor-1α promoter, hemoglobin promoter and creatine kinase promoter. Additionally, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention.
在一些实施方案中，本发明提供了在哺乳动物免疫效应细胞(例如哺乳动物T细胞或哺乳动物NK细胞)中表达本发明的DNA构建体的方法和由此产生的免疫效应细胞。In some embodiments, the invention provides methods of expressing the DNA constructs of the invention in mammalian immune effector cells (eg, mammalian T cells or mammalian NK cells) and immune effector cells generated thereby.
从受试者获得细胞来源(例如，免疫效应细胞，例如，T细胞或NK细胞)。术语“受试者”意在包括可以激发免疫应答的活生物(例如，哺乳动物)。可以从众多来源获得T细胞，包括外周血单个核细胞、骨髓、淋巴结组织、脐带血、胸腺组织、来自感染部位的组织、腹水、胸腔积液、脾组织和肿瘤。A source of cells (eg, immune effector cells, eg, T cells or NK cells) is obtained from the subject. The term "subject" is intended to include living organisms (eg, mammals) that can elicit an immune response. T cells can be obtained from numerous sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors.
可以使用本领域技术人员已知的任何技术(如FicollTM分离法)，从采集自受试者的血液成分中获得T细胞。在一个优选的方面，通过单采血液成分术获得来自个体循环血液的细胞。单采产物一般含有淋巴细胞，包括T细胞、单核细胞、粒细胞、B细胞、其他有核的白细胞、红细胞和血小板。在一个实施方案中，可以洗涤通过单采血液成分术采集的细胞，以除去血浆级分并且以在用于后续加工步骤的适宜缓冲液或培养基中放置细胞。在本发明的一个方面，用磷酸盐缓冲盐水(PBS)洗涤细胞。T cells can be obtained from blood components collected from a subject using any technique known to those skilled in the art, such as Ficoll ™ isolation. In a preferred aspect, cells from the individual's circulating blood are obtained by apheresis. Apheresis products generally contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma fraction and to place the cells in a suitable buffer or culture medium for subsequent processing steps. In one aspect of the invention, cells are washed with phosphate buffered saline (PBS).
可以通过正向或负向选择技术进一步分离特定的T细胞亚群，如CD3+、CD28+、CD4+、CD8+、CD45RA+和CD45RO+T细胞。例如，在一个实施方案中，通过与抗CD3/抗CD28缀合的珠(如M-450 CD3/CD28T)温育一段足够正向选择所需T细胞的时间，分离T细胞。在一些实施方案中，该时间段是约30分钟至36小时之间或更长时间。较长的温育时间可以用来在存在少量T细胞的任何情况下分离T细胞，如用于从肿瘤组织或从免疫受损个体分离肿瘤浸润型淋巴细胞(TIL)。另外，使用较长的温育时间可以增加捕获CD8+T细胞的效率。因此，通过简单地缩短或延长该时间，允许T细胞与CD3/CD28珠结合和/或通过增加或减少珠对T细胞的比率，可以在培养伊始或在培养过程期间的其他时间点偏好地选择T细胞亚群。Specific T cell subsets, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated through positive or negative selection techniques. For example, in one embodiment, by conjugating beads with anti-CD3/anti-CD28 (e.g. M-450 CD3/CD28T) was incubated for a period of time sufficient to positively select the desired T cells, and the T cells were isolated. In some embodiments, the time period is between about 30 minutes and 36 hours or longer. Longer incubation times can be used to isolate T cells in any situation where small numbers of T cells are present, such as for isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or from immunocompromised individuals. Additionally, using longer incubation times can increase the efficiency of capturing CD8+ T cells. Thus, by simply shortening or extending this time, allowing T cells to bind to CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells, one can preferentially select at the beginning of the culture or at other time points during the culture process T cell subsets.
可以用抗体的组合，通过负选择过程完成T细胞群体的富集，其中所述抗体针对负向选择的细胞独有的表面标志物。一种方法是借助负向磁力免疫粘附法或流式细胞术分选和/或选择细胞，所述负向磁力免疫粘附法或流式细胞术使用针对负向选择的细胞上存在的细胞表面标志物的单克隆抗体混合物。Enrichment of T cell populations through a negative selection process can be accomplished using a combination of antibodies directed against surface markers unique to the negatively selected cells. One method is to sort and/or select cells by means of negative magnetic immunoadhesion or flow cytometry, which uses cells present on the negatively selected cells. Mixture of monoclonal antibodies to surface markers.
在一些实施方案中，免疫效应细胞可以是同种异体免疫效应细胞，例如，T细胞或NK细胞。例如，细胞可以是同种异体T细胞，例如，缺少功能性T细胞受体(TCR)和/或人白细胞抗原(HLA)(例如，HLA I类和/或HLA II类)表达的同种异体T细胞。 In some embodiments, the immune effector cells may be allogeneic immune effector cells, such as T cells or NK cells. For example, the cells may be allogeneic T cells, e.g., allogeneic that lack functional T cell receptor (TCR) and/or human leukocyte antigen (HLA) (e.g., HLA class I and/or HLA class II) expression. T cells.
缺少功能性TCR的T细胞可以例如经工程化，从而它在其表面上不表达任何功能性TCR；经工程化，从而它不表达构成功能性TCR的一个或多个亚基(例如，经工程化，从而它不表达或显示出减少表达的TCRα、TCRβ、TCRγ、TCRδ、TCRε和/或TCRζ)；或经工程化，从而它在其表面上产生非常少的功能性TCR。A T cell lacking a functional TCR can, for example, be engineered so that it does not express any functional TCR on its surface; engineered so that it does not express one or more subunits that constitute a functional TCR (e.g., engineered ized such that it does not express or displays reduced expression of TCRα, TCRβ, TCRγ, TCRδ, TCRε and/or TCRζ); or engineered such that it produces very few functional TCRs on its surface.
本文所述的T细胞例如可以如此工程化，从而它不在其表面上表达功能性HLA。例如，本文所述的T细胞可以如此工程化，从而HLA(例如，HLA I类和/或HLA II类)的细胞表面表达下调。在一些方面，可以通过减少或消除β-2微球蛋白(B2M)表达实现HLA的下调。A T cell described herein can, for example, be engineered such that it does not express functional HLA on its surface. For example, T cells described herein can be engineered such that cell surface expression of HLA (e.g., HLA class I and/or HLA class II) is downregulated. In some aspects, HLA downregulation can be achieved by reducing or eliminating beta-2 microglobulin (B2M) expression.
在一些实施方案中，T细胞可以缺少功能性TCR和功能性HLA，例如，HLA I类和/或HLA II类。In some embodiments, T cells may lack functional TCR and functional HLA, e.g., HLA class I and/or HLA class II.
对经体外增殖后获得的共表达CAR和本发明的组成型嵌合细胞因子受体的免疫效应细胞可以如实施例中所述进行效应功能的检测。The effector function of the immune effector cells co-expressing CAR and the constitutive chimeric cytokine receptor of the present invention obtained after in vitro proliferation can be tested as described in the Examples.
V.本发明的药物组合物V. Pharmaceutical composition of the present invention
在一些实施方案中，本发明提供了药物组合物，其包含选自表达本发明的组成型嵌合细胞因子受体的免疫效应细胞(例如，T细胞、NK细胞)、编码所述组成型嵌合细胞因子受体的核酸分子、包含编码所述组成型嵌合细胞因子受体的核酸分子的载体、和它们的任意组合；和任选地可药用辅料。In some embodiments, the invention provides pharmaceutical compositions comprising immune effector cells (e.g., T cells, NK cells) that express the constitutively chimeric cytokine receptors of the invention, cells encoding said constitutively chimeric cytokine receptors, Nucleic acid molecules encoding cytokine receptors, vectors comprising nucleic acid molecules encoding said constitutively chimeric cytokine receptors, and any combination thereof; and optionally pharmaceutically acceptable excipients.
在一些实施方案中，本发明提供了药物组合物，其包含选自表达本发明的组成型嵌合细胞因子受体修饰的CAR多肽的免疫效应细胞(例如，T细胞、NK细胞)、编码所述组成型嵌合细胞因子受体修饰的CAR多肽的核酸分子、包含编码所述组成型嵌合细胞因子受体修饰的CAR多肽的核酸分子的载体、和它们的任意组合；和任选地可药用辅料。进一步地，当所述CAR多肽是分子开关调控型CAR多肽时，所述的药物组合物还包含分子开关，例如抗体分子开关。In some embodiments, the invention provides a pharmaceutical composition comprising an immune effector cell (e.g., T cell, NK cell) selected from the group consisting of expressing a constitutively chimeric cytokine receptor modified CAR polypeptide of the invention, encoding The nucleic acid molecule of the constitutively chimeric cytokine receptor modified CAR polypeptide, a vector comprising the nucleic acid molecule encoding the constitutively chimeric cytokine receptor modified CAR polypeptide, and any combination thereof; and optionally can medical supplements. Further, when the CAR polypeptide is a molecular switch-regulated CAR polypeptide, the pharmaceutical composition further includes a molecular switch, such as an antibody molecular switch.
在一些实施方案中，所述免疫效应细胞是自自体T细胞或同种异体T细胞制备的，例如，所述免疫效应细胞是自人PBMC分离的T细胞制备的。In some embodiments, the immune effector cells are prepared from autologous T cells or allogeneic T cells, for example, the immune effector cells are prepared from T cells isolated from human PBMCs.
本发明的药物组合物可根据常规方法制剂化(例如Remington’s Pharmaceutical Science,最新版,Mark Publishing Company,Easton,U.S.A)。可药用辅料可以例示例如表面活性剂、赋形剂、着色料、香料、防腐剂、稳定剂、缓冲剂、悬浮剂、等张剂、粘合剂、崩解剂、润滑剂、流动促进剂、矫味剂等。进一步地，也可合适地使用其他常用的载剂，例如，轻质无水硅酸、乳糖、结晶纤维素、甘露醇、淀粉、羧甲基纤维素钙、羧甲基纤维素钠、羟丙基纤维素、羟丙基甲基纤维素、聚乙烯缩醛二乙氨基乙酸酯、聚乙烯吡咯啶酮、明胶、中链脂肪酸三甘油酯、聚氧乙烯硬化蓖麻油60、白砂糖、羧甲基纤维素、玉米淀粉、无机盐类等作为载剂，但不限于此。The pharmaceutical composition of the present invention can be formulated according to conventional methods (for example, Remington’s Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, U.S.A.). Pharmaceutically acceptable excipients may include, for example, surfactants, excipients, colorants, flavors, preservatives, stabilizers, buffers, suspending agents, isotonic agents, binders, disintegrants, lubricants, and flow promoters. , flavoring agents, etc. Further, other commonly used carriers may also be suitably used, such as light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carboxymethylcellulose calcium, carboxymethylcellulose sodium, hydroxypropyl Cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain fatty acid triglycerides, polyoxyethylene hardened castor oil 60, white sugar, carboxylic acid Methyl cellulose, corn starch, inorganic salts, etc. are used as carriers, but are not limited to these.
在一些实施方案中，本发明的药物组合物用于治疗癌症，例如表达或过表达CLDN 18.2的癌症。In some embodiments, pharmaceutical compositions of the invention are used to treat cancer, such as cancers that express or overexpress CLDN 18.2.
VI.本发明的药物组合物的用途和使用本发明的药物组合物的治疗方法VI. Uses of the pharmaceutical composition of the present invention and treatment methods using the pharmaceutical composition of the present invention
本发明提供了前述本发明的药物组合物，其用于在受试者中治疗肿瘤(例如癌症)。本发明还涉及治疗受试者的肿瘤(例如癌症)的方法，所述方法包括向所述受试者施用有效量的本发明的药物组合物。在一些实施方案中，所述肿瘤是癌症。在一些实施方案中，本文所述的肿瘤，例如癌症，包括但不限于实体瘤、血液学癌、软组织肿瘤和转移性病灶。 The present invention provides the aforementioned pharmaceutical composition of the present invention for use in treating tumors (eg, cancer) in a subject. The invention also relates to a method of treating a tumor (eg, cancer) in a subject, comprising administering to said subject an effective amount of a pharmaceutical composition of the invention. In some embodiments, the tumor is cancer. In some embodiments, tumors, such as cancers, described herein include, but are not limited to, solid tumors, hematologic cancers, soft tissue tumors, and metastatic lesions.
在一个实施方案中，本发明的药物组合物用于在受试者中治疗表达或过表达CLDN 18.2的癌症，并且能够减轻癌症的至少一种症状或指征的严重性或抑制癌细胞生长。本发明提供了在受试者中治疗癌症(例如，表达或过表达CLDN 18.2的癌症)的方法，其包括向有需要的个体施用治疗有效量的本发明的药物组合物。In one embodiment, the pharmaceutical composition of the invention is used to treat a cancer that expresses or overexpresses CLDN 18.2 in a subject and is capable of reducing the severity of at least one symptom or indication of cancer or inhibiting cancer cell growth. The invention provides methods of treating cancer (e.g., cancers that express or overexpress CLDN 18.2) in a subject, comprising administering to an individual in need thereof a therapeutically effective amount of a pharmaceutical composition of the invention.
本发明提供了前述本发明的药物组合物在制备用于治疗癌症(例如，表达或过表达CLDN18.2的癌症)的药物中的用途。The present invention provides the use of the aforementioned pharmaceutical composition of the present invention in the preparation of medicaments for treating cancer (eg, cancer expressing or overexpressing CLDN18.2).
本发明的药物组合物也可以施用于已经用一种或多种先前疗法治疗癌症但随后复发或转移的个体。Pharmaceutical compositions of the present invention may also be administered to individuals whose cancer has been treated with one or more prior therapies and has subsequently relapsed or metastasized.
本发明的药物组合物可以以合适的剂量施用于受试者。剂量方案将由主治医生和临床因素决定。如医学领域中公知的，用于任何一名患者的剂量取决于许多因素，包括患者的体重、身体表面积、年龄、待施用的特定化合物、性别、施用时间和途径、一般健康状况、和待并行施用的其他药物。The pharmaceutical composition of the present invention can be administered to a subject at an appropriate dose. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, the dosage for any given patient depends on many factors, including the patient's weight, body surface area, age, the specific compound to be administered, sex, time and route of administration, general health, and concurrent medications to be administered. Other medications administered.
在一些实施方案中，向患有癌症的个体施用本发明的药物组合物导致肿瘤的完全消失。在一些实施方案中，向患有癌症的个体施用本发明的药物组合导致肿瘤细胞或肿瘤大小减少至少85％或更多。可以通过本领域已知的任何方法测量肿瘤的减少，例如X-线、正电子发射断层扫描(PET)、计算机断层扫描(CT)、磁共振成像(MRI)、细胞学、组织学或分子遗传分析。In some embodiments, administration of a pharmaceutical composition of the present invention to an individual with cancer results in complete disappearance of the tumor. In some embodiments, administration of a pharmaceutical combination of the invention to an individual with cancer results in a reduction in tumor cells or tumor size of at least 85% or greater. Tumor reduction can be measured by any method known in the art, such as X-ray, positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), cytology, histology, or molecular genetics analyze.
本发明所述的各个实施方案/技术方案以及各个实施方案/技术方案中的特征应当被理解为可以任意进行相互组合，这些相互组合得到的各个方案均包括在本发明的范围内，就如同在本文中具体地且逐一地列出了这些相互组合而得到的方案一样，除非上下文清楚地显示并非如此。It should be understood that the various embodiments/technical solutions and the features of each embodiment/technical solution described in the present invention can be combined with each other arbitrarily, and the various solutions obtained by these mutual combinations are included in the scope of the present invention, just as in Each of these combinations is specifically and individually listed herein, unless the context clearly indicates otherwise.
描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成对本发明的保护范围的限制。The following examples are described to aid understanding of the invention. The examples are not intended and should not be construed in any way as limiting the scope of the invention.
实施例Example
实施例1、M7R的合成、筛选及功能研究Example 1. Synthesis, screening and functional study of M7R
实施例1.1.构建用于表达M7R基因的慢病毒载体Example 1.1. Construction of lentiviral vector for expressing M7R gene
如图2A所示，IL-7与其野生型受体IL-7受体α链(IL7Rα)结合后诱导后者与共同γ信号链异二聚化，激活下游JAK/STAT信号。但当野生型IL7Rα跨膜区发生突变而产生突变的IL7R，所述突变可能诱导所产生的突变IL7R自二聚化，从而能够在不依赖IL-7结合情况下，该自二聚化的突变IL7R组成型激活下游STAT5信号通路。As shown in Figure 2A, IL-7 binds to its wild-type receptor IL-7 receptor α chain (IL7Rα) and induces heterodimerization of the latter with the common γ signal chain, activating downstream JAK/STAT signaling. However, when the transmembrane region of wild-type IL7Rα is mutated to produce a mutated IL7R, the mutation may induce self-dimerization of the mutated IL7R, thereby enabling the self-dimerization of the mutated IL7R without relying on IL-7 binding. IL7R constitutively activates the downstream STAT5 signaling pathway.
如图2B所示，构建病毒表达质粒，用于表达含不同IL7R突变(IL7Rm或M7R)和tCD19组成的嵌合受体tCD19-M7CR(在本说明书中也称为IL7Rm-tCD19)，这些tCD19-M7CR由相同的截短CD19(tCD19，SEQ ID NO:17)构成的胞外结构域(ECD)和不同的IL7R突变体(文中也称IL7Rm或M7R)组成，所述IL7Rm(SEQ ID NO:20-SEQ ID NO:46)由携带不同突变的IL7R跨膜区(IL7R-mutant(TM))(参见序列的加粗部分)和野生型IL7R胞内段(IL7R-wt(ICD)，SEQ ID NO:19)组成。As shown in Figure 2B, a viral expression plasmid was constructed for expressing chimeric receptors tCD19-M7CR (also called IL7Rm-tCD19 in this specification) containing different IL7R mutations (IL7Rm or M7R) and tCD19. These tCD19- M7CR consists of the same extracellular domain (ECD) composed of truncated CD19 (tCD19, SEQ ID NO:17) and different IL7R mutants (also referred to as IL7Rm or M7R in the text), the IL7Rm (SEQ ID NO:20 -SEQ ID NO:46) consists of the IL7R transmembrane region (IL7R-mutant(TM)) carrying different mutations (see the bold part of the sequence) and the wild-type IL7R intracellular segment (IL7R-wt(ICD), SEQ ID NO :19) composition.
所述27个IL7Rm分别命名为IL7Rm1.1、IL7Rm1.2、IL7Rm1.3、IL7Rm1.4、IL7Rm2.1、IL7Rm2.2、IL7Rm2.3、IL7Rm2.4、IL7Rm3.1、IL7Rm3.2、IL7Rm4、IL7Rm5、IL7Rm6、IL7Rm7、IL7Rm8、IL7Rm9、IL7Rm10、IL7Rm11、IL7Rm12、IL7Rm13、IL7Rm14、IL7Rm15、IL7Rm16、 IL7Rm17、IL7Rm18、IL7Rm19、IL7Rm20(参见序列表中的SEQ ID NO:20-SEQ ID NO:46)，其相对应的tCD19-M7CR则分别命名为IL7Rm1.1-tCD19、IL7Rm1.2-tCD19、IL7Rm1.3-tCD19、IL7Rm1.4-tCD19、IL7Rm2.1-tCD19、IL7Rm2.2-tCD19、IL7Rm2.3-tCD19、IL7Rm2.4-tCD19、IL7Rm3.1-tCD19、IL7Rm3.2-tCD19、IL7Rm4-tCD19、IL7Rm5-tCD19、IL7Rm6-tCD19、IL7Rm7-tCD19、IL7Rm8-tCD19、IL7Rm9-tCD19、IL7Rm10-tCD19、IL7Rm11-tCD19、IL7Rm12-tCD19、IL7Rm13-tCD19、IL7Rm14-tCD19、IL7Rm15-tCD19、IL7Rm16-tCD19、IL7Rm17-tCD19、IL7Rm18-tCD19、IL7Rm19-tCD19、IL7Rm20-tCD19；作为对照，IL7R-tCD19构建体包含tCD19胞外结构域和野生型IL7R跨膜区和胞内段(SEQ ID NO:96)，IL7R-WT构建体包含完整的野生型IL7R链(SEQ ID NO:18)。The 27 IL7Rm are named IL7Rm1.1, IL7Rm1.2, IL7Rm1.3, IL7Rm1.4, IL7Rm2.1, IL7Rm2.2, IL7Rm2.3, IL7Rm2.4, IL7Rm3.1, IL7Rm3.2, IL7Rm4, respectively. IL7Rm5, IL7Rm6, IL7Rm7, IL7Rm8, IL7Rm9, IL7Rm10, IL7Rm11, IL7Rm12, IL7Rm13, IL7Rm14, IL7Rm15, IL7Rm16, IL7Rm17, IL7Rm18, IL7Rm19, IL7Rm20 (see SEQ ID NO:20-SEQ ID NO:46 in the sequence listing), and their corresponding tCD19-M7CRs are named IL7Rm1.1-tCD19, IL7Rm1.2-tCD19, and IL7Rm1 respectively. .3-tCD19, IL7Rm1.4-tCD19, IL7Rm2.1-tCD19, IL7Rm2.2-tCD19, IL7Rm2.3-tCD19, IL7Rm2.4-tCD19, IL7Rm3.1-tCD19, IL7Rm3.2-tCD19, IL7Rm4-tCD19 . tCD19, IL7Rm16-tCD19, IL7Rm17 -tCD19, IL7Rm18-tCD19, IL7Rm19-tCD19, IL7Rm20-tCD19; as a control, the IL7R-tCD19 construct contains the tCD19 extracellular domain and the wild-type IL7R transmembrane region and intracellular segment (SEQ ID NO: 96), IL7R- The WT construct contains the complete wild-type IL7R chain (SEQ ID NO: 18).
实施例1.2.构建稳定表达不同M7R的BaF3细胞系Example 1.2. Construction of BaF3 cell lines stably expressing different M7Rs
BaF3(购自南京科佰生物科技有限公司)是鼠源前B淋巴细胞，依赖外源加入的小鼠IL-3(mIL-3)细胞因子(R&D system)生存。当外源基因转入该细胞系使BaF3细胞获得不依赖IL-3的生长特性时，说明转入的外源基因能够传递组成型促细胞生存信号，因此BaF3细胞系是筛选具有组成型促生存信号基因的常用工具细胞系。BaF3 (purchased from Nanjing Kebai Biotechnology Co., Ltd.) is a mouse-derived pre-B lymphocyte that relies on exogenously added mouse IL-3 (mIL-3) cytokine (R&D system) for survival. When exogenous genes are transferred into this cell line, BaF3 cells acquire growth characteristics that are independent of IL-3, indicating that the transferred exogenous genes can transmit constitutive pro-cell survival signals. Therefore, the BaF3 cell line is selected for constitutively pro-survival signals. Commonly used tool cell lines for signaling genes.
为了鉴定实施例1.1中构建的tCD19-M7CR基因导入该BaF3细胞系后，是否能够持续激活STAT5信号，将不同tCD19-M7CR基因通过慢病毒转入BaF3细胞中，根据BaF3细胞是否能够产生IL-3非依赖生长来筛选具有持续激活STAT5功能的M7R基因。具体实验步骤如下，将处于对数生长期的Lenti-X-293T细胞(Takara公司)(3×105个细胞)接种入6孔板中，待细胞贴壁后，分别将27个克隆有不同tCD19-M7CR基因、以及克隆有对照IL7R-tCD19基因的各表达质粒pRK(金维智构建)、包装质粒pMDLg/pRRE(Addgene，12251，购自生物风)、调节质粒pRSV-rev(Addgene，12253，购自生物风)及包膜质粒pMD2G(Addgene，12259，购自生物风)以3:3:2:2的质量比例用PEI转染法转染在含有10％FBS的DMEM培养基中培养的Lenti-X-293T细胞(Takara公司)，转染16小时后，更换含有2％胎牛血清(FBS)的新鲜DEME培养基，继续培养48小时后，收集细胞培养上清，离心去细胞碎片，得到含慢病毒的上清。In order to identify whether the tCD19-M7CR gene constructed in Example 1.1 can continuously activate STAT5 signals after being introduced into the BaF3 cell line, different tCD19-M7CR genes were transferred into BaF3 cells through lentivirus. According to whether the BaF3 cells can produce IL-3 Growth-independent screening of M7R genes with sustained activation of STAT5. The specific experimental steps are as follows. Lenti-X-293T cells (Takara Company) (3×10 5 cells) in the logarithmic growth phase were seeded into a 6-well plate. After the cells adhered to the wall, 27 clones with different tCD19-M7CR gene, and each expression plasmid pRK (constructed by Jin Weizhi) cloned with the control IL7R-tCD19 gene, packaging plasmid pMDLg/pRRE (Addgene, 12251, purchased from Biowind), regulatory plasmid pRSV-rev (Addgene, 12253, purchased from Biowind) Lenti cultured in DMEM medium containing 10% FBS was transfected with PEI transfection method at a mass ratio of 3:3:2:2 -X-293T cells (Takara Company), 16 hours after transfection, replace with fresh DEME medium containing 2% fetal bovine serum (FBS), continue to culture for 48 hours, collect the cell culture supernatant, centrifuge to remove cell debris, and obtain Supernatant containing lentivirus.
用含慢病毒的上清去侵染BaF3细胞24小时，然后用含mIL-3(R&D systems，403-ML)的RPMI 1640完全培养基常规培养48h。The lentivirus-containing supernatant was used to infect BaF3 cells for 24 hours, and then conventionally cultured in RPMI 1640 complete medium containing mIL-3 (R&D systems, 403-ML) for 48 hours.
实施例1.3.检测细胞中M7R的表达Example 1.3. Detection of M7R expression in cells
通过实施例1.2制备的慢病毒侵染BaF3细胞后，采用流式细胞术检测BaF3细胞表面是否表达tCD19来判断M7R是否成功表达。After infecting BaF3 cells with the lentivirus prepared in Example 1.2, flow cytometry was used to detect whether tCD19 was expressed on the surface of BaF3 cells to determine whether M7R was successfully expressed.
具体而言，取实施例1.2制备的慢病毒侵染后的BaF3细胞，用FACS缓冲液洗涤一遍后，用FACS缓冲液重悬BaF3细胞，加入LIVE/DEAD Fixable Dead Cell Stain(Thermo，L34963)，PE-CD19抗体(BD公司，555413)，4℃孵育30至45min。然后用FACS缓冲液洗涤一遍，用FACS缓冲液重悬细胞后，通过流式细胞术检测细胞表面tCD19的表达。Specifically, take the BaF3 cells infected with the lentivirus prepared in Example 1.2, wash them once with FACS buffer, resuspend the BaF3 cells with FACS buffer, add LIVE/DEAD Fixable Dead Cell Stain (Thermo, L34963), PE-CD19 antibody (BD Company, 555413), incubate at 4°C for 30 to 45 minutes. Then, the cells were washed once with FACS buffer, and the cells were resuspended in FACS buffer, and the expression of tCD19 on the cell surface was detected by flow cytometry.
结果如图3A所示，27个包含不同tCD19-M7CR基因的慢病毒侵染BaF3细胞后第4天，BaF3细胞表面均有tCD19的表达，说明了M7R基因在BaF3细胞成功表达。The results are shown in Figure 3A. On the 4th day after BaF3 cells were infected with 27 lentiviruses containing different tCD19-M7CR genes, tCD19 was expressed on the surface of BaF3 cells, indicating that the M7R gene was successfully expressed in BaF3 cells.
实施例1.4.筛选具有激活STAT5功能的M7RExample 1.4. Screening for M7R with the function of activating STAT5
待27个包含不同M7R基因的慢病毒侵染BaF3细胞并测得27个不同M7R基因均在被侵染的BaF3细胞有表达后，将实施例1.2制备的慢病毒侵染后的BaF3细胞(表达不同M7R 基因的每组BaF3细胞)均置于不含mIL-3的RPMI-1640(10％FBS)完全培养基中进行培养，通过观察BaF3细胞是否能够以mIL-3非依赖方式生长，从而判断各M7R是否具有促BaF3细胞mIL-3非依赖生长效应。随后，将筛选出来的具有mIL-3非依赖生长的每组BaF3细胞以相同的细胞数量(即，5×105个细胞/孔)加入24孔板中，通过细胞计数仪对每组细胞进行计数，记录细胞增殖的情况，绘制生长曲线。After 27 lentiviruses containing different M7R genes have infected BaF3 cells and it is determined that 27 different M7R genes are expressed in the infected BaF3 cells, the BaF3 cells infected with the lentiviruses prepared in Example 1.2 (expression Different M7R Each group of BaF3 cells) was cultured in RPMI-1640 (10% FBS) complete medium without mIL-3, and each M7R was judged by observing whether the BaF3 cells could grow in a mIL-3-independent manner. Whether it has the effect of promoting mIL-3-independent growth of BaF3 cells. Subsequently, each group of BaF3 cells selected with mIL-3-independent growth was added to a 24-well plate at the same number of cells (i.e., 5 × 10 5 cells/well), and each group of cells was analyzed by a cell counter. Count, record cell proliferation, and draw a growth curve.
结果如图3B所示，被侵染的BaF3细胞经过不加入外源mIL-3培养后，表达IL7Rm1.1-tCD19、IL7Rm1.3-tCD19、IL7Rm3.1-tCD19、IL7Rm4-tCD19、IL7Rm5-tCD19、IL7Rm6-tCD19、IL7Rm7-tCD19，IL7Rm8-tCD19，IL7Rm9-tCD19，IL7Rm10-tCD19，IL7Rm11-tCD19，IL7Rm12-tCD19，IL7Rm13-tCD19，IL7Rm14-tCD19，IL7Rm15-tCD19，IL7Rm16-tCD19，IL7Rm17-tCD19，IL7Rm18-tCD19，IL7Rm19-tCD19的BaF3细胞中tCD19+细胞的比例上升，剩余组别的BaF3细胞在不加入外源mIL-3培养后不能维持存活，说明IL7Rm1.1，IL7Rm1.3，IL7Rm3.1，IL7Rm4，IL7Rm5，IL7Rm6，IL7Rm7，IL7Rm8，IL7Rm9，IL7Rm10，IL7Rm11，IL7Rm12，IL7Rm13，IL7Rm14，IL7Rm15，IL7Rm16，IL7Rm17，IL7Rm18，IL7Rm19能够提供持续激活的IL7Rα信号，促进BaF3细胞以mIL-3非依赖方式生长。The results are shown in Figure 3B. After being cultured without exogenous mIL-3, the infected BaF3 cells expressed IL7Rm1.1-tCD19, IL7Rm1.3-tCD19, IL7Rm3.1-tCD19, IL7Rm4-tCD19, and IL7Rm5-tCD19. , IL7Rm6-tCD19, IL7Rm7-tCD19, IL7Rm8-tCD19, IL7Rm9-tCD19, IL7Rm10-tCD19, IL7Rm11-tCD19, IL7Rm12-tCD19, IL7Rm13-tCD19, IL7Rm14-tCD19, IL7Rm15-tCD19, IL7Rm16 -tCD19,IL7Rm17-tCD19,IL7Rm18 -tCD19, IL7Rm19-tCD19 The proportion of tCD19 + cells in BaF3 cells increased, and the remaining groups of BaF3 cells could not maintain survival after culture without the addition of exogenous mIL-3, indicating that IL7Rm1.1, IL7Rm1.3, IL7Rm3.1, IL7Rm4, IL7Rm5, IL7Rm6, IL7Rm7, IL7Rm8, IL7Rm9, IL7Rm10, IL7Rm11, IL7Rm12, IL7Rm13, IL7Rm14, IL7Rm15, IL7Rm16, IL7Rm17, IL7Rm18, IL7Rm19 can provide sustained activation of IL7Rα signaling and promote BaF3 cells to mIL -3Grow in an independent way .
在病毒侵染细胞后，从第3天开始不加入外源mIL-3对细胞进行筛选，图3C所示，统计了不加入外源mIL-3筛选BaF3细胞前后CD19+细胞的比例变化，结果表明，不加入外源mIL-3培养BaF3细胞后，在第11天时检测CD19+的比例，有细胞存活的组中CD19+的比例均显著提高。After the virus infected the cells, the cells were screened without adding exogenous mIL-3 starting from the 3rd day. As shown in Figure 3C, the changes in the proportion of CD19 + cells before and after the BaF3 cells were screened without adding exogenous mIL-3 were counted. The results It shows that after culturing BaF3 cells without adding exogenous mIL-3, the proportion of CD19 + was detected on the 11th day, and the proportion of CD19 + in the group with cell survival was significantly increased.
如图3D所示，经过细胞计数，发现IL7Rm1.1、IL7Rm1.3、IL7Rm3.1、IL7Rm4～12、IL7Rm14～18等M7R基因均能够促进BaF3细胞以mIL-3非依赖方式增殖。As shown in Figure 3D, through cell counting, it was found that M7R genes such as IL7Rm1.1, IL7Rm1.3, IL7Rm3.1, IL7Rm4~12, IL7Rm14~18, etc. can promote the proliferation of BaF3 cells in an mIL-3-independent manner.
实施例1.5.BaF3细胞的胞内p-STAT5检测Example 1.5. Detection of intracellular p-STAT5 in BaF3 cells
取未经慢病毒侵染的BaF3细胞(作为对照)和实施例1.4的筛选后能够稳定表达tCD19-M7CR的各组BaF3细胞，用PBS洗涤两遍，重悬至无血清RPMI-1640培养基中过夜。次日，离心收集细胞，用3％多聚甲醛固定细胞，用FACS缓冲液洗涤1遍；用0.1％Triton-X透化细胞，用FACS缓冲液洗涤1遍；将细胞置于95％预冷的甲醇中，置于-20℃过夜；次日，离心收集细胞，用FACS缓冲液洗涤1遍，然后将细胞和AF647-p-STAT5(BD，562076)抗体置于室温共孵育45min，最后通过流式细胞术检测BaF3细胞内p-STAT5的表达水平。Take BaF3 cells that have not been infected by lentivirus (as a control) and BaF3 cells from each group that can stably express tCD19-M7CR after screening in Example 1.4, wash them twice with PBS, and resuspend them in serum-free RPMI-1640 medium. overnight. The next day, collect the cells by centrifugation, fix them with 3% paraformaldehyde, and wash them once with FACS buffer; permeabilize the cells with 0.1% Triton-X, and wash them once with FACS buffer; place the cells in 95% pre-cooling in methanol and placed at -20°C overnight; the next day, cells were collected by centrifugation, washed once with FACS buffer, and then incubated with AF647-p-STAT5 (BD, 562076) antibody at room temperature for 45 minutes, and finally passed Flow cytometry was used to detect the expression level of p-STAT5 in BaF3 cells.
图4中，ISO是指用抗STAT5的同型对照抗体进行染色，+IL3是指在未经侵染的BaF3细胞中加入IL3刺激细胞STAT5激活，作为阳性对照；without IL3是不用IL3刺激未侵染的BaF3细胞，用抗pSTAT5抗体染色，检测细胞中STAT5的基础磷酸化水平。In Figure 4, ISO refers to staining with an anti-STAT5 isotype control antibody; +IL3 refers to adding IL3 to uninfected BaF3 cells to stimulate cell STAT5 activation as a positive control; without IL3 refers to uninfected cells without IL3 stimulation. BaF3 cells were stained with anti-pSTAT5 antibody to detect the basal phosphorylation level of STAT5 in the cells.
如图4所示，未经慢病毒侵染的BaF3细胞(ISO,作为对照)只有在加入外源mIL-3刺激情况下检测到STAT5磷酸化信号；而表达IL7Rm1.1、IL7Rm1.3、IL7Rm3.1、IL7Rm4、IL7Rm5、IL7Rm6、IL7Rm7、IL7Rm8、IL7Rm9、IL7Rm10、IL7Rm11、IL7Rm12、IL7Rm14、IL7Rm15、IL7Rm16、IL7Rm17、IL7Rm18基因的BaF3细胞在不加入外源mIL-3刺激情况下检测到STAT5磷酸化信号，表明这些M7R基因能够组成型激活STAT5信号通路。As shown in Figure 4, BaF3 cells that have not been infected with lentivirus (ISO, as a control) can only detect STAT5 phosphorylation signals when exogenous mIL-3 is added to stimulate them; while expressing IL7Rm1.1, IL7Rm1.3, and IL7Rm3 .1. STAT5 phosphorylation was detected in BaF3 cells of IL7Rm4, IL7Rm5, IL7Rm6, IL7Rm7, IL7Rm8, IL7Rm9, IL7Rm10, IL7Rm11, IL7Rm12, IL7Rm14, IL7Rm15, IL7Rm16, IL7Rm17, and IL7Rm18 genes without the addition of exogenous mIL-3 stimulation. signal, indicating that these M7R genes can constitutively activate the STAT5 signaling pathway.
实施例2、M7R对人T细胞生长的影响Example 2. Effect of M7R on the growth of human T cells
根据实施例1中使用BaF3细胞筛选构建的tCD19-M7CR基因的结果，选取了具有组成型激活功能的M7R序列(分别为IL7Rm1.1、1.3、3.1、4～12、14～18)。 According to the results of screening the constructed tCD19-M7CR gene using BaF3 cells in Example 1, M7R sequences with constitutive activation function were selected (IL7Rm1.1, 1.3, 3.1, 4-12, 14-18 respectively).
为了鉴定所述tCD19-M7CR基因导入T细胞系后，M7R序列是否能够持续激活T细胞中的STAT5信号通路，将不同tCD19-M7CR基因(分别为含IL7Rm1.1、1.3、3.1、4～12、14～18的tCD19-M7CR基因)通过慢病毒转入T细胞中，根据T细胞是否能够产生IL-2非依赖生长来筛选在T细胞中具有持续激活STAT5功能的M7R基因。In order to identify whether the M7R sequence can continuously activate the STAT5 signaling pathway in T cells after the tCD19-M7CR gene is introduced into the T cell line, different tCD19-M7CR genes (containing IL7Rm1.1, 1.3, 3.1, 4-12, respectively) were The tCD19-M7CR gene (14-18 years old) is transferred into T cells through lentivirus, and the M7R gene with the function of continuously activating STAT5 in T cells is screened based on whether the T cells can produce IL-2-independent growth.
慢病毒包装步骤同实施例1.2所述，获得分别包含不同M7R序列(分别为IL7Rm1.1、1.3、3.1、4～12、14～18)的tCD19-M7CR基因的慢病毒上清。采用表达不同tCD19-M7CR基因的慢病毒侵染激活的人T细胞(PBMC信息见表1)以获得稳定表达不同tCD19-M7CR的T细胞。具体步骤如下。The lentivirus packaging steps are the same as those described in Example 1.2, and lentiviral supernatants containing tCD19-M7CR genes containing different M7R sequences (IL7Rm1.1, 1.3, 3.1, 4-12, and 14-18 respectively) are obtained. Lentivirus expressing different tCD19-M7CR genes was used to infect activated human T cells (see Table 1 for PBMC information) to obtain T cells stably expressing different tCD19-M7CR genes. Specific steps are as follows.
T细胞分选和激活步骤：添加注射用重组人白介素-2(国药准字S20040020)至TexMACS GMP Medium(Miltenyi Biotec,170-076-309)中，配制成IL-2浓度为200IU/ml的T细胞培养基。T cell sorting and activation steps: Add recombinant human interleukin-2 for injection (National Drug Approval No. S20040020) to TexMACS GMP Medium (Miltenyi Biotec, 170-076-309) to prepare T cells with an IL-2 concentration of 200IU/ml. Cell culture medium.
