CN117683139A - Constitutive chimeric cytokine receptor, immune cell expressing same and application thereof - Google Patents

Abstract

The present invention relates to a constitutive chimeric cytokine receptor comprising an extracellular domain consisting of an effector molecule having the ability to remodel the tumor microenvironment and a constitutively active IL-7R mutant comprising an IL-7R mutant transmembrane domain and an IL-7R intracellular domain. The invention also relates to the constitutive chimeric cytokine receptor-modified CAR polypeptide or TCR polypeptide, an immune effector cell engineered to express the constitutive chimeric cytokine receptor-modified CAR polypeptide or TCR polypeptide; and a method for preparing the immune effector cell. Immune effector cells expressing the constitutive chimeric cytokine receptor-modified CAR polypeptides or TCR polypeptides of the invention can be used in a subject to treat a tumor.

Description

Constitutive chimeric cytokine receptor, immune cell expressing same and application thereof

Technical Field

The present invention relates generally to the field of genetic engineering and cellular immunology, and in particular, to a constitutive chimeric cytokine receptor for enhancing immune cell expansion and effector function comprising an extracellular domain consisting of an effector molecule having a remodelling tumor microenvironment and a constitutively active IL-7R mutant comprising an IL-7R mutant transmembrane domain and an IL-7R intracellular domain, whereby immune cells (e.g., T cells) express the constitutive chimeric cytokine receptor and thereby have a constitutive IL-7R self-activating signal independent of exogenous cytokine activation and effector molecule efficacy on the extracellular domain for tumor immunotherapy. The invention also relates to combinations of said constitutive chimeric cytokine receptor and chimeric antigen receptor or T cell receptor and uses thereof.

Background

In recent years, great progress has been made in the field of tumor immunotherapy, which has become a cornerstone for clinical treatment of advanced tumors, and among them, adoptive cell immunotherapy (ACT), represented by chimeric antigen receptor T cells (CAR-T), has been attracting attention due to unprecedented clinical therapeutic effects that have been shown in the treatment of refractory hematological tumors. CAR-T and T Cell Receptor (TCR) genetically modified T cells (TCR-T) are both genetically modified cell therapy products: peripheral blood T cells of a tumor patient are collected and activated, and a virus or non-virus vector mediated gene modification is adopted to enable the peripheral blood T cells to carry a CAR or a TCR capable of specifically recognizing tumor cell antigens, so that T cell tumor specific recognition and killing functions are provided. The CAR-T cells have demonstrated unprecedented clinical therapeutic effects in treating refractory hematological tumors, the first CAR-T cell product was approved by the FDA in 2017 for clinical treatment, and 6 CAR-T cell products have been approved by the FDA at present for the treatment of refractory B cell leukemia, lymphoma, myeloma, and the like. Multiple TCR-T cell therapy products are also in a critical registered clinical stage. In addition, there are various types of immune cell therapies in clinical trials, including CAR modified NK cells (CAR-NK), genetically modified tumor infiltrating T cells (TIL), genetically modified γδ T cells, iNKT cells, double negative T cells (DNT), and immune cells (NK, iT) that induce pluripotent stem cell sources (ipscs), etc.

Numerous studies explored the use of CAR-T isogenic modified immune cells in solid tumors, but to date the overall clinical efficacy was very poor. A meta study of 42 solid tumor CAR-T clinical trials found that in 375 CAR-T cell treated patients, the Objective Response Rate (ORR) was only 13.9%, with ORR and persistence of efficacy significantly lower than that of hematological tumors. The poor effect of immune cells in treating solid tumors is mainly caused by the following reasons: the tumor does not secrete matched chemokines, and the genetically modified immune cells cannot effectively enter the tumor tissue part to exert effects; even though these genetically modified immune cells are capable of infiltrating into tumor tissue, many factors rapidly place them in a functionally inactive state, limiting their in vivo expansion and survival, including but not limited to: "unfriendly" Tumor Microenvironments (TMEs) consisting of oxidative stress, nutrient loss, hypoxia, acidic pH, etc.; tumor cells produce large amounts of immunosuppressive cytokines; there are a large number of suppressor immune cells such as regulatory T cells (tregs), myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs) in tumors; upregulation of T cell endogenous negative feedback regulatory mechanisms, such as negative immune regulator expression, e.g., PD-1, results in "depletion" of function. Solid tumor cell antigens express a high degree of heterogeneity, and antigen deletion mutants are easily generated under immune pressure, resulting in immune escape. Therefore, selecting an appropriate therapeutic target, conferring the ability of genetically modified immune cells to persist, expand and be able to remodel an "unfriendly" tumor-suppressing immune microenvironment within a tumor is critical to improving the therapeutic efficacy of solid tumor treatment.

In physiological situations, 3 signals are required for optimal activation of the naive T cells, a first signal provided by the TCR, a second signal provided by co-stimulatory molecules such as CD28, 41BB, etc., and a third signal provided by the cytokine and its receptor binding, wherein the third signal is necessary for optimal proliferation, differentiation into effector cells and development of long-acting memory T cells by the naive T cells.

Genetically modified immune cells (e.g., CAR-T cells), while capable of acquiring the first and second signals through the CAR molecule, lack the third signal, thereby affecting the amplification, survival, and function of CAR-T in vivo. Systemic administration of exogenous cytokines has been reported to promote in vivo expansion and function of CAR-T, TCR-T cells in animals, but systemic administration of cytokines produces serious toxic side effects, and similar toxicities are produced by genetically modified cells in the form of transgenes to autocrine cytokines. Thus, many studies are currently actively exploring other strategies including membrane surface expression of cytokines, expression of cytokine-switching receptors (CSR), or constitutive activation of cytokine receptors or fragments thereof. For example, thomas Shum et al (WO 2018038945A 1) designed CAR-T cells expressing constitutively active IL-7R (i.e., IL-7Rα) mutants (C7R) found naturally in T lymphomas, which C7R lacks the IL-7R native extracellular domain, but which form constitutive dimers due to cysteine or proline mutations in the IL-7R transmembrane region, activate JAK1 kinase and thus downstream STAT5 and other transcriptional effectors, regulate downstream target gene expression, and ultimately promote and maintain T proliferation and survival without reliance on extracellular domain binding to ligands. Compared with unmodified CAR-T cells, the C7R modified CAR-T cells can repeatedly kill tumor cells and reduce the function exhaustion, and have better proliferation, persistence and anti-tumor functions in vivo.

Although the C7R genetically modified CAR-T cells in the prior art confer the CAR-T cells with improved in vivo and in vitro expansion and persistence capabilities, they lack the ability to actively remodel the "unfriendly" tumor-suppressing immune microenvironment, and in the face of solid tumors with severe immunosuppressive TME characteristics, these CAR-ts are still subject to the inhibitory TME, preventing their function.

Summary of The Invention

The present inventors have developed, by research, a set of recombinant polypeptides, a constitutive chimeric cytokine receptor, comprising an extracellular domain consisting of an effector molecule with remodelling of the tumor microenvironment, and a constitutively activated IL-7R mutant. The constitutive chimeric cytokine receptor of the invention endows immune cells with the ability of actively modeling 'unfriendly' TME by utilizing constitutive activated IL-7R mutant to continuously activate STAT5 signals, promote and maintain proliferation and survival of the immune cells, endows the immune cells with the effect of obtaining new extracellular effector molecules, and enables the immune cells to be more favorable for exerting anti-tumor effect by reconstructing TME to change 'cold' tumor into 'hot' tumor. Further, the combined use of the constitutive chimeric cytokine receptor of the present invention and a genetically modified immune cell (e.g., CAR-T cell) including CAR-T cell, TCR-T, CAR-NK, genetically modified TIL, γδ T cell, iNKT cell, DNT, and iPSC-derived iT and iNK cell is expected to produce a synergistic antitumor effect by stimulating or enhancing endogenous antitumor effector mechanisms in vivo.

In a first aspect, the invention provides a constitutive chimeric cytokine receptor comprising an extracellular domain and a constitutively active IL-7R mutant. The constitutively active IL-7R mutant is capable of sustained activation of STAT5 signaling, maintenance of immune effector cell (e.g., T cell) non-exogenous cytokine dependent survival, and the extracellular domain has effector functions of remodelling tumor microenvironment and eliciting an endogenous anti-tumor immune response in the body.

In some embodiments, the invention first compared 27 different constitutively activated IL-7R mutants (also referred to herein as IL7Rm or M7R) consisting of IL7R transmembrane regions (IL 7R-variants (TM)) carrying different mutations (bold portions of SEQ ID NO:20-SEQ ID NO:46 sequence in the sequence Listing) and wild type IL7R (IL 7R-WT) intracellular segments (IL 7R-WT (ICD), SEQ ID NO: 19) in vitro using an exogenous cytokine dependent BaF3 cell line. In some specific embodiments, the extracellular domain tCD19 and the 27 different M7R are combined to construct a constitutive chimeric cytokine receptor, which is also referred to as tCD19-M7CR, identified by detecting positive expression of tCD19 on the cell surface to identify expression of M7R.

The experimental results of the 27M 7R in-vitro maintenance of the non-exogenous cytokine-dependent survival effect of the BaF3 cells show that 19M 7R sequences can maintain the non-exogenous cytokine-dependent survival of the BaF3 cells, and the M7R genes can promote the proliferation of the BaF3 cells in a non-exogenous cytokine-dependent manner. And as the culture time was prolonged, the surviving BaF3 cells were all positive for M7R expression, indicating that only BaF3 cells expressing M7R could survive without the addition of exogenous cytokines. Analysis of IL-7R downstream signaling molecules using intracellular flow cytometry staining found that these 19M 7R molecules (IL7Rm1.1, IL7Rm1.3, IL7Rm3.1, IL Rm 4-19) activated and maintained BaF3 intracellular STAT5 phosphorylation at levels comparable to the addition of exogenous cytokines, suggesting that these 19M 7R activated STAT5 signaling pathways by constitutive, promoting and maintaining BaF3 cell non-exogenous cytokine dependent survival.

In other embodiments, T cells expressing M7R molecules have better viability in vitro, and in particular, the M7R shown by IL7Rm4, IL7Rm5, IL7Rm7, IL7Rm8 has very significant pro-survival effects, compared to T cells that are not transduced or transduced with IL7R-WT, as described above for the transduction and stable expression of the 19 tCD19-M7CR containing different M7R molecules in primary T cells, which expression of said M7R molecules activates STAT5 signals in T cells. On this basis, the present invention designs and constructs a constitutive chimeric cytokine receptor (also referred to herein as M7 CR) of the present invention by fusing an extracellular domain of the constitutive chimeric cytokine receptor with M7R, using a cytokine, an immune effector molecule, an inhibitory molecule antagonist, or an effector molecule targeting NK cell activating receptor. In some embodiments, the M7CR extracellular domain can be IL-12 (IL-12 p40 or IL-12p 70), IL-15 (IL-15 or IL-15FP, the IL-15FP refers to IL-15 and IL-15R alpha (selected from the group consisting of IL-15R alpha or IL-15R alpha (Sushi)) fusion proteins, including IL-15/IL-15R alpha and IL-15R alpha/IL-15 two forms of fusion proteins), IL-21, IL-18, IL-9, IL-23, IL-36 gamma, IFN alpha 2b cytokines, when immune cells (e.g., T cells) express the cytokine containing M7CR gene, has enhanced immune effector function and anti-tumor effect; in some embodiments, the M7CR extracellular domain may also be a 4-1BB targeting molecule moiety (e.g., 4-1BB ligand (4-1 BBL), anti-4-1 BB antibody (α4-1 BB)), CD40 targeting molecule moiety (e.g., CD40 ligand (CD 40L), anti-CD 40 antibody (αcd 40)), CD83 targeting molecule moiety (e.g., anti-CD 83 antibody (αcd 83)), FLT3 targeting molecule moiety (e.g., FLT3 ligand (FTL 3L), anti-FLT 3 antibody (αflt 3)), GITR, ICOS, CD2, ICAM-1, etc. immune effector molecules that activate an endogenous anti-tumor immune response by interacting with an Antigen Presenting Cell (APC) in the body, such as a Dendritic Cell (DC) or a related receptor or ligand on the surface of a macrophage, thereby eliciting an endogenous anti-tumor immune response, thereby producing a synergistic anti-tumor effect with immune cells (e.g., T cells); in some embodiments, the M7CR extracellular domain may also be an anti-PD-L1 antibody, an anti-CD 47 molecule, an anti-IL-4 molecule, an anti-TGF-beta molecule, an anti-PD-1 molecule, an anti-CTLA-4 molecule, an anti-LAG-3 molecule, an anti-TIGIT molecule, an anti-CD 73 molecule, or the like, directed against an inhibitory immunoreceptor or factor, for the purpose of enhancing an anti-tumor immune response by antagonizing the immunosuppressive effect of the inhibitory immunoreceptor or factor, thereby producing a synergistic anti-tumor effect with immune cells (e.g., T cells); in some embodiments, the M7CR extracellular domain may also be a molecular moiety that targets an activating receptor expressed on the surface of NK cells such as NKG2C, NKG2D, NKp, NKp44, NKp46, etc., e.g., anti-NKG 2C, anti-NKG 2D, anti-NKp 30, anti-NKp 44, anti-NKp 46, etc., for the purpose of enhancing an anti-tumor immune effect by activating endogenous NK cells, thereby generating a synergistic anti-tumor effect with immune cells (e.g., T cells).

In a second aspect, the invention provides an M7CR modified CAR or TCR. The novel M7CR 'armed' tumor targeted T cells (for example, the CAR-T cells expressing the M7 CR) obtain 3 signals required by the optimal activation of initial T cells, so that better T cell activation, proliferation, survival and immune effect functions are generated, meanwhile, the effector molecules of the extracellular domain of the M7CR actively remodels 'unfriendly' TME through the mechanisms of activating endogenous T cells of an organism, activating APC, antagonizing immune suppressive receptors or activating inherent immune cells such as NK of the organism and the like, promoting endogenous anti-tumor effect mechanisms and finally generating synergistic anti-tumor immune effects.

