WO2024051831A1 - Récepteur chimérique de cytokine constitutif, cellule immunitaire l'exprimant et utilisation associée - Google Patents

Récepteur chimérique de cytokine constitutif, cellule immunitaire l'exprimant et utilisation associée 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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    • 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
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    • 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

La présente invention concerne un récepteur chimérique de cytokine constitutif, comprenant un domaine extracellulaire et un mutant d'IL-7R activé de manière constitutive. Le domaine extracellulaire est constitué d'une molécule effectrice capable de remodeler le micro-environnement tumoral, et le mutant IL-7R activé de manière constitutive comprend un domaine transmembranaire mutant IL-7R et un domaine intracellulaire IL-7R. La présente invention concerne également un polypeptide CAR ou un polypeptide TCR modifié par le récepteur chimérique de cytokine constitutif, une cellule effectrice immunitaire modifiée pour exprimer le polypeptide CAR ou le polypeptide TCR modifié par le récepteur chimérique de cytokine constitutif, et un procédé de préparation de la cellule effectrice immunitaire. La cellule effectrice immunitaire exprimant le polypeptide CAR ou le polypeptide TCR modifié par le récepteur chimérique de cytokine constitutif selon la présente invention peut être utilisée pour traiter une tumeur chez un sujet.
PCT/CN2023/117778 2022-09-09 2023-09-08 Récepteur chimérique de cytokine constitutif, cellule immunitaire l'exprimant et utilisation associée WO2024051831A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190292533A1 (en) * 2018-03-02 2019-09-26 Allogene Therapeutics, Inc. Inducible chimeric cytokine receptors
CN110892070A (zh) * 2017-03-03 2020-03-17 F1肿瘤医学公司 用于转导和扩增淋巴细胞以及调节其活性的方法及组合物
CN112368298A (zh) * 2018-06-22 2021-02-12 凯德药业股份有限公司 嵌合跨膜蛋白及其用途
CN113383071A (zh) * 2018-11-01 2021-09-10 亘喜生物科技（上海）有限公司 用于t细胞工程化的组合物和方法
CN113543792A (zh) * 2019-03-08 2021-10-22 奥托路斯有限公司 包含工程化嵌合抗原受体和car调节剂的组合物和方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110892070A (zh) * 2017-03-03 2020-03-17 F1肿瘤医学公司 用于转导和扩增淋巴细胞以及调节其活性的方法及组合物
US20190292533A1 (en) * 2018-03-02 2019-09-26 Allogene Therapeutics, Inc. Inducible chimeric cytokine receptors
CN112368298A (zh) * 2018-06-22 2021-02-12 凯德药业股份有限公司 嵌合跨膜蛋白及其用途
CN113383071A (zh) * 2018-11-01 2021-09-10 亘喜生物科技（上海）有限公司 用于t细胞工程化的组合物和方法
CN113543792A (zh) * 2019-03-08 2021-10-22 奥托路斯有限公司 包含工程化嵌合抗原受体和car调节剂的组合物和方法

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