自ORiCELLS获得了多个供者PBMC细胞，具体信息如下表1所示：Multiple donor PBMC cells were obtained from ORiCELLS. The specific information is shown in Table 1 below:
表1.供者PBMC细胞的相关来源信息
Table 1. Relevant source information of donor PBMC cells
第0天使用Pan T Cell Isolation Kit(human)(Miltenyi,130-096-535)对复苏后的各供者PBMC进行分选，获得T细胞，使用T细胞培养基将T细胞重悬至一定的密度(例如，细胞密度1×106个细胞/mL)并添加TransAct(Miltenyi,130-111-160)进行激活；第1天分出一定量细胞不添加慢病毒继续培养，该部分细胞为未转导细胞(UNT细胞,un-transduced T cells)，剩余的细胞添加分别包含不同tCD19-M7CR基因的慢病毒的上清并将T细胞吹打均匀；第2天离心去除包含慢病毒的上清，重悬T细胞至新鲜含IL-2的T细胞培养基中。对UNT细胞无任何操作。37℃，5％CO2细胞培养箱培养48h后，用LIVE/DEAD Fixable Dead Cell Stain(Thermo，L34963)、PE-CD19(BD，555413)、AF647-p-STAT5(BD，562076)抗体组合检测M7R基因在T细胞中的表达水平及T细胞中STAT5的磷酸化水平。然后将相同数量tCD19+T细胞(5×106细胞/孔)加入24孔板中，用RPMI1640完全培养基(不含hIL-2)培养，每2至3天换液一次。每周通过细胞计数仪对总T细胞进行计数，并通过流式细胞术检测tCD19+细胞的比例，并绘制细胞生长曲线。On day 0, Pan T Cell Isolation Kit (human) (Miltenyi, 130-096-535) was used to sort the PBMC of each donor after recovery to obtain T cells, and the T cells were resuspended to a certain concentration using T cell culture medium. density (for example, cell density 1 × 10 6 cells/mL) and add TransAct (Miltenyi, 130-111-160) for activation; on the first day, separate a certain amount of cells and continue culturing without adding lentivirus. This part of the cells is not Transduced cells (UNT cells, un-transduced T cells), add the supernatant of lentivirus containing different tCD19-M7CR genes to the remaining cells and pipet the T cells evenly; centrifuge on the second day to remove the supernatant containing lentivirus. Resuspend T cells in fresh IL-2-containing T cell medium. No manipulation was performed on UNT cells. After culturing for 48 hours in a 5% CO2 cell incubator at 37°C, use LIVE/DEAD Fixable Dead Cell Stain (Thermo, L34963), PE-CD19 (BD, 555413), and AF647-p-STAT5 (BD, 562076) antibody combination for detection. The expression level of M7R gene in T cells and the phosphorylation level of STAT5 in T cells. Then the same number of tCD19+T cells (5×10 6 cells/well) was added to the 24-well plate, cultured in RPMI1640 complete medium (without hIL-2), and the medium was changed every 2 to 3 days. Total T cells were counted weekly by a cell counter, and the proportion of tCD19 + cells was detected by flow cytometry, and the cell growth curve was drawn.
使用供者3的PBMC细胞，待病毒侵染T细胞后48h，检测T细胞上的tCD19表达。结果如图5所示，在感染不同tCD19-M7CR基因、包含野生型IL7Rα跨膜区及胞内段的IL7R-tCD19基因的人T细胞中检测到不同比例tCD19表达，而未转染(UNT)的T细胞及表达野生型IL7Rα(IL7R-WT)的T细胞中未检测到tCD19表达。说明了M7R基因在T细 胞中成功表达。PBMC cells from donor 3 were used, and 48 hours after the virus infected T cells, the expression of tCD19 on T cells was detected. The results are shown in Figure 5. Different proportions of tCD19 expression were detected in human T cells infected with different tCD19-M7CR genes, IL7R-tCD19 genes containing wild-type IL7Rα transmembrane region and intracellular segment, while untransfected (UNT) No tCD19 expression was detected in T cells and T cells expressing wild-type IL7Rα (IL7R-WT). It shows that the M7R gene plays a role in T cells successfully expressed in cells.
使用供者3的PBMC细胞，待病毒侵染T细胞后，培养5天(从第0天开始算分选激活一直到检测时共培养5天)后检测T细胞中STAT5的磷酸化水平。如图6所示，未转染的T细胞(UNT)只有在加入外源IL-2刺激情况下检测到STAT5磷酸化信号；而在不加入外源IL-2刺激情况下，稳定表达不同M7R的而不是表达野生型IL7R跨膜及胞内段(对照IL7R-tCD19)的T细胞中检测到STAT5磷酸化信号，表明这些M7R基因在人T细胞中同样能够组成型激活STAT5信号通路。Use the PBMC cells of donor 3. After the virus infects the T cells, culture them for 5 days (from day 0 counting activation until detection for a total of 5 days) and then detect the phosphorylation level of STAT5 in the T cells. As shown in Figure 6, untransfected T cells (UNT) can only detect STAT5 phosphorylation signals when stimulated by exogenous IL-2; while without stimulation by exogenous IL-2, they stably express different M7Rs. Instead of expressing the wild-type IL7R transmembrane and intracellular segments (control IL7R-tCD19), STAT5 phosphorylation signals were detected in T cells, indicating that these M7R genes can also constitutively activate the STAT5 signaling pathway in human T cells.
使用供者3的PBMC细胞，待病毒侵染T细胞，绘制细胞生长曲线，如图7A和图7B所示，表达IL7R-tCD19的T细胞体外在加入外源IL-2刺激情况下持续扩增2周，此后细胞停止扩增并维持约1周，然后细胞数量显著下降。在不加入外源IL-2刺激情况下，表达IL7R-tCD19的T细胞数量持续下降，2周后所有细胞死亡。Use the PBMC cells of donor 3, wait for the virus to infect the T cells, and draw the cell growth curve, as shown in Figure 7A and Figure 7B. T cells expressing IL7R-tCD19 continue to expand in vitro under the stimulation of exogenous IL-2. 2 weeks, after which the cells stopped expanding and maintained for about 1 week, and then the number of cells dropped significantly. Without the addition of exogenous IL-2 stimulation, the number of T cells expressing IL7R-tCD19 continued to decrease, and all cells died after 2 weeks.
与表达IL7R-tCD19的T细胞相比较，表达IL7Rm4、5、7和8构建体的T细胞在不加入外源IL-2情况下数量能够维持1周，1周后开始缓慢下降，直到4周所有细胞死亡，这些体外实验表明表达M7R基因促进了T细胞的存续并具有维持T细胞存活的能力。Compared with T cells expressing IL7R-tCD19, the number of T cells expressing IL7Rm4, 5, 7 and 8 constructs could be maintained for 1 week without the addition of exogenous IL-2, and began to slowly decrease after 1 week until 4 weeks. All cells died, and these in vitro experiments showed that expression of the M7R gene promoted the survival of T cells and had the ability to maintain T cell survival.
实施例3、构建M7CR修饰的CARExample 3. Construction of M7CR modified CAR
如图8所示，设计了不同的M7CR基因，其中将胞外结构域(ECD)和M7R(IL-7Rα突变体跨膜和胞内信号区组成的融合IL-7R突变体)直接连接组成的组成型嵌合细胞因子受体M7CR；然后通过P2A将M7CR的N端与不同CAR多肽的C端连接，从而构成M7CR修饰的CAR。As shown in Figure 8, different M7CR genes were designed, in which the extracellular domain (ECD) and M7R (the fusion IL-7R mutant consisting of the transmembrane and intracellular signaling regions of the IL-7Rα mutant) were directly connected. Constitutive chimeric cytokine receptor M7CR; the N-terminus of M7CR is then connected to the C-terminus of different CAR polypeptides through P2A to form an M7CR-modified CAR.
在所述M7CR中，位于M7R的N端的ECD包括但不限于tCD19(SEQ ID NO：17)、sIL-15(SEQ ID NO：47)、IL-15/IL-15Rα(SEQ ID NO：140)、IL-15/IL-15Rα(Sushi)(SEQ ID NO：48)、IL-15Rα(Sushi)/IL-15(SEQ ID NO：141)、IL-12-P70(SEQ ID NO：49)、IL-12-p40(SEQ ID NO：50)、IL-21(SEQ ID NO：51)、IL-9(SEQ ID NO：52)、IL-18(SEQ ID NO：53)、IL-23(SEQ ID NO：54)、IL-36γ(SEQ ID NO：55)、IFNα2b(SEQ ID NO：56)、4-1BBL(SEQ ID NO：57)、CD40L(SEQ ID NO：58)、FLT3L(SEQ ID NO：59)、ICOS(SEQ ID NO：60)、GITR(SEQ ID NO：61)、ICAM-1(SEQ ID NO：62)、CD2(SEQ ID NO：63)、抗4-1BB(SEQ ID NO：64)、抗CD40(SEQ ID NO：65)、抗CD83(SEQ ID NO：66)、抗-TGFβ(SEQ ID NO：67)、TGFβRII(SEQ ID NO：198)、dnTGFβRII(SEQ ID NO：199)、抗PD-L1VHH(SEQ ID NO：68)、抗CD47(SEQ ID NO：69)、抗IL-4(SEQ ID NO：70)、抗PD-1(SEQ ID NO：71)、抗CTLA-4(SEQ ID NO：72)、抗LAG-3(SEQ ID NO：73)、抗TIGIT(SEQ ID NO：74)、抗CD73(SEQ ID NO：75)、抗NKG2D(SEQ ID NO：76)、抗NKG2C(SEQ ID NO：77)、抗NKp30(SEQ ID NO：78)和抗NKp46(SEQ ID NO：79)。不同ECD分别和M7R连接后分别构成以下各M7CR分子：tCD19-M7CR、sIL-15-M7CR、IL-15/IL-15Rα-M7CR、IL15/IL15Rα(Sushi)-M7CR、IL15Rα(Sushi)/IL15-M7CR、IL-12-P70-M7CR、IL-12-p40-M7CR、IL-21-M7CR、IL-9-M7CR、IL-18-M7CR、IL-23-M7CR、IL-36γ-M7CR、IFNα2b-M7CR、4-1BBL-M7CR、CD40L-M7CR、FLT3L-M7CR、ICOS-M7CR、GITR-M7CR、ICAM-1-M7CR、CD2-M7CR、抗4-1BB-M7CR、抗CD40-M7CR、抗CD83-M7CR、抗TGFβ-M7CR、TGFβRII-M7CR、dnTGFβRII-M7CR、 抗PD-L1VHH-M7CR、抗CD47-M7CR、抗IL-4-M7CR、抗PD-1-M7CR、抗CTLA-4-M7CR、抗LAG-3-M7CR、抗TIGIT-M7CR、抗CD73-M7CR、抗NKG2D-M7CR、抗NKG2C-M7CR、抗NKp30-M7CR和抗NKp46-M7CR。例如，将不同ECD与M7Rm8连接构建SEQ ID NO：171所示的tCD19-M7CR、SEQ ID NO：172所示的IL-12-M7CR、SEQ ID NO：173所示的IL-15-M7CR、SEQ ID NO：174所示的IL-21-M7CR、SEQ ID NO：175所示的IL-12-p40-M7CR、SEQ ID NO：176所示的IL-9-M7CR、SEQ ID NO：177所示的IL-18-M7CR、SEQ ID NO：178所示的IL-23-M7CR、SEQ ID NO：179所示的IL-36γ-M7CR。In the M7CR, the ECD located at the N-terminus of M7R includes but is not limited to tCD19 (SEQ ID NO: 17), sIL-15 (SEQ ID NO: 47), IL-15/IL-15Rα (SEQ ID NO: 140) , IL-15/IL-15Rα(Sushi)(SEQ ID NO:48), IL-15Rα(Sushi)/IL-15(SEQ ID NO:141), IL-12-P70(SEQ ID NO:49), IL-12-p40 (SEQ ID NO: 50), IL-21 (SEQ ID NO: 51), IL-9 (SEQ ID NO: 52), IL-18 (SEQ ID NO: 53), IL-23 ( SEQ ID NO: 54), IL-36γ (SEQ ID NO: 55), IFNα2b (SEQ ID NO: 56), 4-1BBL (SEQ ID NO: 57), CD40L (SEQ ID NO: 58), FLT3L (SEQ ID NO: 59), ICOS (SEQ ID NO: 60), GITR (SEQ ID NO: 61), ICAM-1 (SEQ ID NO: 62), CD2 (SEQ ID NO: 63), anti-4-1BB (SEQ ID NO: 64), anti-CD40 (SEQ ID NO: 65), anti-CD83 (SEQ ID NO: 66), anti-TGFβ (SEQ ID NO: 67), TGFβRII (SEQ ID NO: 198), dnTGFβRII (SEQ ID NO: 199), anti-PD-L1 VHH (SEQ ID NO: 68), anti-CD47 (SEQ ID NO: 69), anti-IL-4 (SEQ ID NO: 70), anti-PD-1 (SEQ ID NO: 71 ), anti-CTLA-4 (SEQ ID NO: 72), anti-LAG-3 (SEQ ID NO: 73), anti-TIGIT (SEQ ID NO: 74), anti-CD73 (SEQ ID NO: 75), anti-NKG2D (SEQ ID NO:76), anti-NKG2C (SEQ ID NO:77), anti-NKp30 (SEQ ID NO:78) and anti-NKp46 (SEQ ID NO:79). Different ECDs are connected to M7R to form the following M7CR molecules: tCD19-M7CR, sIL-15-M7CR, IL-15/IL-15Rα-M7CR, IL15/IL15Rα(Sushi)-M7CR, IL15Rα(Sushi)/IL15- M7CR, IL-12-P70-M7CR, IL-12-p40-M7CR, IL-21-M7CR, IL-9-M7CR, IL-18-M7CR, IL-23-M7CR, IL-36γ-M7CR, IFNα2b- M7CR, 4-1BBL-M7CR, CD40L-M7CR, FLT3L-M7CR, ICOS-M7CR, GITR-M7CR, ICAM-1-M7CR, CD2-M7CR, anti-4-1BB-M7CR, anti-CD40-M7CR, anti-CD83-M7CR , anti-TGFβ-M7CR, TGFβRII-M7CR, dnTGFβRII-M7CR, Anti-PD-L1 VHH -M7CR, anti-CD47-M7CR, anti-IL-4-M7CR, anti-PD-1-M7CR, anti-CTLA-4-M7CR, anti-LAG-3-M7CR, anti-TIGIT-M7CR, anti-CD73-M7CR , anti-NKG2D-M7CR, anti-NKG2C-M7CR, anti-NKp30-M7CR and anti-NKp46-M7CR. For example, different ECDs are connected to M7Rm8 to construct tCD19-M7CR shown in SEQ ID NO: 171, IL-12-M7CR shown in SEQ ID NO: 172, IL-15-M7CR shown in SEQ ID NO: 173, SEQ IL-21-M7CR shown in ID NO: 174, IL-12-p40-M7CR shown in SEQ ID NO: 175, IL-9-M7CR shown in SEQ ID NO: 176, and IL-9-M7CR shown in SEQ ID NO: 177 IL-18-M7CR, IL-23-M7CR shown in SEQ ID NO: 178, IL-36γ-M7CR shown in SEQ ID NO: 179.
图1显示了所构建的M7CR转导T细胞后，表达所构建的M7CR的T细胞的作用机制。Figure 1 shows the mechanism of action of T cells expressing the constructed M7CR after the constructed M7CR transduces T cells.
实施例4、制备M7CR修饰的CAR-T细胞Example 4. Preparation of M7CR-modified CAR-T cells
实施例4.1.序列合成Example 4.1. Sequence synthesis
人工合成DNA序列，所述DNA序列分别编码HuR968B CAR(SEQ ID NO:15)(下文/附图中有时也简称为“8B”)、H9.1.2 CAR(SEQ ID NO:16)(文中有时也称“H9.1.2”)、H9.2.1-CAR(SEQ ID NO:100)(文中有时也称H9.2.1或H9)、H9.2.1-218-BB-L CAR(SEQ ID NO:144)(文中有时也称H9.2.1-218或H9.2.1-218CAR)、H9.2.1-CD28-L-CAR(SEQ ID NO:142)(文中有时也称“H9.2.1-28”或H9.2.1-28 CAR)、H9.1.2-P2A-tCD19-M7CR(下文中又称H9.1.2-tCD19-M7CR)(SEQ ID NO:80)、H9.1.2-P2A-tCD19-M7CR(CPT)(下文中又称H9.1.2-tCD19-M7CR(CPT))(SEQ ID NO:81)、H9.1.2-P2A-IL-12-P70-M7CR(下文中又称H9.1.2-IL-12-M7CR)(SEQ ID NO:82)、H9.1.2-P2A-IL-15/IL-15Rα-M7CR(下文中又称H9.1.2-IL-15-M7CR)(SEQ ID NO:83)、H9.1.2-P2A-IL-21-M7CR(下文中又称H9.1.2-IL-21-M7CR)(SEQ ID NO:84)、H9.1.2-P2A-CD40L-M7CR(下文中又称H9.1.2-CD40L-M7CR)(SEQ ID NO:85)、H9.1.2-P2A-4-1BBL-M7CR(下文中又称H9.1.2-4-1BBL-M7CR)(SEQ ID NO:86)、H9.1.2-P2A-抗PD-L1VHH-M7CR(下文中又称H9.1.2-抗PD-L1VHH-M7CR)(SEQ ID NO:87)、8B-P2A-tCD19-M7CR(下文中又称8B-tCD19-M7CR)(SEQ ID NO:88)、8B-P2A-tCD19-M7CR(CPT)(下文中又称8B-tCD19-M7CR(CPT))(SEQ ID NO:89)、8B-P2A-IL-15/IL-15Rα-M7CR(下文中又称8B-IL-15-M7CR)(SEQ ID NO:90)、8B-P2A-IL-12-P70-M7CR(下文中又称8B-IL-12-M7CR)(SEQ ID NO:91)、8B-P2A-IL-21-M7CR(下文中又称8B-IL-21-M7CR)(SEQ ID NO:92)、8B-P2A-CD40L-M7CR(下文中又称8B-CD40L-M7CR)(SEQ ID NO:93)、8B-P2A-4-1BBL-M7CR(下文中又称8B-4-1BBL-M7CR)(SEQ ID NO:94)、8B-P2A-抗PD-L1VHH-M7CR(下文中又称8B-抗PD-L1VHH-M7CR)(SEQ ID NO:95)、H9.2.1-P2A-tCD19-M7CR(下文中又称H9.2.1-tCD19-M7CR或H9.2.1-M7CR)(SEQ ID NO:101)、H9.2.1-P2A-IL-12-M7CRin(下文中又称H9.2.1-IL-12-M7CRin)(SEQ ID NO:102)、H9.2.1in-P2A-IL-12-M7CR(下文中又称H9.2.1in-IL-12-M7CR)(SEQ ID NO:103)、H9.2.1-P2A-IL-12-M7CR(下文中又称H9.2.1-IL-12-M7CR)(SEQ ID NO:104)、H9.2.1-P2A-sIL-12(下文中又称H9.2.1-sIL-12)(SEQ ID NO:105)、H9.2.1-P2A-IL-15/IL-15Rα(Sushi)-M7CR(下文中又称H9.2.1-IL-15-M7CR)(SEQ ID NO:136)、H9.2.1-P2A-IL-15/IL-15Rα(Sushi)-M7CRin(下文中又称H9.2.1-IL-15-M7CRin)(SEQ ID NO:135)、H9.2.1in-P2A-IL-15/IL-15Rα(Sushi)-M7CR(下文中又称H9.2.1in-IL-15-M7CR)(SEQ ID  NO:134)、H9.2.1-P2A-sIL-15(下文中又称H9.2.1-sIL-15)(SEQ ID NO:139)、H9.2.1-CD28-P2A-IL-12-P70-M7CR(下文中又称H9.2.1-28-IL-12-M7CR)(SEQ ID NO:133)、H9.2.1-CD28-P2A-IL-15/IL15Rα(Sushi)-M7CR(下文中又称H9.2.1-28-IL-15-M7CR)(SEQ ID NO:137)、H9.2.1-P2A-IL15Rα(Sushi)/IL-15-M7CR(下文中又称H9.2.1-IL-15-M7CR)(SEQ ID NO:138)蛋白。Artificially synthesized DNA sequences, which respectively encode HuR968B CAR (SEQ ID NO: 15) (sometimes also referred to as "8B" in the text and drawings), H9.1.2 CAR (SEQ ID NO: 16) (sometimes also referred to as "8B" in the text and drawings) (sometimes also referred to as "8B" in the text) (called "H9.1.2"), H9.2.1-CAR (SEQ ID NO:100) (sometimes also called H9.2.1 or H9 in the text), H9.2.1-218-BB-L CAR (SEQ ID NO:144) ( Sometimes also called H9.2.1-218 or H9.2.1-218CAR), H9.2.1-CD28-L-CAR (SEQ ID NO: 142) (sometimes called "H9.2.1-28" or H9.2.1- 28 CAR), H9.1.2-P2A-tCD19-M7CR (hereinafter also referred to as H9.1.2-tCD19-M7CR) (SEQ ID NO:80), H9.1.2-P2A-tCD19-M7CR (CPT) (hereinafter also referred to as Referred to as H9.1.2-tCD19-M7CR (CPT)) (SEQ ID NO: 81), H9.1.2-P2A-IL-12-P70-M7CR (hereinafter also referred to as H9.1.2-IL-12-M7CR) (SEQ ID NO:82), H9.1.2-P2A-IL-15/IL-15Rα-M7CR (hereinafter also referred to as H9.1.2-IL-15-M7CR) (SEQ ID NO:83), H9.1.2-P2A- IL-21-M7CR (hereinafter also referred to as H9.1.2-IL-21-M7CR) (SEQ ID NO:84), H9.1.2-P2A-CD40L-M7CR (hereinafter also referred to as H9.1.2-CD40L-M7CR) (SEQ ID NO:85), H9.1.2-P2A-4-1BBL-M7CR (hereinafter also referred to as H9.1.2-4-1BBL-M7CR) (SEQ ID NO:86), H9.1.2-P2A-anti-PD -L1 VHH -M7CR (hereinafter also referred to as H9.1.2-anti-PD-L1 VHH -M7CR) (SEQ ID NO:87), 8B-P2A-tCD19-M7CR (hereinafter also referred to as 8B-tCD19-M7CR) (SEQ ID NO:88), 8B-P2A-tCD19-M7CR(CPT) (hereinafter also referred to as 8B-tCD19-M7CR(CPT)) (SEQ ID NO:89), 8B-P2A-IL-15/IL-15Rα- M7CR (hereinafter also referred to as 8B-IL-15-M7CR) (SEQ ID NO: 90), 8B-P2A-IL-12-P70-M7CR (hereinafter also referred to as 8B-IL-12-M7CR) (SEQ ID NO :91), 8B-P2A-IL-21-M7CR (hereinafter also referred to as 8B-IL-21-M7CR) (SEQ ID NO:92), 8B-P2A-CD40L-M7CR (hereinafter also referred to as 8B-CD40L- M7CR) (SEQ ID NO:93), 8B-P2A-4-1BBL-M7CR (hereinafter also referred to as 8B-4-1BBL-M7CR) (SEQ ID NO:94), 8B-P2A-anti-PD-L1 VHH - M7CR (hereinafter also referred to as 8B-anti-PD-L1 VHH -M7CR) (SEQ ID NO:95), H9.2.1-P2A-tCD19-M7CR (hereinafter also referred to as H9.2.1-tCD19-M7CR or H9.2.1- M7CR) (SEQ ID NO: 101), H9.2.1-P2A-IL-12-M7CRin (hereinafter also referred to as H9.2.1-IL-12-M7CRin) (SEQ ID NO: 102), H9.2.1in-P2A -IL-12-M7CR (hereinafter also referred to as H9.2.1in-IL-12-M7CR) (SEQ ID NO: 103), H9.2.1-P2A-IL-12-M7CR (hereinafter also referred to as H9.2.1- IL-12-M7CR) (SEQ ID NO:104), H9.2.1-P2A-sIL-12 (hereinafter also referred to as H9.2.1-sIL-12) (SEQ ID NO:105), H9.2.1-P2A- IL-15/IL-15Rα (Sushi)-M7CR (hereinafter also referred to as H9.2.1-IL-15-M7CR) (SEQ ID NO: 136), H9.2.1-P2A-IL-15/IL-15Rα (Sushi )-M7CRin (hereinafter also referred to as H9.2.1-IL-15-M7CRin) (SEQ ID NO: 135), H9.2.1in-P2A-IL-15/IL-15Rα (Sushi)-M7CR (hereinafter also referred to as H9.2.1in-IL-15-M7CR)(SEQ ID NO:134), H9.2.1-P2A-sIL-15 (hereinafter also referred to as H9.2.1-sIL-15) (SEQ ID NO:139), H9.2.1-CD28-P2A-IL-12-P70-M7CR (hereinafter also referred to as H9.2.1-28-IL-12-M7CR) (SEQ ID NO: 133), H9.2.1-CD28-P2A-IL-15/IL15Rα(Sushi)-M7CR (hereinafter also referred to as H9. 2.1-28-IL-15-M7CR) (SEQ ID NO: 137), H9.2.1-P2A-IL15Rα(Sushi)/IL-15-M7CR (hereinafter also referred to as H9.2.1-IL-15-M7CR) ( SEQ ID NO:138) protein.
在所述构建体中，HuR968B CAR多肽(SEQ ID NO:15)从N端至C端包含源自CD8的信号肽(CD8-SP)(SEQ ID NO:1)、抗PG抗体VH(SEQ ID NO:9)、G4S接头(SEQ ID NO:5)、抗PG抗体VL(SEQ ID NO:10)、GGGGS铰链、源自CD8的跨膜结构域(CD8TMD)(SEQ ID NO:8)、源自4-1BB的共刺激信号结构域(4-1BB CSD)(SEQ ID NO:11)和源自CD3ζ的刺激信号结构域(CD3ζSSD)(SEQ ID NO:12)。所述CAR多肽通过P2A(SEQ ID NO:3)连接M7CR分子。In the construct, the HuR968B CAR polypeptide (SEQ ID NO: 15) includes a signal peptide (CD8-SP) (SEQ ID NO: 1) derived from CD8 (SEQ ID NO: 1), an anti-PG antibody VH (SEQ ID NO:9), G4S linker (SEQ ID NO:5), anti-PG antibody VL (SEQ ID NO:10), GGGGS hinge, CD8-derived transmembrane domain (CD8TMD) (SEQ ID NO:8), source Costimulatory signaling domain from 4-1BB (4-1BB CSD) (SEQ ID NO: 11) and stimulatory signaling domain from CD3ζ (CD3ζSSD) (SEQ ID NO: 12). The CAR polypeptide is connected to the M7CR molecule through P2A (SEQ ID NO: 3).
在所述构建体中，H9.1.2 CAR分子(SEQ ID NO:16)从N端至C端包含CD8-SP(SEQ ID NO:1)、H9.1.2-VL(SEQ ID NO:13)、(G4S)3接头(SEQ ID NO:4)、H9.1.2-VH(SEQ ID NO:14)、CD8铰链(SEQ ID NO:7)、CD8TMD(SEQ ID NO:8)、4-1BB CSD(SEQ ID NO:11)和CD3ζSSD(SEQ ID NO:12)。所述CAR多肽通过P2A(SEQ ID NO:3)连接M7CR分子。In the construct, the H9.1.2 CAR molecule (SEQ ID NO:16) includes CD8-SP (SEQ ID NO:1), H9.1.2-VL (SEQ ID NO:13), (G4S) 3 connector (SEQ ID NO:4), H9.1.2-VH (SEQ ID NO:14), CD8 hinge (SEQ ID NO:7), CD8TMD (SEQ ID NO:8), 4-1BB CSD ( SEQ ID NO:11) and CD3ζSSD (SEQ ID NO:12). The CAR polypeptide is connected to the M7CR molecule through P2A (SEQ ID NO: 3).
在所述构建体中，H9.2.1 CAR分子(SEQ ID NO:100)从N端至C端包含CD8-SP(SEQ ID NO:1)、H9.2.1-VL(SEQ ID NO:98)、(G4S)3接头(SEQ ID NO:4)、H9.2.1-VH(SEQ ID NO:99)、CD8铰链(SEQ ID NO:7)、CD8TMD(SEQ ID NO:8)、4-1BB CSD(SEQ ID NO:11)和CD3ζSSD(SEQ ID NO:12)。In the construct, the H9.2.1 CAR molecule (SEQ ID NO:100) includes CD8-SP (SEQ ID NO:1), H9.2.1-VL (SEQ ID NO:98), (G4S) 3 connector (SEQ ID NO:4), H9.2.1-VH (SEQ ID NO:99), CD8 hinge (SEQ ID NO:7), CD8TMD (SEQ ID NO:8), 4-1BB CSD ( SEQ ID NO:11) and CD3ζSSD (SEQ ID NO:12).
在所述构建体中，H9.2.1-218 CAR分子(SEQ ID NO:144)从N端至C端包含CD8-SP(SEQ ID NO:1)、H9.2.1-VL(SEQ ID NO:98)、218接头序列(SEQ ID NO:145)、H9.2.1-VH(SEQ ID NO:99)、CD8长铰链(SEQ ID NO:146)、CD8TMD延长(SEQ ID NO:147)、4-1BB CSD(SEQ ID NO:11)和CD3ζSSD(SEQ ID NO:12)。所述CAR多肽通过P2A(SEQ ID NO:3)连接M7CR分子。In the construct, the H9.2.1-218 CAR molecule (SEQ ID NO:144) includes CD8-SP (SEQ ID NO:1), H9.2.1-VL (SEQ ID NO:98) from N-terminus to C-terminus ), 218 linker sequence (SEQ ID NO:145), H9.2.1-VH (SEQ ID NO:99), CD8 long hinge (SEQ ID NO:146), CD8TMD extension (SEQ ID NO:147), 4-1BB CSD (SEQ ID NO:11) and CD3ζSSD (SEQ ID NO:12). The CAR polypeptide is connected to the M7CR molecule through P2A (SEQ ID NO: 3).
在所述的构建体中，H9.2.1-28 CAR分子(SEQ ID NO:142)从N端至C端包含CD8-SP(SEQ ID NO:1)、H9.2.1-VL(SEQ ID NO:98)、218接头序列(SEQ ID NO:145)、H9.2.1-VH(SEQ ID NO:99)、CD8长铰链(SEQ ID NO:146)、CD8TMD延长(SEQ ID NO:147)、CD28CSD(SEQ ID NO:143)和CD3ζSSD(SEQ ID NO:12)。所述CAR多肽通过P2A(SEQ ID NO:3)连接M7CR分子。In the construct, H9.2.1-28 CAR molecule (SEQ ID NO:142) contains CD8-SP (SEQ ID NO:1), H9.2.1-VL (SEQ ID NO: 98), 218 linker sequence (SEQ ID NO:145), H9.2.1-VH (SEQ ID NO:99), CD8 long hinge (SEQ ID NO:146), CD8TMD extension (SEQ ID NO:147), CD28CSD ( SEQ ID NO:143) and CD3ζSSD (SEQ ID NO:12). The CAR polypeptide is connected to the M7CR molecule through P2A (SEQ ID NO: 3).
在所述的构建体中，H9.1.2-P2A-tCD19-M7CR分子(SEQ ID NO:80)从N端至C端包含H9.1.2 CAR(SEQ ID NO:16)、P2A(SEQ ID NO：3)以及tCD19-M7CR(SEQ ID NO：171)。In the construct, H9.1.2-P2A-tCD19-M7CR molecule (SEQ ID NO:80) contains H9.1.2 CAR (SEQ ID NO:16), P2A (SEQ ID NO: 3) and tCD19-M7CR (SEQ ID NO: 171).
在所述的构建体中，H9.1.2-P2A-tCD19-M7CR(CPT)分子(SEQ ID NO:81)从N端至C端包含H9.1.2 CAR(SEQ ID NO:16)、P2A(SEQ ID NO：3)以及tCD19-M7CR(CPT)(SEQ ID NO：182)。In the construct, H9.1.2-P2A-tCD19-M7CR (CPT) molecule (SEQ ID NO:81) contains H9.1.2 CAR (SEQ ID NO:16), P2A (SEQ ID NO: 3) and tCD19-M7CR(CPT) (SEQ ID NO: 182).
在所述的构建体中，H9.1.2-P2A-IL-12-M7CR分子(SEQ ID NO:82)从N端至C端包含H9.1.2 CAR(SEQ ID NO:16)、P2A(SEQ ID NO：3)以及IL-12-P70-M7CR。In the construct, H9.1.2-P2A-IL-12-M7CR molecule (SEQ ID NO:82) contains H9.1.2 CAR (SEQ ID NO:16), P2A (SEQ ID NO:16) from N-terminus to C-terminus. NO: 3) and IL-12-P70-M7CR.
在所述的构建体中，H9.1.2-P2A-IL-15-M7CR分子(SEQ ID NO:83)从N端至C端包含H9.1.2 CAR(SEQ ID NO:16)、P2A(SEQ ID NO：3)以及IL-15/IL-15Rα-M7CR。In the construct, H9.1.2-P2A-IL-15-M7CR molecule (SEQ ID NO:83) contains H9.1.2 CAR (SEQ ID NO:16), P2A (SEQ ID NO:16) from N-terminus to C-terminus. NO: 3) and IL-15/IL-15Rα-M7CR.
在所述的构建体中，H9.1.2-P2A-IL-21-M7CR分子(SEQ ID NO:84)从N端至C端包含 H9.1.2 CAR(SEQ ID NO:16)、P2A(SEQ ID NO：3)以及IL-21-M7CR(SEQ ID NO：174)。In the construct, the H9.1.2-P2A-IL-21-M7CR molecule (SEQ ID NO:84) contains H9.1.2 CAR (SEQ ID NO:16), P2A (SEQ ID NO:3) and IL-21-M7CR (SEQ ID NO:174).
在所述的构建体中，H9.1.2-P2A-CD40L-M7CR分子(SEQ ID NO:85)从N端至C端包含H9.1.2 CAR(SEQ ID NO:16)、P2A(SEQ ID NO：3)以及CD40L-M7CR(SEQ ID NO：183)。In the construct, H9.1.2-P2A-CD40L-M7CR molecule (SEQ ID NO:85) contains H9.1.2 CAR (SEQ ID NO:16), P2A (SEQ ID NO: 3) and CD40L-M7CR (SEQ ID NO: 183).
在所述的构建体中，H9.1.2-P2A-4-1BBL-M7CR分子(SEQ ID NO:86)从N端至C端包含H9.1.2 CAR(SEQ ID NO:16)、P2A(SEQ ID NO：3)以及4-1BBL-M7CR(SEQ ID NO：184)。In the construct, H9.1.2-P2A-4-1BBL-M7CR molecule (SEQ ID NO:86) contains H9.1.2 CAR (SEQ ID NO:16), P2A (SEQ ID NO:16) from N-terminus to C-terminus. NO: 3) and 4-1BBL-M7CR (SEQ ID NO: 184).
在所述的构建体中，H9.1.2-P2A-抗PD-L1VHH-M7CR分子(SEQ ID NO:87)从N端至C端包含H9.1.2 CAR(SEQ ID NO:16)、P2A(SEQ ID NO：3)以及抗PD-L1VHH-M7CR(SEQ ID NO：185)。In the construct, H9.1.2-P2A-anti-PD-L1 VHH -M7CR molecule (SEQ ID NO:87) contains H9.1.2 CAR (SEQ ID NO:16), P2A ( SEQ ID NO: 3) and anti-PD-L1 VHH -M7CR (SEQ ID NO: 185).
在所述的构建体中，8B-P2A-tCD19-M7CR分子(SEQ ID NO:88)从N端至C端包含HuR968B CAR(SEQ ID NO:15)、P2A(SEQ ID NO：3)以及tCD19-M7CR(SEQ ID NO：171)。In the construct, the 8B-P2A-tCD19-M7CR molecule (SEQ ID NO:88) contains HuR968B CAR (SEQ ID NO:15), P2A (SEQ ID NO:3) and tCD19 from N-terminus to C-terminus -M7CR (SEQ ID NO: 171).
在所述的构建体中，8B-P2A-tCD19-M7CR(CPT)分子(SEQ ID NO:89)从N端至C端包含HuR968B CAR(SEQ ID NO:15)、P2A(SEQ ID NO：3)以及tCD19-M7CR(CPT)(SEQ ID NO：182)。In the construct, 8B-P2A-tCD19-M7CR (CPT) molecule (SEQ ID NO:89) contains HuR968B CAR (SEQ ID NO:15), P2A (SEQ ID NO:3) from N-terminus to C-terminus ) and tCD19-M7CR(CPT) (SEQ ID NO: 182).
在所述的构建体中，8B-P2A-IL-15-M7CR分子(SEQ ID NO:90)从N端至C端包含HuR968B CAR(SEQ ID NO:15)、P2A(SEQ ID NO：3)以及IL-15/IL-15Rα-M7CR。In the construct, 8B-P2A-IL-15-M7CR molecule (SEQ ID NO:90) contains HuR968B CAR (SEQ ID NO:15), P2A (SEQ ID NO:3) from N-terminus to C-terminus and IL-15/IL-15Rα-M7CR.
在所述的构建体中，8B-P2A-IL-12-M7CR分子(SEQ ID NO:91)从N端至C端包含HuR968B CAR(SEQ ID NO:15)、P2A(SEQ ID NO：3)以及IL-12-P70-M7CR。In the construct, 8B-P2A-IL-12-M7CR molecule (SEQ ID NO:91) contains HuR968B CAR (SEQ ID NO:15), P2A (SEQ ID NO:3) from N-terminus to C-terminus and IL-12-P70-M7CR.
在所述的构建体中，8B-P2A-IL-21-M7CR分子(SEQ ID NO:92)从N端至C端包含HuR968B CAR(SEQ ID NO:15)、P2A(SEQ ID NO：3)以及IL-21-M7CR(SEQ ID NO：174)。In the described construct, the 8B-P2A-IL-21-M7CR molecule (SEQ ID NO:92) contains HuR968B CAR (SEQ ID NO:15), P2A (SEQ ID NO:3) from N-terminus to C-terminus and IL-21-M7CR (SEQ ID NO: 174).
在所述的构建体中，8B-P2A-CD40L-M7CR分子(SEQ ID NO:93)从N端至C端包含HuR968B CAR(SEQ ID NO:15)、P2A(SEQ ID NO：3)以及CD40L-M7CR(SEQ ID NO：183)。In the construct, the 8B-P2A-CD40L-M7CR molecule (SEQ ID NO:93) contains HuR968B CAR (SEQ ID NO:15), P2A (SEQ ID NO:3) and CD40L from N-terminus to C-terminus -M7CR (SEQ ID NO: 183).
在所述的构建体中，8B-P2A-4-1BBL-M7CR分子(SEQ ID NO:94)从N端至C端包含HuR968B CAR(SEQ ID NO:15)、P2A(SEQ ID NO：3)以及4-1BBL-M7CR(SEQ ID NO：184)。In the described construct, 8B-P2A-4-1BBL-M7CR molecule (SEQ ID NO:94) contains HuR968B CAR (SEQ ID NO:15), P2A (SEQ ID NO:3) from N-terminus to C-terminus and 4-1BBL-M7CR (SEQ ID NO: 184).