In some embodiments, the invention provides fusion proteins that express both a CAR and an M7CR of the invention (e.g., the M7CR has an extracellular domain (ECD) of tCD19, IL-12 (p 40 or p 70), IL-15 FP (including both forms of IL-15/IL-15Rα and IL-15Rα/IL-15, wherein IL-15Rα is selected from IL-15Rα or IL-15Rα (Sushi))IL-21, 4-1BBL, CD40L, anti-PD-L1 nanobody (PD-L1) _VHH ) M7R employs IL7Rm 8) and M7CR modified conventional CAR-T cells (e.g. H9.1.2CAR targeting claudin 18.2) directly targeting tumor antigens or M7CR modified "modular" PG CAR-T cells (e.g. 8B CAR) mediated by P329G mutant antibodies targeting tumors were prepared in vitro and their in vivo and in vitro functions were evaluated.

Bicistronic viral vectors expressing both CAR and M7CR were constructed from P2A self-cleaving peptides, and these virally transduced T cells expressed both CAR and M7CR of the invention, and CAR and M7CR expression of the invention were correlated.

Cell phenotype studies showed that there was no significant change in the proportion of M7R modified CAR-T cell CD4/CD8 cell subsets, and that M7CR modified CAR-T cells had different effects on T cell subsets depending on extracellular domain (ECD). For example, IL-12-M7CR modified CAR-T cells maintained a higher proportion of CD4 cell subsets.

Cell phenotype studies showed that M7R alone (e.g., tCD19-M7CR, extracellular domain tCD19 for examining the effect of M7R) modified CAR-T cells maintained a high proportion of memory cell subsets such as Tscm/Tcm, comparable to the C7R (tCD 19-M7CR (CPT), M7R sequence from C7R) effect as a positive control; m7CR modified CAR-T cells, depending on the extracellular domain (ECD), have different effects on T cell differentiation. For example, IL-15-M7CR modified CAR-T cells have a better memory phenotype, while IL-12-M7CR modification promotes CAR-T cell differentiation.

In some embodiments, the present invention provides a more detailed study of the H9.2.1 CAR-T cell phenotype of IL-15-M7CR (the M7CR extracellular ECD domain is IL15 FP) modified targeted claudin18.2, which suggests that IL-15 (IL-15-M7 CRin, inactivating the M7R signal) or M7R (tCD 19-M7 CR) effects alone significantly promote Tscm memory cell subpopulation maintenance, but IL-15-M7CR has a stronger Tscm-promoting memory cell maintenance effect, suggesting that membrane surface expression of IL-15 and M7R signals produce a synergistic effect.

In some embodiments, the invention investigates M7 CR-modified conventional CAR-T cells by in vitro killing experiments (e.g., targeting claThe killing effect of H9.1.2-BB-L CAR-T cells, H9.2.1-BB-L CAR-T cells, H9.2.1-28-L CAR-T cells, or PG CAR-T cells of udin18.2 (e.g., huR968B CAR-T cells targeted to claudin 18.2) on tumor cells positive for antigen expression indicated that the killing ability of modified CAR-T cells was significantly higher for M7R alone (e.g., tCD19-M7CR, extracellular domain tCD19 for review of the effect of M7R) than for unmodified CAR-T cells. Fusion of extracellular effector molecules on the basis of M7R to form M7CR, M7CR modification can further increase CAR-T cell killing function in vitro. For example, 4-1BBL-M7CR, anti-PD-L1 _VHH The killing capacity of conventional CAR-T cells modified by M7CR, IL-12-M7CR, IL-15-M7CR is significantly higher than that of CAR-T cells modified by unmodified or M7R alone. IL-12-M7CR, IL-15-M7CR modified PG CAR-T cell killing efficacy is enhanced, especially in tumor cells with low expression of antigen (such as SNU-601 ^low ) In this case more pronounced. It is demonstrated that extracellular effector molecules have a combined effect with M7R in promoting killing of CAR-T cells.

In other embodiments, the invention studies the in vitro proliferation capacity of M7CR modified PG CAR-T cells under repeated stimulation of tumor cells by in vitro repeated stimulation experiments, and the results show that the M7R alone (such as tCD19-M7CR, with extracellular domain of tCD19, used for examining the effect of M7R) modified PG CAR-T cells have better sustained proliferation capacity under repeated stimulation of tumor cells. M7CR formed by fusing extracellular effector molecules on the basis of M7R can further increase the in-vitro continuous proliferation capacity of PG CAR-T cells, and the PG CAR-T cells modified by the M7CR such as IL-12-M7CR, IL-15-M7CR and the like have stronger in-vitro continuous proliferation capacity. Further, phenotypic studies have found that IL-12-M7CR modified CAR-T cells have more CD4 under repeated tumor cell stimulation ⁺ T cells. Cytokine detection results showed significant increases in IFN-gamma and TNF release levels from IL-12-M7CR modified CAR-T cells.

In other embodiments, the invention uses tumor cells expressing different levels of claudin18.2 antigen as target cells, studies on the in vitro killing function of M7CR modified conventional CAR-T cells (e.g., H9.2.1-BB-L CAR-T cells, H9.2.1-28-L CAR-T cells targeting claudin 18.2) such as M7R (tCD 19-M7 CR), IL-12-M7CR, IL15-M7CR, etc., showed that the in vitro killing function of CAR-T cells modified by M7R alone (e.g., tCD19-M7CR, extracellular domain is tCD19 for examining the effect of M7R) was enhanced, the enhancement effect of M7CR modification (e.g., IL-12-M7CR, IL15-M7CR modification) was more pronounced, the killing effect of CAR-T cells on target cells with different antigen expression levels was significantly enhanced, and similar effects were observed in CAR-T cells prepared from 2 donors. The results of in vitro repeated killing experiments show that M7CR modified CAR-T cells such as M7R, IL-12-M7CR, IL15-M7CR and the like maintain better in vitro continuous killing function, and similar effects are observed in CAR-T cells prepared from 2 donors by adopting 2 different target ratios.

In some embodiments, the invention provides results from studies of the anti-tumor effect in mice with M7CR modified PG CAR-T cells, which indicate that M7CR modified CAR-T cells have a stronger anti-tumor effect in vivo and that M7CR modified CAR-T cells also have a stronger proliferative capacity than unmodified CAR-T cells.

In other embodiments, the invention provides that M7 CR-modified CAR-T cells have a greater anti-tumor effect in vivo and that M7 CR-modified CAR-T cells also have a greater proliferative capacity than unmodified CAR-T cells, as demonstrated by studying the anti-tumor effect in mice in which M7CR modifies conventional CAR-T cells (e.g., H9.2.1-BB-L CAR-T cells, H9.2.1-28-L CAR-T cells targeted to claudin 18.2).

In a third aspect, the invention provides a nucleic acid molecule encoding an M7CR of the invention or encoding an M7CR modified CAR or TCR of the invention, a vector comprising a nucleic acid molecule encoding an M7CR of the invention or encoding an M7CR modified CAR or TCR of the invention, and a cell comprising a constitutive chimeric cytokine receptor M7CR or M7CR modified CAR polypeptide of the invention, a nucleic acid molecule of the invention, or a vector of the invention, preferably the cell is an autologous T cell or an allogeneic T cell.

In a fourth aspect, the invention provides a method of producing a cell, e.g., an immune effector cell, comprising introducing (e.g., transducing) a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) encoding an M7CR of the invention or encoding an M7 CR-modified CAR or TCR of the invention, or a vector comprising a nucleic acid molecule encoding an M7CR of the invention or encoding an M7 CR-modified CAR or TCR described herein, into an immune effector cell.

In some embodiments, the immune effector cells are T cells, NK cells, e.g., the T cells are autologous T cells or allogeneic T cells, e.g., the immune effector cells are prepared after isolation of T cells, NK cells from human PBMCs.

In a fifth aspect, the invention provides a pharmaceutical composition comprising an immune effector cell (e.g., T cell, NK cell) selected from the group consisting of a constitutive chimeric cytokine receptor of the invention or a constitutive chimeric cytokine receptor modified CAR polypeptide, a nucleic acid molecule encoding a constitutive chimeric cytokine receptor of the invention or a constitutive chimeric cytokine receptor modified CAR polypeptide, a vector of the invention, and any combination thereof; and optionally pharmaceutically acceptable excipients.

In some embodiments, when the CAR polypeptide that expresses the constitutive chimeric cytokine receptor modification of the invention is a molecular switch-regulated CAR polypeptide, the pharmaceutical composition of the invention further comprises a molecular switch, e.g., a molecular switch antibody.

In a sixth aspect, the present invention relates to the use of a pharmaceutical composition according to the fifth aspect for treating a tumor in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to the fifth aspect.

In a seventh aspect, the present invention relates to the use of a pharmaceutical composition according to the fifth aspect for the preparation of a medicament for the treatment of cancer.

In an eighth aspect, the invention provides a method of treating a tumor, the method comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition of the fifth aspect.

Brief Description of Drawings

The preferred embodiments of the present invention described in detail below will be better understood when read in conjunction with the following drawings. For the purpose of illustrating the invention, there is shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

FIG. 1 shows the mechanism of action of T cells expressing M7 CRs and/or CARs of the invention after T cell transduction by the M7 CRs and/or CARs of the invention, in which M7 CRs an effector molecule (e.g., cytokine, immune effector molecule or inhibitory molecule antagonist) having a remodelling tumor microenvironment is fused to M7R, respectively, as an extracellular domain (ECD).

FIG. 2A shows the structure of wild-type IL7R receptor and the structure of engineered mutant IL7R receptor.

FIG. 2B shows schematic structural diagrams of IL7R-tCD19 constructs, IL7R-WT constructs, IL7Rm-tCD19 constructs in the constructed viral expression plasmids. In the figure, IL7Rm refers to the portion consisting of the IL7R transmembrane region (TM region) and the IL7R wild-type intracellular region (ICD) of different mutations.

FIG. 3A shows the results of detecting expression of tCD19 on the surface of BaF3 cells by flow cytometry on day 4 after infection of BaF3 cells with 27 lentiviruses containing tCD19-M7CR genes of different mutated M7R sequences. In the figure, baF3 represents BaF3 cells that have not been infected with lentivirus; IL7R-WT means that BaF3 cells are infected with a lentivirus comprising a wild type IL7R gene; IL7R-tCD19 represents infection of BaF3 cells with a lentivirus comprising the gene for tCD19, the wild-type IL7R transmembrane region and the intracellular region; IL7Rm-tCD19 indicates infection of BaF3 cells with a lentivirus comprising tCD19, a variant IL7R transmembrane region and a gene for the IL7R wild-type intracellular region.

FIG. 3B shows a graph of the results of culturing each lentiviral infected BaF3 cell without the addition of exogenous mIL-3 after infection of the BaF3 cell with a lentiviral comprising the tCD19-M7CR gene of the different mutated M7R sequences, capable of providing a sustained activation of IL7R signal and a different mutated M7R sequence promoting growth of the BaF3 cell. The meaning of each graphic representation in the figure is the same as that of figure 3A.

FIG. 3C shows infection of BaF3 cells with lentiviruses containing the tCD19-M7CR gene of different mutated M7R sequences, without addition of exogenous mIL-3 from day 3, and detection of CD19 expression at day 3 and 11 ⁺ The percentage of BaF3 cells in which CD19 is expressed ⁺ The higher the percentage of BaF3 cells, the more BaF3 cells survive.

FIG. 3D shows the results of cell counting of surviving BaF3 cells after infection of BaF3 cells with lentiviruses comprising the tCD19-M7CR gene of different mutated M7R sequences, and culturing each lentivirus-infected BaF3 cell without the addition of exogenous mIL-3. In the figure, "Parentil BaF3 with IL3" means that BaF3 cells not infected with a virus were cultured in a medium containing IL3, and "parentil BaF3 w/o IL3" means that BaF3 cells not infected with a virus were cultured in a medium containing no IL 3.

FIG. 4 shows the results of measuring the basal phosphorylation levels of STAT5 in BaF3 cells after infection of BaF3 cells with lentiviruses comprising the tCD19-M7CR gene of different mutated M7R sequences, staining BaF3 cells with anti-pSTAT 5 antibodies. In the figure, ISO indicates staining with isotype control antibody against STAT5, +il3 indicates that on BaF3 cells not infected with lentivirus, addition of IL3 stimulated STAT5 activation of the cells, as positive control; "without IL3" means that BaF3 cells not infected with lentivirus are stimulated with IL 3.

FIG. 5 shows the results of detecting T cell surface tCD19 expression by flow cytometry 48 hours after T cell infection with a lentivirus comprising a tCD19-M7CR gene having a different mutated M7R sequence. In the figure, UNT represents T cells not infected with lentivirus; IL7R-WT means that T cells are infected with a lentivirus comprising a wild type IL7R gene; IL7R-tCD19 refers to infection of T cells with a lentivirus comprising the genes for tCD19, the wild-type IL7R transmembrane region, and the intracellular region; IL7Rm-tCD19 refers to infection of T cells with a lentivirus comprising tCD19, a variant IL7R transmembrane region and a gene for the IL7R wild-type intracellular region.

FIG. 6 shows the results of measuring the basal phosphorylation levels of STAT5 in T cells stained with anti-pSTAT 5 antibodies without the addition of exogenous IL-2 stimulation after infection of T cells with lentiviruses comprising the tCD19-M7CR gene of different mutated M7R sequences. In the figure, UNT represents T cells not infected with lentivirus; IL7Rm-tCD19 refers to infection of T cells with a lentivirus comprising tCD19, a variant IL7R transmembrane region and a gene for the IL7R wild-type intracellular region.

FIG. 7A shows the results of counting the number of T cells expressing tCD19-M7CR over time without the addition of exogenous IL-2 stimulation after infection of the T cells with a lentivirus comprising the tCD19-M7CR gene of a different mutated M7R sequence.

FIG. 7B shows the fold change in T cell numbers over time of tCD19-M7CR expressing T cells without the addition of exogenous IL-2 stimulation after infection of the T cells with a lentivirus comprising the tCD19-M7CR gene with different mutated M7R sequences.

Fig. 8 shows the structure of an M7CR modified CAR, wherein M7CR comprises the extracellular domains ECD and IL7Rm. The N-terminus of M7CR is linked to the C-terminus of a different CAR polypeptide via P2A, thereby constituting an M7CR modified CAR.

Fig. 9A shows the expression levels of CAR and M7CR at day 9 after T cell infection with lentiviruses comprising different M7CR modified H9.1.2-BB-L CAR genes, and fig. 9B shows CD4 and CD8 positive cell ratios. In the figure, "UNT" means T cells not infected with lentivirus; "H9.1.2" means H9.1.2-BB-L CAR-T cells, the remainder being tCD19-M7CR, tCD19-M7CR (CPT), IL-15/IL-15Rα -M7CR (labeled IL-15-M7CR in the figure), IL-12-P70-M7CR (labeled IL-12-M7CR in the figure), IL-21-M7CR, CD40L-M7CR, 4-1BBL-M7CR, anti-PD-L1 VHH-M7CR modified H9.1.2-BB-L CAR-T cells.

Fig. 9C shows the expression levels of CAR and M7CR at day 9 after T cell infection with lentiviruses comprising different M7CR modified HuR968B CAR genes, fig. 9D shows CD4 and CD8 positive cell ratios. In the figure, "UNT" means T cells not infected with lentivirus; "8B" means HuR968B CAR-T cells, the remainder tCD19-M7CR, tCD19-M7CR (CPT), IL-15/IL-15Rα -M7CR (labeled IL-15-M7CR in the figure), IL-12-P70-M7CR (labeled IL-12-M7CR in the figure), IL-21-M7CR, CD40L-M7CR, 4-1BBL-M7CR, anti-PD-L1 _VHH -M7CR modified HuR968B CAR-T cells.

FIG. 9E shows total T cells, CD4, in unmodified H9.1.2-BB-L CAR-T samples and M7CR modified H9.1.2-BB-L CAR-T samples ⁺ And CD8 ⁺ T phenotype was detected by flow cytometry. In the figure, "UNT" means T cells not infected with lentivirus; "H9.1.2" means H9.1.2-BB-L CAR-T cells, the remainder being M7CR modified H9.1.2-BB-L CAR-T cells (labeled in the figure as in FIG. 9A).

FIG. 9F shows unmodified HuR968B CAR-T samples and M7CR modificationsTotal T cells, CD4 in HuR968B CAR-T samples ⁺ And CD8 ⁺ T phenotype was detected by flow cytometry. In the figure, "UNT" means T cells not infected with lentivirus; "8B" means HuR968B CAR-T cells, the remainder being M7CR modified HuR968B CAR-T cells (labeled in the figure as in FIG. 9C).

FIGS. 9G and 9H show total T cells, CD4, in unmodified H9.2.1-BB-L CAR-T samples and M7CR modified H9.2.1-BB-L CAR-T samples ⁺ And CD8 ⁺ T phenotype was detected by flow cytometry. Fig. 9G shows PBMC-prepared CAR T cells from donor 15, and fig. 9H shows PBMC-prepared CAR T cells from donor 17. Wherein H9.2.1-BB-L represents H9.2.1-BB-L CAR '-T cells (H9.2.1-BB-L CAR' sequence is shown as SEQ ID NO: 144); H9.2.1-tCD19-M7CR represents tCD19-M7CR modified H9.2.1-BB-L CAR' -T cells; H9.2.1-IL-15-M7CR represents IL-15/IL15Rα (Sushi) -M7CR modified H9.2.1-BB-L CAR' -T cells; h9.2.1in-IL15-M7CR indicates loss of CAR structural function in H9.2.1-IL-15-M7CR cells; H9.2.1-IL15-M7CRin indicates a loss of M7CR intracellular structure (M7R) function in H9.2.1-IL15-M7CR cells; H9.2.1-sIL15 represents a combination of H9.2.1-BB-L CAR' -T cells and soluble IL15, H9.2.1-IL-12-M7CR represents IL-12-p70-M7CR modified H9.2.1-BB-L CAR-T cells (the H9.2.1-BB-L CAR sequence is shown as SEQ ID NO: 100); H9.2.1-28-IL-15-M7CR represents IL-15/IL15Rα (Sushi) -M7CR modified H9.2.1-28-L CAR' cells with a co-stimulatory domain of CD28, and 8E5 represents the CAR sequence from CARsgen CT041 product.

Figures 9I and 9J show that M7R is able to continue to provide an activation signal, activating downstream signaling pathways, in CAR-T cells prepared from PBMCs of donors 15 and 17, respectively. In the figure, H9.2.1-tCD19-M7CR represents tCD19-M7CR modified H9.2.1-BB-L CAR-T cells; H9.2.1-IL-12-M7CR represents IL-12-p70-M7CR modified H9.2.1-BB-L CAR-T cells; H9.2.1in-IL12-M7CR indicates loss of CAR structural function in H9.2.1-IL-12-M7CR cells; H9.2.1-IL12-M7CRin indicates a loss of M7CR intracellular structure (M7R) function in H9.2.1-IL12-M7CR cells; H9.2.1-sIL12 represents a combination of H9.2.1-BB-L CAR-T cells and soluble IL 12.

FIG. 10A shows detection of DANG-G18.2, S by QufikitNU-601 ^high ，SNU-601 ^low Cell surface CLDN 18.2. In the peak, dark portions represent ISO and light portions represent positive cells.

FIG. 10B shows the expression level of CLDN18.2 in DANG18.2, NUGC-4, SNU-620, PANC-1, SNU-601 and Hup-T4 cells, wherein ISO is isotype antibody control, and K562 is CLDN18.2 negative control.

FIGS. 11A, 11B and 11C show the killing effect of each CAR-T cell on target cells when incubated with tumor target cells DAN-G18.2 with unmodified H9.1.2-BB-L CAR-T cells or different M7CR modified H9.1.2-BB-L CAR-T cells, respectively, at E: T of 1:1, 1:3, 1:10, respectively. In the figure, "PC" represents a Positive control (Positive control), target cells are treated with lysate such that all target cells are lysed; "NT" means T cells not infected with lentivirus; "Tumor cell only" means the DAN-G18.2 cell line; "H9.1.2" means H9.1.2-BB-L CAR-T cells, the remainder being M7CR modified H9.1.2-BB-L CAR-T cells (labeled in the figure as in FIG. 9A).

FIG. 11D shows modified HuR968B CAR-T cells with unmodified HuR968B CAR-T cells or different M7CR, respectively, and a P329G mutant A6 antibody (2 nM) and target cell SUN-601 ^high Or SUN-601 ^low Co-incubation, killing of target cells by each CAR-T cell at 1:1 of E: T. In the figure, "PC" represents a Positive control (Positive control), target cells are treated with lysate such that all target cells are lysed; "NT" means T cells not infected with lentivirus; "8B" means unmodified HuR968B CAR-T cells, the remainder being M7CR modified HuR968B CAR-T cells (labeled as in FIG. 9C).

FIGS. 12A, 12C and 12E show modified HuR968B CAR-T cells with unmodified HuR968B CAR-T cells or different M7CR, respectively, and P329G mutant A6 antibody (2 nM) and target cell SUN-601 ^high Co-incubation, with target cell SUN-601 at E:T of 2:1 ^high Results of repeated stimulation for multiple rounds, counting CAR-T cell number and CAR-T cell fold proliferation. In the figure, "8B" represents unmodified HuR968B CAR-T cells, the remainder being M7CR modifiedIs shown in (a) HuR968B CAR-T cells (labeled in the figures as in FIG. 9C).

FIGS. 12B, 12D and 12F show modified HuR968B CAR-T cells with unmodified HuR968B CAR-T cells or different M7CR, respectively, and P329G mutant A6 antibody (2 nM) and target cell SUN-601 ^high Co-incubation, with target cell SUN-601 at E:T of 2:1 ^high Upon repeated stimulation for multiple rounds, CARs in HuR968B CAR-T cells ⁺ Ratio of cells and CAR ⁺ Fold change in cell percentages. In the figure, "8B" represents unmodified HuR968B CAR-T cells, the remainder being M7CR modified HuR968B CAR-T cells (labeled in the figure as in fig. 9C).

FIG. 13A shows CD4 in each of the groups of FIGS. 12A-12F after the first and third rounds of stimulation ⁺ And CD8 ⁺ Representative flow cytometry assays for T cell numbers.

FIG. 13B shows the CD4 pairs in the groups of FIG. 13A ⁺ And CD8 ⁺ Statistics of the proportion of T cells.

FIG. 13C shows modified HuR968B CAR-T cells with unmodified HuR968B CAR-T cells or different M7CR, respectively, and a P329G mutant A6 antibody (2 nM) and target cell SUN-601 ^high Co-incubation, with target cell SUN-601 at E:T of 2:1 ^high Repeatedly stimulating for multiple rounds, using BD ^TM Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit II in culture supernatant, "8B" in the figure means HuR968B CAR-T cells, and the remainder were M7CR modified HuR968B CAR-T cells (the label means the same as in FIG. 9C).

Figures 14A-14D show that killing of CAR-T cells increases with increasing expression levels of CLDN18.2 in target cells with different expression levels of CLDN 18.2. In the figure, H9.2.1-tCD19-M7CR represents tCD19-M7CR modified H9.2.1-BB-L CAR-T cells; H9.2.1-IL-12-M7CR represents IL-12-M7CR modified H9.2.1-BB-L CAR-T cells; H9.2.1in-IL12-M7CR indicates loss of CAR structural function in H9.2.1-IL-12-M7CR cells; H9.2.1-IL-12-M7CRin indicates a loss of M7CR intracellular structure (M7R) function in H9.2.1-IL-12-M7CR cells; H9.2.1-sIL12 represents a combination of H9.2.1-BB-L CAR-T cells and secretion of soluble IL 12.

FIG. 14E shows the killing effect of each CAR-T cell on target cells by three rounds of repeated stimulation with target cells Hup-T4 at E: T of 1:1 and 1:5, incubated with target cells Hup-T4 with unmodified H9.2.1CAR-T cells or IL-12-M7CR modified CAR-T cells, respectively. After three continuous rounds of killing experiments, IL-12-M7CR modified CAR-T cells still have better killing effect on target cells, while unmodified H9.2.1CAR-T cells or single M7R modified H9.2.1CAR-T cells gradually weaken killing effect along with the increase of the number of rounds in multiple rounds of killing. In the figure, H9.2.1 represents H9.2.1-BB-L CAR-T cells; H9.2.1-tCD19-M7CR represents tCD19-M7CR modified H9.2.1-BB-L CAR-T cells; H9.2.1-IL-12-M7CR represents IL-12-M7CR modified H9.2.1-BB-L CAR-T cells.

FIG. 15 shows the detection of tumor burden changes in mice by an IVIS imaging system in the construction of a gastric cancer peritoneal metastasis model by intraperitoneal injection of luciferase-expressing NUGC-4 cells.

Figure 16 shows the anti-tumor effect of PG CAR-T cells expressing a constitutive chimeric cytokine receptor in mice. For tumor-bearing mice, IL-12-M7CR modified PG CAR-T cells (labeled 8B-IL12-M7CR CAR-T in the figures) and tCD19-M7CR modified PG CAR-T cells (labeled 8B-M7R CAR-T in the figures) had better antitumor effects in vivo, as increased over time after administration of the CAR-T cells and the A6 antibody.

Figure 17 shows the level of expansion of PG CAR-T cells expressing a constitutive chimeric cytokine receptor in mice. For tumor-bearing mice, IL-12-M7CR modified PG CAR-T cells (labeled 8B-IL12-M7CR CAR-T in the figures) and tCD19-M7CR modified PG CAR-T cells (labeled 8B-M7R CAR-T in the figures) had higher levels of expansion on days 7 to 28 after administration of PG CAR-T cells and A6 antibodies.

Figure 18 shows the anti-tumor effect of H9.2.1 CAR-T cells expressing a constitutive chimeric cytokine receptor in mice. Two weeks after CAR-T cell administration, 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), with unmodified H9.2.1 CAR T cells having the weakest anti-tumor effect in vivo.

Figure 19 shows the anti-tumor effect of conventional CAR-T cells expressing a constitutive chimeric cytokine receptor in mice. For tumor-bearing mice, IL-12-M7CR modified CAR-T cells (labeled IL12-M7CR-H9.2.1 CAR-T in the figures) and tCD19-M7CR modified CAR-T cells (labeled M7R-H9.2.1 CAR-T in the figures) have better anti-tumor effects in vivo, increasing over time after administration of the CAR-T cells.

Figure 20 shows the level of expansion of conventional CAR-T cells expressing a constitutive chimeric cytokine receptor in mice. For tumor-bearing mice, IL-12-M7CR modified CAR-T cells (labeled IL12-M7CR-H9.2.1 CAR-T in the figures) and tCD19-M7CR modified CAR-T cells (labeled M7R-H9.2.1 CAR-T in the figures) had higher levels of expansion on days 7 to 28 after administration of the CAR-T cells.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

I. Definition of the definition

For purposes of explaining the present specification, the following definitions will be used, and terms used in the singular form may also include the plural, and vice versa, as appropriate. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

The term "about" when used in conjunction with a numerical value is intended to encompass numerical values within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value.

As used herein, the term "and/or" means any one of the selectable items or two or more of the selectable items.

In this document, the terms "comprises" or "comprising" when used herein, unless otherwise indicated, also encompass the circumstance of consisting of the recited elements, integers or steps. For example, when referring to a domain "comprising" a particular sequence, it is also intended to encompass domains consisting of that particular sequence.

"constitutively active IL-7R mutant" refers to a mutant IL-7R produced by mutation of the wild type IL-7 receptor alpha chain (IL 7 Ralpha) transmembrane region, which is capable of dimerizing and activating the downstream STAT5 signaling pathway without reliance on ligand binding by wild type IL7 Ralpha.

The term "autologous" refers to any substance that is derived from the same individual that will later reintroduce the substance to the individual.

The term "allogeneic" refers to any substance derived from a different animal of the same species as the individual into which the substance was 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, the allografts from individuals of the same species may be sufficiently genetically dissimilar to occur antigenic interactions.