在所述的构建体中，8B-P2A-抗PD-L1VHH-M7CR分子(SEQ ID NO:95)从N端至C端包含HuR968B CAR(SEQ ID NO:15)、P2A(SEQ ID NO：3)以及抗PD-L1VHH-M7CR(SEQ ID NO：185)。In the construct, 8B-P2A-anti-PD-L1 VHH -M7CR molecule (SEQ ID NO:95) contains HuR968B CAR (SEQ ID NO:15), P2A (SEQ ID NO: 3) and anti-PD-L1 VHH -M7CR (SEQ ID NO: 185).
在上述“H9.1.2”和“8B”相关构建体中，所有M7CR分子从N端至C端均包含GM-CSFRα-SP(SEQ ID NO:2)、ECD以及M7R，任选地在ECD的C端和M7R的N端之间连接有Flag Tag(下文作为M7R例示了SEQ ID NO:30所示的IL7Rm4、SEQ ID NO:31所示的IL7Rm5、SEQ ID NO:33所示的IL7Rm7、SEQ ID NO：34所示的IL7Rm8和SEQ ID NO:44所示的IL7Rm18)，ECD选自tCD19(SEQ ID NO：17)、IL-12-P70(SEQ ID NO：49)、IL-15/IL-15Rα(SEQ ID NO：140)、IL-21(SEQ ID NO：51)、4-1BBL(SEQ ID NO：57)、CD40L(SEQ ID NO：58)、抗PD-L1VHH(SEQ ID NO：68)。其中IL-12-P70-M7CR、 IL-15/IL-15Rα-M7CR、IL-21-M7CR(SEQ ID NO：174)在ECD序列和M7R序列之间还包含Flag Tag(SEQ ID NO:6)。tCD19-M7CR、IL-12-P70-M7CR、IL-15/IL-15Rα-M7CR、IL-21-M7CR、4-1BBL-M7CR、CD40L-M7CR、抗PD-L1VHH-M7CR分子的M7R部分为IL7Rm8(SEQ ID NO:34)，tCD19-M7CR(CPT)作为对照，其M7R部分为IL7Rm(CPT)(SEQ ID NO:97)(IL7Rm(CPT)是作为对照的M7R分子)。此外，在抗PD-L1VHH-M7CR分子的M7R部分IL7Rm8序列的N端还包括“ESKYGPPCPPCP”序列。In the above "H9.1.2" and "8B" related constructs, all M7CR molecules comprise GM-CSFRa-SP (SEQ ID NO: 2), ECD and M7R from N-terminus to C-terminus, optionally at the ECD A Flag Tag is connected between the C terminus and the N terminus of M7R (hereinafter exemplified as M7R are IL7Rm4 represented by SEQ ID NO:30, IL7Rm5 represented by SEQ ID NO:31, IL7Rm7 represented by SEQ ID NO:33, and SEQ ID NO:33). IL7Rm8 shown in ID NO: 34 and IL7Rm18 shown in SEQ ID NO: 44), ECD selected from tCD19 (SEQ ID NO: 17), IL-12-P70 (SEQ ID NO: 49), IL-15/IL -15Rα (SEQ ID NO: 140), IL-21 (SEQ ID NO: 51), 4-1BBL (SEQ ID NO: 57), CD40L (SEQ ID NO: 58), anti-PD-L1 VHH (SEQ ID NO :68). Among them IL-12-P70-M7CR, IL-15/IL-15Rα-M7CR and IL-21-M7CR (SEQ ID NO: 174) also contain Flag Tag (SEQ ID NO: 6) between the ECD sequence and the M7R sequence. The M7R part of tCD19-M7CR, IL-12-P70-M7CR, IL-15/IL-15Rα-M7CR, IL-21-M7CR, 4-1BBL-M7CR, CD40L-M7CR, anti-PD-L1 VHH -M7CR molecule is IL7Rm8 (SEQ ID NO:34), tCD19-M7CR(CPT) is used as a control, and its M7R part is IL7Rm(CPT) (SEQ ID NO:97) (IL7Rm(CPT) is the M7R molecule used as a control). In addition, the N-terminus of the IL7Rm8 sequence of the M7R part of the anti-PD-L1 VHH -M7CR molecule also includes the "ESKYGPPCPPCP" sequence.
所述H9.2.1-P2A-tCD19-M7CR分子(SEQ ID NO:101)从N端至C端包含H9.2.1 CAR(SEQ ID NO:100)、P2A(SEQ ID NO：3)以及tCD19-M7CR(SEQ ID NO：171)。所述H9.2.1-P2A-IL-12-M7CR分子(SEQ ID NO:104)从N端至C端包含H9.2.1 CAR(SEQ ID NO:100)、P2A(SEQ ID NO：3)以及IL-12-M7CR(SEQ ID NO：172)。所述H9.2.1-P2A-IL-12-M7CRin分子(SEQ ID NO:102)从N端至C端包含H9.2.1 CAR(SEQ ID NO:100)、P2A(SEQ ID NO：3)以及IL-12-M7CRin(SEQ ID NO：181)，其中IL-12-M7CRin表示在IL-12-M7CR的基础上，将IL-7受体胞内Box1结构域(第1060-1071位氨基酸，序列为SEQ ID NO:132所示的PIVWPSLPDHKK)删除及同时设计Y1239F,Y1246F(以IL7Rα(P16871-1)为参照)点突变以失活胞内M7R的信号。所述H9.2.1in-P2A-IL-12-M7CR分子(SEQ ID NO:103)从N端至C端包含H9.2.1in CAR(SEQ ID NO：153)、P2A(SEQ ID NO：3)以及IL-12-M7CR(SEQ ID NO：172)，其中H9.2.1in表示将H9.2.1 CAR(SEQ ID NO:100)分子胞内4-1BB和CD3两个结构域删除(以失活CAR分子胞内信号)、且在C端加上了“KRGR”序列。所述H9.2.1-P2A-sIL-12分子(SEQ ID NO:105)从N端至C端包含H9.2.1 CAR(SEQ ID NO:100)、P2A(SEQ ID NO：3)以及sIL-12，其中sIL-12表示由GM-CSFRα-SP(SEQ ID NO:2)、IL-12-p70(SEQ ID NO:49)组成的、能够表达外分泌可溶性IL-12的基因。上述M7CR分子的M7R部分为IL7Rm8(SEQ ID NO:34)。The H9.2.1-P2A-tCD19-M7CR molecule (SEQ ID NO:101) includes H9.2.1 CAR (SEQ ID NO:100), P2A (SEQ ID NO:3) and tCD19-M7CR from the N-terminus to the C-terminus. (SEQ ID NO: 171). The H9.2.1-P2A-IL-12-M7CR molecule (SEQ ID NO: 104) includes H9.2.1 CAR (SEQ ID NO: 100), P2A (SEQ ID NO: 3) and IL from the N end to the C end. -12-M7CR (SEQ ID NO: 172). The H9.2.1-P2A-IL-12-M7CRin molecule (SEQ ID NO: 102) includes H9.2.1 CAR (SEQ ID NO: 100), P2A (SEQ ID NO: 3) and IL from the N-terminus to the C-terminus. -12-M7CRin (SEQ ID NO: 181), where IL-12-M7CRin means that on the basis of IL-12-M7CR, the IL-7 receptor intracellular Box1 domain (amino acids 1060-1071, the sequence is The PIVWPPSLPDHKK shown in SEQ ID NO:132 was deleted and Y1239F, Y1246F (using IL7Rα (P16871-1) as a reference) point mutations were simultaneously designed to inactivate the intracellular M7R signal. The H9.2.1in-P2A-IL-12-M7CR molecule (SEQ ID NO: 103) contains H9.2.1in CAR (SEQ ID NO: 153), P2A (SEQ ID NO: 3) from the N end to the C end. and IL-12-M7CR (SEQ ID NO: 172), in which H9.2.1in represents the deletion of the two intracellular 4-1BB and CD3 domains of the H9.2.1 CAR (SEQ ID NO: 100) molecule (to inactivate the CAR Molecular intracellular signal), and the "KRGR" sequence is added to the C terminus. The H9.2.1-P2A-sIL-12 molecule (SEQ ID NO: 105) includes H9.2.1 CAR (SEQ ID NO: 100), P2A (SEQ ID NO: 3) and sIL-12 from the N-terminus to the C-terminus. , where sIL-12 represents a gene composed of GM-CSFRα-SP (SEQ ID NO:2) and IL-12-p70 (SEQ ID NO:49) that can express exocrine soluble IL-12. The M7R part of the above-mentioned M7CR molecule is IL7Rm8 (SEQ ID NO: 34).
所述H9.2.1-P2A-IL-15-M7CR分子(SEQ ID NO:136)从N端至C端包含H9.2.1-218 CAR(SEQ ID NO:144)、P2A(SEQ ID NO：3)以及IL-15-M7CR(SEQ ID NO：173)。所述H9.2.1-P2A-IL-15-M7CRin分子(SEQ ID NO:135)从N端至C端包含H9.2.1-218 CAR(SEQ ID NO:144)、P2A(SEQ ID NO：3)以及IL-15-M7CRin(SEQ ID NO：180)，其中IL-15-M7CRin表示在IL-15-M7CR的基础上，将IL-7受体胞内Box1结构域(第1060-1071位氨基酸(272-280(以IL7Rα为参照(P16871-1)))，序列为SEQ ID NO:132所示的PIVWPSLPDHKK)删除及设计Y1239F,Y1246F点突变以失活胞内M7R的信号。所述H9.2.1in-P2A-IL-15-M7CR分子(SEQ ID NO:134)从N端至C端包含H9.2.1-218in CAR(SEQ ID NO：186)、P2A(SEQ ID NO：3)以及IL-15-M7CR，其中H9.2.1-218in CARin表示将H9.2.1-218(SEQ ID NO:144)分子胞内4-1BB和CD3两个结构域删除以失活CAR分子胞内信号。所述H9.2.1-P2A-sIL-15分子(SEQ ID NO:139)从N端至C端包含H9.2.1-218 CAR(SEQ ID NO:144)、P2A(SEQ ID NO：3)以及sIL-15，其中sIL-15表示由GM-CSFRα-SP(SEQ ID NO:2)、IL-15(SEQ ID NO:47)组成的、能够表达外分泌可溶性IL-15的基因。所述H9.2.1-P2A-IL15Rα(Sushi)/IL-15-M7CR分子(SEQ ID NO:138)从N端至C端包含H9.2.1-218 CAR(SEQ ID NO:144)、P2A(SEQ ID NO：3)以及IL15Rα(Sushi)/IL-15-M7CR(SEQ ID NO：187)。上述M7CR分子的M7R部分为IL7Rm8(SEQ ID NO:34)。The H9.2.1-P2A-IL-15-M7CR molecule (SEQ ID NO: 136) contains H9.2.1-218 CAR (SEQ ID NO: 144), P2A (SEQ ID NO: 3) from the N end to the C end. and IL-15-M7CR (SEQ ID NO: 173). The H9.2.1-P2A-IL-15-M7CRin molecule (SEQ ID NO: 135) contains H9.2.1-218 CAR (SEQ ID NO: 144), P2A (SEQ ID NO: 3) from the N end to the C end. And IL-15-M7CRin (SEQ ID NO: 180), where IL-15-M7CRin means that on the basis of IL-15-M7CR, the IL-7 receptor intracellular Box1 domain (amino acids 1060-1071 ( 272-280 (using IL7Rα as reference (P16871-1))), the sequence is PIVWPSLPDHKK shown in SEQ ID NO: 132), delete and design Y1239F, Y1246F point mutations to inactivate the intracellular M7R signal. The H9.2.1in-P2A-IL-15-M7CR molecule (SEQ ID NO: 134) contains H9.2.1-218in CAR (SEQ ID NO: 186), P2A (SEQ ID NO: 3) from the N end to the C end. ) and IL-15-M7CR, where H9.2.1-218in CARin means that the two intracellular 4-1BB and CD3 domains of H9.2.1-218 (SEQ ID NO: 144) are deleted to inactivate the intracellular signal of the CAR molecule . The H9.2.1-P2A-sIL-15 molecule (SEQ ID NO:139) includes H9.2.1-218 CAR (SEQ ID NO:144), P2A (SEQ ID NO:3) and sIL from the N-terminus to the C-terminus. -15, where sIL-15 represents a gene composed of GM-CSFRα-SP (SEQ ID NO: 2) and IL-15 (SEQ ID NO: 47) and capable of expressing exocrine soluble IL-15. The H9.2.1-P2A-IL15Rα(Sushi)/IL-15-M7CR molecule (SEQ ID NO:138) includes H9.2.1-218 CAR (SEQ ID NO:144), P2A (SEQ ID NO: 3) and IL15Rα(Sushi)/IL-15-M7CR (SEQ ID NO: 187). The M7R part of the above-mentioned M7CR molecule is IL7Rm8 (SEQ ID NO: 34).
所述H9.2.1-CD28-P2A-IL-12-M7CR分子(SEQ ID NO:133)从N端至C端包含H9.2.1-28 CAR(SEQ ID NO:142)、序列“RAKR”、P2A(SEQ ID NO：3)以及IL-12-M7CR(SEQ ID NO：172)。所述H9.2.1-CD28-P2A-IL-15-M7CR分子(SEQ ID NO：137)从N端至C端包含H9.2.1-28 CAR(SEQ ID NO:142)、序列“RAKR”、P2A(SEQ ID NO：3)以及IL-15-M7CR(SEQ ID NO：173)。上述M7CR分子的M7R部分为IL7Rm8(SEQ ID NO:34)。The H9.2.1-CD28-P2A-IL-12-M7CR molecule (SEQ ID NO: 133) includes H9.2.1-28 from the N-terminus to the C-terminus. CAR (SEQ ID NO:142), sequence "RAKR", P2A (SEQ ID NO:3) and IL-12-M7CR (SEQ ID NO:172). The H9.2.1-CD28-P2A-IL-15-M7CR molecule (SEQ ID NO: 137) contains H9.2.1-28 CAR (SEQ ID NO: 142), the sequence "RAKR", and P2A from the N-terminus to the C-terminus. (SEQ ID NO: 3) and IL-15-M7CR (SEQ ID NO: 173). The M7R portion of the above M7CR molecule is IL7Rm8 (SEQ ID NO: 34).
在上述与“H9.2.1”和“H9.2.1-CD28”相关的构建体中，所有M7CR分子从N端至C端均包含GM-CSFRα-SP(SEQ ID NO:2)、ECD以及M7R(下文中作为M7R例示了SEQ ID NO:30所示的IL7Rm4、SEQ ID NO:31所示的IL7Rm5、SEQ ID NO:33所示的IL7Rm7、SEQ ID NO：34所示的IL7Rm8和SEQ ID NO:44所示的IL7Rm18)，ECD选自tCD19(SEQ ID NO：17)、IL-12-P70(SEQ ID NO：49)、IL-15/IL-15Rα(Sushi)(SEQ ID NO：48)、IL-15Rα(Sushi)/IL-15(SEQ ID NO：141)。tCD19-M7CR(SEQ ID NO：171)、IL-12-M7CR(SEQ ID NO：172)、IL-15-M7CR(SEQ ID NO：173)和IL-15Rα(Sushi)/IL-15-M7CR(SEQ ID NO：187)分子的M7R部分为IL7Rm8(SEQ ID NO:34)，tCD19-M7CR(CPT)作为对照，其M7R部分为IL7Rm(CPT)(SEQ ID NO:97)(IL7Rm(CPT)是作为对照的M7R分子)。In the above constructs related to "H9.2.1" and "H9.2.1-CD28", all M7CR molecules contain GM-CSFRα-SP (SEQ ID NO: 2), ECD and M7R ( Hereinafter, IL7Rm4 shown in SEQ ID NO:30, IL7Rm5 shown in SEQ ID NO:31, IL7Rm7 shown in SEQ ID NO:33, IL7Rm8 shown in SEQ ID NO:34 and SEQ ID NO: IL7Rm18 shown in 44), ECD is selected from tCD19 (SEQ ID NO: 17), IL-12-P70 (SEQ ID NO: 49), IL-15/IL-15Rα (Sushi) (SEQ ID NO: 48), IL-15Rα(Sushi)/IL-15 (SEQ ID NO: 141). tCD19-M7CR (SEQ ID NO: 171), IL-12-M7CR (SEQ ID NO: 172), IL-15-M7CR (SEQ ID NO: 173) and IL-15Rα (Sushi)/IL-15-M7CR ( SEQ ID NO: 187) The M7R part of the molecule is IL7Rm8 (SEQ ID NO: 34), tCD19-M7CR (CPT) is used as a control, and its M7R part is IL7Rm (CPT) (SEQ ID NO: 97) (IL7Rm (CPT) is M7R molecule as control).
将上述合成的DNA片段插入pRKN慢病毒表达载体(金唯智公司)的EF1α启动子下游，替换原载体中EGFR序列，得到相应的表达质粒(由金唯智公司合成)。The above-mentioned synthesized DNA fragment was inserted into the pRKN lentiviral expression vector (Genewise Company) downstream of the EF1α promoter, and the EGFR sequence in the original vector was replaced to obtain the corresponding expression plasmid (synthesized by Genewise Company).
实施例4.2.慢病毒的制备Example 4.2. Preparation of lentivirus
将实施例4.1获得的表达质粒与结构质粒pMDLg/pRRE(Addgene，12251，购自生物风)、调节质粒pRSV-rev(Addgene，12253，购自生物风)及包膜质粒pMD2G(Addgene，12259，购自生物风)以3:3:2:2的质量比例用PEI转染法转染Lenti-X-293T细胞(Takara公司)，转染16小时后，更换含有2％胎牛血清(FBS)的新鲜DEME培养基，继续培养48小时后，收集细胞上清，离心去细胞碎片，加入PEG8000 4℃孵育16-64小时进行病毒浓缩，再次离心后弃上清，采用T细胞培养基(TexMACs)重悬病毒沉淀物，获得慢病毒浓缩液，分装后-80℃冻存。消化Lenti-X-293T细胞(Takara公司)并用含有8μg/ml Polybrene(Sigma，H9268-5G)的DMEM培养基重悬并加入到24孔板中，加入不同体积上述获得的慢病毒浓缩液培养72小时，进行293T细胞的转导。将转导后的293T细胞消化，用Biotin-SP-conjugated抗Human IgG,F(ab’)2-specific(Jackson ImmunoResearch,109-066-006)以及APC-Streptadvidin(BioLegend,405207)进行染色，并采用细胞流式细胞术检测APC阳性细胞的比例。通过起始细胞量、病毒体积和阳性细胞比例计算出病毒滴度(TU/ml)。The expression plasmid obtained in Example 4.1 was combined with the structural plasmid pMDLg/pRRE (Addgene, 12251, purchased from Biowind), regulatory plasmid pRSV-rev (Addgene, 12253, purchased from Biowind) and envelope plasmid pMD2G (Addgene, 12259, Lenti-X-293T cells (Takara Company) were transfected with PEI transfection method at a mass ratio of 3:3:2:2 (purchased from Biowind). After 16 hours of transfection, the cells were replaced with 2% fetal bovine serum (FBS). Fresh DEME culture medium, continue culturing for 48 hours, collect the cell supernatant, centrifuge to remove cell debris, add PEG8000 and incubate at 4°C for 16-64 hours to concentrate the virus, centrifuge again and discard the supernatant, use T cell culture medium (TexMACs) Resuspend the virus pellet to obtain lentivirus concentrate, aliquot and freeze at -80°C. Digest Lenti-X-293T cells (Takara Company) and resuspend them in DMEM medium containing 8 μg/ml Polybrene (Sigma, H9268-5G) and add them to a 24-well plate. Add different volumes of lentivirus concentrate obtained above and culture for 72 hour, transduction of 293T cells was performed. The transduced 293T cells were digested, stained with Biotin-SP-conjugated anti-Human IgG, F(ab')2-specific (Jackson ImmunoResearch, 109-066-006) and APC-Streptadvidin (BioLegend, 405207), and Cell flow cytometry was used to detect the proportion of APC-positive cells. The virus titer (TU/ml) was calculated from the starting cell volume, virus volume and positive cell proportion.
实施例4.3.T细胞的获得及慢病毒转导Example 4.3. Obtaining T cells and lentiviral transduction
添加注射用重组人白介素-2(国药准字S20040020)至TexMACS GMP Medium(Miltenyi Biotec,170-076-309)中，配制成IL-2浓度为200IU/ml的T细胞培养基。第0天使用Pan T Cell Isolation Kit(human)(Miltenyi,130-096-535)对复苏后的PBMC进行分选，获得T细胞，使用上述T细胞培养基将细胞重悬至一定的密度并添加TransAct(Miltenyi,130-111-160)进行激活；第1天分出一定量细胞不添加慢病毒继续培养，该部分细胞为未转导细胞(UNT细胞,un-transduced T cells)，剩余的细胞按MOI＝1～5)添加不同种类的自上述实施例4.2获得的慢病毒浓缩液并将细胞吹打均匀；第2天离心去除病毒上清，重悬细胞至新鲜T细胞培养基。UNT细胞无任何操作；第3天将所有细胞转移至G-Rex(WILSONWOLF,货号80040S)中，并添加适量新鲜T细胞培养基，放置于37℃CO2培养箱中静置培养；每隔2～3天，弃掉一半细胞培养基上清，补加等体积新鲜含IL-2的T细胞培养基或者补加等体积新鲜T细胞培养 基。将细胞以培养基的半量更换为新鲜培养基或直接补加IL-2，其中，添加IL-2至细胞培养基浓度中IL-2浓度为200IU/ml。当细胞数量扩增约为20-80倍时，满足需求后进行细胞收获，离心去除培养基后CAR-T细胞采用CS10(Stemcell,07930)重悬后分装，程序降温至-80℃进行冻存。Add recombinant human interleukin-2 for injection (National Drug Approval No. S20040020) to TexMACS GMP Medium (Miltenyi Biotec, 170-076-309) to prepare a T cell culture medium with an IL-2 concentration of 200IU/ml. On day 0, use Pan T Cell Isolation Kit (human) (Miltenyi, 130-096-535) to sort the recovered PBMC to obtain T cells. Use the above T cell culture medium to resuspend the cells to a certain density and add TransAct (Miltenyi, 130-111-160) for activation; on the first day, separate a certain amount of cells and continue culturing without adding lentivirus. This part of the cells are untransduced cells (UNT cells, un-transduced T cells), and the remaining cells Add different types of lentivirus concentrates obtained from the above Example 4.2 (according to MOI = 1 to 5) and pipet the cells evenly; centrifuge on the second day to remove the virus supernatant and resuspend the cells in fresh T cell culture medium. UNT cells do not need any operation; on the 3rd day, transfer all cells to G-Rex (WILSONWOLF, Cat. No. 80040S), add an appropriate amount of fresh T cell culture medium, and place them in a 37°C CO 2 incubator for static culture; every 2 ~3 days, discard half of the cell culture medium supernatant, and add an equal volume of fresh T cell culture medium containing IL-2 or add an equal volume of fresh T cell culture medium. base. Replace the cells with half the amount of medium into fresh medium or add IL-2 directly, where IL-2 is added to the concentration of IL-2 in the cell culture medium to be 200 IU/ml. When the number of cells has expanded approximately 20-80 times, the cells are harvested after meeting the demand. After centrifugation to remove the culture medium, the CAR-T cells are used. CS10 (Stemcell, 07930) was resuspended, aliquoted, and programmed to cool to -80°C for freezing.
实施例4.4.CAR表达检测及表型检测Example 4.4. CAR expression detection and phenotypic detection
取适量自上述实施例4.3获得的CAR-T细胞，FACS缓冲液(PBS+2％FBS)洗涤一次，重悬后加入含LIVE/DEAD Fixable Dead Cell Stain(Thermo，L34963)的FACS缓冲液，室温染色10-15min，洗涤两次，加入PE-Cy7-CD45RA(Biolegend，304126)，FITC-CCR7(Biolegend，353216)，BV605-CD4(BD，562658)，BV421-CD8(BD，749366)，APC-Strep(Biolegend，405207)抗体组合，分别用PE-Flag(Biolegend，637310)，PE-CD40L(Biolegend，310806)，PE-41BBL(Biolegend，311504)，PE-IL15(R&D，MAB247-SP)，PE-CD19(BD，555413)，his-PD-L1蛋白和PE-抗his检测不同ECD分子，其中，抗PD-L1VHH作为ECD是用His标记的PD-L1纯化蛋白作为第一染色试剂，然后用PE-抗His的抗体作为第二染色试剂来检测的。4℃染色30～45min；细胞洗涤两次后FACS缓冲液重悬，用流式细胞仪检测。Take an appropriate amount of CAR-T cells obtained from the above Example 4.3, wash once with FACS buffer (PBS+2% FBS), resuspend and add FACS buffer containing LIVE/DEAD Fixable Dead Cell Stain (Thermo, L34963) at room temperature. Stain for 10-15 minutes, wash twice, add PE-Cy7-CD45RA (Biolegend, 304126), FITC-CCR7 (Biolegend, 353216), BV605-CD4 (BD, 562658), BV421-CD8 (BD, 749366), APC- Strep (Biolegend, 405207) antibody combination, using PE-Flag (Biolegend, 637310), PE-CD40L (Biolegend, 310806), PE-41BBL (Biolegend, 311504), PE-IL15 (R&D, MAB247-SP), PE -CD19 (BD, 555413), his-PD-L1 protein and PE-anti-his detect different ECD molecules, where anti-PD-L1 VHH as ECD is used with His-tagged PD-L1 purified protein as the first staining reagent, and then Detected using PE-anti-His antibody as secondary staining reagent. Stain for 30-45 minutes at 4°C; wash the cells twice, resuspend them in FACS buffer, and detect with a flow cytometer.
使用供者5的PBMC细胞，如图9A所示为制备CAR-T后第9天时CAR及M7CR的表达水平。图中，“H9.1.2”为H9.1.2 CAR-T细胞，其余为tCD19-M7CR、tCD19-M7CR(CPT)、IL-15/IL-15Rα-M7CR(图中标记为IL-15-M7CR)、IL-12-M7CR、IL-21-M7CR、CD40L-M7CR、4-1BBL-M7CR、抗PD-L1VHH-M7CR修饰的H9.1.2 CAR-T细胞。Using PBMC cells from donor 5, Figure 9A shows the expression levels of CAR and M7CR on day 9 after CAR-T preparation. In the figure, "H9.1.2" refers to H9.1.2 CAR-T cells, and the others are tCD19-M7CR, tCD19-M7CR (CPT), IL-15/IL-15Rα-M7CR (marked as IL-15-M7CR in the figure) , IL-12-M7CR, IL-21-M7CR, CD40L-M7CR, 4-1BBL-M7CR, anti-PD-L1 VHH -M7CR modified H9.1.2 CAR-T cells.
从图9A中可以看出，在第9天时通过FACS检测CAR多肽和M7CR的表达，CAR多肽能够在所有CAR-T细胞中表达，在H9.1.2中CAR+细胞的比例约为26％，而tCD19-M7CR、tCD19-M7CR(CPT)、IL-15-M7CR、IL-12-M7CR、IL-21-M7CR、CD40L-M7CR、4-1BBL-M7CR和抗PD-L1VHH-M7CR组中的CAR+细胞的比例分别为：12.14％、11.87％、6.81％、6.37％、18.75％、12.97％、11.13％、10.62％。tCD19-M7CR、tCD19-M7CR(CPT)、IL-15-M7CR、IL-12-M7CR、IL-21-M7CR、CD40L-M7CR、4-1BBL-M7CR和抗PD-L1VHH-M7CR组中的M7CR分子均有表达，表达效率分别为：6.6％、6.81％、1.43％、3.59％、4.95％、2.97％、1.03％、7.45％。说明CAR多肽和M7CR分子均能表达，且CAR和各个M7CR分子的表达具有一定的关联性。As can be seen from Figure 9A, the expression of CAR peptide and M7CR was detected by FACS on day 9. The CAR peptide could be expressed in all CAR-T cells. The proportion of CAR + cells in H9.1.2 was approximately 26%, while CAR in tCD19-M7CR, tCD19-M7CR(CPT), IL-15-M7CR, IL-12-M7CR, IL-21-M7CR, CD40L-M7CR, 4-1BBL-M7CR and anti-PD-L1 VHH -M7CR group The proportions of + cells are: 12.14%, 11.87%, 6.81%, 6.37%, 18.75%, 12.97%, 11.13%, 10.62%. tCD19-M7CR, tCD19-M7CR(CPT), IL-15-M7CR, IL-12-M7CR, IL-21-M7CR, CD40L-M7CR, 4-1BBL-M7CR and M7CR in the anti-PD-L1 VHH -M7CR group All molecules were expressed, and the expression efficiencies were: 6.6%, 6.81%, 1.43%, 3.59%, 4.95%, 2.97%, 1.03%, and 7.45%. This shows that both CAR polypeptides and M7CR molecules can be expressed, and there is a certain correlation between the expression of CAR and each M7CR molecule.
图9B中可以看出，CD4和CD8阳性细胞在表达H9.1.2 CAR细胞中的比例分别为59.9％，36.3％；在tCD19-M7CR、tCD19-M7CR(CPT)、IL-15-M7CR、IL-12-M7CR、IL-21-M7CR、CD40L-M7CR、4-1BBL-M7CR和抗PD-L1VHH-M7CR组中的CD4+细胞比例分别为：55.9％、62.5％、60.2％、56.1％、62％、65.7％、60.5％和46.5％；CD8+细胞比例分别为：36.3％、33.5％、26.9％、38％、31.5％、29.8％、32.7％和48.2％。As can be seen in Figure 9B, the proportions of CD4 and CD8 positive cells in cells expressing H9.1.2 CAR were 59.9% and 36.3% respectively; in tCD19-M7CR, tCD19-M7CR(CPT), IL-15-M7CR, IL- The proportions of CD4 + cells in the 12-M7CR, IL-21-M7CR, CD40L-M7CR, 4-1BBL-M7CR and anti-PD-L1 VHH -M7CR groups were respectively: 55.9%, 62.5%, 60.2%, 56.1%, 62 %, 65.7%, 60.5% and 46.5%; the proportions of CD8 + cells were: 36.3%, 33.5%, 26.9%, 38%, 31.5%, 29.8%, 32.7% and 48.2% respectively.
使用供者13的PBMC细胞，待病毒侵染T细胞，如图9C所示为制备CAR-T后第9天时CAR及M7CR的表达水平，图中，“8B”为HuR968B CAR-T细胞，其余为tCD19-M7CR、tCD19-M7CR(CPT)、IL-15/IL-15Rα-M7CR(图中标记为IL-15-M7CR)、IL-12-M7CR、IL-21-M7CR、CD40L-M7CR、4-1BBL-M7CR、抗PD-L1VHH-M7CR修饰的HuR968B CAR-T细胞。Use the PBMC cells of donor 13 and wait for the virus to infect the T cells. Figure 9C shows the expression levels of CAR and M7CR on the 9th day after preparing CAR-T. In the figure, "8B" represents HuR968B CAR-T cells, and the rest For tCD19-M7CR, tCD19-M7CR (CPT), IL-15/IL-15Rα-M7CR (marked as IL-15-M7CR in the figure), IL-12-M7CR, IL-21-M7CR, CD40L-M7CR, 4 -1BBL-M7CR, anti-PD-L1 VHH -M7CR modified HuR968B CAR-T cells.
从图9C中可以看出，在第9天时通过FACS检测CAR多肽和M7CR的表达，CAR多肽能够在所有CAR-T细胞中表达，在8B中CAR+细胞的比例约为39％，其它组tCD19-M7CR、 tCD19-M7CR(CPT)、IL-15-M7CR、IL-12-M7CR、IL-21-M7CR、CD40L-M7CR、4-1BBL-M7CR和抗PD-L1VHH-M7CR的CAR+细胞的比例分别为：20.41％、17.58％、11.29％、12.01％、26.12％、21.86％、20.12％、21.68％。每组中tCD19-M7CR、tCD19-M7CR(CPT)、IL-15-M7CR、IL-12-M7CR、IL-21-M7CR、CD40L-M7CR、4-1BBL-M7CR和抗PD-L1VHH-M7CR分子均有表达，表达效率分别为：6.6％、6.81％、1.43％、3.59％、4.95％、2.97％、1.03％、7.45％。说明了CAR多肽和M7CR分子均能表达，且CAR和各个M7CR分子的表达有一定的关联性。As can be seen from Figure 9C, the expression of CAR peptide and M7CR was detected by FACS on day 9. The CAR peptide could be expressed in all CAR-T cells. The proportion of CAR + cells in 8B was approximately 39%. In other groups, tCD19 -M7CR、 The proportions of CAR + cells for tCD19-M7CR(CPT), IL-15-M7CR, IL-12-M7CR, IL-21-M7CR, CD40L-M7CR, 4-1BBL-M7CR and anti-PD-L1 VHH -M7CR were respectively : 20.41%, 17.58%, 11.29%, 12.01%, 26.12%, 21.86%, 20.12%, 21.68%. tCD19-M7CR, tCD19-M7CR(CPT), IL-15-M7CR, IL-12-M7CR, IL-21-M7CR, CD40L-M7CR, 4-1BBL-M7CR and anti-PD-L1 VHH -M7CR molecules in each group All are expressed, and the expression efficiencies are: 6.6%, 6.81%, 1.43%, 3.59%, 4.95%, 2.97%, 1.03%, and 7.45% respectively. This shows that both CAR polypeptides and M7CR molecules can be expressed, and there is a certain correlation between the expression of CAR and each M7CR molecule.
从图9D中可以看出，CD4和CD8阳性细胞在表达8B CAR细胞中的比例分别为33.2％，61.6％；在tCD19-M7CR、tCD19-M7CR(CPT)、IL-15-M7CR、IL-12-M7CR、IL-21-M7CR、CD40L-M7CR、4-1BBL-M7CR和抗PD-L1VHH-M7CR组中的CD4+细胞比例分别为：31.1％、32.4％、29.8％、38.2％、32.4％、32.2％、32.2％和32.2％；CD8+细胞比例分别为：64.3％、63.2％、65.1％、55.7％、63.5％、63.3％、63.5％和61.1％。As can be seen from Figure 9D, the proportions of CD4 and CD8 positive cells in cells expressing 8B CAR were 33.2% and 61.6% respectively; in tCD19-M7CR, tCD19-M7CR (CPT), IL-15-M7CR, IL-12 The proportions of CD4 + cells in -M7CR, IL-21-M7CR, CD40L-M7CR, 4-1BBL-M7CR and anti-PD-L1 VHH -M7CR groups were: 31.1%, 32.4%, 29.8%, 38.2%, 32.4% respectively , 32.2%, 32.2% and 32.2%; the proportions of CD8 + cells were: 64.3%, 63.2%, 65.1%, 55.7%, 63.5%, 63.3%, 63.5% and 61.1% respectively.
检测了H9.1.2 CAR-T和M7CR修饰的H9.1.2 CAR-T样品中总T细胞，CD4+和CD8+T的表型，结果如图9E所示。代表性流式细胞图显示不同CAR-T细胞CD45RA、CCR7的表达，CD45RA+CCR7+代表初始T细胞或干性记忆性T细胞(TN/TSCM)、CD45RA-CCR7+代表中心记忆性T细胞(TCM)、CD45RA-CCR7-代表效应记忆性T细胞(TEM)、CD45RA+CCR7-代表效应T细胞(Teff)亚群，CAR-T细胞大部分为TN/TSCM、TCM细胞。和UNT相比，H9.1.2的表型没有明显变化。The phenotypes of total T cells, CD4 + and CD8 + T in H9.1.2 CAR-T and M7CR-modified H9.1.2 CAR-T samples were detected, and the results are shown in Figure 9E. Representative flow cytometry shows the expression of CD45RA and CCR7 in different CAR-T cells. CD45RA + CCR7 + represents naive T cells or stem memory T cells (TN/TSCM), and CD45RA-CCR7 + represents central memory T cells ( TCM), CD45RA - CCR7 - represents effector memory T cells (TEM), CD45RA + CCR7 - represents effector T cells (Teff) subsets, and most CAR-T cells are TN/TSCM and TCM cells. Compared with UNT, the phenotype of H9.1.2 did not change significantly.
与未修饰的H9.1.2 CAR-T相比，IL12-M7CR修饰的H9.1.2 CAR-T细胞样品中总T细胞，CD4+和CD8+T细胞发生明显分化，TCM和TN亚群细胞比例降低，TEM和Teff亚群细胞比例上升，其它组中T细胞表型没有发生明显分化。Compared with unmodified H9.1.2 CAR-T, total T cells, CD4 + and CD8 + T cells in IL12-M7CR modified H9.1.2 CAR-T cell samples were significantly differentiated, and the proportion of TCM and TN subpopulation cells was reduced. , the proportion of TEM and Teff subpopulation cells increased, and there was no obvious differentiation of T cell phenotype in other groups.
检测未修饰的HuR968B CAR-T和M7CR修饰的HuR968B CAR-T样品中总T细胞，CD4+和CD8+T的表型，结果如图9F所示。与未修饰的HuR968B CAR相比，IL12-M7CR修饰的HuR968B CAR-T细胞样品中总T细胞，CD4+和CD8+T细胞发生明显分化，TCM和TN亚群细胞比例降低，TEM和Teff亚群细胞比例上升，其它组中T细胞表型没有发生明显分化。进一步检测了H9.2.1-218 CAR-T(其中H9.2.1-218 CAR的序列如SEQ ID NO:144所示)和M7CR修饰的H9.2.1-218 CAR-T样品中总T细胞，CD4+和CD8+T的表型，结果如图9G和图9H所示。在两个供者中均发现，与未修饰的H9.2.1-218 CAR-T相比，IL-15-M7CR、sIL-15或IL-15-M7CRin修饰的H9.2.1-218 CAR-T细胞样品中总T细胞，CD4+和CD8+T细胞TCM和TN亚群细胞比例升高；而IL-12-M7CR修饰的CAR-T细胞样品中总T细胞，CD4+和CD8+T细胞发生部分分化，TCM和TN亚群细胞比例降低，TEM和Teff亚群细胞比例上升。说明不同ECD结构对CAR-T细胞表型会产生不同的影响。The phenotypes of total T cells, CD4 + and CD8 + T in the unmodified HuR968B CAR-T and M7CR-modified HuR968B CAR-T samples were detected, and the results are shown in Figure 9F. Compared with unmodified HuR968B CAR, total T cells, CD4 + and CD8 + T cells in IL12-M7CR modified HuR968B CAR-T cell samples were significantly differentiated, the proportion of TCM and TN subpopulation cells was reduced, and the TEM and Teff subpopulations were reduced. The proportion of cells increased, and there was no obvious differentiation of T cell phenotype in other groups. The total T cells, CD4 + and CD8 + T phenotypes, the results are shown in Figure 9G and Figure 9H . In both donors, IL-15-M7CR, sIL-15, or IL-15-M7CRin-modified H9.2.1-218 CAR-T cells were found to The proportion of total T cells, CD4 + and CD8 + T cells TCM and TN subset cells in the sample increased; while the proportion of total T cells, CD4 + and CD8 + T cells in the IL-12-M7CR modified CAR-T cell sample occurred During differentiation, the proportion of TCM and TN subpopulation cells decreased, while the proportion of TEM and Teff subpopulation cells increased. This shows that different ECD structures will have different effects on the phenotype of CAR-T cells.