The term "xenogeneic" refers to grafts derived from animals of different species.

The term "apheresis" as used herein refers to an art-recognized in vitro method by which a donor or patient's blood is removed from the donor or patient and passed through a device that separates selected specific components and returns the remainder to the donor or patient's circulation, for example, by re-transfusion. Thus, in the context of "apheresis" reference is made to a sample obtained using apheresis.

The term "immune effector cell" refers to a cell that is involved in an immune response, e.g., involved in promoting an immune effector response. Examples of immune effector cells include T cells, e.g., 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.

An "immune effector function", "immune effector response" or "immune effector response" refers to, for example, enhancement of an immune effector cell or promotion of a function or response of an immune attack target cell. For example, immune effector function or response refers to T cell or NK cell characteristics that promote killing or inhibit growth or proliferation of target cells. In the case of T cells, primary stimulation and co-stimulation are examples of immune effector functions or responses.

The term "effector function" refers to a specialized function of a cell. The effector function of T cells may be, for example, cytolytic activity or helper activity, including secretion of cytokines.

The term "T cell activation" refers to one or more cellular responses of T lymphocytes, in particular cytotoxic T lymphocytes, selected from the group consisting of: proliferation, differentiation, cytokine secretion, cytotoxic effector release, cytotoxic activity and expression of activation markers. The chimeric antigen receptor of the invention is capable of inducing T cell activation. Suitable assays for measuring T cell activation are described in the examples and are known in the art.

The term "lentivirus" refers to a genus of the retrovirus family (Retroviridae). Lentiviruses are unique among retroviruses in being able to infect non-dividing cells; they can deliver significant amounts of genetic information to host cells, so they are one of the most efficient methods of gene delivery vehicles. HIV, SIV and FIV are all examples of lentiviruses.

The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome and includes, inter alia, a self-inactivating lentiviral vector as provided in Milone et al, mol. Ther.17 (8): 1453-1464 (2009). Other examples of lentiviral vectors that may be used clinically include, for example, but are not limited to, those from Oxford BioMedica Gene delivery technology, LENTIMAX from Lentigen ^TM Carrier systems, and the like. Non-clinical types of lentiviral vectors are also available and known to those skilled in the art.

The terms "tumor" and "cancer" are used interchangeably herein to encompass solid tumors and liquid tumors.

The terms "cancer" and "cancerous" refer to physiological conditions in a mammal in which cell growth is not regulated.

The term "tumor" refers to all neoplastic (neoplastic) cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer", "cancerous" and "tumor" are not mutually exclusive when referred to herein.

The term "Claudins" is a class of integrin membrane proteins present in tight junctions between epithelium and endothelium, an important component of tight junctions, as found in 1998 by Shoichiro Tsukita et al. There are 24 members of this family. The human Claudin 18 gene has two alternative exons 1, thus yielding two protein subtypes, claudin 18.1 (also referred to herein as "CLDN 18.1") and Claudin 18.2 (also referred to herein as "CLDN 18.2"), which differ in the 1 st extracellular domain by only 7 amino acid residues in the sequence of about 50 amino acids.

There is a significant difference in expression of Claudin18.2 in cancer tissue and normal tissue, which may result from the CpG hypermethylation of the Claudin18.2 promoter region CREB binding site in normal tissue, whereas CpG methylation levels are reduced during cellular canceration, and CREB is involved in activating transcription of Claudin 18.2.

"tumor immune escape" refers to the process by which tumors evade immune recognition and clearance. Thus, as a therapeutic concept, tumor immunity is "treated" when such evasion is reduced, and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage and tumor elimination.

As used herein, the term "bind" or "specifically bind" means that the binding is selective for an antigen and distinguishable from unwanted or non-specific interactions. The ability of an antibody to bind to a particular antigen may be determined by enzyme-linked immunosorbent assay (ELISA), SPR or biofilm layer interference techniques or other conventional binding assays known in the art.

The term "stimulation" refers to a primary response induced by binding of a stimulatory molecule (e.g., a TCR/CD3 complex) to its corresponding ligand, which thus mediates signaling events, such as, but not limited to, signaling via the TCR/CD3 complex. Stimulation may mediate the altered expression of certain molecules, such as down-regulation of TGF-beta and/or reorganization of cytoskeletal structures, and the like.

The term "stimulatory molecule" refers to a molecule expressed by a T cell that provides a primary cytoplasmic signaling sequence that modulates primary activation of the TCR complex in a stimulatory manner in at least some aspect of the T cell signaling pathway. In one embodiment, the primary signal initiates and results in mediating T cell responses including, but not limited to, proliferation, activation, differentiation, etc., e.g., through binding of the TCR/CD3 complex to peptide-loaded MHC molecules.

The term "cd3ζ" is defined as the protein provided by genbank accession No. BAG36664.1 or an equivalent thereof, and "cd3ζ stimulatory signaling domain" is defined as an amino acid residue from the cytoplasmic domain of the cd3ζ chain sufficient to functionally propagate the initial signaling necessary for T cell activation. In one embodiment, the cytoplasmic domain of cd3ζ comprises residues 52 through 164 of GenBank accession No. BAG36664.1 or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.) as functional orthologs thereof. In one embodiment, the "CD3ζ stimulatory signaling domain" is the sequence provided in SEQ ID NO. 12 or a variant thereof.

The term "costimulatory molecule" refers to a corresponding binding partner on a cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response (such as, but not limited to, proliferation) of the cell. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an effective immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activation of NK cell receptors, OX40, CD40, GITR, 4-1BB (i.e., CD 137), CD27, and CD28. In some embodiments, the "costimulatory molecule" is CD28, 4-1BB (i.e., CD 137). The costimulatory signal domain refers to the intracellular portion of a costimulatory molecule.

The term "4-1BB" refers to a TNFR superfamily member having the amino acid sequence provided as GenBank accession No. AAA62478.2 or an equivalent residue 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 No. AAA62478.2 or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.). In one embodiment, a "4-1BB co-stimulatory domain" is a sequence provided as SEQ ID NO. 11 or an equivalent residue from a non-human species (e.g., mouse, rodent, monkey, ape, etc.).

The term "signaling pathway" refers to a biochemical relationship between a plurality of signaling molecules that function in propagating a signal from one portion of a cell to another portion of the cell.

When referring to the extracellular domain of a constitutive chimeric cytokine receptor, the extracellular domain may be a cytokine, and the "cytokine" is a generic term for proteins released by one cell population that act as an intercellular mediator to another cell. Examples of such cytokines are lymphokines, monokines, interleukins (IL), such as IL-1, IL-1. Alpha., IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL15, IL-21, IL-18, IL-9, IL-23, IL-36. Gamma; tumor necrosis factors such as TNF- α or TNF- β; and other polypeptide factors, including interferons. In some embodiments, the "cytokine" that is the extracellular domain of a constitutive chimeric cytokine receptor of the invention is selected from any of IL-12 (e.g., IL-12p40 or IL-12p 70), IL-15 (e.g., IL-15 or IL-15FP, which refers to a fusion protein of IL-15 and IL-15Rα (selected from IL-15Rα or IL-15Rα (Sushi)), including fusion proteins of both forms of IL-15/IL-15Rα and IL-15Rα/IL-15), IL-21, IL-18, IL-9, IL-23, IL-36 γ, and IFNα 2b.

When referring to the extracellular domain of a constitutive chimeric cytokine receptor, the extracellular domain may be an immune effector molecule, and the "immune effector molecule" may be selected from the group consisting of: (i) A molecule that enhances antigen presentation (e.g., tumor antigen presentation); (ii) Molecules that enhance effector cell responses (e.g., activate and/or mobilize B cells and/or T cells). The "immune effector molecule" is, for example, the following molecule or an agonist thereof: GITR, OX40, ICOS, SLAM (e.g., SLAMF 7), HVEM, LIGHT, CD, CD27, CD28, CDs, ICAM1, LFA-1 (CD 11a/CD 18), ICOS (CD 278), 4-1BB (CD 137), CD30, CD40, BAFFR, CD7, CD160, B7-H3, or CD83. In some embodiments, the "immune effector molecule" that is an extracellular domain of a constitutive chimeric cytokine receptor of the invention is selected from any of the 4-1BB targeting molecule moieties (e.g., 4-1BB ligand, anti-4-1 BB antibody), CD40 targeting molecule moieties (e.g., CD40 ligand, anti-CD 40 antibody), CD83 targeting molecule moieties (e.g., anti-CD 83 antibody), FLT3 ligand, GITR, ICOS, CD2, and ICAM1.

When referring to the extracellular domain of a constitutive chimeric cytokine receptor, the extracellular domain may be an inhibitory molecular antagonist, and the "inhibitory molecular antagonist" is an agent that reduces tumor immunosuppression. Such inhibitory molecules include, but are not limited to, PD-1, PD-L1, CD47, TIM-3, IL-4, TGF beta, LAG-3, VISTA, B7-H4, CTLA-4, CD73, or TIGIT. In some embodiments, the "inhibitory molecular antagonist" that is an extracellular domain of a constitutive chimeric cytokine receptor of the invention is selected from any one of an anti-PD-L1 molecule, an anti-CD 47 molecule, an anti-IL-4 molecule, an anti-TGF-beta molecule, an anti-PD-1 molecule, an anti-CTLA-4 molecule, an anti-LAG-3 molecule, an anti-TIGIT molecule, and an anti-CD 73 molecule.

When referring to the extracellular domain of a constitutive chimeric cytokine receptor, the extracellular domain may be an effector molecule targeting an NK cell activating receptor, and the "effector molecule targeting an NK cell activating receptor" is a class of molecules capable of activating NK cells upon binding to an NK cell activating receptor. Such NK cell-activating receptors include, but are not limited to, NKG2C, NKG2D, NKp, NKp44 and NKp46 on NK cells. In some embodiments, the "effector molecule targeting NK cell activating receptor" that is the extracellular domain of the constitutive chimeric cytokine receptor of the invention is selected from molecules targeting NK cell activating receptors NKG2C, NKG2D, NKp, NKp44 and NKp46, e.g., anti-NKG 2C, anti-NKG 2D, anti-NKp 30, anti-NKp 44, anti-NKp 46, by activating endogenous NK cells, an enhanced anti-tumor immune effect is obtained.

The term "antibody" is used herein in its broadest sense to refer to a protein comprising an antigen binding site, and encompasses natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single chain antibodies, intact antibodies, and antibody fragments.

An "antibody fragment" or "antigen-binding fragment" is used interchangeably herein to refer to a molecule that is different from an intact antibody, which comprises a portion of the intact antibody and binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, fab ', F (ab') 2, fv, single chain Fab, diabody (diabody).

The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are continuously linked, optionally via a flexible short polypeptide linker, and are capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. As used herein, an scFv may have a VL variable region and a VH variable region in any order (e.g., with respect to the N-terminus and C-terminus of the polypeptide), unless otherwise indicated, an scFv may comprise a VL-linker-VH or may comprise a VH-linker-VL.

"complementarity determining regions" or "CDR regions" or "CDRs" or "hypervariable regions" are regions of an antibody variable domain that are hypervariable in sequence and form structurally defined loops ("hypervariable loops") and/or contain antigen-contacting residues ("antigen-contacting points"). CDRs are mainly responsible for binding to the epitope. CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. CDRs located within the antibody heavy chain variable domain are referred to as CDR H1, CDR H2 and CDR H3, while CDRs located within the antibody light chain variable domain are referred to as CDR L1, CDR L2 and CDR L3. In a given light chain variable region or heavy chain variable region amino acid sequence, the exact amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example: chothia (Chothia et al, (1989) Nature 342:877-883, al-Lazikani et al, "Standard conformations for the canonical structures of immunoglobulins", journal of Molecular Biology,273,927-948 (1997)) based on the three-dimensional structure of antibodies and topology of CDR loops, kabat (Kabat et al, sequences of Proteins of Immunological Interest, 4 th edition, U.S. Pat. No. of Health and Human Services, national Institutes of Health (1987)), abM (University of Bath), contact (University College London), international ImMunoGeneTics database (IMGT) (world Wide Web IMGT. Cines. Fr /), and North CDR definitions based on neighbor-propagating clusters (affinity propagation clustering) using a large number of crystal structures.

In the present invention, unless otherwise indicated, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above-described ways.

CDRs may also be determined based on having the same Kabat numbering positions as the reference CDR sequences (e.g., any of the CDRs of the examples of the invention). In the present invention, when referring to the antibody variable region and specific CDR sequences (including heavy chain variable region residues), reference is made to numbering positions according to the Kabat numbering system.

Although CDRs vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. Using at least two of the Kabat, chothia, abM and Contact methods, the minimum overlap region can be determined, thereby providing a "minimum binding unit" for antigen binding. The minimum binding unit may be a sub-portion of the CDR. As will be apparent to those skilled in the art, the residues in the remainder of the CDR sequences can be determined by the structure of the antibody and the protein folding. Thus, the present invention also contemplates variants of any of the CDRs presented herein. For example, in a variant of one CDR, the amino acid residues of the smallest binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia or AbM may be replaced by conserved amino acid residues.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies generally have similar structures, with each domain comprising four conserved Framework Regions (FR) and three Complementarity Determining Regions (CDRs). (see, e.g., kindt et al Kuby Immunology,6 ^th ed., w.h. freeman and co.91 page (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which region comprises at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In certain embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carbonyl terminus of the heavy chain. However, the C-terminal lysine (Lys 447) of the Fc region may or may not be present. Unless otherwise indicated, 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 set forth in Kabat et al Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,MD,1991. The term "functional variant" refers to a polypeptide having substantial or significant sequence identity or similarity to a polypeptide encoded by a nucleic acid sequence of the invention, which functional variant retains the biological activity of the polypeptide encoded by the nucleic acid sequence of the invention. Functional variants may, for example, comprise amino acid substitutions having at least one conservation in the amino acid sequence of a polypeptide encoded by a nucleic acid sequence of the invention.