实施例4.5 CAR-T细胞STAT5信号激活Example 4.5 STAT5 signal activation of CAR-T cells
通过FACS检测胞内p-STAT5的表达水平，研究STAT5的激活情况。实验步骤如下，取1E6的T细胞，用PBS洗涤一遍后，重悬于无血清的RPMI1640培养基中，过夜。次日，将用IL-2(200UI/mL)刺激UNT细胞20min后，FACS缓冲液洗涤一遍。然后加入AF647-p-STAT5(BD，562076)抗体进行胞内染色，具体步骤同实施例1.5。如图9I和图9J所示，UNT在IL-2的刺激下，p-STAT5水平升高，H9.2.1 CAR-T细胞中不论CAR+还是CAR-，p-STAT5均不上升。而表达M7R的CAR+细胞中，p-STAT5的平均表达水平上升，其中对M7R进行失活突变的H9.2.1-IL-12-M7CRin中，p-STAT5的表达水平没有上升。以上结果表明，在CAR-T 细胞中M7R能够持续提供激活信号，激活下游信号通路。The expression level of intracellular p-STAT5 was detected by FACS to study the activation of STAT5. The experimental steps are as follows. Take 1E6 T cells, wash them once with PBS, and resuspend them in serum-free RPMI1640 medium overnight. The next day, UNT cells were stimulated with IL-2 (200UI/mL) for 20 minutes and then washed once with FACS buffer. Then add AF647-p-STAT5 (BD, 562076) antibody for intracellular staining. The specific steps are the same as Example 1.5. As shown in Figure 9I and Figure 9J, UNT increased the level of p-STAT5 under the stimulation of IL-2, but p-STAT5 did not increase in H9.2.1 CAR-T cells regardless of CAR+ or CAR-. In CAR+ cells expressing M7R, the average expression level of p-STAT5 increased. Among them, in H9.2.1-IL-12-M7CRin with an inactivating mutation of M7R, the expression level of p-STAT5 did not increase. The above results show that in CAR-T M7R in cells can continuously provide activation signals and activate downstream signaling pathways.
实施例5、Qufikit定量分析细胞表面抗原分子数Example 5. Qufikit quantitative analysis of the number of cell surface antigen molecules
用饱和浓度的小鼠抗人CLDN18抗体与细胞系(SNU-601high，SNU-601low，和DAN-G18.2细胞系购自南京科佰生物科技公司)进行孵育，随后qufikit试剂盒(Agilent company)定量测量细胞表面上CLDN18的抗原数目。Mouse anti-human CLDN18 antibody at saturating concentrations was incubated with cell lines (SNU-601 high , SNU-601 low , and DAN-G18.2 cell lines purchased from Nanjing Kebai Biotechnology Co., Ltd.), followed by qufikit kit (Agilent company) quantitatively measured the number of CLDN18 antigens on the cell surface.
具体而言，取DAN-G18.2和SNU-601细胞(1E5)于96孔V底板中，丢弃上清。随后添加FACS缓冲液进行重悬洗涤，再次离心去除上清。使用FACS缓冲液配置饱和的小鼠抗人CLDN18.2混合液，于孔内添加100μL的上述混合液。于孔内添加100μL的FACS缓冲液。4℃避光孵育30min。300g离心5min，弃上清。对qufikit试剂盒中的标准品的孔分别添加震荡混匀后的40ul beads(Vial 1和Vial 2)，300g离心5min，弃上清。使用FACS缓冲液配制二抗(Alexa488AffiniPure Goat抗Mouse IgG,F(ab')2fragment specific,1:100),于每个孔内添加100μL的二抗染料，4℃避光孵育30min。使用FACS缓冲液重悬，300g离心5min，丢弃上清。添加FACS缓冲液150μL进行重悬，上机FACS分析。Specifically, DAN-G18.2 and SNU-601 cells (1E5) were placed in a 96-well V-bottom plate, and the supernatant was discarded. Then add FACS buffer for resuspension and washing, and centrifuge again to remove the supernatant. Use FACS buffer to prepare a saturated mouse anti-human CLDN18.2 mixture, and add 100 μL of the above mixture into the well. Add 100 μL of FACS buffer to the well. Incubate in the dark for 30 minutes at 4°C. Centrifuge at 300g for 5 minutes and discard the supernatant. Add 40ul of beads (Vial 1 and Vial 2) after shaking and mixing to the standard wells in the qufikit kit, centrifuge at 300g for 5 minutes, and discard the supernatant. Use FACS buffer to prepare secondary antibodies (Alexa 488AffiniPure Goat anti-Mouse IgG, F(ab')2 fragment specific, 1:100), add 100 μL of secondary antibody dye to each well, and incubate at 4°C in the dark for 30 minutes. Resuspend in FACS buffer, centrifuge at 300g for 5 minutes, and discard the supernatant. Add 150 μL of FACS buffer to resuspend, and run FACS analysis on the machine.
如图10A所示，SNU-601high细胞，SNU-601low细胞，和DAN-G18.2细胞表面CLDN18的表达量具有差异，其中DAN-G18.2的表达水平最高。As shown in Figure 10A, there are differences in the expression levels of CLDN18 on the surface of SNU-601 high cells, SNU-601 low cells, and DAN-G18.2 cells, among which DAN-G18.2 has the highest expression level.
表2
Table 2
如表2所示，通过Qufikit定量分析计算细胞表面分子数量，SNU-601high，SNU-601low，和DANG-18.2high表面CLDN18分子数量分别为：87427、24360和655891个。As shown in Table 2, the number of cell surface molecules was calculated through Qufikit quantitative analysis. The numbers of CLDN18 molecules on the surface of SNU-601high, SNU-601low, and DANG-18.2high were: 87427, 24360, and 655891, respectively.
通过抗体标记不同靶细胞表面的CLDN18.2，然后用流式细胞术检测靶细胞表面CLDN18.2的表达水平。具体而言，用A6抗体与靶细胞4℃共孵育30min；然后FACS缓冲液洗涤一遍后，用APC标记的抗人Fc抗体与细胞4℃共孵育30min；最后用FACS缓冲液洗涤一遍后，上机检测。如图10B所示，峰状图表示每种细胞中CLDN18.2的表达量，通过计算MFI对表达量进行定量。其中，DANG18.2，NUGC-4细胞中CLDN18.2表达量较高，SNU-601和Hup-T4为中表达，SNU-620和PANC-1为低表达，ISO为同型抗体对照，K562为CLDN18.2阴性对照细胞。Antibodies were used to label CLDN18.2 on the surface of different target cells, and then flow cytometry was used to detect the expression level of CLDN18.2 on the target cell surface. Specifically, the A6 antibody was used to incubate the target cells with the target cells for 30 minutes at 4°C; then, after washing once with FACS buffer, the cells were incubated with the APC-labeled anti-human Fc antibody for 30 minutes at 4°C; finally, after washing once with FACS buffer, the cells were plated. Machine detection. As shown in Figure 10B, the peak diagram represents the expression level of CLDN18.2 in each cell type, and the expression level was quantified by calculating the MFI. Among them, CLDN18.2 expression is higher in DANG18.2 and NUGC-4 cells, SNU-601 and Hup-T4 are medium expression, SNU-620 and PANC-1 are low expression, ISO is the isotype antibody control, and K562 is CLDN18 .2 Negative control cells.
实施例6、体外动态杀伤实验Example 6. In vitro dynamic killing experiment
用xCELLigence RTCA MP仪器(Agilent company)动态实时检测CAR-T细胞对靶细胞的杀伤情况。在E-Plates板中添加50μL培养基，仪器读取基准值后，添加50μL肿瘤靶细胞，然后置于机器内对细胞生长情况进行动态监测。复苏实施例4.3制备的UNT细胞以及CAR-T细胞(T细胞来自供者5，供者13的PBMC细胞)，置于37℃细胞培养箱中过夜。次日，按实验需要的E:T比例将CAR-T加入对应组别E-Plates孔中，同时，针对HuR968B CAR-T细胞及M7CR修饰的HuR968B CAR-T细胞所对应的孔内添加含P329G突变A6抗体，利用P329G突变A6抗体的VH/VL结构域结合肿瘤靶细胞、Fc端P329G突变结合HuR968B CAR-T细胞的胞外结合区域，从而激活HuR968B CAR-T细胞或M7CR修饰的HuR968B CAR-T细 胞的靶向肿瘤杀伤功能。xCELLigence RTCA MP仪器系统动态监测CAR-T细胞对靶细胞杀伤情况，持续48-96小时。The xCELLigence RTCA MP instrument (Agilent company) was used to dynamically detect the killing of target cells by CAR-T cells in real time. Add 50 μL of culture medium to the E-Plates plate. After the instrument reads the baseline value, add 50 μL of tumor target cells, and then place them in the machine to dynamically monitor the cell growth. Resuscitate the UNT cells and CAR-T cells prepared in Example 4.3 (T cells are from PBMC cells of donor 5 and donor 13), and place them in a 37°C cell culture incubator overnight. The next day, add CAR-T into the E-Plates wells of the corresponding group according to the E:T ratio required for the experiment. At the same time, add P329G into the wells corresponding to HuR968B CAR-T cells and M7CR-modified HuR968B CAR-T cells. Mutated A6 antibody uses the VH/VL domain of the P329G mutated A6 antibody to bind to tumor target cells and the Fc-terminal P329G mutation to bind to the extracellular binding region of HuR968B CAR-T cells, thereby activating HuR968B CAR-T cells or M7CR-modified HuR968B CAR- T thin Targeted tumor killing function of cells. The xCELLigence RTCA MP instrument system dynamically monitors the killing of target cells by CAR-T cells for 48-96 hours.
如图11A所示，分别用H9.1.2 CAR-T细胞或不同M7CR修饰的H9.1.2 CAR-T细胞与肿瘤靶细胞DAN-G18.2共孵育，在E:T分别为1:1、1:3、1:10时，tCD19-M7CR修饰的H9.1.2 CAR-T细胞的杀伤作用相当且均优于H9.1.2 CAR-T细胞，其它组中CAR-T细胞的杀伤作用和H9.1.2 CAR-T细胞基本上相当。As shown in Figure 11A, H9.1.2 CAR-T cells or H9.1.2 CAR-T cells modified with different M7CR were co-incubated with the tumor target cell DAN-G18.2. The E:T ratio was 1:1 and 1 respectively. :3. At 1:10, the killing effect of tCD19-M7CR modified H9.1.2 CAR-T cells is equivalent and better than that of H9.1.2 CAR-T cells. The killing effect of CAR-T cells in other groups is similar to that of H9.1.2 CAR-T cells are basically equivalent.
如图11B所示，在E:T分别为1:1、1:3、1:10时，4-1BBL-M7CR修饰的H9.1.2 CAR-T细胞的杀伤作用强于tCD19-M7CR修饰的H9.1.2 CAR-T细胞。As shown in Figure 11B, when E:T are 1:1, 1:3, and 1:10 respectively, the killing effect of 4-1BBL-M7CR modified H9.1.2 CAR-T cells is stronger than that of tCD19-M7CR modified H9 .1.2 CAR-T cells.
如图11C所示，在E:T分别为1:1、1:3、1:10时，抗PD-L1VHH-M7CR修饰的H9.1.2 CAR-T细胞的杀伤作用均强于tCD19-M7CR修饰的H9.1.2 CAR-T细胞和未修饰的H9.1.2 CAR-T细胞，且这种优势随E:T比例的降低而更明显。说明抗PD-L1VHH和M7R组合修饰使得经修饰的H9.1.2 CAR-T细胞对靶细胞的杀伤效果具有协同促进的作用。As shown in Figure 11C, when E:T were 1:1, 1:3, and 1:10 respectively, the killing effect of anti-PD-L1 VHH -M7CR-modified H9.1.2 CAR-T cells was stronger than that of tCD19-M7CR Modified H9.1.2 CAR-T cells and unmodified H9.1.2 CAR-T cells, and this advantage becomes more obvious as the E:T ratio decreases. This shows that the combined modification of anti-PD-L1 VHH and M7R enables the modified H9.1.2 CAR-T cells to synergistically promote the killing effect of target cells.
如图11D所示，用HuR968B CAR-T细胞和M7CR修饰的HuR968B CAR-T细胞和A6抗体(2nM)及靶细胞SUN-601high或SUN-601low共孵育(E:T＝1)。IL-12-M7CR修饰的HuR968B CAR-T细胞对SNU-601high和SNU-601low的杀伤作用优于未修饰的HuR968B CAR-T、tCD19-M7CR修饰的HuR968B CAR-T细胞及其它M7CR修饰的HuR968B CAR-T细胞，且这种效果在SNU-601low中更明显。另外，IL-15/IL-15Rα-M7CR(图11D中标记为IL-15-M7CR)修饰的HuR968B CAR-T细胞对SNU-601low的杀伤作用略优于未修饰的HuR968B CAR-T细胞和tCD19-M7CR修饰的HuR968B CAR-T细胞。As shown in Figure 11D, HuR968B CAR-T cells and M7CR-modified HuR968B CAR-T cells were co-incubated with A6 antibody (2nM) and target cells SUN-601 high or SUN-601 low (E:T=1). The killing effect of IL-12-M7CR modified HuR968B CAR-T cells on SNU-601 high and SNU-601 low is better than that of unmodified HuR968B CAR-T, tCD19-M7CR modified HuR968B CAR-T cells and other M7CR modified cells. HuR968B CAR-T cells, and this effect is more obvious in SNU-601 low . In addition, the killing effect of IL-15/IL-15Rα-M7CR (labeled IL-15-M7CR in Figure 11D)-modified HuR968B CAR-T cells on SNU-601 low was slightly better than that of unmodified HuR968B CAR-T cells and tCD19-M7CR modified HuR968B CAR-T cells.
实施例7、体外肿瘤细胞反复刺激实验Example 7. Experiment on repeated stimulation of tumor cells in vitro
第0天将SNU-601high细胞(2.5E5)加入24孔板中，待其贴壁过夜，同时复苏实施例4.3制备的不同组别HuR968B CAR-T细胞(使用供者13的PBMC细胞)。第1天调整CAR-T细胞的CAR阳性率并使其一致，然后将以E:T＝2向每孔中分别加入不同组别HuR968B CAR-T细胞，并加入含P329G突变A6抗体，使其最终浓度为2nM。第2天，收集100μL上清用于细胞因子检测实验。第3天，第5天对每组进行半量换液。第7天将SNU-601high细胞(2×105)加入一块新的24孔板中(记为下一轮刺激的第0天)，通过流式细胞术检测每组中CAR-T细胞的比例及表型，通过细胞计数仪检测T细胞数量。第8天取出5E5个CAR-T细胞加入新的24孔板中(记为下一轮刺激的第1天)。每轮刺激结束，增殖倍数(CAR-T细胞)＝CAR-T细胞数(第7天)/5E5，累计增殖倍数＝增殖倍数Round1×增值倍数Round2×…。On day 0, SNU-601 high cells (2.5E5) were added to the 24-well plate and allowed to adhere overnight. At the same time, different groups of HuR968B CAR-T cells prepared in Example 4.3 (using PBMC cells from donor 13) were recovered. On the first day, adjust the CAR positive rate of CAR-T cells to make it consistent, then add different groups of HuR968B CAR-T cells to each well at E:T=2, and add the P329G mutant A6 antibody to make it The final concentration is 2nM. On the second day, 100 μL of supernatant was collected for cytokine detection experiments. On the 3rd and 5th day, half of the medium was changed in each group. On day 7, SNU-601 high cells (2×10 5 ) were added to a new 24-well plate (recorded as day 0 of the next round of stimulation), and the number of CAR-T cells in each group was detected by flow cytometry. Proportion and phenotype, and the number of T cells was detected by a cell counter. On day 8, 5E5 CAR-T cells were taken out and added to a new 24-well plate (recorded as day 1 of the next round of stimulation). At the end of each round of stimulation, proliferation multiple (CAR-T cells) = number of CAR-T cells (day 7)/5E5, cumulative proliferation multiple = proliferation multiple Round1 × proliferation multiple Round2 ×….
如图12A所示，前7天，所有组中的HuR968B CAR-T细胞在靶细胞的刺激下增殖，第7天至第21天，IL-15/IL-15Rα-M7CR(图12A中标记为IL-15-M7CR)和IL-12-M7CR修饰的组中HuR968B CAR-T细胞持续扩增，增殖倍数均高于未修饰的HuR968B CAR-T细胞组和tCD19-M7CR修饰的HuR968BCAR-T细胞。其中，IL-12-M7CR修饰的组中的CAR-T细胞经过21天的刺激后累计扩增倍数最高(约44倍)。以上表明在靶细胞反复刺激下，IL-12-M7CR修饰能够提高CAR-T细胞的增殖能力。As shown in Figure 12A, in the first 7 days, HuR968B CAR-T cells in all groups proliferated under the stimulation of target cells, and from days 7 to 21, IL-15/IL-15Rα-M7CR (marked in Figure 12A The HuR968B CAR-T cells in the IL-15-M7CR) and IL-12-M7CR modified groups continued to expand, and the proliferation multiples were higher than those in the unmodified HuR968B CAR-T cell group and tCD19-M7CR modified HuR968BCAR-T cells. Among them, the CAR-T cells in the IL-12-M7CR modified group had the highest cumulative expansion fold after 21 days of stimulation (approximately 44 times). The above shows that IL-12-M7CR modification can improve the proliferation ability of CAR-T cells under repeated stimulation of target cells.
如图12B所示，在经过靶细胞多轮刺激后，第14天时HuR968B CAR-T细胞中的CAR+细胞比例为53.3％，和第1天时相比略有上升。而经修饰的HuR968B CAR-T细胞中CAR+细 胞的比例显著上升，在第21天时tCD19-M7CR、tCD19-M7CR(CPT)、IL15-M7CR、IL-12-M7CR和IL-21-M7CR修饰的组中CAR+细胞比例分别是：76.5％、78.6％、90.1％、79.52％、51.39％。As shown in Figure 12B, after multiple rounds of stimulation of target cells, the proportion of CAR + cells in HuR968B CAR-T cells was 53.3% on day 14, which was slightly higher than that on day 1. However, CAR + cells in modified HuR968B CAR-T cells The proportion of CAR + cells increased significantly. On day 21, the proportion of CAR + cells in the tCD19-M7CR, tCD19-M7CR (CPT), IL15-M7CR, IL-12-M7CR and IL-21-M7CR modified groups were respectively: 76.5%, 78.6%, 90.1%, 79.52%, 51.39%.
如图12C和图12D所示，经过靶细胞刺激后，第7天时经4-1BBL-M7CR、CD40L-M7CR和tCD19-M7CR修饰过的CAR-T细胞增殖倍数显著高于HuR968B CAR-T细胞。第7天至第14天，经过靶细胞刺激后所有组中CAR-T细胞的增殖能力均开始下降。As shown in Figure 12C and Figure 12D, after target cell stimulation, the proliferation fold of CAR-T cells modified with 4-1BBL-M7CR, CD40L-M7CR and tCD19-M7CR was significantly higher than that of HuR968B CAR-T cells on day 7. From day 7 to day 14, the proliferation ability of CAR-T cells in all groups began to decline after target cell stimulation.
如图12E和图12F所示，在经过靶细胞多轮刺激后，抗PD-L1VHH-M7CR修饰的HuR968B CAR-T细胞增殖倍数略高于tCD19-M7CR修饰的HuR968B CAR-T细胞和未经修饰的HuR968B CAR-T细胞。第14天时HuR968B CAR-T细胞中的CAR+比例为53.3％，和第1天时相比略有上升。而经过tCD19-M7CR、CD40L-M7CR、4-1BBl-M7CR和抗PD-L1VHH-M7CR修饰的HuR968B CAR-T细胞中CAR+比例显著上升，至第14天时CAR+的比例分别为：77.4％、71.8％、66.3％、75.1％。As shown in Figure 12E and Figure 12F, after multiple rounds of stimulation of target cells, the proliferation fold of anti-PD-L1 VHH -M7CR-modified HuR968B CAR-T cells was slightly higher than that of tCD19-M7CR-modified HuR968B CAR-T cells and those without. Modified HuR968B CAR-T cells. The CAR + proportion in HuR968B CAR-T cells was 53.3% on day 14, which was slightly higher than that on day 1. The proportion of CAR + in HuR968B CAR-T cells modified by tCD19-M7CR, CD40L-M7CR, 4-1BBl-M7CR and anti-PD-L1 VHH -M7CR increased significantly. By day 14, the proportion of CAR + was: 77.4%. , 71.8%, 66.3%, 75.1%.
图13A显示了图12A－图12F中经靶细胞第一轮和第三轮刺激后，各组中CD4+和CD8+T细胞数的代表性流式细胞术测定结果图。如图13A所示，实验起始时各组中CD4+和CD8+T细胞的比例基本维持在1:2。经过多轮实验后，IL-12-M7CR修饰能够提高CD4+和CD8+T细胞的比例(约为1:1)，而其它组中CD8+T细胞比例显著增加，CD4+T细胞比例明显降低。表明在靶细胞的刺激下，IL-12-M7CR修饰能够促进CD4+T细胞的扩增。Figure 13A shows representative flow cytometry results of the numbers of CD4 + and CD8 + T cells in each group after the first and third rounds of stimulation of target cells in Figures 12A to 12F. As shown in Figure 13A, the ratio of CD4 + and CD8 + T cells in each group was basically maintained at 1:2 at the beginning of the experiment. After multiple rounds of experiments, IL-12-M7CR modification was able to increase the ratio of CD4 + and CD8 + T cells (approximately 1:1), while the proportion of CD8 + T cells in other groups increased significantly and the proportion of CD4 + T cells decreased significantly. . It shows that IL-12-M7CR modification can promote the expansion of CD4 + T cells under the stimulation of target cells.
图13B显示了对图13A中各组中CD4+和CD8+T细胞的比例的统计结果。如图所示，经过多轮刺激后，IL-12-M7CR修饰能够提高CD4+和CD8+T细胞的比例至1：1左右，而其它组中CD8+T细胞比例显著增加，CD4+T细胞比例明显降低。Figure 13B shows the statistical results of the proportion of CD4 + and CD8 + T cells in each group in Figure 13A. As shown in the figure, after multiple rounds of stimulation, IL-12-M7CR modification can increase the ratio of CD4 + and CD8 + T cells to about 1:1, while the ratio of CD8 + T cells in other groups increased significantly, and the ratio of CD4 + T cells increased significantly. The ratio is significantly reduced.
实施例8、细胞因子检测实验Example 8. Cytokine detection experiment
使用BDTM Cytometric Bead Array(CBA)Human Th1/Th2 Cytokine Kit II对细胞因子进行检测。等体积混匀Capture Beads，以25μL/孔进行铺板。添加等体积的体外肿瘤细胞反复刺激实验中的上清液或上清稀释液或标准品。混匀后添加25μL等体积的人Th1/Th2PE检测试剂，室温避光孵育3h。使用洗涤缓冲液洗涤两次后重悬，通过流式细胞仪PE通道MFI值计算细胞因子浓度。Cytokines were detected using BD TM Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit II. Mix the Capture Beads in an equal volume and plate at 25 μL/well. Add an equal volume of supernatant or supernatant dilution or standard from the repeated stimulation experiment with in vitro tumor cells. After mixing, add 25 μL of equal volume of human Th1/Th2PE detection reagent and incubate at room temperature in the dark for 3 hours. Wash twice with wash buffer and resuspend, and calculate the cytokine concentration through the MFI value of the PE channel of the flow cytometer.
如图13C所示，通过CBA检测第1轮刺激时效应细胞加入后24h上清中IL-2、IFN-γ和TNF细胞因子的水平。发现IL-15/IL-15Rα-M7CR(图13C中标记为IL-15-M7CR)修饰组(＜2000pg/mL)上清中的IL-2水平最低，IL-12-M7CR修饰和tCD19-M7CR(CPT)修饰组(2000-4000pg/mL)次之，未修饰的HuR968B CAR组，tCD19-M7CR、IL-21-M7CR、CD40L-M7CR、4-1BBl-M7CR和αPD-L1VHH-M7CR修饰的HuR968B CAR组(＞6000pg/mL)较高。IL-12-M7CR修饰组(＞10000pg/mL)上清中IFN-γ水平最高，其它组上清中IFN-γ水平均低于5000pg/mL。IL-12-M7CR修饰组(＞1000pg/mL)上清中TNF的水平最高，其它组上清中TNF水平均处于较低水平。As shown in Figure 13C, the levels of IL-2, IFN-γ, and TNF cytokines in the supernatant 24 h after the addition of effector cells in the first round of stimulation were detected by CBA. It was found that the IL-2 level in the supernatant of the IL-15/IL-15Rα-M7CR (labeled IL-15-M7CR in Figure 13C) modified group (<2000pg/mL) was the lowest, IL-12-M7CR modified and tCD19-M7CR (CPT) modified group (2000-4000pg/mL) followed by unmodified HuR968B CAR group, tCD19-M7CR, IL-21-M7CR, CD40L-M7CR, 4-1BBl-M7CR and αPD-L1VHH-M7CR modified HuR968B The CAR group (>6000pg/mL) was higher. The level of IFN-γ in the supernatant of the IL-12-M7CR modified group (>10000pg/mL) was the highest, while the levels of IFN-γ in the supernatant of other groups were all lower than 5000pg/mL. The level of TNF in the supernatant of the IL-12-M7CR modified group (>1000pg/mL) was the highest, while the TNF levels in the supernatant of other groups were all at lower levels.
实施例9、IL-12-M7CR/IL-15-M7CR修饰的H9.2.1 CAR-T细胞的体外杀伤作用Example 9. In vitro killing effect of IL-12-M7CR/IL-15-M7CR modified H9.2.1 CAR-T cells
进一步地，针对不同Claudin18.2表达水平的靶细胞实施了体外杀伤实验，研究IL-12-M7CR/IL-15-M7CR修饰的H9.2.1 CAR-T在体外对靶细胞的杀伤作用。Furthermore, an in vitro killing experiment was conducted on target cells with different Claudin18.2 expression levels to study the killing effect of IL-12-M7CR/IL-15-M7CR modified H9.2.1 CAR-T on target cells in vitro.
用xCELLigence RTCA MP仪器(Agilent company)动态实时检测CAR-T细胞对靶细胞 的杀伤情况。实验方法如前面所述。选取CLDN18.2表达水平不同的细胞系作为靶细胞。其中PANC-1为CLDN18.2低表达细胞系，SNU-601和Hup-T4是中高表达细胞系，DAN-G18.2为高表达细胞系。使用来自供者15和供者17的PBMC制备CAR-T细胞。其中，H9.2.1-IL12-M7CR表示IL-12-P70修饰的H9.2.1 CAR-T细胞；H9.2.1in-IL12-M7CR表示H9.2.1CAR-T中的4-1BB共刺激结构域和CD3ζ信号传导结构域缺失，进而达到CAR结构功能缺失的目的；H9.2.1-IL12-M7CRin表示将M7CR胞内Box1结构域缺失，且引入Y449F，Y456F(以IL7Rα(P16871-1)为参照)突变进而达到失活M7CR胞内结构功能的目的；H9.2.1-sIL12表示H9.2.1 CAR-T细胞和可溶性IL12的组合。如图14A－图14D所示，在CLDN18.2的不同表达水平的靶细胞中，CAR-T细胞的杀伤作用随CLDN18.2的表达水平升高而增强，说明H9.2.1 CAR-T细胞的杀伤作用具有抗原依赖性。在不同效靶比下CAR-T细胞的杀伤能力从低到高依次为H9.2.1in-IL-12-M7CR CAR-T，其次为H9.2.1 CAR-T细胞，tCD19-M7CR修饰的H9.2.1 CAR-T细胞和H9.2.1-IL-12-M7CRin CAR-T细胞、H9.2.1-sIL12 CAR-T细胞相当，IL-12-M7CR修饰的H9.2.1 CAR-T细胞杀伤作用最强。说明IL-12-P70和M7R在促进CAR-T细胞的杀伤作用上具有联合效应。Dynamic real-time detection of CAR-T cells against target cells using xCELLigence RTCA MP instrument (Agilent company) The killing situation. The experimental method was as described previously. Cell lines with different expression levels of CLDN18.2 were selected as target cells. Among them, PANC-1 is a low-expression cell line of CLDN18.2, SNU-601 and Hup-T4 are medium-high-expression cell lines, and DAN-G18.2 is a high-expression cell line. CAR-T cells were prepared using PBMC from donor 15 and donor 17. Among them, H9.2.1-IL12-M7CR represents IL-12-P70 modified H9.2.1 CAR-T cells; H9.2.1in-IL12-M7CR represents the 4-1BB costimulatory domain and The CD3ζ signaling domain is deleted, thereby achieving the purpose of loss of CAR structure and function; H9.2.1-IL12-M7CRin means that the M7CR intracellular Box1 domain is deleted, and Y449F, Y456F (using IL7Rα (P16871-1) as a reference) mutation is introduced To achieve the purpose of inactivating the intracellular structure and function of M7CR; H9.2.1-sIL12 represents the combination of H9.2.1 CAR-T cells and soluble IL12. As shown in Figure 14A-Figure 14D, in target cells with different expression levels of CLDN18.2, the killing effect of CAR-T cells increases with the increase in the expression level of CLDN18.2, indicating that the effect of H9.2.1 CAR-T cells The killing effect is antigen-dependent. The killing ability of CAR-T cells under different efficacy-target ratios from low to high is H9.2.1in-IL-12-M7CR CAR-T, followed by H9.2.1 CAR-T cells, and tCD19-M7CR modified H9. 2.1 CAR-T cells are equivalent to H9.2.1-IL-12-M7CRin CAR-T cells and H9.2.1-sIL12 CAR-T cells, and IL-12-M7CR-modified H9.2.1 CAR-T cells have the strongest killing effect. This shows that IL-12-P70 and M7R have a combined effect in promoting the killing effect of CAR-T cells.
类似地，使用H9.2.1 CAR-T细胞实施了对靶细胞Hup-T4的反复杀伤实验。Similarly, repeated killing experiments on target cells Hup-T4 were performed using H9.2.1 CAR-T cells.
用Xcelligence仪器检测CAR-T细胞对靶细胞Hup-T4的杀伤作用。具体实验步骤如下，day0将靶细胞Hup-T4(4×104个/孔)加入Eplate中，放入Xcelligence仪器过夜。待Cell index升至1左右，进行实验。所述Cell index是Xcelligence仪器的读出(read out)，对于使用该型号仪器进行杀伤实验，用cell index数值来表示活细胞量为通用标准。按E：T＝1：1和E：T＝1：5，向孔中加入不同组别的CAR-T细胞，PC组中加入裂解缓冲液，UNT组中加入未转染CAR的来自同一供者的T细胞。当Cell index稳定不再降低时，结束本轮杀伤。然后将Eplate取出，离心去除上清，用新鲜RPMI 1640完全培养基重悬细胞，加入至前一天铺好的含有靶细胞的Eplate中，进行第二轮实验。共进行三轮实验。如图14E所示，持续三轮杀伤实验后，IL-12-M7CR修饰的CAR-T细胞仍然具有较好的杀伤作用，而未经修饰的H9.2.1CAR-T细胞在多轮杀伤中，随轮数增加杀伤效果逐渐减弱。以上结果说明，在反复杀伤实验中IL-12-M7CR分子能够提高H9.2.1 CAR-T细胞的杀伤能力，及持续杀伤的作用。The Xcelligence instrument was used to detect the killing effect of CAR-T cells on the target cell Hup-T4. The specific experimental steps are as follows. On day0, add the target cells Hup-T4 (4×10 4 cells/well) to the Eplate and place it in the Xcelligence instrument overnight. After the Cell index rises to about 1, conduct the experiment. The Cell index is the read out of the Xcelligence instrument. For killing experiments using this type of instrument, the cell index value is used to represent the amount of viable cells as a universal standard. According to E:T=1:1 and E:T=1:5, add different groups of CAR-T cells into the wells, add lysis buffer to the PC group, and add untransfected CAR cells from the same donor to the UNT group. of the patient’s T cells. When the Cell index is stable and no longer decreases, the killing round ends. Then remove the Eplate, centrifuge to remove the supernatant, resuspend the cells in fresh RPMI 1640 complete medium, and add them to the Eplate containing target cells spread the day before to conduct the second round of experiments. A total of three rounds of experiments were conducted. As shown in Figure 14E, after three rounds of killing experiments, the IL-12-M7CR-modified CAR-T cells still had a good killing effect, while the unmodified H9.2.1 CAR-T cells failed in multiple rounds of killing. As the number of rounds increases, the killing effect gradually weakens. The above results show that IL-12-M7CR molecule can improve the killing ability and sustained killing effect of H9.2.1 CAR-T cells in repeated killing experiments.
实施例10、表达组成型嵌合细胞因子受体的CAR-T细胞在小鼠体内的抗肿瘤作用Example 10. Anti-tumor effect of CAR-T cells expressing constitutive chimeric cytokine receptors in mice
通过动物实验，研究了表达组成型嵌合细胞因子受体的CAR-T细胞在小鼠体内的抗肿瘤作用，检测是否IL-12-M7CR分子能够促进PG CAR-T细胞在体内的抗肿瘤作用及增殖能力。实验方法具体如下，选用NOG小鼠(购自维通利华)，Day-7给小鼠腹腔注射NUGC-4-Gluc细胞(每只1×106)进行造模，通过小动物活体成像系统对小鼠造模情况进行检测，至Day0时肿瘤负荷为1×109p/s(为IVIS成像系统中对于肿瘤产生光子数的计算值和单位)时对动物进行分组(每组5只)。然后通过尾静脉注射给予每只小鼠5×105个CAR-T细胞，UNT组给予的UNT细胞数量和CAR阳性率最低组小鼠输注的总T细胞数量一致。每周对小鼠负荷进行成像，外周血中CAR-T细胞的数量。Through animal experiments, we studied the anti-tumor effect of CAR-T cells expressing constitutive chimeric cytokine receptors in mice, and tested whether IL-12-M7CR molecules can promote the anti-tumor effect of PG CAR-T cells in vivo. and proliferation ability. The specific experimental method is as follows. NOG mice (purchased from Viton Lever) were selected. On Day-7, the mice were intraperitoneally injected with NUGC-4-Gluc cells (1 × 10 6 each) to create a model. Through the small animal in vivo imaging system The mouse modeling conditions were tested, and the animals were divided into groups (5 animals in each group) when the tumor burden was 1×10 9 p/s (the calculated value and unit for the number of photons generated by the tumor in the IVIS imaging system) on Day 0. . Each mouse was then given 5 × 10 5 CAR-T cells via tail vein injection. The number of UNT cells given to the UNT group was consistent with the total number of T cells infused into the mice with the lowest CAR positivity rate. Mice burden, and the number of CAR-T cells in peripheral blood, were imaged weekly.
如图15及图16所示，将基于PG CAR-T细胞(HuR968B CAR-T细胞)构建的表达M7CR的CAR-T细胞，通过尾静脉注射进造模小鼠体内，同时注射0.3mg/kg含P329G突变A6抗体，通过小动物活体成像发现，随时间增加IL-12-M7CR和tCD19-M7CR修饰的PG CAR-T 细胞在体内具有更好的抗肿瘤作用，且如图17所示，在第7天至第28天PG CAR-T细胞具有较高的扩增水平，而未经修饰的PG CAR-T细胞在体内具有较弱的抗肿瘤作用及较差的扩增能力。以上体内结果说明，M7CR修饰在体内具有促进PG CAR-T细胞扩增和提高CAR-T细胞抗肿瘤的作用。As shown in Figure 15 and Figure 16, the M7CR-expressing CAR-T cells constructed based on PG CAR-T cells (HuR968B CAR-T cells) were injected into the model mice through the tail vein, and 0.3 mg/kg was injected at the same time. Antibody containing P329G mutation A6, found through small animal in vivo imaging, increased IL-12-M7CR and tCD19-M7CR modified PG CAR-T over time The cells have better anti-tumor effects in vivo, and as shown in Figure 17, PG CAR-T cells have higher expansion levels from days 7 to 28, while unmodified PG CAR-T cells have It has weak anti-tumor effect and poor amplification ability in vivo. The above in vivo results show that M7CR modification can promote the expansion of PG CAR-T cells and improve the anti-tumor effect of CAR-T cells in vivo.
如图18所示，将基于传统CAR-T细胞(H9.2.1 CAR-T细胞，下文中示例的H9.2.1 CAR序列如SEQ ID NO:100所示)构建的表达M7CR的CAR-T细胞通过尾静脉注射进造模小鼠体内，通过小动物活体成像发现，随时间增加，IL-12-M7CR和tCD19-M7CR修饰的CAR-T细胞在体内具有更好的抗肿瘤作用，且在Day13时，IL-12-M7CR修饰的CAR-T细胞能够使得造模小鼠体内肿瘤完全消除。如图19所示，该图是对IVIS影像的量化统计，同样可以发现，与UNT组相比，H9.2.1 CAR-T细胞、M7R H9.2.1 CAR-T细胞和IL-12-M7CR CAR-T细胞在造模小鼠体内具有较好的抗肿瘤作用。其中，M7R修饰能够提高H9.2.1 CAR-T细胞的抗肿瘤作用，IL-12修饰能够进一步提高M7R H9.2.1 CAR-T细胞的抗肿瘤作用。如图20所示，通过流式细胞术检测小鼠外周血中总人T细胞和CAR-T细胞的数量，在一次注射CAR-T细胞后，随着时间增加，除UNT组外，其它组别中总T细胞和CAR-T细胞均增加，其中IL-12-M7CR组中CAR-T细胞扩增数量最多，其次为M7R组，最后为H9.2.1组。以上体内结果表明，M7CR修饰在体内具有促进传统CAR-T细胞扩增和提高CAR T细胞抗肿瘤的作用。As shown in Figure 18, the M7CR-expressing CAR-T cells constructed based on traditional CAR-T cells (H9.2.1 CAR-T cells, the H9.2.1 CAR sequence example below is shown in SEQ ID NO: 100) are passed After tail vein injection into model mice, it was found through small animal in vivo imaging that IL-12-M7CR and tCD19-M7CR-modified CAR-T cells had better anti-tumor effects in vivo as time went by, and on Day 13 , IL-12-M7CR modified CAR-T cells can completely eliminate tumors in model mice. As shown in Figure 19, this figure is a quantitative statistics of IVIS images. It can also be found that compared with the UNT group, H9.2.1 CAR-T cells, M7R H9.2.1 CAR-T cells and IL-12-M7CR CAR- T cells have good anti-tumor effects in model mice. Among them, M7R modification can improve the anti-tumor effect of H9.2.1 CAR-T cells, and IL-12 modification can further improve the anti-tumor effect of M7R H9.2.1 CAR-T cells. As shown in Figure 20, the number of total human T cells and CAR-T cells in the peripheral blood of mice was detected by flow cytometry. After a single injection of CAR-T cells, the number increased with time. Except for the UNT group, the number of other groups Both total T cells and CAR-T cells increased in each group, with the largest number of CAR-T cell expansions in the IL-12-M7CR group, followed by the M7R group, and finally the H9.2.1 group. The above in vivo results show that M7CR modification can promote the expansion of traditional CAR-T cells and improve the anti-tumor effect of CAR T cells in vivo.