The terms "conservative sequence modification", "conservative sequence change" are used interchangeably to refer to an amino acid modification or change that does not significantly affect or alter the biological activity of a polypeptide comprising an amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications may be introduced into the polypeptides of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative substitutions are amino acid substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a polypeptide of the invention may be replaced with other amino acid residues from the same side chain family, and altered polypeptides may be tested for biological activity using the functional assays described herein.

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

The term "fluorescence activated cell sorting" or "FACS" refers to a specialized type of flow cytometry. It provides a method of sorting a heterogeneous mixture of biological cells into two or more containers one cell at a time according to specific light scattering and fluorescence characteristics of each cell (flowmetric. "Sorting Out Fluorescence Activated Cell Sorting". 2017-11-09). The apparatus for performing FACS is known to those skilled in the art and commercially available to the public. Examples of such instruments include FACS Star Plus, FACScan and FACSort instruments from Becton Dickinson (Foster City, calif.), epics C from Coulter Epics Division (Hialeah, FL) and MoFlo from Cytomation (Colorado Springs, colorado).

The term "pharmaceutically acceptable adjuvant" refers to diluents, adjuvants (e.g. Freund's adjuvant (complete and incomplete)), excipients, buffers or stabilizers etc. for administration with the active substance.

As used herein, "treating" refers to slowing, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Desirable therapeutic effects include, but are not limited to, preventing occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, improving or moderating the disease state, and alleviating or improving prognosis.

"therapeutically effective amount" means an amount effective to achieve the desired therapeutic result at the desired dosage and for the desired period of time. The therapeutically effective amount can vary depending on a variety of factors such as the disease state, age, sex and weight of the individual. The "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 50%, 60% or 70% and still more preferably by at least about 80% or 90% relative to an untreated subject. The ability of a compound to inhibit a measurable parameter (e.g., cancer) can be evaluated in an animal model system that predicts efficacy in human tumors.

II. Constitutive chimeric cytokine receptors of the invention

The present invention relates to a constitutive chimeric cytokine receptor capable of sustained activation of STAT5 signaling, maintenance of immune effector cell (e.g., T cell) exogenous cytokine independent survival, and having effector molecules that remodel the tumor microenvironment. In particular, the constitutive chimeric cytokine receptor of the present invention comprises:

(i) An extracellular domain consisting of an effector molecule having a remodelling tumor microenvironment; and

(ii) Constitutively active IL-7R mutants consist of IL7R transmembrane regions (IL 7R-mutants (TM)) and an intracellular segment of IL7R alpha carrying various mutations.

In some embodiments, the (i) extracellular domain of a constitutive chimeric cytokine receptor of the invention is selected from the group consisting of a cytokine, an immune effector molecule, an inhibitory molecular antagonist, or an effector molecule targeting an NK cell activating receptor.

When the extracellular domain of the constitutive chimeric cytokine receptor of the present invention is a cytokine, the cytokine may be IL-12 (IL-12-P40 or IL-12-P70), IL-15 (IL-15 or IL-15FP, which refers to a fusion protein of IL-15 and IL-15Rα (selected from IL-15Rα or IL-15Rα (Sushi)), including two forms of fusion protein of IL-15/IL-15Rα and IL-15Rα/IL-15), IL-21, IL-18, IL-9, IL-23, IL-36 γ, IFN α 2b, and the like, and immune cells genetically modified with these cytokines have enhanced immune effector functions and antitumor effects.

When the (i) extracellular domain of the constitutive chimeric cytokine receptor of the invention is an immune effector molecule, the immune effector molecule may be a 4-1BB targeting molecule moiety (e.g., 4-1BB ligand (4-1 BBL), anti-4-1 BB antibody (α4-1 BB)), a CD40 targeting molecule moiety (e.g., CD40 ligand (CD 40L), anti-CD 40 antibody (αcd 40)), a CD83 targeting molecule moiety (e.g., anti-CD 83 antibody (αcd83)), FLT3 ligand (FTL 3L), GITR, ICOS, CD, ICAM1, etc., which activates an APC by interacting with a related receptor or ligand on the surface of a professional Antigen Presenting Cell (APC) in vivo, such as a Dendritic Cell (DC), thereby eliciting an endogenous anti-tumor immune response.

When the (i) extracellular domain of the constitutive chimeric cytokine receptor of the invention is an inhibitory molecular antagonist, the inhibitory molecular antagonist may be an antibody moiety against an inhibitory immune receptor or factor, e.g., an anti-PD-L1 molecule, an anti-CD 47 molecule, an anti-IL-4 molecule, an anti-TGF-beta molecule, an anti-PD-1 molecule, an anti-CTLA-4 molecule, an anti-LAG-3 molecule, an anti-TIGIT molecule, an anti-CD 73 molecule, etc., against an inhibitory immune receptor or factor, e.g., an anti-PD-L1 molecule _VHH The aim of enhancing the anti-tumor immune response is achieved by antagonizing the immunosuppressive effect of the inhibitory immune receptor or factor.

In some embodiments, the (i) extracellular domain of the constitutive chimeric cytokine receptor of the invention is selected from the group consisting of a molecular moiety that targets an activating receptor expressed on the surface of NK cells such as NKG2C, NKG2D, NKp, NKp44, NKp46, e.g., an anti-NKG 2C, anti-NKG 2D, anti-NKp 30, anti-NKp 44, anti-NKp 46, etc., antibody moiety, by activating endogenous NK cells, with the aim of enhancing an anti-tumor immune effect.

In some embodiments, the (ii) constitutively activated IL-7R mutant of a constitutive chimeric cytokine receptor of the invention comprises any amino acid sequence selected from the group consisting of SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:28, SEQ ID NO:30 to SEQ ID NO:45, preferably the constitutively activated IL-7R mutant comprises any amino acid sequence selected from the group consisting of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33 and SEQ ID NO:34, most preferably the constitutively activated IL-7R mutant comprises the amino acid sequence shown in SEQ ID NO: 34.

The constitutive chimeric cytokine receptor is a constitutive dimer, and can activate intracellular signaling of IL-7R independent of combination of IL-7R and ligand thereof and independent of combination of a common gamma signal chain (gammac), activate JAK1 kinase, further phosphorylate transcription effector factors such as downstream STAT5 and the like, regulate and control downstream target gene expression, and finally promote and maintain T proliferation and survival.

The constitutive chimeric cytokine receptor of the invention remodels the tumor microenvironment by comprising said (i) extracellular domain as effector molecule on the basis of comprising said (ii) constitutively activated IL-7R mutant.

Co-expression of the constitutive chimeric cytokine receptor and CAR polypeptide of the invention

When the constitutive chimeric cytokine receptor and the Chimeric Antigen Receptor (CAR) polypeptide are coexpressed in T cells, the constitutive chimeric cytokine receptor comprises an extracellular domain and a constitutive activated IL-7R mutant, the constitutive activated IL-7R mutant continuously activates STAT5 signals, promotes and maintains proliferation and survival of immune cells, and endows the immune cells with new extracellular effector molecule efficacy through the extracellular domain, so that the modified immune cells (such as CAR-T cells) have the capability of actively modeling an 'unfriendly' Tumor Microenvironment (TME), and the modified immune cells (such as CAR-T cells) are in a more 'friendly' TME by reconstructing the TME, thereby being more beneficial to exerting an anti-tumor effect.

In some embodiments, the CAR polypeptide is a traditional CAR polypeptide that directly targets one or more cancer-associated antigens. For example, the cancer-associated antigen (also referred to 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 (GD 2); TNF receptor family member B Cell Maturation (BCMA); prostate Specific Membrane Antigen (PSMA); fms-like tyrosine kinase 3 (FLT 3); tumor-associated glycoprotein 72 (TAG 72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD 276); KIT (CD 117); interleukin 13 receptor subunit alpha-2 (IL-13 Ra2 or CD213 A2); mesothelin; interleukin 11 receptor alpha (IL-11 Ra); prostate Stem Cell Antigen (PSCA); protease serine 21; vascular endothelial growth factor receptor 2 (VEGFR 2); lewis (Y) antigen; platelet-derived growth factor receptor beta (PDGFR-beta); stage specific embryonic antigen-4 (SSEA-4); folate receptor alpha; receptor tyrosine protein kinase ERBB2 (Her 2/neu); cell surface associated mucin 1 (MUC 1); epidermal Growth Factor Receptor (EGFR); neural Cell Adhesion Molecules (NCAM); prostatectomy phosphatase (PAP); mutated elongation factor 2 (ELF 2M); liver accessory protein B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor); ephrin-type a receptor 2 (EphA 2); fucosyl GM1; sialic acid based lewis adhesion molecules (sLe); transglutaminase 5 (TGS 5); high Molecular Weight Melanoma Associated Antigen (HMWMAA); o-acetyl GD2 ganglioside (OAcGD 2); folate receptor beta; tumor vascular endothelial marker 1 (TEM 1/CD 248); tumor vascular endothelial marker 7-associated (TEM 7R); claudin 6 (CLDN 6); CLDN18.2; thyroid Stimulating Hormone Receptor (TSHR); g protein coupled receptor group C, member D (GPRC 5D); x chromosome open reading frame 61 (CXORF 61); CD97; CD179a; anaplastic Lymphoma Kinase (ALK); polysialic acid; placenta-specific 1 (PLAC 1); hexose moiety of globoH glucoside ceramide (globoH); breast differentiation antigen (NY-BR-1); uroplakin 2 (UPK 2); hepatitis a virus cell receptor 1 (HAVCR 1); adrenergic receptor beta 3 (ADRB 3); pannexin 3 (PANX 3); g protein-coupled receptor 20 (GPR 20); lymphocyte antigen 6 complex, locus K9 (LY 6K); olfactory receptor 51E2 (OR 51E 2); tcrγ alternate reading frame protein (TARP); a wilms tumor protein (WT 1); cancer/testis antigen 1 (NY-ESO-1); cancer/testis antigen 2 (age-1A); melanoma-associated antigen 1 (MAGE-A1); ETS translocation mutant gene 6, located on chromosome 12p (ETV 6-AML); sperm protein 17 (SPA 17); x antigen family, member 1A (XAGE 1); angiogenin binds to cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); FOS-associated antigen 1; tumor protein p53 (p 53); a p53 mutant; prostein; survivin; telomerase; prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), melanoma antigen 1 (MelanA or MART 1) recognized by T cells; rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; a melanoma inhibitory agent of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS 2) ETS fusion gene); n-acetylglucosaminyl transferase V (NA 17); pairing box protein Pax-3 (Pax 3); androgen receptor; cyclin B1; a V-myc avian myeloblastosis virus oncogene neuroblastosis derived homolog (MYCN); ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450 1B1 (CYP 1B 1); CCCTC binding factor (zinc finger protein) like (BORIS or brother of regulator of imprinting sites), squamous cell carcinoma antigen 3 (SART 3) recognized by T cells; pairing box protein Pax-5 (Pax 5); the acrosin zymogen binding protein sp32 (OY-TES 1); lymphocyte-specific protein tyrosine kinase (LCK); a kinase anchored protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX 2); advanced glycation end product receptor (RAGE-1); renin 1 (RU 1); renin 2 (RU 2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; mutant heat shock protein 70-2 (mut hosp 70-2); CD79a; CD79b; CD72; leukocyte associated immunoglobulin-like receptor 1 (LAIR 1); an Fc fragment of IgA receptor (FCAR or CD 89); leukocyte immunoglobulin-like receptor subfamily a member 2 (LILRA 2); CD300 molecular-like family member f (CD 300 LF); c lectin domain family 12 member a (CLEC 12A); bone marrow stromal cell antigen 2 (BST 2); containing EGF-like module mucin-like hormone receptor-like 2 (EMR 2); lymphocyte antigen 75 (LY 75); glypican-3 (GPC 3); fc receptor like 5 (FCRL 5); and immunoglobulin lambda-like polypeptide 1 (IGLL 1).

In some embodiments, the cancer-associated antigen directly targeted by the conventional CAR polypeptide comprising, from N-terminus to C-terminus, a signal peptide, a cancer-associated antigen binding domain, a transmembrane domain, a costimulatory signaling domain, and a primary signaling domain is CLDN 18.2.

In some embodiments, the cancer-associated antigen binding domain of the encoded CAR polypeptide comprises an antibody, antibody fragment, scFv, fv, fab, (Fab') 2, single Domain Antibody (SDAB), VH or VL domain, or a camelidae VHH domain, directed against a cancer-associated antigen.

In some embodiments, the transmembrane domain of the CAR polypeptide comprises a transmembrane domain selected from the group consisting of: the transmembrane domain of α, β, or ζ of a T cell receptor, CD28, CD3 ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD 11a, CD 18), ICOS (CD 278), 4-1BB (CD 137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF 1), CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49 626, VLA-6 the transmembrane domain of CD49f, ITGAD, CD11D, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11B, ITGAX, CD C, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD 226), SLAMF4 (CD 244, 2B 4), CD84, CD96 (Tactive), CEACAM1, CRTAM, ly9 (CD 229), CD160 (BY 55), PSGL1, CD100 (SEMA 4D), SLAMF6 (NTB-A, ly), SLAM (SLAMF 1, CD150, IPO-3), BLASME (SLAMF 8), SELPLG (CD 162), LTBR, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, and/or NKG 2C.

In certain embodiments, the transmembrane domain of the CAR polypeptide comprises the amino acid sequence of the CD8 transmembrane domain having the sequence of 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.

In certain embodiments, the cancer-associated antigen binding domain is linked to the transmembrane domain by a hinge region. In one embodiment, the hinge region comprises the amino acid sequence of a CD8 hinge, e.g., SEQ ID NO. 7, or a sequence having one, two or three amino acid modifications with SEQ ID NO. 7.

In other embodiments, the CAR polypeptide comprises an intracellular signaling domain, such as a primary signaling domain (primary signaling domain) and/or a costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises a primary signaling domain. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises a primary signaling domain and a co-stimulatory signaling domain.

In certain embodiments, the primary signaling domain comprises a functional signaling domain of a protein selected from the group consisting of cd3ζ, cd3γ, cd3δ, cd3ε, common fcrγ (FCER 1G), fcrβ (fcεr1b), CD79a, CD79b, fcγriia, DAP10, and DAP 12.