实施例11、构建M7CR修饰的CARExample 11. Construction of M7CR modified CAR
如图21所示，将不同分子的胞外结构域(ECD)和M7Rm8(SEQ ID NO：34)直接连接构建组成型嵌合细胞因子受体M7CRm8；然后通过P2A将M7CRm8的N端与H9.2.1-218 CAR(SEQ ID NO：144)或H9.2.1-28 CAR(SEQ ID NO：142)或H9.2.1 CAR(SEQ ID NO：100)多肽C端连接，从而构成M7CR修饰的CAR。所构建的H9.2.1-tCD19-M7CR的序列如SEQ ID NO：101所示，所构建的H9.2.1-IL-12-M7CR的序列如SEQ ID NO：104所示，所构建的H9.2.1-IL-15-M7CRin的序列如SEQ ID NO：135所示，所构建的H9.2.1in-IL-15-M7CR的序列如SEQ ID NO：134所示，所构建的H9.2.1-IL-15-M7CR的序列如SEQ ID NO：136所示，所构建的H9.2.1-28-IL-15-M7CR的序列如SEQ ID NO：137所示。As shown in Figure 21, the extracellular domain (ECD) of different molecules and M7Rm8 (SEQ ID NO: 34) were directly connected to construct a constitutive chimeric cytokine receptor M7CRm8; then the N-terminus of M7CRm8 was connected to H9 through P2A. The C-terminus of the 2.1-218 CAR (SEQ ID NO: 144) or H9.2.1-28 CAR (SEQ ID NO: 142) or H9.2.1 CAR (SEQ ID NO: 100) polypeptide is connected to form an M7CR-modified CAR. The sequence of the constructed H9.2.1-tCD19-M7CR is shown in SEQ ID NO: 101. The sequence of the constructed H9.2.1-IL-12-M7CR is shown in SEQ ID NO: 104. The constructed H9.2.1 -The sequence of IL-15-M7CRin is shown in SEQ ID NO: 135. The sequence of the constructed H9.2.1in-IL-15-M7CR is shown in SEQ ID NO: 134. The constructed H9.2.1-IL- The sequence of 15-M7CR is shown in SEQ ID NO: 136, and the sequence of the constructed H9.2.1-28-IL-15-M7CR is shown in SEQ ID NO: 137.
在所述M7CR中，ECD包括tCD19(SEQ ID NO：17)、IL-15/IL-15Rα(Sushi)(SEQ ID NO：48)、IL-12-P70(SEQ ID NO：49)；上述不同ECD和M7Rm8连接后构成tCD19-M7CR(SEQ ID NO：171)、IL-12-M7CR(SEQ ID NO：172)、IL-15-M7CR(SEQ ID NO：173)。同时设计对照分子，包括M7Rm8胞内信号缺失的IL-15-M7CRin(SEQ ID NO：180)、胞内信号缺失的H9.2.1-218in CAR(SEQ ID NO：186)以及分泌型sIL-15的H9.2.1-sIL-15(SEQ ID NO：139)。In the M7CR, ECD includes tCD19 (SEQ ID NO: 17), IL-15/IL-15Rα (Sushi) (SEQ ID NO: 48), IL-12-P70 (SEQ ID NO: 49); the above differences ECD and M7Rm8 are connected to form tCD19-M7CR (SEQ ID NO: 171), IL-12-M7CR (SEQ ID NO: 172), and IL-15-M7CR (SEQ ID NO: 173). At the same time, control molecules were designed, including IL-15-M7CRin (SEQ ID NO: 180) with missing intracellular signal of M7Rm8, H9.2.1-218in CAR (SEQ ID NO: 186) with missing intracellular signal, and secreted sIL-15. H9.2.1-sIL-15 (SEQ ID NO: 139).
实施例12、制备M7CR修饰的CAR-T细胞Example 12. Preparation of M7CR-modified CAR-T cells
实施例12.1.序列合成Example 12.1. Sequence synthesis
人工合成DNA序列，所述DNA序列分别编码H9.2.1 CAR(SEQ ID NO:100)、H9.2.1-218CAR(SEQ ID NO:144)、H9.2.1-28 CAR(SEQ ID NO:142)、H9.2.1-tCD19-M7CR(SEQ ID NO:101)、H9.2.1-IL-15-M7CRin(SEQ ID NO:135)、H9.2.1in-IL-15-M7CR(SEQ ID NO:134)、 H9.2.1-IL-15-M7CR(SEQ ID NO:136)、H9.2.1-sIL-15(SEQ ID NO:139)、H9.2.1-IL-12-M7CR(SEQ ID NO:104)、H9.2.1-28-IL-15-M7CR(SEQ ID NO:137)及对照8E5 CAR(SEQ ID NO:188)。Artificially synthesized DNA sequences encode H9.2.1 CAR (SEQ ID NO: 100), H9.2.1-218 CAR (SEQ ID NO: 144), H9.2.1-28 CAR (SEQ ID NO: 142), H9.2.1-tCD19-M7CR (SEQ ID NO:101), H9.2.1-IL-15-M7CRin (SEQ ID NO:135), H9.2.1in-IL-15-M7CR (SEQ ID NO:134), H9.2.1-IL-15-M7CR (SEQ ID NO:136), H9.2.1-sIL-15 (SEQ ID NO:139), H9.2.1-IL-12-M7CR (SEQ ID NO:104), H9 .2.1-28-IL-15-M7CR (SEQ ID NO:137) and control 8E5 CAR (SEQ ID NO:188).
将合成的DNA片段插入pRKN慢病毒表达载体(金唯智公司)的EF1α启动子下游，替换原载体中EGFR序列，得到相应的表达质粒(由金唯智公司合成)。The synthesized DNA fragment was inserted into the pRKN lentiviral expression vector (Genewise Company) downstream of the EF1α promoter, and the EGFR sequence in the original vector was replaced to obtain the corresponding expression plasmid (synthesized by Genewise Company).
实施例12.2.T细胞的获得及慢病毒转导Example 12.2. Obtaining T cells and lentiviral transduction
慢病毒制备方法同实施例4.2，CAR-T或M7CR修饰的CAR-T细胞制备方法同实施例4.3。使用供者15和供者17的T细胞，如图22所示为CAR-T或M7CR修饰的CAR-T细胞制备第9天的扩增倍数统计柱状图，可以看出不同供者来源的T细胞制备出来的各CAR-T扩增情况存在差异，总体扩增倍数在约20-80倍之间，满足实验需求。The lentivirus preparation method is the same as Example 4.2, and the preparation method of CAR-T or M7CR-modified CAR-T cells is the same as Example 4.3. Using T cells from donor 15 and donor 17, Figure 22 shows the statistical histogram of amplification folds on the 9th day of preparation of CAR-T or M7CR-modified CAR-T cells. It can be seen that T cells derived from different donors The amplification status of each CAR-T prepared from the cells is different, and the overall amplification factor is between about 20-80 times, which meets the experimental needs.
实施例12.3.CAR表达检测及CAR-T细胞表型检测Example 12.3. CAR expression detection and CAR-T cell phenotype detection
使用供者15和供者17的T细胞制备CAR‐T细胞或M7CR修饰的CAR‐T细胞，并进行检测，检测方法同实施例4.4。图23A、图23B显示制备的CAR-T细胞的CAR或M7CR的表达，图23A为代表性流式细胞散点图，图23B为对CAR阳性细胞的统计柱状图。Use the T cells of Donor 15 and Donor 17 to prepare CAR-T cells or M7CR-modified CAR-T cells and perform detection. The detection method is the same as in Example 4.4. Figure 23A and Figure 23B show the expression of CAR or M7CR in the prepared CAR-T cells. Figure 23A is a representative flow cytometry scatter plot, and Figure 23B is a statistical histogram of CAR-positive cells.
结果表明，CAR多肽或M7CR修饰的CAR多肽均能够在各CAR-T细胞中表达；同时采用PE标记的抗p40抗体能够检测到H9.2.1-IL12-M7CR CAR-T细胞表达胞外IL-12；当使用供者15的T细胞转导后，H9.2.1组中CAR+细胞的比例约为56％，而H9.2.1-tCD19-M7CR、H9.2.1-IL-15-M7CRin、H9.2.1in-IL-15-M7CR、H9.2.1-IL-15-M7CR、H9.2.1-28-IL-15-M7CR、H9.2.1-sIL-15、H9.2.1-IL12-M7CR、8E5组中的CAR+细胞的比例分别为：38.6％、7.99％、24.2％、26.1％、34.5％、17.6％、3.17％、59.8％。当使用供者17的T细胞转导后，H9.2.1组中CAR+细胞的比例约为44.3％，而H9.2.1-tCD19-M7CR、H9.2.1-IL-15-M7CRin、H9.2.1in-IL-15-M7CR、H9.2.1-IL-15-M7CR、H9.2.1-28-IL-15-M7CR、H9.2.1-sIL-15、H9.2.1-IL12-M7CR、8E5组中的CAR+细胞的比例分别为：17.7％、5.09％、21.9％、30.5％、33.7％、16.2％、3.89％、54.4％。使用相同的CAR分子转导后，在两个供者来源的细胞中获得的CAR+细胞的阳性率接近。The results show that both CAR polypeptides or M7CR-modified CAR polypeptides can be expressed in each CAR-T cell; at the same time, the PE-labeled anti-p40 antibody can detect the expression of extracellular IL-12 in H9.2.1-IL12-M7CR CAR-T cells. ; When transduced with T cells from donor 15, the proportion of CAR + cells in the H9.2.1 group was approximately 56%, while H9.2.1-tCD19-M7CR, H9.2.1-IL-15-M7CRin, H9.2.1 in-IL-15-M7CR, H9.2.1-IL-15-M7CR, H9.2.1-28-IL-15-M7CR, H9.2.1-sIL-15, H9.2.1-IL12-M7CR, 8E5 group The proportions of CAR + cells were: 38.6%, 7.99%, 24.2%, 26.1%, 34.5%, 17.6%, 3.17%, 59.8%. When transduced with T cells from donor 17, the proportion of CAR + cells in the H9.2.1 group was approximately 44.3%, while H9.2.1-tCD19-M7CR, H9.2.1-IL-15-M7CRin, H9.2.1in -IL-15-M7CR, H9.2.1-IL-15-M7CR, H9.2.1-28-IL-15-M7CR, H9.2.1-sIL-15, H9.2.1-IL12-M7CR, CAR in the 8E5 group The proportions of + cells were: 17.7%, 5.09%, 21.9%, 30.5%, 33.7%, 16.2%, 3.89%, 54.4%. The positivity rate of CAR + cells obtained in cells derived from both donors after transduction with the same CAR molecule was close.
图23C、图23D显示，使用供者15的T细胞转导H9.1.2 CAR后，CD4和CD8阳性细胞在表达H9.1.2 CAR细胞中的比例分别为38.4％，55.8％；在H9.2.1-tCD19-M7CR、H9.2.1-IL-15-M7CRin、H9.2.1in-IL-15-M7CR、H9.2.1-IL-15-M7CR、H9.2.1-28-IL-15-M7CR、H9.2.1-sIL-15、H9.2.1-IL12-M7CR、8E5组中的CD4+细胞比例分别为：37.8％、31.4％、37.1％、38.3％、39.4％、29.7％、53.6％和41.3％；CD8+细胞比例分别为：56.5％、63.9％、59.1％、57.3％、56.3％、64.9％、40.2％和55％。使用供者17的T细胞转导H9.1.2 CAR后，CD4和CD8阳性细胞在表达H9.1.2 CAR细胞中的比例分别为18.6％，73.6％；在H9.2.1-tCD19-M7CR、H9.2.1-IL-15-M7CRin、H9.2.1in-IL-15-M7CR、H9.2.1-IL-15-M7CR、H9.2.1-28-IL-15-M7CR、H9.2.1-sIL-15、H9.2.1-IL12-M7CR、8E5组中的CD4+细胞比例分别为：18.9％、19％、24.4％、23.5％、27.2％、18％、29.7％和22.7％；CD8+细胞比例分别为：72.5％、72.7％、68.3％、67.5％、63.8％、73％、64.2％和72％。Figure 23C and Figure 23D show that after using donor 15's T cells to transduce H9.1.2 CAR, the proportions of CD4 and CD8 positive cells in cells expressing H9.1.2 CAR were 38.4% and 55.8% respectively; in H9.2.1- tCD19-M7CR, H9.2.1-IL-15-M7CRin, H9.2.1in-IL-15-M7CR, H9.2.1-IL-15-M7CR, H9.2.1-28-IL-15-M7CR, H9.2.1 The proportions of CD4 + cells in -sIL-15, H9.2.1-IL12-M7CR, and 8E5 groups were: 37.8%, 31.4%, 37.1%, 38.3%, 39.4%, 29.7%, 53.6%, and 41.3% respectively; CD8 + The cell proportions were: 56.5%, 63.9%, 59.1%, 57.3%, 56.3%, 64.9%, 40.2% and 55%. After using T cells from donor 17 to transduce H9.1.2 CAR, the proportions of CD4 and CD8 positive cells in cells expressing H9.1.2 CAR were 18.6% and 73.6% respectively; in H9.2.1-tCD19-M7CR, H9.2.1 -IL-15-M7CRin, H9.2.1in-IL-15-M7CR, H9.2.1-IL-15-M7CR, H9.2.1-28-IL-15-M7CR, H9.2.1-sIL-15, H9. The proportions of CD4 + cells in the 2.1-IL12-M7CR and 8E5 groups were: 18.9%, 19%, 24.4%, 23.5%, 27.2%, 18%, 29.7% and 22.7% respectively; the proportions of CD8 + cells were: 72.5% respectively. , 72.7%, 68.3%, 67.5%, 63.8%, 73%, 64.2% and 72%.
检测制备第7天和第9天M7CR修饰CAR-T细胞表型，结果如图23E所示，CD45RA+CCR7+代表初始T细胞或干性记忆性T细胞(TN/TSCM)、CD45RA-CCR7+代表中心记忆性T细胞(TCM)、CD45RA-CCR7-代表效应记忆性T细胞(TEM)、CD45RA+CCR7-代表效应T细胞(Teff)亚群，CAR-T细胞大部分为TN/TSCM、TCM细胞。如图23E、图23F所示，和NT相比，H9.2.1 CAR-T细胞表型及对照8E5 CAR-T细胞表型在制备第7天 TN/TSCM亚群细胞比例降低，第9天降低更显著。而H9.2.1-tCD19-M7CR、H9.2.1-IL-15-M7CRin、H9.2.1in-IL-15-M7CR、H9.2.1-IL-15-M7CR、H9.2.1-28-IL-15-M7CR、H9.2.1-sIL-15 CAR-T细胞相比较H9.2.1及对照8E5 CAR-T细胞，维持了较高比例TN/TSCM细胞，H9.2.1-IL-15-M7CR中TN/TSCM细胞比例甚至与NT相当；而H9.2.1-IL12-M7CR促进了CAR-T细胞分化，TN/TSCM细胞比例明显降低。The phenotype of M7CR modified CAR-T cells was detected on days 7 and 9 of preparation. The results are shown in Figure 23E. CD45RA + CCR7 + represents initial T cells or stem memory T cells (TN/TSCM), CD45RA-CCR7 + Represents central memory T cells (TCM), CD45RA - CCR7 - represents effector memory T cells (TEM), CD45RA + CCR7 - represents effector T cells (Teff) subsets, and most CAR-T cells are TN/TSCM, TCM cell. As shown in Figure 23E and Figure 23F, compared with NT, the phenotype of H9.2.1 CAR-T cells and the phenotype of control 8E5 CAR-T cells on the 7th day of preparation The ratio of TN/TSCM subpopulation cells decreased, and the decrease was more significant on the 9th day. And H9.2.1-tCD19-M7CR, H9.2.1-IL-15-M7CRin, H9.2.1in-IL-15-M7CR, H9.2.1-IL-15-M7CR, H9.2.1-28-IL-15- Compared with H9.2.1 and control 8E5 CAR-T cells, M7CR and H9.2.1-sIL-15 CAR-T cells maintained a higher proportion of TN/TSCM cells. TN/TSCM cells in H9.2.1-IL-15-M7CR The ratio is even comparable to that of NT; while H9.2.1-IL12-M7CR promotes CAR-T cell differentiation, and the ratio of TN/TSCM cells is significantly reduced.
实施例12.4.CAR-T细胞胞内磷酸化STAT5信号Example 12.4. Intracellular phosphorylated STAT5 signal in CAR-T cells
通过FACS检测胞内p-STAT5的表达水平，研究STAT5信号激活情况。实验步骤如下，取1E6的CAR-T细胞，用PBS洗涤一遍后，重悬于无血清的RPMI1640培养基中，过夜。次日取出细胞，FACS缓冲液洗涤一遍。然后加入AF647-p-STAT5(BD，目录号562076)抗体进行胞内染色，具体步骤同实施例2。The expression level of intracellular p-STAT5 was detected by FACS to study the activation of STAT5 signal. The experimental steps are as follows. Take 1E6 CAR-T cells, wash them once with PBS, and resuspend them in serum-free RPMI1640 medium overnight. The next day, cells were taken out and washed once with FACS buffer. Then add AF647-p-STAT5 (BD, catalog number 562076) antibody for intracellular staining. The specific steps are the same as Example 2.
结果如图23G和图23H所示。在不使用IL-2刺激的情况下，与NT相比，H9.2.1-CAR-T细胞中不论CAR+还是CAR，p-STAT5均不上升。而H9.2.1-tCD19-M7CR、H9.2.1in-IL-15-M7CR、H9.2.1-IL-15-M7CR、H9.2.1-28-IL-15-M7CR CAR-T细胞相比较非CAR-T细胞p-STAT5水平明显增加，尤以H9.2.1in-IL-15-M7CR、H9.2.1-IL-15-M7CR显著，而H9.2.1-IL-15-M7CRin CAR-T细胞p-STAT5水平轻微升高，推测膜表面表达的IL-15通过顺式或反式作用激活STAT5信号，而H9.2.1-sIL-15 CAR-T细胞p-STAT5水平未有升高。The results are shown in Figure 23G and Figure 23H. Without IL-2 stimulation, compared with NT, p-STAT5 did not increase in either CAR + or CAR in H9.2.1-CAR-T cells. Compared with non-CAR- T cell p-STAT5 levels increased significantly, especially H9.2.1in-IL-15-M7CR and H9.2.1-IL-15-M7CR, while H9.2.1-IL-15-M7CRin CAR-T cell p-STAT5 The level was slightly increased. It is speculated that IL-15 expressed on the membrane surface activates STAT5 signaling through cis or trans action, while the level of p-STAT5 in H9.2.1-sIL-15 CAR-T cells did not increase.
实施例13、CAR-T激活实验Example 13. CAR-T activation experiment
H9.2.1 CAR-T细胞、8E5 CAR-T细胞或不同M7CR修饰的H9.2.1 CAR-T细胞分别与CLDN18.2低表达的PANC-1或CLDN18.2中高表达的HUP-T4在E:T为1:1时进行共孵育培养。24h后收集100μL上清用于后续细胞因子检测实验(实施例14)。收获细胞，FACS缓冲液(PBS+2％FBS)洗涤一次，重悬后加入含LIVE/DEAD Fixable Dead Cell Stain(Thermo，L34963)和Biotin-SP-conjugated AffiniPure F(ab)2Fragment Goat Anti-human IgG,F(ab)2fragment specific的FACS缓冲液，室温染色30min，洗涤两次，加入APC-Cy7-CD25(Biolegend，302614)，PE-CD69(Biolegend，310906)，Percp-Cy5.5-CD3(Biolegend，300430)，APC-Strep(Biolegend，405207)抗体，作为第二染色试剂用于检测，4℃染色30～45min；细胞洗涤两次后FACS缓冲液重悬，用流式细胞仪检测。结果如图24A和图24B所示。H9.2.1 CAR-T cells, 8E5 CAR-T cells or H9.2.1 CAR-T cells with different M7CR modifications were compared with PANC-1 with low expression in CLDN18.2 or HUP-T4 with high expression in CLDN18.2 in E:T Co-incubate culture at 1:1. After 24 hours, 100 μL of supernatant was collected for subsequent cytokine detection experiments (Example 14). Harvest cells, wash once with FACS buffer (PBS+2% FBS), resuspend and add LIVE/DEAD Fixable Dead Cell Stain (Thermo, L34963) and Biotin-SP-conjugated AffiniPure F(ab)2Fragment Goat Anti-human IgG , F(ab)2fragment specific FACS buffer, stain for 30 minutes at room temperature, wash twice, add APC-Cy7-CD25 (Biolegend, 302614), PE-CD69 (Biolegend, 310906), Percp-Cy5.5-CD3 (Biolegend , 300430), APC-Strep (Biolegend, 405207) antibody was used as the second staining reagent for detection, staining at 4°C for 30 to 45 minutes; cells were washed twice and resuspended in FACS buffer, and detected by flow cytometry. The results are shown in Figures 24A and 24B.
T细胞激活后上调CD25和CD69表达，因此CD25+CD69+代表一群激活的T细胞。从图24A和图24C可以看出，供者15和供者17T细胞来源的不同CAR-T细胞与PANC-1细胞共孵育24h后，CD25和CD69表达上调。使用供者15来源的CAR-T细胞，H9.2.1、H9.2.1-tCD19-M7CR、H9.2.1-IL-15-M7CRin、H9.2.1in-IL-15-M7CR、H9.2.1-IL-15-M7CR、H9.2.1-28-IL-15-M7CR、H9.2.1-sIL-15、H9.2.1-IL12-M7CR、8E5组中的CD25+CD69+细胞比例分别为：3.58％、5.24％、0.43％、0.39％、5.84％、10.3％、0.69％、16.9％和3.91％。使用供者17来源的CAR-T细胞，H9.2.1、H9.2.1-tCD19-M7CR、H9.2.1-IL-15-M7CRin、H9.2.1in-IL-15-M7CR、H9.2.1-IL-15-M7CR、H9.2.1-28-IL-15-M7CR、H9.2.1-sIL-15、H9.2.1-IL12-M7CR、8E5组中的CD25+CD69+细胞比例分别为：1.67％、2.33％、0.51％、1.12％、7.44％、10.4％、1.47％、16.1％和1.67％。H9.2.1-IL-15-M7CR、H9.2.1-28-IL-15-M7CR、H9.2.1-IL12-M7CR相比较H9.2.1CAR-T细胞激活明显增加，说明IL-15-M7CR、IL-12-M7CR能够促进CAR-T在低抗原刺激后的激活。 T cells upregulate CD25 and CD69 expression after activation, so CD25 + CD69 + represents a population of activated T cells. As can be seen from Figure 24A and Figure 24C, after 24 hours of co-incubation of different CAR-T cells derived from donor 15 and donor 17 T cells with PANC-1 cells, the expressions of CD25 and CD69 were up-regulated. Use CAR-T cells derived from donor 15, H9.2.1, H9.2.1-tCD19-M7CR, H9.2.1-IL-15-M7CRin, H9.2.1in-IL-15-M7CR, H9.2.1-IL- The proportions of CD25 + CD69 + cells in the 15-M7CR, H9.2.1-28-IL-15-M7CR, H9.2.1-sIL-15, H9.2.1-IL12-M7CR, and 8E5 groups were: 3.58% and 5.24% respectively. , 0.43%, 0.39%, 5.84%, 10.3%, 0.69%, 16.9% and 3.91%. Use CAR-T cells derived from donor 17, H9.2.1, H9.2.1-tCD19-M7CR, H9.2.1-IL-15-M7CRin, H9.2.1in-IL-15-M7CR, H9.2.1-IL- The proportions of CD25 + CD69 + cells in the 15-M7CR, H9.2.1-28-IL-15-M7CR, H9.2.1-sIL-15, H9.2.1-IL12-M7CR, and 8E5 groups were: 1.67% and 2.33% respectively. , 0.51%, 1.12%, 7.44%, 10.4%, 1.47%, 16.1% and 1.67%. H9.2.1-IL-15-M7CR, H9.2.1-28-IL-15-M7CR, and H9.2.1-IL12-M7CR significantly increased activation of H9.2.1 CAR-T cells compared with H9.2.1, indicating that IL-15-M7CR, IL -12-M7CR can promote the activation of CAR-T after low antigen stimulation.
从图24B和图24D可以看出，供者15和供者17T来源的不同CAR-T细胞与HUP-T4细胞共孵育24h后，CD25和CD69表达上调。使用供者15来源的CAR-T细胞，H9.2.1、H9.2.1-tCD19-M7CR、H9.2.1-IL-15-M7CRin、H9.2.1in-IL-15-M7CR、H9.2.1-IL-15-M7CR、H9.2.1-28-IL-15-M7CR、H9.2.1-sIL-15、H9.2.1-IL12-M7CR、8E5组中的CD25+CD69+细胞比例分别为：19.4％、18.1％、1.29％、1.33％、13.6％、19.5％、5.31％、31.5％和26.9％。使用供者17来源的CAR-T细胞，H9.2.1、H9.2.1-tCD19-M7CR、H9.2.1-IL-15-M7CRin、H9.2.1in-IL-15-M7CR、H9.2.1-IL-15-M7CR、H9.2.1-28-IL-15-M7CR、H9.2.1-sIL-15、H9.2.1-IL12-M7CR、8E5组中的CD25+CD69+细胞比例分别为：19.9％、14.1％、2.08％、1.49％、16.8％、24.6％、8.93％、34.1％和27.5％。H9.2.1-IL12-M7CR相比较H9.2.1CAR-T细胞激活明显增加，说明IL-12-M7CR能够促进CAR-T在中高抗原刺激后的激活。It can be seen from Figure 24B and Figure 24D that after 24 hours of co-incubation of different CAR-T cells derived from donor 15 and donor 17T with HUP-T4 cells, the expression of CD25 and CD69 was up-regulated. Use CAR-T cells derived from donor 15, H9.2.1, H9.2.1-tCD19-M7CR, H9.2.1-IL-15-M7CRin, H9.2.1in-IL-15-M7CR, H9.2.1-IL- The proportions of CD25 + CD69 + cells in the 15-M7CR, H9.2.1-28-IL-15-M7CR, H9.2.1-sIL-15, H9.2.1-IL12-M7CR, and 8E5 groups were: 19.4% and 18.1% respectively. , 1.29%, 1.33%, 13.6%, 19.5%, 5.31%, 31.5% and 26.9%. Use CAR-T cells derived from donor 17, H9.2.1, H9.2.1-tCD19-M7CR, H9.2.1-IL-15-M7CRin, H9.2.1in-IL-15-M7CR, H9.2.1-IL- The proportions of CD25 + CD69 + cells in the 15-M7CR, H9.2.1-28-IL-15-M7CR, H9.2.1-sIL-15, H9.2.1-IL12-M7CR, and 8E5 groups were: 19.9% and 14.1% respectively. , 2.08%, 1.49%, 16.8%, 24.6%, 8.93%, 34.1% and 27.5%. H9.2.1-IL12-M7CR significantly increased the activation of CAR-T cells compared with H9.2.1, indicating that IL-12-M7CR can promote the activation of CAR-T after medium and high antigen stimulation.
实施例14、细胞因子检测实验Example 14. Cytokine detection experiment
使用BDTM Cytometric Bead Array(CBA)Human Th1/Th2 Cytokine Kit II对实施例13中收集的细胞培养上清进行细胞因子检测，实验步骤同实施例8。通过CBA检测CAR-T激活后上清中IL-2、IFN-γ和TNFα细胞因子水平。结果如图25A和图25B所示。The cell culture supernatant collected in Example 13 was used to detect cytokines using BD TM Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit II. The experimental procedures were the same as those in Example 8. The levels of IL-2, IFN-γ and TNFα cytokines in the supernatant after CAR-T activation were detected by CBA. The results are shown in Figures 25A and 25B.
由图25A和图25B可见，与PANC-1相比，各组CAR-T在与HUP-T4共孵育24h后细胞因子分泌增加。对于供者15和供者17来源的CAR-T细胞而言，表达相同CAR分子的细胞与肿瘤细胞共孵育后显示出接近的细胞因子分泌量，显示实验的可重复性高。As can be seen from Figure 25A and Figure 25B, compared with PANC-1, CAR-T in each group increased cytokine secretion after co-incubation with HUP-T4 for 24 hours. For CAR-T cells derived from donor 15 and donor 17, cells expressing the same CAR molecule showed similar amounts of cytokine secretion after co-incubation with tumor cells, indicating high reproducibility of the experiment.
图25A和图25B的结果还显示，使用PANC-1细胞刺激后，与H9.2.1CAR-T细胞相比较，H9.2.1-IL-15-M7CR、H9.2.1-28-IL-15-M7CR、H9.2.1-IL12-M7CR CAR-T细胞IL-2、IFN-γ分泌明显增加，这与上述激活结果一致。使用HUP-T4细胞刺激后，与H9.2.1CAR-T细胞相比较，H9.2.1-IL-15-M7CR、H9.2.1-28-IL-15-M7CR、H9.2.1-IL12-M7CR CAR-T细胞IL-2、IFN-γ和TNFα分泌明显增加，说明IL-15-M7CR、IL-12-M7CR能够促进CAR-T细胞分泌效应细胞因子。H9.2.1in-IL-15-M7CR CAR-T细胞分泌很少细胞因子，说明IL-15-M7CR增加CAR-T细胞因子分泌依赖CAR信号。The results in Figure 25A and Figure 25B also show that after stimulation with PANC-1 cells, compared with H9.2.1CAR-T cells, H9.2.1-IL-15-M7CR and H9.2.1-28-IL-15-M7CR , H9.2.1-IL12-M7CR CAR-T cells secreted IL-2 and IFN-γ significantly increased, which is consistent with the above activation results. After stimulation with HUP-T4 cells, compared with H9.2.1 CAR-T cells, H9.2.1-IL-15-M7CR, H9.2.1-28-IL-15-M7CR, H9.2.1-IL12-M7CR CAR- The secretion of IL-2, IFN-γ and TNFα by T cells increased significantly, indicating that IL-15-M7CR and IL-12-M7CR can promote the secretion of effector cytokines by CAR-T cells. H9.2.1in-IL-15-M7CR CAR-T cells secrete very few cytokines, indicating that IL-15-M7CR increases CAR-T cytokine secretion dependent on CAR signaling.
实施例15、CAR-T增殖实验Example 15. CAR-T proliferation experiment
如图26A和图26B所示，首先采用NT细胞调整各CAR-T阳性率一致，然后对CAR-T细胞进行CTV(Thermo，C34557)标记，将CTV标记的CAR-T细胞与CLDN18.2低表达PANC-1细胞和CLDN18.2高表达SNU620细胞按照1:1E:T共孵育培养，每2天换一次液，5天后收获细胞，FACS缓冲液(PBS+2％FBS)洗涤一次，重悬后加入含LIVE/DEAD Fixable Dead Cell Stain(Thermo，L34965)和Biotin-SP-conjugated AffiniPure F(ab)2Fragment Goat Anti-human IgG,F(ab)2fragment specific的FACS缓冲液，室温染色30min，洗涤两次，APC-Strep(Biolegend，405207)抗体，作为第二染色试剂来检测，4℃染色30～45min；细胞洗涤两次后FACS缓冲液重悬，用流式细胞仪检测。As shown in Figure 26A and Figure 26B, NT cells were first used to adjust the positive rate of each CAR-T to be consistent, and then the CAR-T cells were labeled with CTV (Thermo, C34557), and the CTV-labeled CAR-T cells were compared with CLDN18.2 low PANC-1-expressing cells and CLDN18.2 high-expressing SNU620 cells were co-incubated and cultured according to 1:1E:T. The medium was changed every 2 days. The cells were harvested after 5 days, washed once with FACS buffer (PBS+2% FBS), and resuspended. Then add FACS buffer containing LIVE/DEAD Fixable Dead Cell Stain (Thermo, L34965) and Biotin-SP-conjugated AffiniPure F(ab)2Fragment Goat Anti-human IgG, F(ab)2fragment specific, stain for 30 minutes at room temperature, and wash for two Second, APC-Strep (Biolegend, 405207) antibody was used as the second staining reagent for detection, staining at 4°C for 30 to 45 minutes; cells were washed twice and resuspended in FACS buffer, and detected with a flow cytometer.
图26A的结果显示，PANC-1细胞刺激后，H9.2.1-IL-15-M7CR、H9.2.1-28-IL-15-M7CR、H9.2.1-IL12-M7CR相比较H9.2.1CAR-T细胞增殖明显增加，这与上述CAR-T激活、细胞因子结果一致，说明IL-15-M7CR、IL-12-M7CR能够促进CAR-T在低抗原刺激后的增殖。The results in Figure 26A show that after PANC-1 cell stimulation, H9.2.1-IL-15-M7CR, H9.2.1-28-IL-15-M7CR, H9.2.1-IL12-M7CR compared with H9.2.1CAR-T Cell proliferation increased significantly, which was consistent with the above CAR-T activation and cytokine results, indicating that IL-15-M7CR and IL-12-M7CR can promote the proliferation of CAR-T after low antigen stimulation.
图26B的结果显示，HUP-T4细胞刺激后，H9.2.1-tCD19-M7CR、H9.2.1-IL-15-M7CR、 H9.2.1-28-IL-15-M7CR、H9.2.1-IL12-M7CR相比较H9.2.1CAR-T细胞增殖明显增加，说明tCD19-M7CR、IL-15-M7CR、IL-12-M7CR能够促进CAR-T在中高抗原刺激后的增殖，IL-15-M7CR、IL-12-M7CR相比较tCD19-M7CR效应更明显，说明IL-12、IL-15等ECD与M7R产生相加或协同效应。The results in Figure 26B show that after HUP-T4 cell stimulation, H9.2.1-tCD19-M7CR, H9.2.1-IL-15-M7CR, H9.2.1-28-IL-15-M7CR and H9.2.1-IL12-M7CR significantly increased the proliferation of H9.2.1 CAR-T cells, indicating that tCD19-M7CR, IL-15-M7CR and IL-12-M7CR can promote In the proliferation of CAR-T after medium and high antigen stimulation, the effect of IL-15-M7CR and IL-12-M7CR is more obvious than that of tCD19-M7CR, indicating that IL-12, IL-15 and other ECDs have additive or synergistic effects with M7R.
实施例16、IL-15-M7CR修饰的CAR-T细胞在小鼠体内的抗肿瘤作用Example 16. Anti-tumor effect of IL-15-M7CR modified CAR-T cells in mice
具体实验步骤同实施例10。选用NOG小鼠，Day-7给小鼠腹腔注射NUGC-4-Gluc细胞进行造模，通过小动物活体成像系统对小鼠造模情况进行检测，至Day0时肿瘤负荷为1×109p/s时对动物进行分组(每组5只)，通过尾静脉注射给予小鼠1×106，1×105，1×104个H9.2.1-IL-15-M7CR CAR-T细胞或H9.2.1-CD28-IL-15-M7CR CAR-T细胞，对照小鼠给予NT细胞(剂量与1×106个CAR-T细胞组总T细胞数一致)。通过每周成像对小鼠肿瘤负荷进行检测，每周取外周血检测CAR-T体内扩增。The specific experimental steps are the same as in Example 10. NOG mice were selected, and NUGC-4-Gluc cells were intraperitoneally injected into the mice on Day-7 to create a model. The mouse modeling was detected through a small animal in vivo imaging system. By Day 0, the tumor load was 1×10 9 p/ At s, the animals were divided into groups (5 in each group), and 1×10 6 , 1×10 5 , and 1×10 4 H9.2.1-IL-15-M7CR CAR-T cells or H9 were administered to the mice via tail vein injection. .2.1-CD28-IL-15-M7CR CAR-T cells, control mice were given NT cells (the dose was consistent with the total number of T cells in the 1×10 6 CAR-T cell group). The tumor burden of mice was detected through weekly imaging, and peripheral blood was collected weekly to detect CAR-T amplification in vivo.
如图27A及图27B所示，H9.2.1-IL-15-M7CR CAR-T细胞或H9.2.1-CD28-IL-15-M7CR CAR-T细胞回输7后产生显著抗肿瘤效应，并呈现剂量依赖性。如图27C所示，CAR-T回输2周后高剂量组治疗小鼠体重显著下降，推测由于小鼠正常胃表皮细胞表达CLDN18.2产生了“在靶”毒性。As shown in Figure 27A and Figure 27B, H9.2.1-IL-15-M7CR CAR-T cells or H9.2.1-CD28-IL-15-M7CR CAR-T cells produced significant anti-tumor effects after infusion 7 and showed dose dependent. As shown in Figure 27C, the body weight of the mice treated in the high-dose group decreased significantly 2 weeks after CAR-T reinfusion. It is speculated that the expression of CLDN18.2 in the normal gastric epithelial cells of the mice resulted in "on-target" toxicity.
如图27D及图27E所示，H9.2.1-IL-15-M7CR和H9.2.1-CD28-IL-15-M7CR CAR-T细胞在小鼠体内产生显著扩增，并呈现剂量依赖性。As shown in Figure 27D and Figure 27E, H9.2.1-IL-15-M7CR and H9.2.1-CD28-IL-15-M7CR CAR-T cells significantly expanded in mice in a dose-dependent manner.
实施例17、构建细胞因子-M7CR修饰的CARExample 17. Construction of cytokine-M7CR modified CAR
如图28所示，将不同细胞因子作为胞外结构域(ECD)和M7Rm8(SEQ ID NO：34)直接连接构建组成型嵌合细胞因子受体M7CRm8；然后通过P2A将M7CRm8的N端与H9.2.1 CAR多肽的C端连接，从而构成细胞因子-M7CR修饰的H9.2.1 CAR。图28中构建的CAR的序列信息如下。H9.2.1 CAR具有SEQ ID NO：100所示的氨基酸序列；H9.2.1-tCD19-M7CR具有SEQ ID NO：101所示的氨基酸序列；H9.2.1-IL-18-M7CR具有SEQ ID NO：148所示的氨基酸序列；H9.2.1-IL-9-M7CR具有SEQ ID NO：149所示的氨基酸序列；H9.2.1-IL-36-M7CR具有SEQ ID NO：150所示的氨基酸序列；H9.2.1-IL-23-M7CR具有SEQ ID NO：151所示的氨基酸序列；H9.2.1-IL12-p40-M7CR具有SEQ ID NO：152所示的氨基酸序列；H9.2.1-IL-12-M7CR具有SEQ ID NO：104所示的氨基酸序列；H9.2.1-218 CAR具有SEQ ID NO：144所示的氨基酸序列。As shown in Figure 28, different cytokines are directly connected as extracellular domains (ECD) and M7Rm8 (SEQ ID NO: 34) to construct a constitutive chimeric cytokine receptor M7CRm8; then the N-terminus of M7CRm8 is connected to H9 through P2A .2.1 The C-terminus of the CAR polypeptide is connected to form the cytokine-M7CR modified H9.2.1 CAR. The sequence information of the CAR constructed in Figure 28 is as follows. H9.2.1 CAR has the amino acid sequence shown in SEQ ID NO: 100; H9.2.1-tCD19-M7CR has the amino acid sequence shown in SEQ ID NO: 101; H9.2.1-IL-18-M7CR has SEQ ID NO: 148 The amino acid sequence shown; H9.2.1-IL-9-M7CR has the amino acid sequence shown in SEQ ID NO: 149; H9.2.1-IL-36-M7CR has the amino acid sequence shown in SEQ ID NO: 150; H9. 2.1-IL-23-M7CR has the amino acid sequence shown in SEQ ID NO: 151; H9.2.1-IL12-p40-M7CR has the amino acid sequence shown in SEQ ID NO: 152; H9.2.1-IL-12-M7CR has The amino acid sequence shown in SEQ ID NO: 104; H9.2.1-218 CAR has the amino acid sequence shown in SEQ ID NO: 144.