In one embodiment, the primary signaling domain of the CAR polypeptide comprises a functional signaling domain of cd3ζ. The CD3 zeta 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.

In some embodiments, the intracellular signaling domain of the CAR polypeptide comprises a primary signaling domain and a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain comprises a functional signaling domain of a protein selected from one or more of the following: CD27, CD28, 4-1BB (CD 137), OX40, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B-H3, ligand that specifically binds CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF 1), CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2 Rbeta, IL2 Rgamma, IL7 Ralpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD D, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11B, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNRANFR 2, TRANCE/KL, DNAM1 (CD 226), SLAMF4 (CD 244, 2B 4), CD84, CD96 (TactiM 1, CEMA 9, CD229, SLAMF 9, SLAMF 35, SLAMG 6, SLAMG 35 (SLSLSLSLSLGL 6, SLSLSLSLGL 6, SLAMP 6, SLSLSLSLSLSLSL35 (SLSLSLSLSLCD 6).

In some embodiments, the costimulatory signaling domain of the CAR polypeptide comprises a 1, 2, or 3 amino acid modification having the amino acid sequence of SEQ ID No. 11. In some embodiments, the encoded costimulatory signaling domain comprises the sequence of SEQ ID NO. 11.

In some embodiments, the CAR further comprises a signal peptide sequence. In one embodiment, the signal peptide sequence comprises the sequence of SEQ ID NO. 1.

In certain embodiments, the cancer-associated antigen binding domain of the CAR polypeptide has 10 for a cancer-associated antigen ^-4 M to 10 ^-8 Binding affinity K of M _D 。

In some embodiments, the legacy CAR polypeptide comprises a legacy CLDN18.2 CAR polypeptide.

In one embodiment, the conventional CLDN18.2 CAR polypeptide comprises:

(1) H9.1.2 antibody scFv sequences which specifically bind to CLDN18.2 molecules, comprising a heavy chain variable region and a light chain variable region,

wherein:

the heavy chain variable region comprises a CDR H1 represented by the amino acid sequence SYNIH (SEQ ID NO: 106) according to Kabat numbering, or a variant of said CDR H1 having NO more than 2 amino acid changes or NO more than 1 amino acid change; CDR H2 represented by amino acid sequence YIAPFQGDARYNQKFKG (SEQ ID NO: 107), or a variant of said CDR H2 that does not vary by more than 2 amino acids or does not vary by more than 1 amino acid; and amino acid sequence LNRGQSLDY (SEQ ID NO: 108), or a variant of said CDR H3 that does not vary by more than 2 amino acids or does not vary by more than 1 amino acid; the light chain variable region comprises a CDR L1 as set forth in amino acid sequence KSSQSLFNAGNQRNYLT (SEQ ID NO: 109) according to Kabat numbering, or a variant of said CDR L1 having NO more than 2 amino acid changes or NO more than 1 amino acid change; CDR L2 represented by amino acid sequence WASTRES (SEQ ID NO: 110), or a variant of said CDR L2 that does not vary by more than 2 amino acids or does not vary by more than 1 amino acid; and amino acid sequence QNNYIYPLT (SEQ ID NO: 111), or a variant of said CDR L3 that does not vary by more than 2 amino acids or does not vary by more than 1 amino acid;

Wherein the amino acid change is an addition, deletion or substitution of an amino acid;

for example, the heavy chain variable region comprises the sequence of SEQ ID No. 14 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the light chain variable region comprises the sequence of SEQ ID No. 13 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;

(2) A hinge region selected from the group consisting of the CD8 hinge region (SEQ ID NO: 7), or a hinge region thereof having at least 80% sequence identity.

(3) A transmembrane region (TM) 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 a variant thereof having 1-2 amino acid modifications;

(4) A Costimulatory Signal Domain (CSD) selected from the group consisting of a 4-1BB costimulatory domain or a variant thereof having 1-5 amino acid modifications, e.g.the sequence shown in SEQ ID NO. 11 or a variant thereof having 1-2 amino acid modifications;

(5) A Stimulatory Signaling Domain (SSD) being a CD3 zeta signaling domain or a variant thereof having 1-10 amino acid modifications, e.g. the sequence shown in SEQ ID NO. 12 or a variant thereof having 1-10, 1-5 amino acid modifications

Optionally, the conventional CLDN18.2 CAR polypeptide further comprises a signal peptide sequence at the N-terminus, e.g., a signal peptide sequence as shown in SEQ ID NO. 1,

in a specific embodiment, the traditional CLDN18.2 CAR polypeptide comprises, for example, a H9.1.2-BB-L CAR (SEQ ID NO: 16) as described herein.

In one embodiment, the conventional CLDN18.2 CAR polypeptide comprises:

(1) A H9.2.1 antibody scFv sequence that specifically binds to CLDN18.2 molecule comprising a heavy chain variable region and a light chain variable region, wherein:

the heavy chain variable region comprises a CDR H1 represented by the amino acid sequence SYNIH (SEQ ID NO: 112) according to Kabat numbering, or a variant of said CDR H1 having NO more than 2 amino acid changes or NO more than 1 amino acid change; CDR H2 represented by amino acid sequence YIAPFQGDARYNQKFKG (SEQ ID NO: 113), or a variant of said CDR H2 that does not vary by more than 2 amino acids or does not vary by more than 1 amino acid; and amino acid sequence LNRGNALDY (SEQ ID NO: 114), or a variant of said CDR H3 that does not vary by more than 2 amino acids or does not vary by more than 1 amino acid; the light chain variable region comprises a CDR L1 as set forth in amino acid sequence KSSQSLFQSGNQRNYLT (SEQ ID NO: 115) according to Kabat numbering, or a variant of said CDR L1 having NO more than 2 amino acid changes or NO more than 1 amino acid change; CDR L2 represented by amino acid sequence WASTRES (SEQ ID NO: 116), or a variant of said CDR L2 that does not vary by more than 2 amino acids or does not vary by more than 1 amino acid; and amino acid sequence QNNYIYPLT (SEQ ID NO: 117), or a variant of said CDR L3 that does not vary by more than 2 amino acids or does not vary by more than 1 amino acid;

Wherein the amino acid change is an addition, deletion or substitution of an amino acid;

for example, the heavy chain variable region comprises or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID No. 99, and the light 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. 98;

(2) A hinge region selected from the group consisting of a CD8 hinge region (SEQ ID NO:7 or SEQ ID NO: 146), or a hinge region having at least 80% sequence identity.

(3) A transmembrane region (TM) 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 SEQ ID NO. 147 or a variant thereof having 1-2 amino acid modifications;

(4) A Costimulatory Signal Domain (CSD) selected from the group consisting of a 4-1BB costimulatory domain or a variant thereof having 1-5 amino acid modifications, e.g.the sequence shown in SEQ ID NO. 11 or a variant thereof having 1-2 amino acid modifications; or a variant thereof selected from the group consisting of the CD28 co-stimulatory domain or having 1-5 amino acid modifications, e.g. the sequence shown in SEQ ID NO 143 or a variant thereof having 1-2 amino acid modifications;

(5) A Stimulatory Signaling Domain (SSD) being a CD3 zeta signaling domain or a variant thereof having 1-10 amino acid modifications, e.g. the sequence shown in SEQ ID NO. 12 or a variant thereof having 1-10, 1-5 amino acid modifications

Optionally, the conventional CLDN18.2 CAR polypeptide further comprises a signal peptide sequence at the N-terminus, e.g., a signal peptide sequence as shown in SEQ ID NO. 1,

in a specific embodiment, the traditional CLDN18.2 CAR polypeptide comprises, for example, a H9.2.1-BB-L CAR (SEQ ID NO: 100) as described herein.

In a specific embodiment, the traditional CLDN18.2 CAR polypeptide comprises, for example, H9.2.1-BB-L CAR' (SEQ ID NO: 144) described herein.

In a specific embodiment, the traditional CLDN18.2 CAR polypeptide comprises a H9.2.1-28-L CAR (SEQ ID NO: 142) as described herein, for example.

In some embodiments, the CAR polypeptide is a molecular switch-regulated CAR polypeptide that does not directly target one or more cancer-associated antigens, but rather targets one or more cancer-associated antigens via a "molecular switch. For example, a CAR molecule capable of specifically binding to an antibody comprising a P329G mutant Fc domain but not to an antibody comprising a P329G mutant Fc domain is constructed by mutating Pro329Gly (the Fc segment of the antibody is mutated to glycine, abbreviated as P329G according to EU numbering) as a "molecular switch", whereby an immune effector cell (e.g., T cell, NK cell) expressing the CAR is used for treating a tumor by combining with a P329G mutant antibody that targets a cancer-associated antigen as a "molecular switch".

In one embodiment, the molecular switch regulated CLDN18.2 CAR polypeptide comprises:

(1) A humanized anti-P329G mutant scFv sequence, wherein the scFv sequence comprises the following sequence capable of specifically binding to an antibody Fc domain comprising a P329G mutation, but not to an unmutated parent antibody Fc domain:

(i) A heavy chain variable region comprising a sequence numbered according to Kabat

(a) A heavy chain complementarity determining region CDR H1 represented by the amino acid sequence RYWMN (SEQ ID NO: 118), or a variant of said CDR H1 that does not vary by more than 2 amino acids or does not vary by more than 1 amino acid;

(b) CDR H2 represented by amino acid sequence EITPDSSTINYAPSLKG (SEQ ID NO: 119), or a variant of said CDR H2 that does not vary by more than 2 amino acids or does not vary by more than 1 amino acid; and

(c) CDR H3 as set forth in amino acid sequence PYDYGAWFAS (SEQ ID NO: 120), or a variant of said CDR H3 that does not vary by more than 2 amino acids or by more than 1 amino acid; and

(ii) A light chain variable region comprising a sequence numbered according to Kabat

(d) A light chain complementarity determining region (CDR L) 1 represented by amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 121), or a variant of said CDR L1 having NO more than 2 amino acid changes or NO more than 1 amino acid change;

(e) CDR L2 as represented by amino acid sequence GTNKRAP (SEQ ID NO: 122), or a variant of said CDR L2 having NO more than 2 amino acid changes or NO more than 1 amino acid change; and

(f) CDR L3 as set forth in amino acid sequence ALWYSNHWV (SEQ ID NO: 123), or a variant of said CDR L3 that does not vary by more than 2 amino acids or by more than 1 amino acid;

wherein the amino acid change is an addition, deletion or substitution of an amino acid

For example, the heavy chain variable region comprises the sequence of SEQ ID No. 9 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the light chain variable region comprises the sequence of SEQ ID No. 10 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;

(2) A hinge region selected from the group consisting of a CD8 hinge region (SEQ ID NO: 7), or a hinge region thereof having at least 80% sequence identity;

(3) A transmembrane region (TM) 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 a variant thereof having 1-2 amino acid modifications;

(4) A Costimulatory Signal Domain (CSD) selected from the group consisting of a 4-1BB costimulatory domain or a variant thereof having 1-5 amino acid modifications, e.g.the sequence shown in SEQ ID NO. 11 or a variant thereof having 1-2 amino acid modifications;

(5) A Stimulatory Signaling Domain (SSD) is a CD3 zeta signaling domain or a variant thereof having 1-10 amino acid modifications, e.g., the sequence shown in SEQ ID NO. 12 or a variant thereof having 1-10, 1-5 amino acid modifications.

Optionally, the molecular switch regulated CLDN18.2 CAR polypeptide further comprises a signal peptide sequence at the N-terminus, e.g., a signal peptide sequence set forth in SEQ ID No. 1.

In a specific embodiment, the molecular switch regulated CLDN18.2 CAR polypeptide comprises a HuR968B CAR (SEQ ID NO: 15) as described herein, for example

In one embodiment, the P329G mutant antibody that targets a cancer-associated antigen as a "molecular switch" comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises a CDR H1 represented by the amino acid sequence synms (SEQ ID NO: 124) according to Kabat numbering, or a variant of said CDR H1 having NO more than 2 amino acid changes or NO more than 1 amino acid change; CDR H2 represented by amino acid sequence TISHSGGSTYYADSVKG (SEQ ID NO: 125), or a variant of said CDR H2 that does not vary by more than 2 amino acids or does not vary by more than 1 amino acid; and amino acid sequence DAPYYDILTGYRY (SEQ ID NO: 126), or a variant of said CDR H3 that does not vary by more than 2 amino acids or does not vary by more than 1 amino acid; the light chain variable region comprises a CDR L1 as set forth in amino acid sequence RASQSISSWLA (SEQ ID NO: 127) according to Kabat numbering, or a variant of said CDR L1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; CDR L2 as represented by amino acid sequence KASSLES (SEQ ID NO: 128), or a variant of said CDR L2 having NO more than 2 amino acid changes or NO more than 1 amino acid change; and amino acid sequence QQYNSYSYT (SEQ ID NO: 129), or a variant of said CDR L3 that does not vary by more than 2 amino acids or does not vary by more than 1 amino acid;

Wherein the amino acid change is an addition, deletion or substitution of an amino acid;

for example, the heavy chain variable region comprises the sequence of SEQ ID No. 130 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the light chain variable region comprises the sequence of SEQ ID No. 131 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

In some embodiments, the combination of a CAR-T cell expressing a molecular switch-regulated CAR polypeptide (e.g., a HuR968B CAR shown in SEQ ID NO: 15) with a P329G mutated anti-CLDN 18.2 antibody (e.g., a P329G mutated HB37A6 PG Ab, also referred to herein as an A6 antibody,) (see chinese application No. 202111416497.1) exhibits the ability to sustain killing of tumor cells and maintains the specificity of the CAR molecule, and the T cell expressing the molecular switch-regulated CAR polypeptide can be activated, proliferated, secrete effector cytokines and exert a killing effect on tumor cells expressing or overexpressing CLDN18.2 only in the presence of the P329G mutated antibody in a co-culture experiment with the tumor cells.

In order to co-express the constitutive chimeric cytokine receptor and Chimeric Antigen Receptor (CAR) polypeptide of the invention on immune effector cells, the constitutive chimeric cytokine receptor may be constructed on one construct, the CAR polypeptide on another construct, and both constructs introduced together into immune effector cells for expression.

Alternatively, a nucleic acid encoding a constitutive chimeric cytokine receptor-modified CAR polypeptide comprising a constitutive chimeric cytokine receptor of the invention at the N-terminus or the C-terminus of the CAR polypeptide is constructed on one nucleic acid construct with a self-cleaving peptide between the constitutive chimeric cytokine receptor and the CAR polypeptide such that the nucleic acid construct produces a polypeptide comprising the constitutive chimeric cytokine receptor of the invention and the CAR polypeptide linked by the self-cleaving peptide without any external cleavage activity, cleavage of the polypeptide produced by the nucleic acid construct into a separate constitutive chimeric cytokine receptor and a separate CAR polypeptide.

By "self-cleaving peptide" is meant a peptide that functions such that when a fusion polypeptide comprising a first polypeptide, a self-cleaving peptide and a second polypeptide from the N-terminus to the C-terminus is produced, the fusion polypeptide is cleaved into the unique and discrete first and second polypeptides without any external cleavage activity. The self-cleaving peptide may be a 2A self-cleaving peptide from an orotic or cardioviral virus.

In some embodiments, the self-cleaving peptide is P2A as shown in SEQ ID NO. 3 or a variant thereof having 1-5 amino acid modifications.

In some embodiments, the constitutive chimeric cytokine receptor-modified CAR polypeptide of the invention contains, from N-terminus to C-terminus, a CAR polypeptide, a self-cleaving peptide, and a constitutive chimeric cytokine receptor.

Nucleic acid molecules, vectors and expression cells encoding the constitutive chimeric cytokine receptor of the invention or encoding the constitutive chimeric cytokine receptor modified CAR polypeptides of the invention

The invention provides nucleic acid molecules encoding the constitutive chimeric cytokine receptor of the invention or encoding the constitutive chimeric cytokine receptor modified CAR polypeptides of the invention. In one embodiment, the nucleic acid molecule is provided as a DNA construct.

IV.1. DNA constructs encoding constitutive chimeric cytokine receptors of the invention

In some embodiments, a DNA construct encoding a constitutive chimeric cytokine receptor of the invention comprises, from N-terminus to C-terminus, a polynucleotide encoding a signal peptide, a polynucleotide encoding an extracellular domain consisting of an effector molecule having a remodelling tumor microenvironment, a polynucleotide encoding an IL-7R mutant transmembrane domain, and an IL-7R intracellular domain. Optionally, a polynucleotide encoding a hinge region, such as the Flag Tag shown in SEQ ID NO. 6 or a functional variant thereof, is present between the polynucleotide encoding the extracellular domain and the polynucleotide encoding the IL-7R mutant transmembrane domain and the IL-7R intracellular domain.

In some embodiments, the signal peptide comprises the sequence of SEQ ID NO. 2 or a functional variant thereof.

In some embodiments, the polynucleotide encoding the IL-7R mutant transmembrane domain and IL-7R intracellular domain comprises a polynucleotide encoding any one of the amino acid sequences selected from the group consisting of SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 28, SEQ ID NO. 30 to SEQ ID NO. 45, preferably comprises a polynucleotide encoding any one of the amino acid sequences selected from the group consisting of SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 33 and SEQ ID NO. 34, most preferably comprises a polynucleotide encoding the amino acid sequence shown in SEQ ID NO. 34.

In some embodiments, the extracellular domain consisting of an effector molecule with a remodelling tumor microenvironment is a cytokine, which may be, for example, IL-12 (IL-12-P40 or IL-12-P70), IL-15 (IL-15 or IL-15FP, which refers to a fusion protein of IL-15 and IL-15Rα (selected from IL-15Rα or IL-15Rα (Sushi)), including both forms of IL-15/IL-15Rα and IL-15Rα/IL-15, IL-21, IL-18, IL-9, IL-23, IL-36 γ, IFN α 2b, and the like, and immune cells (e.g., T cells) express the M7CR gene comprising the cytokine with enhanced immune effector function and anti-tumor effect. In some embodiments, the cytokine is SEQ ID NO:47 or a functional variant thereof; SEQ ID NO:48 (IL 15/IL15rα (Sushi) fusion protein) or a functional variant thereof; SEQ ID NO:140 (IL 15/IL15 ra fusion protein) or a functional variant thereof; SEQ ID NO:141 (IL 15rα (Sushi)/IL 15 fusion protein) or a functional variant thereof; SEQ ID NO:49 or a functional variant thereof; SEQ ID NO:50 or a functional variant thereof; SEQ ID NO:51 or a functional variant thereof; SEQ ID NO:52 or a functional variant thereof; SEQ ID NO:53 or a functional variant thereof; SEQ ID NO:54 or a functional variant thereof; SEQ ID NO:55 or a functional variant thereof; SEQ ID NO:56 or a functional variant thereof.

In some embodiments, the extracellular domain consisting of an effector molecule with a remodelling tumor microenvironment is an immune effector molecule that can be, for example, a 4-1BB targeting molecule moiety (e.g., 4-1BB ligand (4-1 BBL), anti-4-1 BB antibody (α4-1 BB)), a CD40 targeting molecule moiety (e.g., CD40 ligand (CD 40L), anti-CD 40 antibody (αcd 40)), a CD83 targeting molecule moiety (e.g., anti-CD 83 antibody (αcd83)), FLT3 ligand (FTL 3L), GITR, ICOS, CD2, ICAM1, etc., that activates an APC by interacting with a related receptor or ligand on the surface of a professional Antigen Presenting Cell (APC) in vivo, such as a Dendritic Cell (DC), thereby eliciting an endogenous anti-tumor immune response, thereby producing a synergistic anti-tumor efficacy with immune cells (e.g., T cells). In some embodiments, the immune effector molecule is SEQ ID NO:57 or a functional variant thereof; SEQ ID NO:58 or a functional variant thereof; SEQ ID NO:59 or a functional variant thereof; SEQ ID NO: ICOS shown at 60 or a functional variant thereof; SEQ ID NO:61 or a functional variant thereof; SEQ ID NO:62 or a functional variant thereof; SEQ ID NO:63 or a functional variant thereof; SEQ ID NO:64 against 4-1BB or a functional variant thereof; SEQ ID NO:65 or a functional variant thereof; SEQ ID NO:66 or a functional variant thereof.

In some embodiments, the extracellular domain comprised of an effector molecule having a remodelling tumor microenvironment is an inhibitory molecular antagonist, which may be, for example, an anti-PD-L1 molecule, an anti-CD 47 molecule, an anti-IL-4 molecule, an anti-tgfβ molecule, an anti-PD-1 molecule, an anti-CTLA-4 molecule, an anti-LAG-3 molecule, an anti-TIGIT molecule, an anti-CD 73 molecule, or the like, antibody moiety directed against an inhibitory immune receptor or factor that achieves the purpose of enhancing the anti-tumor immune response by antagonizing the immunosuppressive effects of the inhibitory immune receptor or factor, thereby producing a synergistic anti-tumor effect with immune cells (e.g., T cells). In some embodiments, the inhibitory molecular antagonist is SEQ ID NO:67 or a functional variant thereof;SEQ ID NO: anti-PD-L1 as shown at 68 _VHH Or a functional variant thereof; SEQ ID NO:69 or a functional variant thereof; SEQ ID NO:70 or a functional variant thereof; SEQ ID NO:71 or a functional variant thereof; SEQ ID NO:72 or a functional variant thereof; SEQ ID NO:73 or a functional variant thereof; SEQ ID NO:74 or a functional variant thereof; SEQ ID NO:75 or a functional variant thereof.

In some embodiments, the extracellular domain consisting of an effector molecule with a remodelling tumor microenvironment is an effector molecule targeting NK cell-activating receptors, which may be a molecular moiety targeting NK cell surface expressed activating receptors such as NKG2C, NKG2D, NKp, NKp44, NKp46, e.g. anti-NKG 2C, anti-NKG 2D, anti-NKp 30, anti-NKp 44, anti-NKp 46, etc. antibody moiety, by activating endogenous NK cells, achieving the objective of enhancing anti-tumor immune effects, thereby generating synergistic anti-tumor efficacy with immune cells (e.g. T cells). In some embodiments, the NK cell activating molecule is SEQ ID NO:76 or a functional variant thereof; SEQ ID NO:77 or a functional variant thereof; SEQ ID NO:78 or a functional variant thereof; SEQ ID NO:79 or a functional variant thereof.

IV.2. DNA constructs encoding the constitutive chimeric cytokine receptor-modified CAR polypeptides of the invention

In some embodiments, the DNA construct encoding a constitutive chimeric cytokine receptor-modified CAR polypeptide of the invention comprises, from N-terminus to C-terminus, a polynucleotide encoding a constitutive chimeric cytokine receptor, a polynucleotide encoding a self-cleaving peptide, and a polynucleotide encoding a CAR polypeptide.

In some embodiments, the DNA construct encoding a constitutive chimeric cytokine receptor-modified CAR polypeptide of the invention comprises, from N-terminus to C-terminus, a polynucleotide encoding a CAR polypeptide, a polynucleotide encoding a self-cleaving peptide, and a polynucleotide encoding a constitutive chimeric cytokine receptor.

The polynucleotide encoding the constitutive chimeric cytokine receptor is as described above.

The polynucleotide encoding the self-cleaving peptide is, for example, a polynucleotide encoding P2A shown in SEQ ID NO. 3 or a variant thereof having 1-5 amino acid modifications.

The polynucleotide encoding the CAR polypeptide may be a polynucleotide encoding any CAR polypeptide known in the art.

In some embodiments, the CAR polypeptide is a traditional CAR polypeptide that directly targets one or more of the cancer-associated antigens described above. In some embodiments, the CAR polypeptide is a traditional CAR polypeptide that directly targets CLDN18.2, comprising, from N-terminus to C-terminus, a signal peptide, a cancer-associated antigen binding domain, a transmembrane domain, a costimulatory signaling domain, and a primary signaling domain.

In a specific embodiment, the conventional CAR polypeptide comprises, from N-terminus to C-terminus: a CD8 signal peptide as set forth in SEQ ID No. 1 or a variant thereof having 1-5 amino acid modifications; VL- (G) shown in SEQ ID NO. 13 ₄ S) _n Peptide linker-VH shown in SEQ ID NO. 14, wherein "n" is an integer from 1 to 10, e.g. an integer from 2 to 4, e.g. the sequence shown in SEQ ID NO. 4, SEQ ID NO. 5; the CD8 hinge region shown in SEQ ID NO. 7 or a variant thereof having 1-5 amino acid modifications; a transmembrane domain shown in SEQ ID NO. 8 or a variant thereof having 1-5 amino acid modifications; a costimulatory signaling domain shown in SEQ ID NO. 11 or a variant thereof with 1-5 amino acid modifications; the main signaling domain shown in SEQ ID NO. 12 or a variant thereof having 1-5 amino acid modifications. In a specific embodiment, the conventional CAR polypeptide is a H9.1.2-BB-L CAR having the amino acid sequence shown in SEQ ID NO. 16.

In a specific embodiment, the conventional CAR polypeptide comprises, from N-terminus to C-terminus: a CD8 signal peptide as set forth in SEQ ID No. 1 or a variant thereof having 1-5 amino acid modifications; VL- (G) shown in SEQ ID NO. 98 ₄ S) _n Peptide linker-VH shown in SEQ ID NO 99, wherein "n" is an integer from 1 to 10, e.g. an integer from 2 to 4, e.g. the sequence shown in SEQ ID NO 4, SEQ ID NO 5; CD8 hinge region shown in SEQ ID NO. 7Or a variant thereof having 1-5 amino acid modifications; a transmembrane domain shown in SEQ ID NO. 8 or a variant thereof having 1-5 amino acid modifications; a costimulatory signaling domain shown in SEQ ID NO. 11 or a variant thereof with 1-5 amino acid modifications; the main signaling domain shown in SEQ ID NO. 12 or a variant thereof having 1-5 amino acid modifications. In a specific embodiment, the conventional CAR polypeptide is a H9.2.1-BB-L CAR having the amino acid sequence shown in SEQ ID NO. 100.

In a specific embodiment, the conventional CAR polypeptide comprises, from N-terminus to C-terminus: a CD8 signal peptide as set forth in SEQ ID No. 1 or a variant thereof having 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 the sequence shown in SEQ ID NO. 145; the CD8 hinge region shown in SEQ ID NO. 147 or a variant thereof having 1-5 amino acid modifications; a transmembrane domain shown in SEQ ID NO. 148 or a variant thereof having 1-5 amino acid modifications; a costimulatory signaling domain shown in SEQ ID NO. 11 or a variant thereof with 1-5 amino acid modifications; the main signaling domain shown in SEQ ID NO. 12 or a variant thereof having 1-5 amino acid modifications. In a specific embodiment, the conventional CAR polypeptide is H9.2.1-BB-L CAR' having the amino acid sequence shown in SEQ ID NO. 144.

In a specific embodiment, the conventional CAR polypeptide comprises, from N-terminus to C-terminus: a CD8 signal peptide as set forth in SEQ ID No. 1 or a variant thereof having 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 the sequence shown in SEQ ID NO. 145; the CD8 hinge region shown in SEQ ID NO. 147 or a variant thereof having 1-5 amino acid modifications; a transmembrane domain shown in SEQ ID NO. 148 or a variant thereof having 1-5 amino acid modifications; a costimulatory signaling domain shown in SEQ ID NO. 143 or a variant thereof with 1-5 amino acid modifications; the main signaling domain shown in SEQ ID NO. 12 or a variant thereof having 1-5 amino acid modifications. In a specific embodiment, the conventional CAR polypeptide is a H9.2.1-28-L CAR having the amino acid sequence shown in SEQ ID NO: 142.