在所述M7CR中，位于M7Rm8的N端的细胞因子ECD包括但不限于IL-18(SEQ ID NO：53)、IL-9(SEQ ID NO：52)、IL-36γ(SEQ ID NO：55)、IL-23(SEQ ID NO：54)、IL-12-P70(SEQ ID NO：49)、IL-12-P40(SEQ ID NO：50)。上述不同细胞因子作为ECD和M7Rm8连接后构成IL18-M7CR(SEQ ID NO：177)、IL9-M7CR(SEQ ID NO：176)、IL36γ-M7CR(SEQ ID NO：179)(下文中也称IL36-M7CR)、IL23-M7CR(SEQ ID NO：178)、IL12-M7CR(SEQ ID NO：172)、IL12-p40-M7CR(SEQ ID NO：175)。使用tCD19-M7CR作为对照分子。In the M7CR, the cytokine ECD located at the N-terminus of M7Rm8 includes but is not limited to IL-18 (SEQ ID NO: 53), IL-9 (SEQ ID NO: 52), IL-36γ (SEQ ID NO: 55) , IL-23 (SEQ ID NO: 54), IL-12-P70 (SEQ ID NO: 49), IL-12-P40 (SEQ ID NO: 50). The above different cytokines serve as ECD and are connected to M7Rm8 to form IL18-M7CR (SEQ ID NO: 177), IL9-M7CR (SEQ ID NO: 176), IL36γ-M7CR (SEQ ID NO: 179) (hereinafter also referred to as IL36- M7CR), IL23-M7CR (SEQ ID NO: 178), IL12-M7CR (SEQ ID NO: 172), IL12-p40-M7CR (SEQ ID NO: 175). tCD19-M7CR was used as a control molecule.
实施例18、制备细胞因子-M7CR修饰的CAR-T细胞 Example 18. Preparation of cytokine-M7CR modified CAR-T cells
实施例18.1.序列合成Example 18.1. Sequence synthesis
人工合成DNA序列，所述DNA序列分别编码H9.2.1-CAR(SEQ ID NO:100)、H9.2.1-tCD19-M7CR(SEQ ID NO:101)、H9.2.1-IL8-M7CR(SEQ ID NO:148)、H9.2.1-IL9-M7CR(SEQ ID NO:149)、H9.2.1-IL36-M7CR(SEQ ID NO:150)、H9.2.1-IL23-M7CR(SEQ ID NO:151)、H9.2.1-p40-M7CR(SEQ ID NO:152)、H9.2.1-IL12-M7CR)(SEQ ID NO:104)、H9.2.1-218-CAR(SEQ ID NO:153)。Artificially synthesized DNA sequences encoding H9.2.1-CAR (SEQ ID NO:100), H9.2.1-tCD19-M7CR (SEQ ID NO:101), H9.2.1-IL8-M7CR (SEQ ID NO :148), H9.2.1-IL9-M7CR (SEQ ID NO:149), H9.2.1-IL36-M7CR (SEQ ID NO:150), H9.2.1-IL23-M7CR (SEQ ID NO:151), H9 .2.1-p40-M7CR (SEQ ID NO:152), H9.2.1-IL12-M7CR) (SEQ ID NO:104), H9.2.1-218-CAR (SEQ ID NO:153).
在所述构建体中，H9.2.1-IL-18-M7CR分子(SEQ ID NO:148)从N端到C端包含所述H9.2.1CAR分子(SEQ ID NO:100)、P2A(SEQ ID NO：3)、SP(SEQ ID NO：2)以及IL-18-M7CR(SEQ ID NO：177)。所述H9.2.1-IL-9-M7CR分子(SEQ ID NO:149)从N端到C端包含所述H9.2.1 CAR分子(SEQ ID NO:100)、P2A(SEQ ID NO：3)、SP(SEQ ID NO：2)以及IL-9-M7CR(SEQ ID NO：176)。所述H9.2.1-IL-36-M7CR分子(SEQ ID NO:150)从N端到C端包含所述H9.2.1 CAR分子(SEQ ID NO:100)、P2A(SEQ ID NO：3)、SP(SEQ ID NO：2)以及IL36γ-M7CR(SEQ ID NO：179)。所述H9.2.1-IL-23-M7CR分子(SEQ ID NO:151)从N端到C端包含所述H9.2.1 CAR分子(SEQ ID NO:100)、P2A(SEQ ID NO：3)、SP(SEQ ID NO：2)以及IL-23-M7CR(SEQ ID NO：178)。所述H9.2.1-IL12-p40-M7CR分子(SEQ ID NO:152)从N端到C端包含所述H9.2.1 CAR分子(SEQ ID NO:100)、P2A(SEQ ID NO：3)、SP(SEQ ID NO：2)以及IL-12p40-M7CR(SEQ ID NO：175)；所述H9.2.1-IL-12-M7CR分子(SEQ ID NO:104)从N端到C端包含所述H9.2.1-BB-L CAR分子(SEQ ID NO:100)、P2A(SEQ ID NO：3)、SP(SEQ ID NO：2)以及IL-12-M7CR(SEQ ID NO：172)。In the construct, H9.2.1-IL-18-M7CR molecule (SEQ ID NO:148) includes the H9.2.1CAR molecule (SEQ ID NO:100), P2A (SEQ ID NO: 3), SP (SEQ ID NO: 2) and IL-18-M7CR (SEQ ID NO: 177). The H9.2.1-IL-9-M7CR molecule (SEQ ID NO: 149) includes the H9.2.1 CAR molecule (SEQ ID NO: 100), P2A (SEQ ID NO: 3), SP (SEQ ID NO: 2) and IL-9-M7CR (SEQ ID NO: 176). The H9.2.1-IL-36-M7CR molecule (SEQ ID NO:150) includes the H9.2.1 CAR molecule (SEQ ID NO:100), P2A (SEQ ID NO:3), SP (SEQ ID NO: 2) and IL36γ-M7CR (SEQ ID NO: 179). The H9.2.1-IL-23-M7CR molecule (SEQ ID NO:151) includes the H9.2.1 CAR molecule (SEQ ID NO:100), P2A (SEQ ID NO:3), SP (SEQ ID NO: 2) and IL-23-M7CR (SEQ ID NO: 178). The H9.2.1-IL12-p40-M7CR molecule (SEQ ID NO:152) includes the H9.2.1 CAR molecule (SEQ ID NO:100), P2A (SEQ ID NO:3), SP (SEQ ID NO: 2) and IL-12p40-M7CR (SEQ ID NO: 175); the H9.2.1-IL-12-M7CR molecule (SEQ ID NO: 104) contains the H9.2.1-BB-L CAR molecules (SEQ ID NO:100), P2A (SEQ ID NO:3), SP (SEQ ID NO:2) and IL-12-M7CR (SEQ ID NO:172).
在上述细胞因子-M7CR相关构建体中，所有M7CR分子从N端至C端均包含GM-CSFRα-SP(SEQ ID NO:2)、细胞因子ECD以及M7R(下文中，对本实施例的相关构建体，作为M7R例示了SEQ ID NO：34所示的IL7Rm8)。In the above cytokine-M7CR related constructs, all M7CR molecules include GM-CSFRα-SP (SEQ ID NO: 2), cytokine ECD and M7R from the N-terminus to the C-terminus (hereinafter, the related construction of this embodiment body, IL7Rm8) shown in SEQ ID NO: 34 is exemplified as M7R.
将上述合成的DNA片段插入pRKN慢病毒表达载体(金唯智公司)的EF1α启动子下游，替换原载体中EGFR序列，得到相应的表达质粒(由金唯智公司合成)。The above-mentioned synthesized DNA fragment was inserted into the pRKN lentiviral expression vector (Genewise Company) downstream of the EF1α promoter, and the EGFR sequence in the original vector was replaced to obtain the corresponding expression plasmid (synthesized by Genewise Company).
实施例18.2.T细胞的获得及慢病毒转导Example 18.2. Obtaining T cells and lentiviral transduction
慢病毒制备方法同实施例4.2，CAR-T制备方法同实施例4.3。使用供者16和供者6，11，17的T细胞，如图29A和图29B所示为制备的CAR-T细胞第1天到第9天的扩增情况，图29C为供者6，11，17来源的CAR-T细胞制备第9天的扩增倍数统计柱状图，可以看出不同供者来源的T细胞制备出来的CAR-T细胞扩增情况存在差异，总体扩增倍数在20-80倍之间，满足需求。The lentivirus preparation method is the same as Example 4.2, and the CAR-T preparation method is the same as Example 4.3. Using T cells from donor 16 and donors 6, 11, and 17, Figure 29A and Figure 29B show the expansion of the prepared CAR-T cells from day 1 to day 9, and Figure 29C shows donor 6, The statistical histogram of the expansion fold of CAR-T cells from 11,17 on the 9th day of preparation shows that there are differences in the expansion of CAR-T cells prepared from T cells from different donors. The overall expansion fold is between 20 and 20 -80 times to meet the needs.
实施例18.3.CAR表达检测及CAR-T细胞表型检测Example 18.3. CAR expression detection and CAR-T cell phenotype detection
取适量自上述实施例18.2获得的CAR-T细胞，染色步骤见实施例4.4，染色结束后，细胞洗涤两次后FACS缓冲液重悬，用流式细胞仪检测。Take an appropriate amount of CAR-T cells obtained from the above Example 18.2. The staining procedure is as shown in Example 4.4. After the staining is completed, the cells are washed twice and resuspended in FACS buffer, and detected with a flow cytometer.
图30A和图30B所示为供者16来源CAR-T细胞后第7和第9天时CAR和或作为ECD的细胞因子表达情况。在第9天时通过FACS检测CAR多肽表达，CAR多肽能够在所有CAR-T细胞中表达，使用供者16的T细胞，H9.2.1组中CAR+细胞的比例约为39.8％，H9.2.1-218组中CAR+细胞的比例约为41.2％。可见接头选择G4SLinker和218Linker对CAR+率影响不大。而H9.2.1-IL18-M7CR、H9.2.1-IL9-M7CR、H9.2.1-IL23-M7CR、H9.2.1-p40-M7CR、 H9.2.1-IL36-M7CR组中的CAR+细胞的比例分别为：20.2％、19.5％、13.6％、19.0％、21.1％。使用PE标记IL-12P40抗体也检测到H9.2.1-IL23-M7CR、H9.2.1-p40-M7CR细胞中胞外细胞因子表达。Figure 30A and Figure 30B show the cytokine expression of CAR and/or ECD on days 7 and 9 after CAR-T cells derived from donor 16. CAR polypeptide expression was detected by FACS on day 9. The CAR polypeptide could be expressed in all CAR-T cells. Using T cells from donor 16, the proportion of CAR + cells in the H9.2.1 group was approximately 39.8%, and H9.2.1- The proportion of CAR + cells in the 218 group was approximately 41.2%. It can be seen that the selection of connectors G4SLinker and 218Linker has little impact on the CAR + rate. And H9.2.1-IL18-M7CR, H9.2.1-IL9-M7CR, H9.2.1-IL23-M7CR, H9.2.1-p40-M7CR, The proportions of CAR+ cells in the H9.2.1-IL36-M7CR group were: 20.2%, 19.5%, 13.6%, 19.0%, and 21.1% respectively. The expression of extracellular cytokines in H9.2.1-IL23-M7CR and H9.2.1-p40-M7CR cells was also detected using PE-labeled IL-12P40 antibody.
图30C和图30D所示为供者6，11，17来源CAR-T细胞后第9天时CAR表达情况。使用供者6的T细胞制备CAR-T细胞时，H9.2.1组中CAR+细胞的比例约为54.6％，而H9.2.1-tCD19-M7CR、H9.2.1-IL12-M7CR、H9.2.1-IL9-M7CR、H9.2.1-IL18-M7CR、H9.2.1-IL23-M7CR、H9.2.1-IL36-M7CR、H9.2.1-p40-M7CR组中的CAR+细胞的比例分别为：28.5％、14.7％、27.3％、25.3％、5.84％、13.7％、14.3％。使用供者11的T细胞制备CAR-T细胞时，H9.2.1组中CAR+细胞的比例约为54.7％，而H9.2.1-tCD19-M7CR、H9.2.1-IL12-M7CR、H9.2.1-IL9-M7CR、H9.2.1-IL18-M7CR、H9.2.1-IL23-M7CR、H9.2.1-IL36-M7CR、H9.2.1-p40-M7CR组中的CAR+细胞的比例分别为：36.1％、18.7％、30.9％、30.0％、8.45％、16.9％、19.0％。使用供者17的T细胞制备CAR-T细胞时，H9.2.1组中CAR+细胞的比例约为59.7％，而H9.2.1-tCD19-M7CR、H9.2.1-IL12-M7CR、H9.2.1-IL9-M7CR、H9.2.1-IL18-M7CR、H9.2.1-IL23-M7CR、H9.2.1-IL36-M7CR、H9.2.1-p40-M7CR组中的CAR+细胞的比例分别为：34.0％、16.7％、29.1％、30.2％、8.17％、14.6％、19.8％。Figure 30C and Figure 30D show the CAR expression on day 9 after CAR-T cells derived from donors 6, 11, and 17. When CAR-T cells were prepared using T cells from donor 6, the proportion of CAR + cells in the H9.2.1 group was approximately 54.6%, while H9.2.1-tCD19-M7CR, H9.2.1-IL12-M7CR, H9.2.1- The proportions of CAR+ cells in the IL9-M7CR, H9.2.1-IL18-M7CR, H9.2.1-IL23-M7CR, H9.2.1-IL36-M7CR, and H9.2.1-p40-M7CR groups were: 28.5% and 14.7% respectively. , 27.3%, 25.3%, 5.84%, 13.7%, 14.3%. When CAR-T cells were prepared using T cells from donor 11, the proportion of CAR + cells in the H9.2.1 group was approximately 54.7%, while H9.2.1-tCD19-M7CR, H9.2.1-IL12-M7CR, H9.2.1- The proportions of CAR+ cells in the IL9-M7CR, H9.2.1-IL18-M7CR, H9.2.1-IL23-M7CR, H9.2.1-IL36-M7CR, and H9.2.1-p40-M7CR groups were: 36.1% and 18.7% respectively. , 30.9%, 30.0%, 8.45%, 16.9%, 19.0%. When CAR-T cells were prepared using T cells from donor 17, the proportion of CAR + cells in the H9.2.1 group was approximately 59.7%, while H9.2.1-tCD19-M7CR, H9.2.1-IL12-M7CR, H9.2.1- The proportions of CAR+ cells in the IL9-M7CR, H9.2.1-IL18-M7CR, H9.2.1-IL23-M7CR, H9.2.1-IL36-M7CR, and H9.2.1-p40-M7CR groups were: 34.0% and 16.7% respectively. , 29.1%, 30.2%, 8.17%, 14.6%, 19.8%.
如图30E所示，使用供者16的T细胞制备的CAR-T细胞在第9天时，CD4阳性细胞和CD8阳性细胞在表达H9.1.2 CAR细胞中的比例分别为43.6％，49.1％；在H9.2.1-218、H9.2.1-IL18-M7CR、H9.2.1-IL9-M7CR、H9.2.1-IL23-M7CR、H9.2.1-p40-M7CR、H9.2.1-IL36-M7CR组中的CD4+细胞比例分别为：47.9％、45.8％、40％、51.4％、49.1％和45.9％；CD8+细胞比例分别为：44.6％、47.9％、54.5％、42.2％、44.6％和48.1％。各CAR分子间CD4和CD8比例差异不大。As shown in Figure 30E, on day 9 of CAR-T cells prepared using T cells from donor 16, the proportions of CD4-positive cells and CD8-positive cells in H9.1.2-expressing CAR cells were 43.6% and 49.1% respectively; CD4 + in H9.2.1-218, H9.2.1-IL18-M7CR, H9.2.1-IL9-M7CR, H9.2.1-IL23-M7CR, H9.2.1-p40-M7CR, H9.2.1-IL36-M7CR group The cell proportions were: 47.9%, 45.8%, 40%, 51.4%, 49.1% and 45.9% respectively; the CD8 + cell proportions were: 44.6%, 47.9%, 54.5%, 42.2%, 44.6% and 48.1% respectively. There is little difference in the ratio of CD4 and CD8 between CAR molecules.
如图30F和图30G所示，使用供者6，11，17的T细胞制备CAR-T细胞时，H9.2.1 CAR组在各供者中CD4和CD8阳性细胞比例与NT组相比无显著变化，且CD4和CD8阳性细胞在表达各细胞因子-M7CR CAR分子细胞中的比例与H9.2.1相比也无明显变化，可见细胞因子-M7CR分子未影响CD4/CD8细胞亚群比例。As shown in Figure 30F and Figure 30G, when T cells from donors 6, 11, and 17 were used to prepare CAR-T cells, the proportion of CD4 and CD8 positive cells in each donor in the H9.2.1 CAR group was not significantly different from that in the NT group. changes, and the proportion of CD4 and CD8 positive cells in cells expressing each cytokine-M7CR CAR molecule has no significant change compared with H9.2.1. It can be seen that the cytokine-M7CR molecule does not affect the proportion of CD4/CD8 cell subpopulations.
如图30H和图30I所示，检测制备第7和第9天供者16来源CAR-T细胞分化表型，相比较第7天，所有CAR-T细胞在第9天TN/TSCM比例轻微下降，TCM比例轻微增加。相比较H9.2.1，H9.2.1-IL9-M7CR、H9.2.1-p40-M7CR、H9.2.1-IL36-M7CR维持更多比例TN/TSCM细胞。As shown in Figure 30H and Figure 30I, the differentiation phenotype of donor 16-derived CAR-T cells on days 7 and 9 of preparation was detected. Compared with day 7, the TN/TSCM ratio of all CAR-T cells decreased slightly on day 9. , the TCM ratio increases slightly. Compared with H9.2.1, H9.2.1-IL9-M7CR, H9.2.1-p40-M7CR, and H9.2.1-IL36-M7CR maintained a higher proportion of TN/TSCM cells.
如图30J和图30K所示，检测制备第9天供者6，11，17来源CAR-T细胞分化表型，相比较NT、H9.2.1和H9.2.1-IL12-M7CR，H9.2.1-tCD19-M7CR、H9.2.1-IL18-M7CR、H9.2.1-IL9-M7CR、H9.2.1-IL23-M7CR、H9.2.1-p40-M7CR、H9.2.1-IL36-M7CR维持更多比例TN/TSCM细胞。As shown in Figure 30J and Figure 30K, the differentiation phenotype of CAR-T cells derived from donors 6, 11, and 17 on day 9 of preparation was detected, and compared with NT, H9.2.1 and H9.2.1-IL12-M7CR, H9.2.1- tCD19-M7CR, H9.2.1-IL18-M7CR, H9.2.1-IL9-M7CR, H9.2.1-IL23-M7CR, H9.2.1-p40-M7CR, H9.2.1-IL36-M7CR maintain a higher ratio of TN/TSCM cell.
如图30L和图30M所示，检测制备第9天供者6，11，17来源CAR-T细胞分化表型，相比较NT、H9.2.1和H9.2.1-IL12-M7CR，H9.2.1-tCD19-M7CR、H9.2.1-IL18-M7CR、H9.2.1-IL9-M7CR、H9.2.1-IL23-M7CR、H9.2.1-p40-M7CR、H9.2.1-IL36-M7CR维持更高CD45RA表达。As shown in Figure 30L and Figure 30M, the differentiation phenotype of CAR-T cells derived from donors 6, 11, and 17 on day 9 of preparation was detected, and compared with NT, H9.2.1 and H9.2.1-IL12-M7CR, H9.2.1- tCD19-M7CR, H9.2.1-IL18-M7CR, H9.2.1-IL9-M7CR, H9.2.1-IL23-M7CR, H9.2.1-p40-M7CR, H9.2.1-IL36-M7CR maintained higher CD45RA expression.
如图30N和图30O所示，检测制备第9天供者6，11，17来源CAR-T细胞分化表型，相比较H9.2.1和H9.2.1-IL12-M7CR，H9.2.1-tCD19-M7CR、H9.2.1-IL18-M7CR、 H9.2.1-IL9-M7CR、H9.2.1-IL23-M7CR、H9.2.1-p40-M7CR、H9.2.1-IL36-M7CR维持更高CCR7表达。上述结果表明，细胞因子-M7CR修饰的CAR-T细胞具有更好的记忆性表型。As shown in Figure 30N and Figure 30O, the differentiation phenotype of CAR-T cells derived from donors 6, 11, and 17 on day 9 of preparation was detected. Compared with H9.2.1 and H9.2.1-IL12-M7CR, H9.2.1-tCD19- M7CR, H9.2.1-IL18-M7CR, H9.2.1-IL9-M7CR, H9.2.1-IL23-M7CR, H9.2.1-p40-M7CR, and H9.2.1-IL36-M7CR maintained higher CCR7 expression. The above results indicate that cytokine-M7CR-modified CAR-T cells have a better memory phenotype.
实施例19、构建不同信号强度M7CR CARExample 19. Construction of M7CR CARs with different signal strengths
如图31所示，构建病毒表达质粒，用于表达含不同信号强度嵌合受体M7CR。tCD19和IL7R突变(IL7Rm或M7R)组成嵌合受体tCD19-M7CR，截短的CD34(tCD34)和IL7R突变(IL7Rm或M7R)组成嵌合受体tCD34-M7CR，这些tCD19-M7CR或tCD34-M7CR由相同的tCD19(SEQ ID NO:17)或tCD34(SEQ ID NO:189)构成的胞外结构域(ECD)和不同的IL7R突变体(文中也称IL7Rm或M7R)组成，选取的4个IL7Rm能够递送不同强度的信号(见实施例4.5)。通过P2A将不同信号强度tCD19-M7CR的N端与H9.2.CAR多肽的C端连接，从而构成tCD19-M7CR修饰的H9.2.1 CAR。通过P2A将不同信号强度tCD34-M7CR的N端与BB2121 CAR多肽的C端连接，从而构成tCD34-M7CR修饰的BB2121CAR。As shown in Figure 31, a viral expression plasmid was constructed for expressing chimeric receptor M7CR containing different signal strengths. tCD19 and IL7R mutations (IL7Rm or M7R) constitute the chimeric receptor tCD19-M7CR, and truncated CD34 (tCD34) and IL7R mutations (IL7Rm or M7R) constitute the chimeric receptor tCD34-M7CR. These tCD19-M7CR or tCD34-M7CR It consists of an extracellular domain (ECD) composed of the same tCD19 (SEQ ID NO:17) or tCD34 (SEQ ID NO:189) and different IL7R mutants (also called IL7Rm or M7R in the text). The four selected IL7Rm Able to deliver signals of different strengths (see Example 4.5). The N-terminus of tCD19-M7CR with different signal strengths is connected to the C-terminus of the H9.2.CAR polypeptide through P2A to form a tCD19-M7CR modified H9.2.1 CAR. The N-terminus of tCD34-M7CR with different signal strengths is connected to the C-terminus of BB2121 CAR polypeptide through P2A to form a tCD34-M7CR modified BB2121CAR.
所述4个IL7Rm分别为IL7Rm5(SEQ ID NO:31)、IL7Rm7(SEQ ID NO:33)、IL7Rm8(SEQ ID NO:34)、IL7Rm18(SEQ ID NO:44)，其相对应的tCD19-M7CR则分别命名为tCD19-M7CR(5)(SEQ ID NO:190)、tCD19-M7CR(7)(SEQ ID NO:191)、tCD19-M7CR(8)(即文中tCD19-M7CR，SEQ ID NO:171)、tCD19-M7CR(18)(SEQ ID NO:192)，tCD19-M7CR(CPT)作为阳性对照。其相对应的tCD34-M7CR则分别命名为tCD34-M7CR(5)(SEQ ID NO:194)、tCD34-M7CR(7)(SEQ ID NO:195)、tCD34-M7CR(8)(SEQ ID NO:196)、tCD34-M7CR(18)(SEQ ID NO:197)，tCD34-M7CR(CPT)作为阳性对照(SEQ ID NO:193)。The four IL7Rm are IL7Rm5 (SEQ ID NO:31), IL7Rm7 (SEQ ID NO:33), IL7Rm8 (SEQ ID NO:34), IL7Rm18 (SEQ ID NO:44), and their corresponding tCD19-M7CR Then they were named tCD19-M7CR(5)(SEQ ID NO:190), tCD19-M7CR(7)(SEQ ID NO:191), tCD19-M7CR(8) (i.e. tCD19-M7CR, SEQ ID NO:171 in the text ), tCD19-M7CR(18)(SEQ ID NO:192), tCD19-M7CR(CPT) were used as positive controls. The corresponding tCD34-M7CR are named tCD34-M7CR(5)(SEQ ID NO:194), tCD34-M7CR(7)(SEQ ID NO:195), tCD34-M7CR(8)(SEQ ID NO: 196), tCD34-M7CR(18) (SEQ ID NO:197), tCD34-M7CR(CPT) as a positive control (SEQ ID NO:193).
实施例20、制备不同信号强度M7CR修饰CAR-T细胞Example 20. Preparation of M7CR modified CAR-T cells with different signal intensities
实施例20.1.序列合成Example 20.1. Sequence synthesis
人工合成DNA序列，所述DNA序列分别编码H9.2.1CAR(SEQ ID NO:100)、tCD19-M7CR(CPT)(SEQ ID NO:154)、H9.2.1-tCD19-M7CR(5)(SEQ ID NO:155)、H9.2.1-tCD19-M7CR(7)(SEQ ID NO:156)、H9.2.1-tCD19-M7CR(8)(SEQ ID NO:101)、H9.2.1-tCD19-M7CR(18))(SEQ ID NO:157)、BB2121-CAR(SEQ ID NO:158)、BB2121-tCD34-M7CR(CPT)(SEQ ID NO:159)、BB2121-tCD34-M7CR(5))(SEQ ID NO:160)、BB2121-tCD34-M7CR(7)(SEQ ID NO:161)、BB2121-tCD34-M7CR(8)(SEQ ID NO:162)、BB2121-tCD34-M7CR(18)(SEQ ID NO:163)。Artificially synthesized DNA sequences encoding H9.2.1CAR (SEQ ID NO:100), tCD19-M7CR (CPT) (SEQ ID NO:154), H9.2.1-tCD19-M7CR (5) (SEQ ID NO:155), H9.2.1-tCD19-M7CR(7)(SEQ ID NO:156), H9.2.1-tCD19-M7CR(8)(SEQ ID NO:101), H9.2.1-tCD19-M7CR(18 ))(SEQ ID NO:157), BB2121-CAR(SEQ ID NO:158), BB2121-tCD34-M7CR(CPT)(SEQ ID NO:159), BB2121-tCD34-M7CR(5))(SEQ ID NO :160), BB2121-tCD34-M7CR(7)(SEQ ID NO:161), BB2121-tCD34-M7CR(8)(SEQ ID NO:162), BB2121-tCD34-M7CR(18)(SEQ ID NO:163 ).
在所述构建体中，H9.2.1 CAR分子(SEQ ID NO:100)和BB2121-CAR分子(SEQ ID NO:158)多肽分别通过P2A(SEQ ID NO:3)连接M7CR分子。In the construct, H9.2.1 CAR molecule (SEQ ID NO: 100) and BB2121-CAR molecule (SEQ ID NO: 158) polypeptides are connected to the M7CR molecule through P2A (SEQ ID NO: 3) respectively.
所述H9.2.1-tCD19-M7CR(CPT)分子(SEQ ID NO:154)从N端到C端包含所述H9.2.1 CAR分子(SEQ ID NO:100)、P2A(SEQ ID NO：3)、SP(SEQ ID NO：2)以及tCD19-M7CR(CPT)(SEQ ID NO:182)。所述H9.2.1-tCD19-M7CR(5)分子(SEQ ID NO:155)从N端到C端包含所述H9.2.1 CAR分子(SEQ ID NO:100)、P2A(SEQ ID NO：3)、SP(SEQ ID NO：2)以及tCD19-M7CR(5)(SEQ ID NO:190)。所述H9.2.1-tCD19-M7CR(7)分子(SEQ ID NO:156)从N端到C端包含所述H9.2.1 CAR分子(SEQ ID NO:100)、P2A(SEQ ID NO：3)、SP (SEQ ID NO：2)以及tCD19-M7CR(7)(SEQ ID NO:191)。所述H9.2.1-tCD19-M7CR(8)分子(SEQ ID NO:101)从N端到C端包含所述H9.2.1 CAR分子(SEQ ID NO:100)、P2A(SEQ ID NO：3)、SP(SEQ ID NO：2)以及tCD19-M7CR(8)(SEQ ID NO:171)。所述H9.2.1-tCD19-M7CR(18)分子(SEQ ID NO:157)从N端到C端包含所述H9.2.1 CAR分子(SEQ ID NO:100)、P2A(SEQ ID NO：3)、SP(SEQ ID NO：2)以及tCD19-M7CR(18)(SEQ ID NO:192)。The H9.2.1-tCD19-M7CR (CPT) molecule (SEQ ID NO: 154) includes the H9.2.1 CAR molecule (SEQ ID NO: 100) and P2A (SEQ ID NO: 3) from the N-terminus to the C-terminus. , SP (SEQ ID NO: 2) and tCD19-M7CR (CPT) (SEQ ID NO: 182). The H9.2.1-tCD19-M7CR(5) molecule (SEQ ID NO:155) includes the H9.2.1 CAR molecule (SEQ ID NO:100) and P2A (SEQ ID NO:3) from the N-terminus to the C-terminus. , SP (SEQ ID NO:2) and tCD19-M7CR(5) (SEQ ID NO:190). The H9.2.1-tCD19-M7CR (7) molecule (SEQ ID NO: 156) includes the H9.2.1 CAR molecule (SEQ ID NO: 100) and P2A (SEQ ID NO: 3) from the N end to the C end. , SP (SEQ ID NO:2) and tCD19-M7CR(7) (SEQ ID NO:191). The H9.2.1-tCD19-M7CR (8) molecule (SEQ ID NO: 101) includes the H9.2.1 CAR molecule (SEQ ID NO: 100) and P2A (SEQ ID NO: 3) from the N end to the C end. , SP (SEQ ID NO:2) and tCD19-M7CR(8) (SEQ ID NO:171). The H9.2.1-tCD19-M7CR (18) molecule (SEQ ID NO: 157) includes the H9.2.1 CAR molecule (SEQ ID NO: 100) and P2A (SEQ ID NO: 3) from the N end to the C end. , SP (SEQ ID NO:2) and tCD19-M7CR(18) (SEQ ID NO:192).
所述BB2121-tCD34-M7CR(CPT)分子(SEQ ID NO:159)从N端到C端包含所述BB2121CAR分子(SEQ ID NO:158)、P2A(SEQ ID NO：3)、SP(SEQ ID NO：2)以及tCD34-M7CR(CPT)(SEQ ID NO:193)。所述BB2121-tCD34-M7CR(5)分子(SEQ ID NO:160)从N端到C端包含所述BB2121 CAR分子(SEQ ID NO:158)、P2A(SEQ ID NO：3)、SP(SEQ ID NO：2)以及tCD34-M7CR(5)(SEQ ID NO:194)。所述BB2121-tCD34-M7CR(7)分子(SEQ ID NO:161)从N端到C端包含所述BB2121 CAR分子(SEQ ID NO:158)、P2A(SEQ ID NO：3)、SP(SEQ ID NO：2)以及tCD34-M7CR(7)(SEQ ID NO:195)。所述BB2121-tCD34-M7CR(8)分子(SEQ ID NO:162)从N端到C端包含所述BB2121 CAR分子(SEQ ID NO:158)、P2A(SEQ ID NO：3)、SP(SEQ ID NO：2)以及tCD34-M7CR(8)(SEQ ID NO:196)。所述BB2121-tCD34-M7CR(18)分子(SEQ ID NO:163)从N端到C端包含所述BB2121 CAR分子(SEQ ID NO:158)、P2A(SEQ ID NO：3)、SP(SEQ ID NO：2)以及tCD34-M7CR(18)(SEQ ID NO:197)。The BB2121-tCD34-M7CR (CPT) molecule (SEQ ID NO: 159) includes the BB2121CAR molecule (SEQ ID NO: 158), P2A (SEQ ID NO: 3), SP (SEQ ID NO: 3) from the N end to the C end. NO: 2) and tCD34-M7CR(CPT) (SEQ ID NO: 193). The BB2121-tCD34-M7CR(5) molecule (SEQ ID NO:160) includes the BB2121 CAR molecule (SEQ ID NO:158), P2A (SEQ ID NO:3), SP (SEQ ID NO: 2) and tCD34-M7CR(5) (SEQ ID NO: 194). The BB2121-tCD34-M7CR(7) molecule (SEQ ID NO:161) includes the BB2121 CAR molecule (SEQ ID NO:158), P2A (SEQ ID NO:3), SP (SEQ ID NO: 2) and tCD34-M7CR(7) (SEQ ID NO: 195). The BB2121-tCD34-M7CR(8) molecule (SEQ ID NO:162) includes the BB2121 CAR molecule (SEQ ID NO:158), P2A (SEQ ID NO:3), SP (SEQ ID NO: 2) and tCD34-M7CR(8) (SEQ ID NO: 196). The BB2121-tCD34-M7CR (18) molecule (SEQ ID NO: 163) includes the BB2121 CAR molecule (SEQ ID NO: 158), P2A (SEQ ID NO: 3), SP (SEQ ID NO: 2) and tCD34-M7CR(18) (SEQ ID NO: 197).
在上述不同信号强度M7CR相关构建体中，所有M7CR分子从N端至C端均包含GM-CSFRα-SP(SEQ ID NO:2)、tCD19(SEQ ID NO:17)或tCD34(SEQ ID NO:189)构成的胞外结构域(ECD)和不同的IL7R突变体。In the above M7CR-related constructs with different signal strengths, all M7CR molecules contain GM-CSFRα-SP (SEQ ID NO: 2), tCD19 (SEQ ID NO: 17) or tCD34 (SEQ ID NO:) from N-terminus to C-terminus. 189) and different IL7R mutants.
将上述合成的DNA片段插入pCKW慢病毒表达载体(金唯智公司)的EF1α启动子下游，替换原载体中EGFR序列，得到相应的表达质粒(由金唯智公司合成)。The above-mentioned synthesized DNA fragment was inserted into the downstream of the EF1α promoter of the pCKW lentiviral expression vector (Genewise Company), and the EGFR sequence in the original vector was replaced to obtain the corresponding expression plasmid (synthesized by Genewise Company).
实施例20.2.慢病毒及CAR-T细胞制备Example 20.2. Preparation of lentivirus and CAR-T cells
慢病毒制备方法同实施例4.2，CAR-T制备方法同实施例4.3。图32显示供者15来源CAR-T细胞第1天到第9天的扩增动力学，各CAR-T总体扩增接近，第9天收获时扩增倍数约在90-120倍之间。图40A显示供者13、16、17来源CAR-T细胞第1天到第9天的扩增动力学，图40B显示第9天收获时扩增倍数，结果显示，除M7CR(5)外，其他M7CR修饰促进了CAR-T细胞体外扩增。The lentivirus preparation method is the same as Example 4.2, and the CAR-T preparation method is the same as Example 4.3. Figure 32 shows the expansion kinetics of CAR-T cells derived from donor 15 from day 1 to day 9. The overall expansion of each CAR-T is close, and the expansion fold when harvested on day 9 is approximately 90-120 times. Figure 40A shows the expansion kinetics of CAR-T cells derived from donors 13, 16, and 17 from day 1 to day 9. Figure 40B shows the expansion fold at harvest on day 9. The results show that, except for M7CR(5), Other M7CR modifications promote CAR-T cell expansion in vitro.
实施例20.3.CAR表达检测及CAR-T细胞表型检测Example 20.3. CAR expression detection and CAR-T cell phenotype detection
取适量自上述实施例20.2获得的CAR-T细胞，对细胞进行CAR和细胞表型染色，具体实验步骤同实施例4.4，用流式细胞仪检测。图33A和图33B显示供者15来源CAR-T细胞的CAR表达水平。CAR分子能够在所有CAR-T细胞中表达，H9.2.1组中CAR+细胞的比例约为53.5％，而H9.2.1-tCD19-M7CR(CPT)、H9.2.1-tCD19-M7CR(5)、H9.2.1-tCD19-M7CR(7)、H9.2.1-tCD19-M7CR(8)、H9.2.1-tCD19-M7CR(18)组中的CAR+细胞的比例分别为：26.6％、24.9％、29％、28.7％、30.8％。BB2121中CAR+细胞的比例约为83.7％，而BB2121-tCD19-M7CR(CPT)、BB2121-tCD19-M7CR(5)、BB2121-tCD19-M7CR(7)、BB2121-tCD19-M7CR(8)、BB2121-tCD19-M7CR(18)中的CAR+细胞的比例分别为：79.4％、80.4％、82.5％、82.2％、85.7％。图33C为统计柱状图。图41A和图41B显示供者13、16、 17来源CAR-T细胞CAR和CD19表达水平，除M7CR(5)外，H9.2.1及其他M7CR修饰CAR-T细胞CAR表达阳性率在20-40％之间，M7CR修饰CAR-T细胞中CAR和CD19表达较一致。Take an appropriate amount of CAR-T cells obtained from the above Example 20.2, and perform CAR and cell phenotype staining on the cells. The specific experimental steps are the same as those in Example 4.4, and detected by flow cytometry. Figures 33A and 33B show the CAR expression levels of donor 15-derived CAR-T cells. CAR molecules can be expressed in all CAR-T cells. The proportion of CAR+ cells in the H9.2.1 group is approximately 53.5%, while H9.2.1-tCD19-M7CR(CPT), H9.2.1-tCD19-M7CR(5), H9 The proportions of CAR+ cells in the .2.1-tCD19-M7CR(7), H9.2.1-tCD19-M7CR(8), and H9.2.1-tCD19-M7CR(18) groups were: 26.6%, 24.9%, 29%, 28.7%, 30.8%. The proportion of CAR+ cells in BB2121 is approximately 83.7%, while BB2121-tCD19-M7CR(CPT), BB2121-tCD19-M7CR(5), BB2121-tCD19-M7CR(7), BB2121-tCD19-M7CR(8), BB2121- The proportions of CAR+ cells in tCD19-M7CR(18) were: 79.4%, 80.4%, 82.5%, 82.2%, and 85.7% respectively. Figure 33C is a statistical histogram. Figures 41A and 41B show donors 13, 16, The expression levels of CAR and CD19 in CAR-T cells from 17 sources. In addition to M7CR (5), the positive rate of CAR expression in H9.2.1 and other M7CR-modified CAR-T cells is between 20-40%. The CAR in M7CR-modified CAR-T cells The expression is more consistent with CD19.
图34A和图34B分别显示供者15来源H9.2.1和BB2121及各M7CR修饰的CAR-T细胞中CD4和CD8细胞亚群代表性流式细胞图。如图34C所示，各CAR-T细胞之间CD4和CD8细胞亚群比例接近。Figure 34A and Figure 34B show representative flow cytometry diagrams of CD4 and CD8 cell subsets in CAR-T cells derived from H9.2.1 and BB2121 derived from donor 15 and each M7CR, respectively. As shown in Figure 34C, the proportions of CD4 and CD8 cell subsets among each CAR-T cell were close.
图42A显示供者13、16、17来源CAR-T细胞中CD4和CD8细胞亚群代表性流式细胞图。如图42B所示，各CAR-T细胞之间CD4和CD8细胞亚群比例接近。Figure 42A shows representative flow cytometry diagrams of CD4 and CD8 cell subsets in CAR-T cells derived from donors 13, 16, and 17. As shown in Figure 42B, the proportions of CD4 and CD8 cell subsets among each CAR-T cell were close.
图35A和图35B分别显示供者15来源H9.2.1和BB2121及各M7CR修饰CAR-T细胞CD45RA和CCR7表达代表性流式细胞图。如图35A所示，H9.2.1、H9.2.1-tCD19-M7CR(CPT)、H9.2.1-tCD19-M7CR(5)、H9.2.1-tCD19-M7CR(7)、H9.2.1-tCD19-M7CR(8)、H9.2.1-tCD19-M7CR(18)中的TN/TSCM细胞的比例分别为：57.3％、72.2％、68.7％、68.8％、68.3％、71.4％。如图35B所示，BB2121、BB2121-tCD19-M7CR(CPT)、BB2121-tCD19-M7CR(5)、BB2121-tCD19-M7CR(7)、BB2121-tCD19-M7CR(8)、BB2121-tCD19-M7CR(18)CAR-T细胞中TN/TSCM细胞的比例分别为：78.3％、84.3％、81.8％、82.9％、82.5％、85.5％。相比较H9.2.1或BB2121 CAR-T细胞，M7CR修饰CAR-T细胞维持更高比例CD45RA+CCR7+TN/TSCM记忆性细胞。图35C显示供者15来源H9.2.1和BB2121及各M7CR修饰CAR-T细胞的CD45RA+CCR7+细胞比例统计图。Figure 35A and Figure 35B respectively show representative flow cytometry diagrams of CD45RA and CCR7 expression in H9.2.1 and BB2121 derived from donor 15 and each M7CR modified CAR-T cell. As shown in Figure 35A, H9.2.1, H9.2.1-tCD19-M7CR(CPT), H9.2.1-tCD19-M7CR(5), H9.2.1-tCD19-M7CR(7), H9.2.1-tCD19-M7CR (8) and the proportions of TN/TSCM cells in H9.2.1-tCD19-M7CR (18) were: 57.3%, 72.2%, 68.7%, 68.8%, 68.3%, and 71.4% respectively. As shown in Figure 35B, BB2121, BB2121-tCD19-M7CR(CPT), BB2121-tCD19-M7CR(5), BB2121-tCD19-M7CR(7), BB2121-tCD19-M7CR(8), BB2121-tCD19-M7CR( 18) The proportions of TN/TSCM cells in CAR-T cells are: 78.3%, 84.3%, 81.8%, 82.9%, 82.5%, 85.5% respectively. Compared with H9.2.1 or BB2121 CAR-T cells, M7CR-modified CAR-T cells maintained a higher proportion of CD45RA + CCR7 + TN/TSCM memory cells. Figure 35C shows a statistical diagram of the proportion of CD45RA+CCR7+ cells derived from H9.2.1 and BB2121 from donor 15 and each M7CR modified CAR-T cell.
图43A显示供者13、16、17来源H9.2.1及各M7CR修饰CAR-T细胞CD45RA和CCR7表达代表性流式细胞图。如图43B所示，H9.2.1、H9.2.1-tCD19-M7CR(CPT)、H9.2.1-tCD19-M7CR(5)、H9.2.1-tCD19-M7CR(7)、H9.2.1-tCD19-M7CR(8)、H9.2.1-tCD19-M7CR(18)CAR-T细胞中CD45RA+CCR7+TN/TSCM记忆性细胞比例分别约为：80.0％、85.7％、83.7％、86.7％、81.6％、83.4％。相比较H9.2.1 CAR-T细胞，各M7CR修饰的CAR-T细胞维持更高比例的CD45RA+CCR7+TN/TSCM记忆性细胞。Figure 43A shows representative flow cytometry diagrams of CD45RA and CCR7 expression in H9.2.1 and each M7CR modified CAR-T cells derived from donors 13, 16, and 17. As shown in Figure 43B, H9.2.1, H9.2.1-tCD19-M7CR(CPT), H9.2.1-tCD19-M7CR(5), H9.2.1-tCD19-M7CR(7), H9.2.1-tCD19-M7CR (8), H9.2.1-tCD19-M7CR (18) The proportions of CD45RA + CCR7 + TN/TSCM memory cells in CAR-T cells are approximately: 80.0%, 85.7%, 83.7%, 86.7%, 81.6%, 83.4 respectively %. Compared with H9.2.1 CAR-T cells, each M7CR-modified CAR-T cell maintained a higher proportion of CD45RA + CCR7 + TN/TSCM memory cells.
如图43C和图43D所示，检测供者13、16、17来源H9.2.1及各M7CR修饰CAR-T细胞分化表型，相比较H9.2.1，各M7CR修饰CAR-T细胞维持更高CD45RA表达。As shown in Figure 43C and Figure 43D, the differentiation phenotypes of H9.2.1 and each M7CR-modified CAR-T cell derived from donors 13, 16, and 17 were detected. Compared with H9.2.1, each M7CR-modified CAR-T cell maintained higher CD45RA Express.
如图43E和图43F所示，检测供者13、16、17来源H9.2.1及各M7CR修饰CAR-T细胞分化表型，相比较H9.2.1，各M7CR修饰CAR-T细胞维持更高CCR7表达。上述结果表明，不同信号强度M7CR修饰的CAR-T细胞维持更好的记忆性细胞表型。As shown in Figure 43E and Figure 43F, the differentiation phenotypes of H9.2.1 and each M7CR-modified CAR-T cell derived from donors 13, 16, and 17 were detected. Compared with H9.2.1, each M7CR-modified CAR-T cell maintained higher CCR7 Express. The above results indicate that M7CR-modified CAR-T cells with different signal strengths maintain a better memory cell phenotype.
实施例20.4.CAR-T细胞胞内磷酸化STAT5信号Example 20.4. Intracellular phosphorylated STAT5 signal in CAR-T cells
具体实验步骤同实施例12.4。如图36A和图36B分别显示制备第5天的CAR-T细胞和冻存复苏后CAR-T细胞胞内磷酸化STAT5(pSTAT5)表达，与NT或H9.2.1 CAR-T细胞、BB2121 CAR-T细胞比较，各M7CR修饰的CAR-T细胞具有更高的pSTAT5表达水平，说明M7CR修饰激活了CAR-T细胞中STAT5信号。根据激活STAT5信号的强度，M7CR排序为：M7CR(8)≥M7CR(7)>M7CR(5)>M7CR(18)。The specific experimental steps are the same as those in Example 12.4. Figure 36A and Figure 36B respectively show the expression of intracellular phosphorylated STAT5 (pSTAT5) in CAR-T cells on the 5th day of preparation and after cryopreservation and recovery, compared with NT or H9.2.1 CAR-T cells, BB2121 CAR- Comparing T cells, each M7CR-modified CAR-T cell had a higher pSTAT5 expression level, indicating that M7CR modification activated STAT5 signaling in CAR-T cells. According to the intensity of the activated STAT5 signal, the M7CR order is: M7CR(8)≥M7CR(7)>M7CR(5)>M7CR(18).
实施例21、不同信号强度M7CR修饰的CAR-T细胞激活实验Example 21. Activation experiment of M7CR-modified CAR-T cells with different signal intensities
如图37A所示，H9.2.1 CAR-T细胞及各M7CR修饰CAR-T细胞与PANC1或NUGC-4肿瘤细胞在E:T为1:1时进行共孵育培养。24h后收集100μL上清用于细胞因子检测。收获细胞并染色CD25和CD69，细胞染色方法及步骤见实施例13。 As shown in Figure 37A, H9.2.1 CAR-T cells and each M7CR-modified CAR-T cell were co-incubated with PANC1 or NUGC-4 tumor cells when E:T was 1:1. After 24 h, 100 μL of supernatant was collected for cytokine detection. The cells were harvested and stained for CD25 and CD69. See Example 13 for cell staining methods and procedures.
图37A显示供者15来源的H9.2.1 CAR-T细胞及各M7CR修饰CAR-T细胞与NUGC-4肿瘤细胞培养24h后CD25和CD69表达的代表性流式细胞图。图37B显示统计结果，与NT相比，H9.2.1 CAR-T细胞与NUGC-4共孵育24h后显著激活。与H9.2.1 CAR-T细胞相比，各M7CR修饰的CAR-T细胞中CD25+CD69+细胞比例增加，增加强度与上述M7CR激活STAT5信号强度较一致，说明M7CR通过激活STAT5信号促进CAR-T细胞在抗原刺激后的激活。Figure 37A shows representative flow cytometry diagrams of CD25 and CD69 expression after H9.2.1 CAR-T cells derived from donor 15 and each M7CR-modified CAR-T cell were cultured with NUGC-4 tumor cells for 24 hours. Figure 37B shows the statistical results. Compared with NT, H9.2.1 CAR-T cells were significantly activated after incubation with NUGC-4 for 24 hours. Compared with H9.2.1 CAR-T cells, the proportion of CD25+CD69 + cells in each M7CR-modified CAR-T cell increased, and the intensity of the increase was consistent with the above-mentioned M7CR-activated STAT5 signal intensity, indicating that M7CR promotes CAR-T by activating STAT5 signal Activation of cells following antigen stimulation.
如图44A显示供者13、16、17来源的H9.2.1 CAR-T细胞及各M7CR修饰CAR-T细胞与PANC1肿瘤细胞培养24h后CAR+或CAR-细胞中CD25+CD69+细胞比例统计图。与H9.2.1CAR-T细胞相比，各M7CR修饰的CAR+细胞中CD25+CD69+细胞比例增加，而CAR-细胞中CD25+CD69+细胞比例未有明显增加，说明M7CR表达提高了低抗原表达靶细胞刺激后的CAR-T细胞激活。如图44B显示供者13、16、17来源的H9.2.1 CAR-T细胞及各M7CR修饰CAR-T细胞与NUGC-4肿瘤细胞培养24h后CAR+或CAR-细胞中CD25+CD69+细胞比例统计图。与H9.2.1 CAR-T细胞相比，除了M7CR(5)，各M7CR修饰的CAR+细胞中CD25+CD69+细胞比例增加；而CAR-细胞中CD25+CD69+细胞比例未有明显增加，说明M7CR表达提高了中高抗原表达靶细胞刺激后的CAR-T细胞激活。Figure 44A shows the statistical chart of the proportion of CD25 + CD69 + cells in CAR + or CAR - cells after H9.2.1 CAR-T cells derived from donors 13, 16, and 17 and each M7CR-modified CAR-T cell were cultured with PANC1 tumor cells for 24 hours. . Compared with H9.2.1 CAR-T cells, the proportion of CD25+CD69 + cells in each M7CR-modified CAR + cell increased, while the proportion of CD25+CD69 + cells in CAR- cells did not increase significantly, indicating that M7CR expression increased the low antigen CAR-T cell activation after stimulation of expressing target cells. Figure 44B shows the proportion of CD25 + CD69 + cells in CAR + or CAR - cells after H9.2.1 CAR-T cells derived from donors 13, 16, and 17 and each M7CR-modified CAR-T cell were cultured with NUGC-4 tumor cells for 24 hours. summary graph. Compared with H9.2.1 CAR-T cells, except for M7CR(5), the proportion of CD25+CD69 + cells in each M7CR-modified CAR + cell increased; while the proportion of CD25+CD69 + cells in CAR - cells did not increase significantly, indicating that M7CR expression improves CAR-T cell activation after stimulation of medium and high antigen-expressing target cells.
实施例22、不同信号强度M7CR修饰CAR-T杀伤活性Example 22. M7CR modified CAR-T killing activity with different signal intensities
进一步地，通过与表达萤光素酶的NUGC-4细胞进行共孵育，研究供者15来源的CAR-T细胞体外对靶细胞的杀伤作用。取表达GFP-萤光素酶的NUGC-4细胞，与CAR-T细胞在96孔白板中以1:1的E:T比进行共孵育，24h后加入one-GloTM试剂(Promega，E6120)，并在30min内上机器检测。如下计算对靶细胞的细胞毒性％：Furthermore, the killing effect of CAR-T cells derived from donor 15 on target cells in vitro was studied by co-incubation with NUGC-4 cells expressing luciferase. NUGC-4 cells expressing GFP-luciferase were co-incubated with CAR-T cells in a 96-well white plate at an E:T ratio of 1:1. After 24 hours, one-Glo TM reagent (Promega, E6120) was added. , and put it on the machine for inspection within 30 minutes. The % cytotoxicity to target cells was calculated as follows:
细胞毒性％＝(NT组的发光–受试组的发光)/NT组的发光Cytotoxicity % = (luminescence of NT group – luminescence of test group)/luminescence of NT group
结果如图38所示，与H9.2.1 CAR-T相比，M7CR修饰轻微增加了CAR-T细胞对于靶细胞杀伤作用。The results are shown in Figure 38. Compared with H9.2.1 CAR-T, M7CR modification slightly increased the killing effect of CAR-T cells on target cells.
实施例23、细胞因子检测实验Example 23. Cytokine detection experiment
使用BDTM Cytometric Bead Array(CBA)Human Th1/Th2 Cytokine Kit II对实施例21中收获的细胞培养上清进行细胞因子检测，实验步骤见实施例8。The cell culture supernatant harvested in Example 21 was used to detect cytokines using BD TM Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit II. The experimental procedures are shown in Example 8.
图39显示肿瘤细胞激活CAR-T细胞24h后上清中IL-2、IFN-γ和TNFα细胞因子的水平。结果显示，与NT相比，各组CAR-T细胞在与NUGC-4共孵育24h后，上清中IL-2，IFN-γ，TNF-α浓度显著增加。与H9.2.1 CAR-T相比，M7CR修饰的CAR-T细胞分泌IL-2，IFN-γ，TNF-α细胞因子明显提高，说明M7CR修饰促进CAR-T细胞分泌效应细胞因子。Figure 39 shows the levels of IL-2, IFN-γ and TNFα cytokines in the supernatant after tumor cells activated CAR-T cells for 24 hours. The results showed that compared with NT, the concentrations of IL-2, IFN-γ, and TNF-α in the supernatant of CAR-T cells in each group increased significantly after incubation with NUGC-4 for 24 hours. Compared with H9.2.1 CAR-T, M7CR-modified CAR-T cells secreted IL-2, IFN-γ, and TNF-α cytokines significantly increased, indicating that M7CR modification promotes CAR-T cells to secrete effector cytokines.
实施例24、不同信号强度H9.2.1M7CR-CAR-T体内药效实验Example 24. In vivo efficacy experiment of H9.2.1M7CR-CAR-T with different signal intensities
通过动物实验，研究不同信号强度M7CR-CAR-T细胞在小鼠体内的抗肿瘤作用。具体实验步骤同实施例10。选用NOG小鼠，Day-5给小鼠腹腔注射NUGC-4-Gluc细胞(每只小鼠1×106个NUGC-4-Gluc细胞)进行造模，通过小动物活体成像系统对小鼠造模情况进行检测，至Day0时肿瘤负荷为1×109p/s时对动物进行分组(每组3只)。然后通过腹腔注射给予每只小鼠5×105个CAR-T细胞(供者15来源)，对照小鼠给予NT细胞(剂量与最大总T细 胞数一致)。每周对小鼠负荷进行成像，外周血中CAR-T细胞的数量。Through animal experiments, the anti-tumor effects of M7CR-CAR-T cells with different signal strengths in mice were studied. The specific experimental steps are the same as in Example 10. NOG mice were selected, and NUGC-4-Gluc cells (1 × 10 6 NUGC-4-Gluc cells per mouse) were injected intraperitoneally on Day-5 to create a model. The mice were modeled through a small animal in vivo imaging system. The model condition was tested, and the animals were divided into groups (3 animals in each group) when the tumor burden was 1×10 9 p/s on Day 0. Each mouse was then given 5 × 10 5 CAR-T cells (from donor 15) via intraperitoneal injection, and control mice were given NT cells (dose related to maximum total T cells The number of cells is the same). Mice burden, and the number of CAR-T cells in peripheral blood, were imaged weekly.
如图45A、图45B及图45C所示，相比较H9.2.1 CAR-T细胞，M7CR修饰CAR-T细胞体内具有更好更快的抗肿瘤作用，但CAR-T细胞回输2周后，各M7CR修饰CAR-T治疗组小鼠体重呈现不同程度下降，推测由于小鼠正常胃表皮细胞表达CLDN18.2产生了“在靶”毒性。As shown in Figure 45A, Figure 45B and Figure 45C, compared with H9.2.1 CAR-T cells, M7CR-modified CAR-T cells have better and faster anti-tumor effects in vivo, but after 2 weeks of reinfusion of CAR-T cells, The weight of mice in each M7CR-modified CAR-T treatment group decreased to varying degrees. It is speculated that the expression of CLDN18.2 in the normal gastric epithelial cells of the mice resulted in "on-target" toxicity.
如图45D及图45E所示，相比较H9.2.1 CAR-T细胞，除了M7CR(5)，其他M7CR修饰CAR-T细胞在小鼠体内扩增有不同程度提高。As shown in Figure 45D and Figure 45E, compared with H9.2.1 CAR-T cells, except for M7CR(5), the expansion of other M7CR-modified CAR-T cells in mice was improved to varying degrees.
实施例25、BB2121M7CR-CAR-T体内药效实验Example 25. In vivo efficacy experiment of BB2121M7CR-CAR-T
通过动物实验，研究BB2121M7CR-CAR-T细胞在小鼠体内的抗肿瘤作用。选用NOG小鼠，Day-9给小鼠皮下注射H929骨髓瘤细胞(每只小鼠2×106个H929骨髓瘤细胞)进行造模，至Day0时肿瘤体积为100-200mm3，对动物进行随机分组(每组5只)。然后通过静脉注射给予每只小鼠1×106个BB2121 CAR-T细胞或M7CR(8)修饰的CAR-T细胞(供者17来源)。每周对小鼠肿瘤体积进行测量，并检测外周血中CAR-T细胞数量。Through animal experiments, the anti-tumor effect of BB2121M7CR-CAR-T cells in mice was studied. NOG mice were selected, and H929 myeloma cells (2×10 6 H929 myeloma cells per mouse) were subcutaneously injected into the mice on Day-9 for modeling. By Day 0, the tumor volume was 100-200mm 3 , and the animals were subjected to Randomly group (5 animals per group). Each mouse was then given 1 × 10 6 BB2121 CAR-T cells or M7CR(8)-modified CAR-T cells (from donor 17) via intravenous injection. The tumor volume of mice was measured weekly, and the number of CAR-T cells in peripheral blood was detected.
如图46A所示，相比较BB2121 CAR-T细胞，M7CR(8)修饰CAR-T细胞体内具有更强抗肿瘤作用。如图46B所示，CAR-T细胞治疗后，小鼠体重未有明显下降，说明上述NUGC-4肿瘤模型中M7CR修饰CAR-T细胞产生的毒性与靶点有关。As shown in Figure 46A, compared with BB2121 CAR-T cells, M7CR(8) modified CAR-T cells have stronger anti-tumor effects in vivo. As shown in Figure 46B, after CAR-T cell treatment, the body weight of mice did not decrease significantly, indicating that the toxicity produced by M7CR-modified CAR-T cells in the above-mentioned NUGC-4 tumor model is related to the target.
实施例26、构建TGFβRII-M7CR CARExample 26. Construction of TGFβRII-M7CR CAR
如图47所示，构建病毒表达质粒，用于表达含TGFβRII胞外结构域(ECD)(SEQ ID NO:198)和IL7R突变体(文中也称IL7Rm或M7R)组成的嵌合受体TGFβRII-M7CR。文中选取IL7Rm7(SEQ ID NO：33)，构建TGFβRII-M7CR(7)(SEQ ID NO:200)。作为对照，同时设计tCD19-M7CR(SEQ ID NO:191)、显性负性TGFβRII突变体(文中又称dnTGFβRII)(SEQ ID NO:199)、TGFβRII ECD(SEQ ID NO:198)和CD28 CSD(SEQ ID NO:143)组成的嵌合受体TGFβRII-CD28(SEQ ID NO:201)、TGFβRII ECD(SEQ ID NO:198)和4-1BB CSD(SEQ ID NO:11)组成的嵌合受体TGFβRII-41BB(SEQ ID NO:202)。通过P2A将TGFβRII-M7CR(7)、dnTGFβRII、TGFβRII-CD28和TGFβRII-41BB等嵌合受体N端与H9.2.1或H9.2.1-28 CAR多肽的C端连接，从而构成TGFβR-M7CR等嵌合受体修饰的CAR。所构建的H9.2.1 CAR的序列如SEQ ID NO：100所示，所构建的H9.2.1-TGFβRII-M7CR(7)的序列如SEQ ID NO：164所示，所构建的H9.2.1--dnTGFβRII的序列如SEQ ID NO：165所示，所构建的H9.2.1-TGFβRII-CD28的序列如SEQ ID NO：166所示，所构建的H9.2.1-28 CAR的序列如SEQ ID NO：142所示，所构建的H9.2.1-28-tCD19-M7CR(7)的序列如SEQ ID NO：167所示，所构建的H9.2.1-28-TGFβRII-M7CR(7)的序列如SEQ ID NO：168所示，所构建的H9.2.1-28-dnTGFβRII的序列如SEQ ID NO：169所示，所构建的H9.2.1-28-TGFβRII-BB的序列如SEQ ID NO：170所示。As shown in Figure 47, a viral expression plasmid was constructed for expressing the chimeric receptor TGFβRII-containing TGFβRII extracellular domain (ECD) (SEQ ID NO: 198) and IL7R mutant (also called IL7Rm or M7R in the text). M7CR. In this article, IL7Rm7 (SEQ ID NO: 33) was selected to construct TGFβRII-M7CR(7) (SEQ ID NO: 200). As a control, tCD19-M7CR (SEQ ID NO:191), dominant negative TGFβRII mutant (also called dnTGFβRII in the article) (SEQ ID NO:199), TGFβRII ECD (SEQ ID NO:198) and CD28 CSD ( SEQ ID NO:143) chimeric receptor composed of TGFβRII-CD28 (SEQ ID NO:201), TGFβRII ECD (SEQ ID NO:198) and 4-1BB CSD (SEQ ID NO:11) TGFβRII-41BB (SEQ ID NO:202). The N-terminus of chimeric receptors such as TGFβRII-M7CR(7), dnTGFβRII, TGFβRII-CD28 and TGFβRII-41BB is connected to the C-terminus of H9.2.1 or H9.2.1-28 CAR polypeptide through P2A to form chimeric receptors such as TGFβR-M7CR. receptor-modified CAR. The sequence of the constructed H9.2.1 CAR is shown in SEQ ID NO: 100, the sequence of the constructed H9.2.1-TGFβRII-M7CR(7) is shown in SEQ ID NO: 164, the constructed H9.2.1-- The sequence of dnTGFβRII is shown in SEQ ID NO: 165, the sequence of the constructed H9.2.1-TGFβRII-CD28 is shown in SEQ ID NO: 166, and the sequence of the constructed H9.2.1-28 CAR is shown in SEQ ID NO: 142 As shown, the sequence of the constructed H9.2.1-28-tCD19-M7CR(7) is shown in SEQ ID NO: 167, and the sequence of the constructed H9.2.1-28-TGFβRII-M7CR(7) is shown in SEQ ID NO. : 168, the constructed sequence of H9.2.1-28-dnTGFβRII is shown in SEQ ID NO: 169, and the constructed sequence of H9.2.1-28-TGFβRII-BB is shown in SEQ ID NO: 170.
实施例27、制备TGFβRII-M7CR CAR-T细胞Example 27. Preparation of TGFβRII-M7CR CAR-T cells
实施例27.1.序列合成Example 27.1. Sequence synthesis
人工合成DNA序列，所述DNA序列分别为H9.2.1 CAR(SEQ ID NO:100)、 H9.2.1-tCD19-M7CR(7)(SEQ ID NO:156)、H9.2.1-dnTGFβRII-M7CR(7)(SEQ ID NO:164)、H9.2.1-dnTGFβRII(SEQ ID NO:165)、H9.2.1-dnTGFβRII-CD28(SEQ ID NO:166)、H9.2.1-28-CAR(SEQ ID NO:142)、H9.2.1-28-tCD19-M7CR(7)(SEQ ID NO:167)、H9.2.1-28-dnTGFβRII-M7CR(7)(SEQ ID NO:168)、H9.2.1-28-dnTGFβRII)(SEQ ID NO:169)、H9.2.1-28-dnTGFβRII-BB(SEQ ID NO:170)Artificially synthesized DNA sequences, the DNA sequences are H9.2.1 CAR (SEQ ID NO: 100), H9.2.1-tCD19-M7CR(7)(SEQ ID NO:156), H9.2.1-dnTGFβRII-M7CR(7)(SEQ ID NO:164), H9.2.1-dnTGFβRII(SEQ ID NO:165), H9 .2.1-dnTGFβRII-CD28 (SEQ ID NO:166), H9.2.1-28-CAR (SEQ ID NO:142), H9.2.1-28-tCD19-M7CR(7) (SEQ ID NO:167), H9 .2.1-28-dnTGFβRII-M7CR(7) (SEQ ID NO:168), H9.2.1-28-dnTGFβRII) (SEQ ID NO:169), H9.2.1-28-dnTGFβRII-BB (SEQ ID NO:170 )
所述H9.2.1-tCD19-M7CR(7)分子(SEQ ID NO:156)从N端到C端包含所述H9.2.1 CAR分子(SEQ ID NO:100)、P2A(SEQ ID NO：3)、SP(SEQ ID NO：2)以及tCD19-M7CR(7)(SEQ ID NO:191)。所述H9.2.1-TGFβRII-M7CR(7)分子(SEQ ID NO:164)从N端到C端包含所述H9.2.1 CAR分子(SEQ ID NO:100)、“RAKR”序列、P2A(SEQ ID NO：3)以及TGFβRII-M7CR(7)(SEQ ID NO:200)。所述H9.2.1-dnTGFβRII分子(SEQ ID NO:165)从N端到C端包含所述H9.2.1 CAR分子(SEQ ID NO:100)、“RAKR”序列、P2A(SEQ ID NO：3)以及dnTGFβRII(SEQ ID NO:199)。所述H9.2.1-TGFβRII-28分子(SEQ ID NO:166)从N端到C端包含所述H9.2.1 CAR分子(SEQ ID NO:100)、“RAKR”序列、P2A(SEQ ID NO：3)以及TGFβRII-CD28(SEQ ID NO:201)。The H9.2.1-tCD19-M7CR(7) molecule (SEQ ID NO:156) includes the H9.2.1 CAR molecule (SEQ ID NO:100), P2A (SEQ ID NO:3) from N-terminus to C-terminus , SP (SEQ ID NO: 2) and tCD19-M7CR (7) (SEQ ID NO: 191). The H9.2.1-TGFβRII-M7CR(7) molecule (SEQ ID NO:164) contains the H9.2.1 CAR molecule (SEQ ID NO:100), "RAKR" sequence, P2A (SEQ ID NO: 3) and TGFβRII-M7CR (7) (SEQ ID NO: 200). The H9.2.1-dnTGFβRII molecule (SEQ ID NO: 165) includes the H9.2.1 CAR molecule (SEQ ID NO: 100), the "RAKR" sequence, and P2A (SEQ ID NO: 3) from the N-terminus to the C-terminus. and dnTGFβRII (SEQ ID NO:199). The H9.2.1-TGFβRII-28 molecule (SEQ ID NO:166) includes the H9.2.1 CAR molecule (SEQ ID NO:100), "RAKR" sequence, P2A (SEQ ID NO: 3) and TGFβRII-CD28 (SEQ ID NO: 201).
所述H9.2.1-28-tCD19-M7CR(7)分子(SEQ ID NO:167)从N端到C端包含所述H9.2.1-28CAR分子(SEQ ID NO:142)、“RAKR”序列、P2A(SEQ ID NO：3)、SP(SEQ ID NO：2)以及tCD19-M7CR(7)(SEQ ID NO:191)。所述H9.2.1-28-TGFβRII-M7CR(7)分子(SEQ ID NO:168)从N端到C端包含所述H9.2.1-28 CAR分子(SEQ ID NO:142)、“RAKR”序列、P2A(SEQ ID NO：3)以及TGFβRII-M7CR(7)(SEQ ID NO:200)。所述H9.2.1-28-dnTGFβRII分子(SEQ ID NO:169)从N端到C端包含所述H9.2.1 CAR分子(SEQ ID NO:142)、“RAKR”序列、P2A(SEQ ID NO：3)以及dnTGFβRII(SEQ ID NO:199)。所述H9.2.1-28-TGFβRII-BB分子(SEQ ID NO:170)从N端到C端包含所述H9.2.1 CAR分子(SEQ ID NO:142)、“RAKR”序列、P2A(SEQ ID NO：3)以及TGFβRII-BB(SEQ ID NO:202)。The H9.2.1-28-tCD19-M7CR(7) molecule (SEQ ID NO:167) includes the H9.2.1-28CAR molecule (SEQ ID NO:142), "RAKR" sequence, P2A (SEQ ID NO: 3), SP (SEQ ID NO: 2), and tCD19-M7CR(7) (SEQ ID NO: 191). The H9.2.1-28-TGFβRII-M7CR(7) molecule (SEQ ID NO:168) includes the H9.2.1-28 CAR molecule (SEQ ID NO:142) and the "RAKR" sequence from N-terminus to C-terminus , P2A (SEQ ID NO: 3) and TGFβRII-M7CR (7) (SEQ ID NO: 200). The H9.2.1-28-dnTGFβRII molecule (SEQ ID NO:169) includes the H9.2.1 CAR molecule (SEQ ID NO:142), "RAKR" sequence, P2A (SEQ ID NO: 3) and dnTGFβRII (SEQ ID NO: 199). The H9.2.1-28-TGFβRII-BB molecule (SEQ ID NO:170) includes the H9.2.1 CAR molecule (SEQ ID NO:142), "RAKR" sequence, P2A (SEQ ID NO: 3) and TGFβRII-BB (SEQ ID NO: 202).
将上述合成的DNA片段插入pCKW慢病毒表达载体(金唯智公司)的EF1α启动子下游，替换原载体中EGFR序列，得到相应的表达质粒(由金唯智公司合成)。The above-mentioned synthesized DNA fragment was inserted into the downstream of the EF1α promoter of the pCKW lentiviral expression vector (Genewise Company), and the EGFR sequence in the original vector was replaced to obtain the corresponding expression plasmid (synthesized by Genewise Company).
实施例27.2.慢病毒及CAR-T细胞制备Example 27.2. Preparation of lentivirus and CAR-T cells
慢病毒制备方法同实施例4.2，CAR-T制备方法同实施例4.3。图48显示供者5，10，18来源的CAR-T细胞扩增动力学，总体上各CAR-T细胞扩增倍数在15-60之间，供者18来源的H9.2.1-dnTGFβRII、H9.2.1-28-dnTGFβRII扩增较低。The lentivirus preparation method is the same as Example 4.2, and the CAR-T preparation method is the same as Example 4.3. Figure 48 shows the expansion kinetics of CAR-T cells derived from donors 5, 10, and 18. Overall, the expansion fold of each CAR-T cell is between 15 and 60. H9.2.1-dnTGFβRII and H9 derived from donor 18 .2.1-28-dnTGFβRII amplification is low.
实施例27.3.CAR及TGFβRII ECD的表达检测及CAR-T细胞表型检测Example 27.3. Expression detection of CAR and TGFβRII ECD and CAR-T cell phenotype detection
取适量自上述实施例28.2获得的CAR-T细胞，FACS缓冲液(PBS+2％FBS)洗涤一次，重悬后加入含LIVE/DEAD Fixable Dead Cell Stain(Thermo，L34963)的FACS缓冲液，室温染色10-15min，洗涤两次，加入BV605-CD4(BD，562658)，BV421-CD8(BD，749366)，APC-Strep(Biolegend，405207)，PE-TGFβRII(R&D，FAB2411P-100)抗体组合，其中PE-TGFβRII用于ECD染色，具体实验步骤同实施例4.4，用流式细胞仪检测。图49A显示第7天制备的CAR-T细胞CAR和TGFβRII ECD表达的代表性流式散点图，所有CAR-T细胞都能检测到CAR表达，图49B显示CAR阳性率约在20-80％之间。在H9.2.1-TGFβRII-M7CR、H9.2.1-dnTGFβRII、H9.2.1-TGFβRII-CD28、H9.2.1-28-TGFβRII-M7CR、 H9.2.1-28-dnTGFβRII、H9.2.1-28-TGFβRII-BB CAR-T细胞中能同时检测到TGFβRII ECD表达，其中，TGFβRII-M7CR修饰的CAR-T细胞中ECD与CAR表达一致性较高，其次是dnTGFβRII，TGFβRII-CD28、TGFβRII-BB表达较差。Take an appropriate amount of CAR-T cells obtained from the above Example 28.2, wash once with FACS buffer (PBS+2% FBS), resuspend and add FACS buffer containing LIVE/DEAD Fixable Dead Cell Stain (Thermo, L34963) at room temperature. Stain for 10-15 minutes, wash twice, and add BV605-CD4 (BD, 562658), BV421-CD8 (BD, 749366), APC-Strep (Biolegend, 405207), PE-TGFβRII (R&D, FAB2411P-100) antibody combination, PE-TGFβRII was used for ECD staining. The specific experimental steps were the same as those in Example 4.4, and flow cytometry was used for detection. Figure 49A shows a representative flow scatter plot of CAR and TGFβRII ECD expression in CAR-T cells prepared on day 7. CAR expression can be detected in all CAR-T cells. Figure 49B shows that the CAR positivity rate is approximately 20-80%. between. In H9.2.1-TGFβRII-M7CR, H9.2.1-dnTGFβRII, H9.2.1-TGFβRII-CD28, H9.2.1-28-TGFβRII-M7CR, TGFβRII ECD expression can be detected simultaneously in H9.2.1-28-dnTGFβRII and H9.2.1-28-TGFβRII-BB CAR-T cells. Among them, the consistency between ECD and CAR expression in TGFβRII-M7CR-modified CAR-T cells is high. , followed by dnTGFβRII, TGFβRII-CD28, and TGFβRII-BB expression was poor.
图50A和图50B显示CAR-T细胞中CD4和CD8亚群代表性流式散点图和统计图。相比较H9.2.1或H9.2.1-28 CAR-T细胞，表达TGFβRII-M7CR轻微增加了CD4T细胞比例。Figure 50A and Figure 50B show representative flow scatter plots and statistical diagrams of CD4 and CD8 subpopulations in CAR-T cells. Compared with H9.2.1 or H9.2.1-28 CAR-T cells, expression of TGFβRII-M7CR slightly increased the proportion of CD4 T cells.
实施例28、TGFβRII-M7CR CAR-T细胞的长期杀伤功能Example 28. Long-term killing function of TGFβRII-M7CR CAR-T cells
进一步地，采用Claudin18.2中高表达水平HUP-T4作为靶细胞，实施了体外低效靶比长期杀伤实验，研究TGFβRII-M7CR修饰的CAR-T体外对靶细胞的杀伤作用。Furthermore, HUP-T4 with medium and high expression levels of Claudin18.2 was used as the target cell, and an in vitro low-efficiency target ratio long-term killing experiment was carried out to study the killing effect of TGFβRII-M7CR modified CAR-T on target cells in vitro.
用xCELLigence RTCA MP仪器(Agilent company)动态实时检测CAR-T细胞对靶细胞的杀伤情况。在E-Plates板中添加50μL培养基，仪器读取基准值后，添加50μL肿瘤靶细胞，然后置于机器内对细胞生长情况进行动态监测。复苏实施例28.3制备的NT细胞以及CAR-T细胞(供者5、10来源CAR-T细胞)，置于37℃细胞培养箱中过夜。次日，按1:50的E:T比例将CAR-T加入对应组别E-Plates孔中，在重悬CAR-T时，加入TGFβ1至终浓度为10ng/ml。之后每两天进行一次半体积换液，小心吸去上清，补充等体积新鲜培养基，并补加TGFβ1，直至实验结束。如图51A－图51D所示，随着时间延长，在低效靶比实验条件下，CAR-T逐渐发挥杀伤功能。结果显示，在TGFβ1存在情况下，相比较H9.2.1或H9.2.1-28CAR-T细胞，H9.2.1-TGFβRII-M7CR、H9.2.1-dnTGFβRII、H9.2.1-TGFβRII-CD28、H9.2.1-28-TGFβRII-M7CR、H9.2.1-28-dnTGFβRII、H9.2.1-28-TGFβRII-BB CAR-T细胞具有更好的杀伤效应，其中TGFβRII-M7CR修饰CAR-T细胞具有最佳的长期杀伤功能，2个供者来源CAR-T细胞中结果类似。The xCELLigence RTCA MP instrument (Agilent company) was used to dynamically detect the killing of target cells by CAR-T cells in real time. Add 50 μL of culture medium to the E-Plates plate. After the instrument reads the baseline value, add 50 μL of tumor target cells, and then place them in the machine to dynamically monitor the cell growth. Resuscitate the NT cells and CAR-T cells (CAR-T cells derived from donors 5 and 10) prepared in Example 28.3 and place them in a 37°C cell culture incubator overnight. The next day, add CAR-T into the E-Plates wells of the corresponding group at an E:T ratio of 1:50. When resuspending the CAR-T, add TGFβ1 to a final concentration of 10ng/ml. After that, a half-volume medium change was performed every two days, the supernatant was carefully aspirated, and an equal volume of fresh culture medium was added, and TGFβ1 was added until the end of the experiment. As shown in Figure 51A-Figure 51D, as time prolongs and under low-efficiency target ratio experimental conditions, CAR-T gradually exerts its killing function. The results showed that in the presence of TGFβ1, compared with H9.2.1 or H9.2.1-28 CAR-T cells, H9.2.1-TGFβRII-M7CR, H9.2.1-dnTGFβRII, H9.2.1-TGFβRII-CD28, H9.2.1- 28-TGFβRII-M7CR, H9.2.1-28-dnTGFβRII, H9.2.1-28-TGFβRII-BB CAR-T cells have better killing effect, among which TGFβRII-M7CR modified CAR-T cells have the best long-term killing function , the results were similar in the CAR-T cells derived from the two donors.
以上描述了本发明的示例性实施方案，本领域技术人员应当理解的是，这些公开内容仅是示例性的，在本发明的范围内可以进行各种其它替换、适应和修改。因此，本发明不限于文中列举的具体实施方案。Exemplary embodiments of the present invention have been described above. It will be understood by those skilled in the art that these disclosures are exemplary only, and various other substitutions, adaptations and modifications can be made within the scope of the present invention. Therefore, the invention is not limited to the specific embodiments set forth herein.
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Claims (22)

  1. 组成型嵌合细胞因子受体，其包含胞外结构域和组成型激活的IL-7R突变体，所述胞外结构域由具有重塑肿瘤微环境能力的效应分子组成，例如，所述胞外结构域选自细胞因子、免疫效应分子、抑制性分子拮抗剂、或靶向NK细胞激活性受体的效应分子；所述组成型激活的IL-7R突变体包含IL-7R突变跨膜结构域和IL-7R胞内结构域。A constitutively chimeric cytokine receptor comprising an extracellular domain consisting of effector molecules with the ability to reshape the tumor microenvironment and a constitutively activated IL-7R mutant, e.g. The external domain is selected from the group consisting of cytokines, immune effector molecules, inhibitory molecule antagonists, or effector molecules targeting NK cell activating receptors; the constitutively activated IL-7R mutant includes an IL-7R mutant transmembrane structure domain and IL-7R intracellular domain.
  2. 根据权利要求1所述的组成型嵌合细胞因子受体，其中所述组成型激活的IL-7R突变体包含在对应于SEQ ID NO:18的第237-269位的跨膜区突变，The constitutively chimeric cytokine receptor of claim 1, wherein the constitutively activated IL-7R mutant comprises a transmembrane region mutation corresponding to positions 237-269 of SEQ ID NO: 18,
    例如包含在对应于SEQ ID NO:18的第237-269位的任一选自SEQ ID NO:203-221的跨膜区突变，For example, any transmembrane region mutation selected from SEQ ID NO: 203-221 contained in positions 237-269 corresponding to SEQ ID NO: 18,
    例如，所述组成型激活的IL-7R突变体包含任一选自SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:28、SEQ ID NO:30至SEQ ID NO:45所示的氨基酸序列，For example, the constitutively activated IL-7R mutant includes any one selected from the group consisting of SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:28, SEQ ID NO:30 to SEQ ID NO:45. amino acid sequence,
    优选地，所述组成型激活的IL-7R突变体包含任一选自SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:34和SEQ ID NO:44所示的氨基酸序列，Preferably, the constitutively activated IL-7R mutant comprises any one selected from the group consisting of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:44. The amino acid sequence of
    最优选地，所述组成型激活的IL-7R突变体包含SEQ ID NO:34所示的氨基酸序列。Most preferably, the constitutively activated IL-7R mutant comprises the amino acid sequence shown in SEQ ID NO: 34.
  3. 根据权利要求1或2所述的组成型嵌合细胞因子受体，其中The constitutive chimeric cytokine receptor according to claim 1 or 2, wherein
    作为胞外结构域的细胞因子选自任一IL-12(例如，IL-12p40或IL-12p70)、IL15(例如，IL-15或IL-15 FP，其中所述IL-15 FP是IL-15和IL-15Rα的融合蛋白，由此，所述IL-15 FP包括IL-15/IL-15Rα和IL-15Rα/IL-15两种形式的融合蛋白，所述IL-15Rα选自IL-15Rα或IL-15Rα(Sushi))、IL-21、IL-18、IL-9、IL-23、IL-36γ和IFNα2b；The cytokine as the extracellular domain is selected from any of IL-12 (e.g., IL-12p40 or IL-12p70), IL15 (e.g., IL-15 or IL-15 FP, wherein the IL-15 FP is IL-12p40 or IL-12p70). 15 and IL-15Rα fusion protein, whereby the IL-15 FP includes two forms of fusion proteins: IL-15/IL-15Rα and IL-15Rα/IL-15, and the IL-15Rα is selected from IL-15Rα. 15Rα or IL-15Rα(Sushi)), IL-21, IL-18, IL-9, IL-23, IL-36γ and IFNα2b;
    例如，所述细胞因子是SEQ ID NO：47所示的IL-15或其功能变体；SEQ ID NO：48所示的IL-15FP(IL15/IL15Rα(Sushi)融合蛋白)或其功能变体；SEQ ID NO：140所示的IL-15FP(IL15/IL15Rα融合蛋白)或其功能变体；SEQ ID NO：141所示的IL-15FP(IL15 Rα(Sushi)/IL15融合蛋白)或其功能变体；SEQ ID NO：49所示的IL-12-P70或其功能变体；SEQ ID NO：50所示的IL-12-p40或其功能变体；SEQ ID NO：51所示的IL-21或其功能变体；SEQ ID NO：52所示的IL-9或其功能变体；SEQ ID NO：53所示的IL-18或其功能变体；SEQ ID NO：54所示的IL-23或其功能变体；SEQ ID NO：55所示的IL-36γ或其功能变体；SEQ ID NO：56所示的IFNα2b或其功能变体；For example, the cytokine is IL-15 or a functional variant thereof shown in SEQ ID NO: 47; IL-15FP (IL15/IL15Rα (Sushi) fusion protein) shown in SEQ ID NO: 48 or a functional variant thereof ; IL-15FP (IL15/IL15Rα fusion protein) shown in SEQ ID NO: 140 or its functional variant; IL-15FP (IL15 Rα (Sushi)/IL15 fusion protein) shown in SEQ ID NO: 141 or its function Variant; IL-12-P70 or functional variant thereof shown in SEQ ID NO: 49; IL-12-p40 or functional variant thereof shown in SEQ ID NO: 50; IL shown in SEQ ID NO: 51 -21 or its functional variant; IL-9 or its functional variant shown in SEQ ID NO: 52; IL-18 or its functional variant shown in SEQ ID NO: 53; SEQ ID NO: 54 IL-23 or its functional variant; IL-36γ or its functional variant shown in SEQ ID NO: 55; IFNα2b or its functional variant shown in SEQ ID NO: 56;
    作为胞外结构域的免疫效应分子选自任一4-1BB靶向分子部分(例如，4-1BB配体、抗4-1BB抗体)、CD40靶向分子部分(例如，CD40配体、抗CD40抗体)、CD83靶向分子部分(例如，抗CD83抗体)、FLT3靶向分子部分(例如，FLT3配体(FTL3L)、抗FLT3抗体(αFLT3))、GITR、ICOS、CD2和ICAM-1；The immune effector molecule as the extracellular domain is selected from any 4-1BB targeting molecule moiety (e.g., 4-1BB ligand, anti-4-1BB antibody), CD40 targeting molecule moiety (e.g., CD40 ligand, anti-CD40 Antibodies), CD83-targeting molecule moieties (e.g., anti-CD83 antibodies), FLT3-targeting molecule moieties (e.g., FLT3 ligand (FTL3L), anti-FLT3 antibodies (αFLT3)), GITR, ICOS, CD2, and ICAM-1;
    例如，所述免疫效应分子是SEQ ID NO：57所示的4-1BBL或其功能变体；SEQ ID NO：58所示的CD40L或其功能变体；SEQ ID NO：59所示的FLT3L或其功能变体；SEQ ID NO：60所示的ICOS或其功能变体；SEQ ID NO：61所示的GITR或其功能变体；SEQ ID NO：62所示的ICAM-1或其功能变体；SEQ ID NO：63所示的CD2或其功能变体；SEQ ID NO：64所示的抗4-1BB或其功能变体；SEQ ID NO：65所示的抗CD40或其功能变体；SEQ ID NO： 66所示的抗CD83或其功能变体；For example, the immune effector molecule is 4-1BBL shown in SEQ ID NO: 57 or a functional variant thereof; CD40L shown in SEQ ID NO: 58 or a functional variant thereof; FLT3L shown in SEQ ID NO: 59 or Its functional variant; ICOS shown in SEQ ID NO: 60 or its functional variant; SEQ ID NO: 61 shown GITR or its functional variant; SEQ ID NO: 62 shown ICAM-1 or its functional variant body; CD2 shown in SEQ ID NO: 63 or a functional variant thereof; anti-4-1BB shown in SEQ ID NO: 64 or a functional variant thereof; anti-CD40 shown in SEQ ID NO: 65 or a functional variant thereof ;SEQ ID NO: The anti-CD83 or functional variant thereof shown in 66;
    作为胞外结构域的抑制性分子拮抗剂选自任一抗PD-L1分子、抗CD47分子、抗IL-4分子、TGFβ结合分子(例如，抗TGFβ1分子、dnTGFβRII、TGFβRII)、抗PD-1分子、抗CTLA-4分子、抗LAG-3分子、抗TIGIT分子和抗CD73分子；The inhibitory molecule antagonist of the extracellular domain is selected from any anti-PD-L1 molecule, anti-CD47 molecule, anti-IL-4 molecule, TGFβ binding molecule (eg, anti-TGFβ1 molecule, dnTGFβRII, TGFβRII), anti-PD-1 molecules, anti-CTLA-4 molecules, anti-LAG-3 molecules, anti-TIGIT molecules and anti-CD73 molecules;
    例如，所述抑制性分子拮抗剂是SEQ ID NO：67所示的抗TGFβ分子或其功能变体；SEQ ID NO：198所示的TGFβRII ECD或其功能变体；SEQ ID NO：199所示的TGFβRII或其功能变体；SEQ ID NO：68所示的抗PD-L1VHH或其功能变体；SEQ ID NO：69所示的抗CD47分子或其功能变体；SEQ ID NO：70所示的抗IL-4分子或其功能变体；SEQ ID NO：71所示的抗PD-1分子或其功能变体；SEQ ID NO：72所示的抗CTLA-4分子或其功能变体；SEQ ID NO：73所示的抗LAG-3分子或其功能变体；SEQ ID NO：74所示的抗TIGIT分子或其功能变体；SEQ ID NO：75所示的抗CD73分子或其功能变体；For example, the inhibitory molecule antagonist is the anti-TGFβ molecule shown in SEQ ID NO: 67 or its functional variant; the TGFβRII ECD shown in SEQ ID NO: 198 or its functional variant; SEQ ID NO: 199 TGFβRII or functional variant thereof; anti-PD-L1 VHH shown in SEQ ID NO: 68 or functional variant thereof; anti-CD47 molecule shown in SEQ ID NO: 69 or functional variant thereof; SEQ ID NO: 70 The anti-IL-4 molecule shown in SEQ ID NO: 71 or its functional variant; the anti-PD-1 molecule shown in SEQ ID NO: 71 or its functional variant; the anti-CTLA-4 molecule shown in SEQ ID NO: 72 or its functional variant ; The anti-LAG-3 molecule shown in SEQ ID NO: 73 or its functional variant; the anti-TIGIT molecule shown in SEQ ID NO: 74 or its functional variant; the anti-CD73 molecule shown in SEQ ID NO: 75 or its functional variant; Functional variants;
    作为胞外结构域的靶向NK细胞激活性受体的效应分子选自靶向NK细胞激活性受体NKG2C、NKG2D、NKp30、NKp44和NKp46的分子，例如，抗NKG2C、抗NKG2D、抗NKp30、抗NKp44、抗NKp46，通过激活内源性NK细胞，获得增强的抗肿瘤免疫效应；Effector molecules targeting NK cell activating receptors as extracellular domains are selected from molecules targeting NK cell activating receptors NKG2C, NKG2D, NKp30, NKp44 and NKp46, for example, anti-NKG2C, anti-NKG2D, anti-NKp30, Anti-NKp44 and anti-NKp46 can achieve enhanced anti-tumor immune effects by activating endogenous NK cells;
    例如，所述NK细胞激活分子是SEQ ID NO：76所示的抗NKG2D或其功能变体；SEQ ID NO：77所示的抗NKG2C或其功能变体；SEQ ID NO：78所示的抗NKp30或其功能变体；SEQ ID NO：79所示的抗NKp46或其功能变体。For example, the NK cell activating molecule is anti-NKG2D or a functional variant thereof shown in SEQ ID NO: 76; anti-NKG2C or a functional variant thereof shown in SEQ ID NO: 77; anti-NKG2D shown in SEQ ID NO: 78 NKp30 or functional variants thereof; anti-NKp46 or functional variants thereof shown in SEQ ID NO:79.
  4. 根据权利要求1或2所述的组成型嵌合细胞因子受体，其中所述胞外结构域选自IL-12(例如，IL-12p40或IL-12p70)、IL15(例如，IL-15或IL-15 FP，其中所述IL-15 FP是IL-15和IL-15Rα的融合蛋白，由此，所述IL-15 FP包括IL-15/IL-15Rα和IL-15Rα/IL-15两种形式的融合蛋白，所述IL-15Rα选自IL-15Rα或IL-15Rα(Sushi))、IL-18、IL-9、IL-21、IL-36γ、IL-23、TGFβRII ECD或其功能变体、4-1BB配体、CD40配体或抗PD-L1纳米抗体；The constitutive chimeric cytokine receptor according to claim 1 or 2, wherein the extracellular domain is selected from the group consisting of IL-12 (eg, IL-12p40 or IL-12p70), IL15 (eg, IL-15 or IL-15 FP, wherein the IL-15 FP is a fusion protein of IL-15 and IL-15Rα, whereby the IL-15 FP includes both IL-15/IL-15Rα and IL-15Rα/IL-15 A form of fusion protein, the IL-15Rα is selected from IL-15Rα or IL-15Rα (Sushi)), IL-18, IL-9, IL-21, IL-36γ, IL-23, TGFβRII ECD or its function variants, 4-1BB ligands, CD40 ligands or anti-PD-L1 Nanobodies;
    例如，所述组成型嵌合细胞因子受体具有SEQ ID NO：172所示的序列或其功能变体、SEQ ID NO：173所示的序列或其功能变体、SEQ ID NO：174所示的序列或其功能变体、SEQ ID NO：175所示的序列或其功能变体、SEQ ID NO：176所示的序列或其功能变体、SEQ ID NO：177所示的序列或其功能变体、SEQ ID NO：178所示的序列或其功能变体、SEQ ID NO：179所示的序列或其功能变体、SEQ ID NO：183所示的序列或其功能变体、SEQ ID NO：184所示的序列或其功能变体、SEQ ID NO：185所示的序列或其功能变体、SEQ ID NO：187所示的序列或其功能变体、SEQ ID NO：191所示的序列或其功能变体、SEQ ID NO：192所示的序列或其功能变体、SEQ ID NO：194所示的序列或其功能变体、SEQ ID NO：195所示的序列或其功能变体、SEQ ID NO：196所示的序列或其功能变体、SEQ ID NO：197所示的序列或其功能变体、SEQ ID NO：200所示的序列或其功能变体。For example, the constitutive chimeric cytokine receptor has the sequence shown in SEQ ID NO: 172 or its functional variant, the sequence shown in SEQ ID NO: 173 or its functional variant, the sequence shown in SEQ ID NO: 174 The sequence or its functional variant, the sequence shown in SEQ ID NO: 175 or its functional variant, the sequence shown in SEQ ID NO: 176 or its functional variant, the sequence shown in SEQ ID NO: 177 or its function Variant, the sequence shown in SEQ ID NO: 178 or its functional variant, the sequence shown in SEQ ID NO: 179 or its functional variant, the sequence shown in SEQ ID NO: 183 or its functional variant, SEQ ID The sequence shown in NO: 184 or its functional variant, the sequence shown in SEQ ID NO: 185 or its functional variant, the sequence shown in SEQ ID NO: 187 or its functional variant, the sequence shown in SEQ ID NO: 191 The sequence or its functional variant, the sequence shown in SEQ ID NO: 192 or its functional variant, the sequence shown in SEQ ID NO: 194 or its functional variant, the sequence shown in SEQ ID NO: 195 or its function Variant, the sequence shown in SEQ ID NO: 196 or its functional variant, the sequence shown in SEQ ID NO: 197 or its functional variant, the sequence shown in SEQ ID NO: 200 or its functional variant.
  5. 核酸分子，其编码权利要求1至4中任一项所述的组成型嵌合细胞因子受体。Nucleic acid molecule encoding the constitutive chimeric cytokine receptor of any one of claims 1 to 4.
  6. 载体，其包含权利要求5所述的核酸分子，例如，所述载体选自DNA载体、RNA载体、质粒、慢病毒载体、腺病毒载体或逆转录病毒载体。 A vector comprising the nucleic acid molecule of claim 5, for example, the vector is selected from the group consisting of DNA vectors, RNA vectors, plasmids, lentiviral vectors, adenoviral vectors or retroviral vectors.
  7. 细胞，其包含权利要求1至4中任一项所述的组成型嵌合细胞因子受体、权利要求5所述的核酸分子、或权利要求6所述的载体，所述细胞是例如免疫效应细胞，例如，所述免疫效应细胞是T细胞、NK细胞，例如，所述T细胞是自体T细胞或同种异体T细胞，例如，所述免疫效应细胞是自人PBMC分离T细胞、NK细胞后制备的。Cells comprising the constitutive chimeric cytokine receptor of any one of claims 1 to 4, the nucleic acid molecule of claim 5, or the vector of claim 6, the cell being, for example, an immune effector For example, the immune effector cells are T cells and NK cells. For example, the T cells are autologous T cells or allogeneic T cells. For example, the immune effector cells are T cells and NK cells isolated from human PBMC. prepared later.
  8. 药物组合物，其包含Pharmaceutical compositions comprising
    选自表达权利要求1至4中任一项所述的组成型嵌合细胞因子受体的免疫效应细胞(例如，T细胞、NK细胞)、编码权利要求1至4中任一项所述的组成型嵌合细胞因子受体的核酸分子、权利要求6的载体、和它们的任意组合；和Selected from immune effector cells (for example, T cells, NK cells) expressing the constitutive chimeric cytokine receptor according to any one of claims 1 to 4, encoding the constitutive chimeric cytokine receptor according to any one of claims 1 to 4 The nucleic acid molecule of a constitutive chimeric cytokine receptor, the vector of claim 6, and any combination thereof; and
    任选地可药用辅料；Optionally pharmaceutical excipients;
    例如，所述免疫效应细胞是自自体T细胞或同种异体T细胞制备的表达权利要求1至4中任一项所述的组成型嵌合细胞因子受体的T细胞，例如，所述免疫效应细胞是自人PBMC分离的T细胞制备的表达权利要求1至4中任一项所述的组成型嵌合细胞因子受体的T细胞。For example, the immune effector cells are T cells prepared from autologous T cells or allogeneic T cells expressing the constitutive chimeric cytokine receptor according to any one of claims 1 to 4, for example, the immune effector cells Effector cells are T cells expressing the constitutive chimeric cytokine receptor of any one of claims 1 to 4 prepared from T cells isolated from human PBMC.
  9. 根据权利要求8所述的药物组合物的用途，用于在受试者中增强抗肿瘤免疫效应，例如，使免疫效应细胞激活、增殖、生存和发挥免疫效应功能。The use of the pharmaceutical composition according to claim 8 is used to enhance the anti-tumor immune effect in a subject, for example, to activate, proliferate, survive and exert immune effector functions of immune effector cells.
  10. 根据权利要求8所述的药物组合物在制备用于治疗肿瘤的药物中的用途。Use of the pharmaceutical composition according to claim 8 in the preparation of medicaments for treating tumors.
  11. 权利要求1至4中任一项所述的组成型嵌合细胞因子受体修饰的CAR多肽或TCR多肽，其包含位于CAR多肽或TCR多肽的N端或者C端的权利要求1至4中任一项所述的组成型嵌合细胞因子受体，且所述组成型嵌合细胞因子受体与CAR多肽或TCR多肽之间具有自切割肽或IRES序列，例如，所述自切割肽是来自口蹄病毒或心脏病毒的2A自切割肽，例如，SEQ ID NO:3所示的P2A。The constitutively chimeric cytokine receptor modified CAR polypeptide or TCR polypeptide according to any one of claims 1 to 4, which comprises any one of claims 1 to 4 located at the N-terminus or C-terminus of the CAR polypeptide or TCR polypeptide. The constitutive chimeric cytokine receptor described in the item, and there is a self-cleaving peptide or IRES sequence between the constitutive chimeric cytokine receptor and the CAR polypeptide or TCR polypeptide, for example, the self-cleaving peptide is from oral 2A self-cleaving peptide of hoofvirus or cardiovirus, for example, P2A shown in SEQ ID NO:3.
  12. 根据权利要求11所述的组成型嵌合细胞因子受体修饰的CAR多肽，所述CAR多肽直接靶向或通过“分子开关”靶向一种或多种癌相关抗原，例如，直接靶向癌相关抗原的CAR多肽从N端至C端包含信号肽、抗原结合结构域、跨膜结构域、胞内信号结构域；通过“分子开关”靶向癌相关抗原的CAR多肽从N端至C端包含信号肽、P329G突变结合结构域、跨膜结构域、胞内信号结构域，所述CAR多肽结合“分子开关”的P329G突变，继而通过“分子开关”靶向癌相关抗原。The constitutively chimeric cytokine receptor-modified CAR polypeptide according to claim 11, which targets one or more cancer-associated antigens directly or through a "molecular switch", for example, directly targeting cancer The CAR polypeptide of related antigens contains signal peptide, antigen-binding domain, transmembrane domain, and intracellular signaling domain from N-terminus to C-terminus; the CAR polypeptide that targets cancer-related antigens through a "molecular switch" includes from N-terminus to C-terminus Containing a signal peptide, a P329G mutation binding domain, a transmembrane domain, and an intracellular signaling domain, the CAR polypeptide binds to the P329G mutation of the "molecular switch" and then targets cancer-related antigens through the "molecular switch".
  13. 根据权利要求11或12所述的组成型嵌合细胞因子受体修饰的CAR多肽，所述CAR多肽直接靶向或通过“分子开关”靶向一种或多种癌相关抗原，The constitutively chimeric cytokine receptor-modified CAR polypeptide according to claim 11 or 12, which targets one or more cancer-related antigens directly or through a "molecular switch",
    例如，所述癌相关抗原选自以下一种或多种：CD19；CD20；CD22；CD24；CD30；CD123；CD171；CD33表皮生长因子受体变体III(EGFRvIII)；神经节苷脂G2(GD2)；TNF受体家族成员B细胞成熟(BCMA)；前列腺特异性膜抗原(PSMA)；Fms样酪氨酸激酶3(FLT3)；肿瘤相关的糖蛋白72(TAG72)；CD38；CD44v6；癌胚抗原(CEA)；上皮细胞粘附分子(EPCAM)；B7H3(CD276)；KIT(CD117)；白介素13受体亚基α-2(IL-13Ra2或CD213A2)；间皮素；白介 素11受体α(IL-11Ra)；前列腺干细胞抗原(PSCA)；蛋白酶丝氨酸21；血管内皮生长因子受体2(VEGFR2)；路易斯(Y)抗原；血小板衍生生长因子受体β(PDGFR-β)；阶段特异性胚胎抗原-4(SSEA-4)；叶酸受体α；受体酪氨酸蛋白激酶ERBB2(Her2/neu)；细胞表面相关的粘蛋白1(MUC1)；表皮生长因子受体(EGFR)；神经细胞粘附分子(NCAM)；前列腺酸性磷酸酶(PAP)；突变的延伸因子2(ELF2M)；肝配蛋白B2；成纤维细胞活化蛋白α(FAP)；胰岛素样生长因子1受体(IGF-I受体)；肝配蛋白A型受体2(EphA2)；岩藻糖基GM1；唾液酸基路易斯粘附分子(sLe)；转谷氨酰胺酶5(TGS5)；高分子量黑素瘤相关抗原(HMWMAA)；邻乙酰基GD2神经节苷脂(OAcGD2)；叶酸受体β；肿瘤血管内皮标记1(TEM1/CD248)；肿瘤血管内皮标记7相关的(TEM7R)；Claudin 6(CLDN6)；CLDN18.2；促甲状腺激素受体(TSHR)；G蛋白偶联受体C类5组，成员D(GPRC5D)；X染色体开放阅读框61(CXORF61)；CD97；CD179a；间变性淋巴瘤激酶(ALK)；聚唾液酸；胎盘特异性1(PLAC1)；globoH葡糖苷神经酰胺的己糖部分(GloboH)；乳腺分化抗原(NY-BR-1)；uroplakin 2(UPK2)；甲型肝炎病毒细胞受体1(HAVCR1)；肾上腺素受体β3(ADRB3)；pannexin 3(PANX3)；G蛋白偶联受体20(GPR20)；淋巴细胞抗原6复合物，基因座K9(LY6K)；嗅觉受体51E2(OR51E2)；TCRγ交替阅读框蛋白(TARP)；肾母细胞瘤蛋白(WT1)；癌/睾丸抗原1(NY-ESO-1)；癌症/睾丸抗原2(LAGE-1A)；黑素瘤相关抗原1(MAGE-A1)；ETS易位变异基因6，位于染色体12p(ETV6-AML)；精子蛋白17(SPA17)；X抗原家族，成员1A(XAGE1)；血管生成素结合细胞表面受体2(Tie2)；黑素瘤癌睾丸抗原-1(MAD-CT-1)；黑素瘤癌睾丸抗原-2(MAD-CT-2)；FOS相关抗原1；肿瘤蛋白质p53(p53)；p53突变体；prostein；存活蛋白；端粒酶；前列腺癌肿瘤抗原-1(PCTA-1或半乳凝素8)，由T细胞识别的黑素瘤抗原1(MelanA或MART1)；大鼠肉瘤(Ras)突变体；人端粒酶逆转录酶(hTERT)；肉瘤易位断点；细胞凋亡的黑素瘤抑制剂(ML-IAP)；ERG(跨膜蛋白酶，丝氨酸2(TMPRSS2)ETS融合基因)；N-乙酰葡糖胺基转移酶V(NA17)；配对盒蛋白Pax-3(PAX3)；雄激素受体；细胞周期蛋白B1；V-myc鸟髓细胞瘤病病毒癌基因神经母细胞瘤衍生的同源物(MYCN)；Ras同源物家族成员C(RhoC)；酪氨酸酶相关蛋白2(TRP-2)；细胞色素P450 1B1(CYP1B1)；CCCTC结合因子(锌指蛋白)样(BORIS或印记位点的调节物的兄弟)，由T细胞识别的鳞状细胞癌抗原3(SART3)；配对盒蛋白Pax-5(PAX5)；顶体蛋白酶原结合蛋白sp32(OY-TES1)；淋巴细胞特异性蛋白酪氨酸激酶(LCK)；A激酶锚定蛋白4(AKAP-4)；滑膜肉瘤，X断点2(SSX2)；高级糖化终产物受体(RAGE-1)；肾泛素1(RU1)；肾泛素2(RU2)；豆荚蛋白酶；人类乳头瘤病毒E6(HPV E6)；人类乳头瘤病毒E7(HPV E7)；肠羧基酯酶；突变的热休克蛋白70-2(mut hosp 70-2)；CD79a；CD79b；CD72；白细胞相关免疫球蛋白样受体1(LAIR1)；IgA受体的Fc片段(FCAR或CD89)；白细胞免疫球蛋白样受体亚家族A成员2(LILRA2)；CD300分子样家族成员f(CD300LF)；C型凝集素结构域家族12成员A(CLEC12A)；骨髓基质细胞抗原2(BST2)；含有EGF样模块粘蛋白样激素受体样2(EMR2)；淋巴细胞抗原75(LY75)；磷脂酰肌醇蛋白聚糖-3(GPC3)；Fc受体样5(FCRL5)；与免疫球蛋白λ样多肽1(IGLL1)For example, the cancer-associated antigen is selected from one or more of the following: CD19; CD20; CD22; CD24; CD30; CD123; CD171; CD33 epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2 ); TNF receptor family member B cell maturation (BCMA); prostate-specific membrane antigen (PSMA); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic Antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin 13 receptor subunit α-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin IL-11Ra; prostate stem cell antigen (PSCA); protease serine 21; vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; platelet-derived growth factor receptor beta (PDGFR-β) ); stage-specific embryonic antigen-4 (SSEA-4); folate receptor alpha; receptor tyrosine protein kinase ERBB2 (Her2/neu); cell surface-associated mucin 1 (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostatic acid phosphatase (PAP); mutated elongation factor 2 (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 Receptor (IGF-I receptor); ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); transglutaminase 5 (TGS5); high Molecular weight melanoma-associated antigen (HMWMAA); o-acetyl GD2 ganglioside (OAcGD2); folate receptor beta; tumor endothelial marker 1 (TEM1/CD248); tumor endothelial marker 7-related (TEM7R); Claudin 6 (CLDN6); CLDN18.2; thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; Anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexose portion of globoH glucosylceramide (GloboH); breast differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K9 (LY6K ); Olfactory receptor 51E2 (OR51E2); TCRγ alternative reading frame protein (TARP); Wilms tumor protein (WT1); Cancer/testis antigen 1 (NY-ESO-1); Cancer/testis antigen 2 (LAGE-1A ); Melanoma-associated antigen 1 (MAGE-A1); ETS translocation variant gene 6, located on chromosome 12p (ETV6-AML); Sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); Angiopoietin Binds cell surface receptor 2 (Tie2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); FOS-related antigen 1; tumor protein p53 (p53); p53 mutants; prostein; survivin; telomerase; prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), melanoma antigen 1 recognized by T cells (MelanA or MART1) ; Rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; V-myc avian myelocytoma Viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450 1B1 (CYP1B1); CCCTC binding factor (zinc finger protein)-like (brother of BORIS or regulator of imprinted loci), squamous cell carcinoma antigen 3 (SART3) recognized by T cells; paired box protein Pax-5 (PAX5); acrosome protease-binding Protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase-anchored protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); advanced glycation end products affected body (RAGE-1); renal ubiquitin 1 (RU1); renal ubiquitin 2 (RU2); leguminase; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxyl esterase ; Mutated heat shock protein 70-2 (mut hosp 70-2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); Leukocyte immunity Globulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); Bone marrow stromal cell antigen 2 (BST2); contains EGF-like Module mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1(IGLL1)
    例如，所述癌相关抗原选自CLDN18.2和BCMA。For example, the cancer-associated antigen is selected from CLDN18.2 and BCMA.
  14. 根据权利要求13所述的组成型嵌合细胞因子受体修饰的CAR多肽，所述组成型嵌合细胞因子受体的胞外结构域选自IL-12(例如，IL-12p40或IL-12p70)、IL15(例如，IL-15 或IL-15 FP，其中所述IL-15 FP是IL-15和IL-15Rα的融合蛋白，由此，所述IL-15 FP包括IL-15/IL-15Rα和IL-15Rα/IL-15两种形式的融合蛋白，所述IL-15Rα选自IL-15Rα或IL-15Rα(Sushi))、IL-18、IL-9、IL-21、IL-36γ、IL-23、TGFβRII ECD或其功能变体、4-1BB配体、CD40配体或抗PD-L1纳米抗体；The CAR polypeptide modified by a constitutive chimeric cytokine receptor according to claim 13, wherein the extracellular domain of the constitutive chimeric cytokine receptor is selected from the group consisting of IL-12 (e.g., IL-12p40 or IL-12p70 ), IL15 (e.g., IL-15 Or IL-15 FP, wherein the IL-15 FP is a fusion protein of IL-15 and IL-15Rα, whereby the IL-15 FP includes IL-15/IL-15Rα and IL-15Rα/IL-15 Two forms of fusion protein, the IL-15Rα is selected from IL-15Rα or IL-15Rα (Sushi)), IL-18, IL-9, IL-21, IL-36γ, IL-23, TGFβRII ECD or its Functional variants, 4-1BB ligands, CD40 ligands or anti-PD-L1 Nanobodies;
    例如，所述组成型嵌合细胞因子受体具有SEQ ID NO：172所示的序列或其功能变体、SEQ ID NO：173所示的序列或其功能变体、SEQ ID NO：174所示的序列或其功能变体、SEQ ID NO：175所示的序列或其功能变体、SEQ ID NO：176所示的序列或其功能变体、SEQ ID NO：177所示的序列或其功能变体、SEQ ID NO：178所示的序列或其功能变体、SEQ ID NO：179所示的序列或其功能变体、SEQ ID NO：183所示的序列或其功能变体、SEQ ID NO：184所示的序列或其功能变体、SEQ ID NO：185所示的序列或其功能变体、SEQ ID NO：187所示的序列或其功能变体、SEQ ID NO：191所示的序列或其功能变体、SEQ ID NO：192所示的序列或其功能变体、SEQ ID NO：194所示的序列或其功能变体、SEQ ID NO：195所示的序列或其功能变体、SEQ ID NO：196所示的序列或其功能变体、SEQ ID NO：197所示的序列或其功能变体、SEQ ID NO：200所示的序列或其功能变体；For example, the constitutive chimeric cytokine receptor has the sequence shown in SEQ ID NO: 172 or its functional variant, the sequence shown in SEQ ID NO: 173 or its functional variant, the sequence shown in SEQ ID NO: 174 The sequence or its functional variant, the sequence shown in SEQ ID NO: 175 or its functional variant, the sequence shown in SEQ ID NO: 176 or its functional variant, the sequence shown in SEQ ID NO: 177 or its function Variant, the sequence shown in SEQ ID NO: 178 or its functional variant, the sequence shown in SEQ ID NO: 179 or its functional variant, the sequence shown in SEQ ID NO: 183 or its functional variant, SEQ ID The sequence shown in NO: 184 or its functional variant, the sequence shown in SEQ ID NO: 185 or its functional variant, the sequence shown in SEQ ID NO: 187 or its functional variant, the sequence shown in SEQ ID NO: 191 The sequence or its functional variant, the sequence shown in SEQ ID NO: 192 or its functional variant, the sequence shown in SEQ ID NO: 194 or its functional variant, the sequence shown in SEQ ID NO: 195 or its function Variant, the sequence shown in SEQ ID NO: 196 or its functional variant, the sequence shown in SEQ ID NO: 197 or its functional variant, the sequence shown in SEQ ID NO: 200 or its functional variant;
    所述CAR多肽直接靶向或通过“分子开关”靶向所述一种或多种癌相关抗原；The CAR polypeptide targets the one or more cancer-related antigens directly or through a "molecular switch";
    例如，所述CAR多肽直接靶向或通过“分子开关”靶向癌相关抗原CLDN18.2(例如，直接靶向CLDN18.2的CAR多肽从N端至C端包含信号肽、CLDN18.2抗原结合结构域、跨膜结构域、胞内信号结构域；通过“分子开关”靶向CLDN18.2的CAR多肽从N端至C端包含信号肽、P329G突变结合结构域、跨膜结构域、胞内信号结构域，所述CAR多肽结合“分子开关”的P329G突变，继而通过“分子开关”的靶向癌相关抗原CLDN18.2)；For example, the CAR polypeptide directly targets or targets the cancer-associated antigen CLDN18.2 through a "molecular switch" (for example, a CAR polypeptide that directly targets CLDN18.2 includes a signal peptide from the N-terminus to the C-terminus, a CLDN18.2 antigen-binding structural domain, transmembrane domain, and intracellular signaling domain; the CAR polypeptide targeting CLDN18.2 through a "molecular switch" contains a signal peptide, a P329G mutation binding domain, a transmembrane domain, and an intracellular signaling domain from the N-terminus to the C-terminus. Signaling domain, the CAR polypeptide binds to the P329G mutation of the "molecular switch", and then targets the cancer-associated antigen CLDN18.2) through the "molecular switch";
    例如，所述CAR多肽直接靶向或通过“分子开关”靶向癌相关抗原BCMA(例如，直接靶向BCMA的CAR多肽从N端至C端包含信号肽、BCMA抗原结合结构域、跨膜结构域、胞内信号结构域；通过“分子开关”靶向BCMA的CAR多肽从N端至C端包含信号肽、P329G突变结合结构域、跨膜结构域、胞内信号结构域，所述CAR多肽结合“分子开关”的P329G突变，继而通过“分子开关”的靶向癌相关抗原BCMA)。For example, the CAR polypeptide directly targets the cancer-associated antigen BCMA or targets the cancer-associated antigen BCMA through a "molecular switch" (for example, a CAR polypeptide that directly targets BCMA includes a signal peptide, a BCMA antigen-binding domain, and a transmembrane structure from the N-terminus to the C-terminus. domain, intracellular signaling domain; the CAR polypeptide that targets BCMA through a "molecular switch" includes a signal peptide, a P329G mutation binding domain, a transmembrane domain, and an intracellular signaling domain from the N-terminus to the C-terminus. The CAR polypeptide Combined with the P329G mutation of the "molecular switch", the cancer-associated antigen BCMA is then targeted through the "molecular switch".
  15. 核酸分子，其编码权利要求11-14中任一项所述的组成型嵌合细胞因子受体修饰的CAR多肽或TCR多肽。A nucleic acid molecule encoding the constitutively chimeric cytokine receptor modified CAR polypeptide or TCR polypeptide of any one of claims 11-14.
  16. 载体，其包含权利要求15所述的核酸分子，例如，所述载体选自DNA载体、RNA载体、质粒、慢病毒载体、腺病毒载体或逆转录病毒载体。A vector comprising the nucleic acid molecule of claim 15, for example, the vector is selected from the group consisting of DNA vectors, RNA vectors, plasmids, lentiviral vectors, adenoviral vectors or retroviral vectors.
  17. 细胞，其表达(1)CAR多肽或TCR多肽；和(2)权利要求1至4中任一项所述的组成型嵌合细胞因子受体；Cells expressing (1) a CAR polypeptide or a TCR polypeptide; and (2) the constitutive chimeric cytokine receptor of any one of claims 1 to 4;
    例如，其包含权利要求11-14中任一项所述的组成型嵌合细胞因子受体修饰的CAR多肽或TCR多肽、权利要求15所述的核酸分子、或权利要求16所述的载体；或者，其包含权利要求5的核酸分子的核酸构建体和表达CAR多肽或TCR多肽的核酸构建体； For example, it includes the constitutive chimeric cytokine receptor modified CAR polypeptide or TCR polypeptide of any one of claims 11-14, the nucleic acid molecule of claim 15, or the vector of claim 16; Alternatively, it comprises a nucleic acid construct of the nucleic acid molecule of claim 5 and a nucleic acid construct expressing a CAR polypeptide or a TCR polypeptide;
    所述细胞是例如免疫效应细胞，例如，所述免疫效应细胞是T细胞、NK细胞，例如，所述T细胞是自体T细胞或同种异体T细胞，例如，所述免疫效应细胞是自人PBMC分离T细胞、NK细胞后制备的。The cells are, for example, immune effector cells. For example, the immune effector cells are T cells or NK cells. For example, the T cells are autologous T cells or allogeneic T cells. For example, the immune effector cells are from human Prepared by separating T cells and NK cells from PBMC.
  18. 药物组合物，其包含Pharmaceutical compositions comprising
    选自(1)权利要求17所述的细胞；(2)编码权利要求15所述的组成型嵌合细胞因子受体修饰的CAR多肽的核酸分子；(3)权利要求16的载体；(4)权利要求5的核酸分子的核酸构建体和表达CAR多肽或TCR多肽的核酸构建体；和(5)所述(1)至(4)的任意组合；和任选地可药用辅料；Selected from (1) the cell of claim 17; (2) a nucleic acid molecule encoding the constitutively chimeric cytokine receptor modified CAR polypeptide of claim 15; (3) the vector of claim 16; (4) ) The nucleic acid construct of the nucleic acid molecule of claim 5 and the nucleic acid construct expressing the CAR polypeptide or TCR polypeptide; and (5) any combination of (1) to (4); and optionally pharmaceutically acceptable excipients;
    例如，所述细胞是自体T细胞或同种异体T细胞制备的，例如，所述细胞是自人PBMC分离的T细胞制备的。For example, the cells are prepared from autologous T cells or allogeneic T cells, for example, the cells are prepared from T cells isolated from human PBMCs.
  19. 根据权利要求18所述的药物组合物，其中，当所述CAR多肽是分子开关调控型CAR多肽时，所述药物组合物还包含分子开关。The pharmaceutical composition according to claim 18, wherein when the CAR polypeptide is a molecular switch-regulated CAR polypeptide, the pharmaceutical composition further comprises a molecular switch.
  20. 根据权利要求18或19所述的药物组合物的用途，用于在受试者中治疗肿瘤，包括向受试者施用治疗有效量的权利要求18或19所述的药物组合物。The use of the pharmaceutical composition according to claim 18 or 19 for treating tumors in a subject includes administering to the subject a therapeutically effective amount of the pharmaceutical composition according to claim 18 or 19.
  21. 根据权利要求18或19所述的药物组合物在制备用于治疗肿瘤的药物中的用途。Use of the pharmaceutical composition according to claim 18 or 19 in the preparation of medicaments for treating tumors.
  22. 一种治疗肿瘤的方法，所述方法包括向受试者施用治疗有效量的权利要求8、权利要求18和19中任一项所述的药物组合物。 A method of treating tumors, the method comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition according to any one of claims 8, 18 and 19.
PCT/CN2023/117778 2022-09-09 2023-09-08 Constitutive chimeric cytokine receptor, immune cell expressing same, and use thereof WO2024051831A1 (en)

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