In some embodiments, the CAR polypeptide is molecular switch-regulatedA CAR polypeptide of type. In some embodiments, the CAR polypeptide is targeted to a cancer-associated antigen by an antibody combination with a P329G mutation as a molecular switch (the P329G mutation is also simply referred to as "PG") anti-cancer-associated antigen comprising, from the N-terminus to the C-terminus, a signal peptide, an anti-PG antibody scFv sequence, a transmembrane domain, a costimulatory signaling domain, and a primary signaling domain. In particular embodiments, the molecular switch-regulated CAR polypeptide comprises, from N-terminus to C-terminus: a CD8 signal peptide as set forth in SEQ ID No. 1 or a variant thereof having 1-5 amino acid modifications; anti-PG antibody VH- (G) shown in SEQ ID NO 9 ₄ S) _n The peptide linker-anti-PG antibody VL shown in SEQ ID NO. 10, wherein "n" is an integer of 1 to 10, for example an integer of 2 to 4, for example the sequences shown in SEQ ID NO. 4, SEQ ID NO. 5; GGGGS hinge; a transmembrane domain shown in SEQ ID NO. 8 or a variant thereof having 1-5 amino acid modifications; a costimulatory signaling domain shown in SEQ ID NO. 11 or a variant thereof with 1-5 amino acid modifications; the main signaling domain shown in SEQ ID NO. 12 or a variant thereof having 1-5 amino acid modifications. In a specific embodiment, the molecular switch regulated CAR polypeptide is a HuR968B CAR having the amino acid sequence shown in SEQ ID No. 15.

In some embodiments, the DNA constructs encoding the constitutive chimeric cytokine receptor-modified CAR polypeptides of the invention comprise polynucleotides encoding the amino acid sequences of any one of SEQ ID NO:80-SEQ ID NO:95, SEQ ID NO:101, SEQ ID NO:104, SEQ ID NO:133, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, and functional variants thereof.

The invention also provides vectors into which the DNA constructs of the invention are inserted. Expression of the polypeptide encoded by the polynucleotide on the DNA construct is achieved by inserting the nucleic acid encoding the DNA construct of the invention into an expression vector operably linked to a promoter. Vectors may be suitable for replication and integration in eukaryotes. Common cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters for regulating the expression of the desired nucleic acid sequence.

Numerous virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene may be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus may then be isolated and delivered to cells of the subject in vivo or ex vivo. Numerous retroviral systems are known in the art. In some embodiments, lentiviral vectors are used.

Vectors derived from retroviruses (e.g., lentiviruses) are suitable tools for achieving long-term gene transfer, as they allow long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have the additional advantage over vectors derived from cancer-retroviruses (e.g., murine leukemia virus) in that they can transduce non-proliferative cells, such as hepatocytes. They also have the additional advantage of low immunogenicity. The retroviral vector may also be, for example, a gamma retroviral vector. The gamma retroviral vector may, for example, comprise a promoter, a packaging signal (ψ), a Primer Binding Site (PBS), one or more (e.g., two) Long Terminal Repeats (LTRs), and a transgene of interest, e.g., a gene encoding a constitutive chimeric cytokine receptor of the invention or a gene encoding a constitutive chimeric cytokine receptor modified CAR polypeptide of the invention. The gamma retroviral vector may lack viral structural genes such as gag, pol and env.

An example of a promoter capable of expressing the transgene of the invention in mammalian T cells is the EF1a promoter. The native EF1a promoter drives the expression of the alpha subunit of the elongation factor-1 complex, which is responsible for the enzymatic delivery of aminoacyl tRNA to the ribosome. The EF1a promoter has been widely used in mammalian expression plasmids and has been shown to be effective in driving expression of transgenes cloned into lentiviral vectors. See, e.g., milone et al mol. Ther.17 (8): 1453-1464 (2009).

Another example of a promoter is the immediate early Cytomegalovirus (CMV) promoter sequence. This promoter sequence is a constitutive strong promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked thereto. However, other constitutive promoter sequences may also be used, including but not limited to monkey virus 40 (SV 40) 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 actin promoter, myosin promoter, elongation factor-1 alpha promoter, hemoglobin promoter, and creatine kinase promoter. In addition, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention.

In some embodiments, the invention provides methods of expressing the DNA constructs of the invention in mammalian immune effector cells (e.g., mammalian T cells or mammalian NK cells) and immune effector cells produced thereby.

A cell source (e.g., an immune effector cell, e.g., a T cell or NK cell) is obtained from a subject. The term "subject" is intended to include living organisms (e.g., mammals) that can elicit an immune response. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors.

Any technique known to those skilled in the art (e.g., ficoll ^TM Isolation) to obtain T cells from blood components collected from a subject. In a preferred aspect, cells from circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes and platelets. In one embodiment, cells collected by apheresis may be washed to remove plasma fractions and to place the cells in a suitable buffer or medium for subsequent processing steps. In one aspect of the invention, the cells are washed with Phosphate Buffered Saline (PBS).

Specific T cell subsets, such as cd3+, cd28+, cd4+, cd8+, C, can be further isolated by positive or negative selection techniques D45ra+ and cd45ro+ T cells. For example, in one embodiment, the conjugate is provided by a bead conjugated to an anti-CD 3/anti-CD 28 (e.g.M-450CD3/CD 28T) for a period of time sufficient to positively select the desired T cells, and isolating the T cells. In some embodiments, the period of time is between about 30 minutes and 36 hours or more. Longer incubation times can be used to isolate T cells in any situation where a small number of T cells are present, such as for isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or from immunocompromised individuals. In addition, the use of longer incubation times can increase the efficiency of cd8+ T cell capture. Thus, by simply shortening or lengthening this time, allowing T cells to bind to CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells, T cell subsets can be preferentially selected at the beginning of the culture or at other points in time during the culture process.

Enrichment of the T cell population can be accomplished by a negative selection process with 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 a negative magnetic immunoadhesion method or flow cytometry using a monoclonal antibody mixture directed against cell surface markers present on the negatively selected cells.

In some embodiments, the immune effector cell may be an allogeneic immune effector cell, e.g., a T cell or NK cell. For example, the cells may be allogeneic T cells, e.g., allogeneic T cells lacking functional T Cell Receptors (TCRs) and/or expression of Human Leukocyte Antigens (HLA) (e.g., HLA class I and/or HLA class II).

A T cell lacking a functional TCR may, 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 make up a functional TCR (e.g., engineered so that it does not express or exhibit reduced expression of tcra, tcrp, tcrγ, tcrδ, tcrε, and/or tcrζ); or engineered so that it produces very few functional TCRs on its surface.

The T cell described herein may, for example, be engineered such that it does not express a functional HLA on its surface. For example, T cells described herein can be engineered such that cell surface expression of HLA (e.g., HLA class I and/or HLA class II) is down-regulated. In some aspects, down-regulation of HLA can be achieved by reducing or eliminating beta-2 microglobulin (B2M) expression.

In some embodiments, T cells may lack a functional TCR and a functional HLA, e.g., HLA class I and/or HLA class II.

Immune effector cells co-expressing the CAR and the constitutive chimeric cytokine receptor of the invention obtained after in vitro proliferation can be tested for effector function as described in the examples.

V. pharmaceutical composition of the invention

In some embodiments, the invention provides pharmaceutical compositions comprising an immune effector cell (e.g., T cell, NK cell) selected from the group consisting of a constitutive chimeric cytokine receptor of the invention, a nucleic acid molecule encoding the constitutive chimeric cytokine receptor, a vector comprising a nucleic acid molecule encoding the constitutive chimeric cytokine receptor, and any combination thereof; and optionally pharmaceutically acceptable excipients.

In some embodiments, the invention provides a pharmaceutical composition comprising an immune effector cell (e.g., T cell, NK cell) selected from the group consisting of a constitutive chimeric cytokine receptor-modified CAR polypeptide of the invention, a nucleic acid molecule encoding the constitutive chimeric cytokine receptor-modified CAR polypeptide, a vector comprising a nucleic acid molecule encoding the constitutive chimeric cytokine receptor-modified CAR polypeptide, and any combination thereof; and optionally pharmaceutically acceptable excipients. Further, when the CAR polypeptide is a molecular switch-regulated CAR polypeptide, the pharmaceutical composition further comprises a molecular switch, such as an antibody molecular switch.

In some embodiments, the immune effector cells are prepared from autologous T cells or allogeneic T cells, e.g., the immune effector cells are prepared from T cells isolated from human PBMCs.

The pharmaceutical compositions of the invention may be formulated according to conventional methods (e.g., remington's Pharmaceutical Science, latest edition, mark Publishing Company, easton, u.s.a.). Examples of pharmaceutically acceptable excipients include surfactants, excipients, colorants, fragrances, preservatives, stabilizers, buffers, suspending agents, isotonic agents, binders, disintegrants, lubricants, flow promoters, flavoring agents, and the like. Further, other commonly used carriers such as light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carboxymethylcellulose calcium, carboxymethylcellulose sodium, hydroxypropylcellulose, hydroxypropylmethyl cellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain fatty acid triglycerides, polyoxyethylene hardened castor oil 60, white granulated sugar, carboxymethylcellulose, corn starch, inorganic salts and the like may also be suitably used as the carrier, but are not limited thereto.

In some embodiments, the pharmaceutical compositions of the invention are used to treat cancer, e.g., a cancer that expresses or overexpresses CLDN 18.2.

Use of the pharmaceutical composition of the invention and methods of treatment using the pharmaceutical composition of the invention

The present invention provides the aforementioned pharmaceutical composition of the invention for use in treating a tumor (e.g., cancer) in a subject. The invention also relates to a method of treating a tumor (e.g., cancer) in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition of the invention. In some embodiments, the tumor is a cancer. In some embodiments, the tumors described herein, such as cancers, include, but are not limited to, solid tumors, hematological cancers, soft tissue tumors, and metastatic lesions.

In one embodiment, the pharmaceutical composition of the invention is used to treat a cancer that expresses or overexpresses CLDN 18.2 in a subject and is capable of reducing the severity of at least one symptom or indication of cancer or inhibiting cancer cell growth. The invention provides a method of treating cancer (e.g., a cancer that expresses or overexpresses 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 invention provides the use of the aforementioned pharmaceutical composition of the invention in the manufacture of a medicament for treating cancer (e.g., CLDN 18.2 expressing or overexpressing cancer).

The pharmaceutical compositions of the invention may also be administered to individuals who have been treated for cancer with one or more previous therapies but subsequently relapsed or metastasized.

The pharmaceutical composition of the invention may be administered to a subject in a suitable dosage. 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 one patient depends on many factors, including the patient's weight, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs to be administered concurrently.

In some embodiments, administration of the pharmaceutical composition of the invention to an individual suffering from cancer results in complete disappearance of the tumor. In some embodiments, administration of a pharmaceutical combination of the invention to an individual having cancer results in a reduction in tumor cell or tumor size of at least 85% or more. The reduction of tumors may 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 genetic analysis.

The various embodiments/technical solutions described herein and features in the various embodiments/technical solutions should be understood to be arbitrarily combined with each other, and the various solutions obtained by these combinations are included in the scope of the present invention as if the combinations were specifically and individually listed herein unless the context clearly indicates otherwise.

The following examples are described to aid in the understanding of the present invention. The examples are not intended to, and should not be construed in any way as, limiting the scope of the invention.

Examples

Example 1 Synthesis, screening and functional study of M7R

EXAMPLE 1.1 construction of lentiviral vectors for expression of the M7R Gene

As shown in FIG. 2A, IL-7 binds to its wild-type receptor IL-7 receptor alpha chain (IL 7Rα) and induces heterodimerization of the latter with a common gamma signal chain, activating the downstream JAK/STAT signal. However, when wild-type IL7R alpha transmembrane region is mutated to produce a mutated IL7R, the mutation may induce the resulting mutated IL7R to self-dimerize, thereby enabling constitutive activation of downstream STAT5 signaling pathways by the self-dimerized mutated IL7R without reliance on IL-7 binding.

As shown in FIG. 2B, viral expression plasmids were constructed for expression of chimeric receptors tCD19-M7CR (also referred to herein as IL7Rm-tCD 19) comprising different IL7R mutations (IL 7Rm or M7R) and tCD19, these tCD19-M7CR consisting of the extracellular domain (ECD) of the same truncated CD19 (tCD 19, SEQ ID NO: 17) and different IL7R mutants (also referred to herein as IL7Rm or M7R), said IL7Rm (SEQ ID NO:20-SEQ ID NO: 46) consisting of an IL7R transmembrane region (IL 7R-mutant (TM)) carrying different mutations (see the bolded part of the sequence) and a wild-type IL7R intracellular segment (IL 7R-wt (ICD), SEQ ID NO: 19).

The 27 IL7Rm are designated as IL7Rm1.1, IL7Rm1.2, IL7Rm1.3, IL7Rm1.4, IL7Rm2.1, IL7Rm2.2, IL7Rm2.3, IL7Rm2.4, IL7Rm3.1, IL7Rm3.2, IL7Rm4, IL7Rm5, IL7Rm6, IL7Rm7, IL7Rm8, IL7Rm9, IL7Rm10, IL7Rm11, IL7Rm12, IL7Rm13, IL7Rm14, IL7Rm15, IL7Rm16, IL7Rm17, IL7Rm18, IL7Rm19, IL7Rm20, respectively (see SEQ ID NO in the sequence listing: 20-SEQ ID NO: 46) are designated IL7Rm1.1-tCD19, IL7Rm1.2-tCD19, IL7Rm1.3-tCD19, IL7Rm1.4-tCD19, IL7Rm2.1-tCD19, IL7Rm2.2-tCD19, IL7Rm2.3-tCD19, IL7Rm2.4-tCD19, IL7Rm3.1-tCD19, IL7Rm3.2-tCD19, IL7Rm4-tCD19, IL7Rm5-tCD19, IL7Rm6-tCD19, IL7Rm7-tCD19, IL7Rm8-tCD19, IL7Rm9-tCD19, IL7 10-tCD19, IL7Rm11-tCD19, IL7Rm12-tCD19, IL7Rm13-tCD19, IL 7-tCD 14-CD 19, IL 7-tCD19, IL 7-tCD19, IL7 t19-tCD 19, IL7 t19 tCD19; as a control, the IL7R-tCD19 construct comprises the tCD19 extracellular domain and the wild-type IL7R transmembrane region and intracellular segment (SEQ ID NO: 96), and the IL7R-WT construct comprises the complete wild-type IL7R chain (SEQ ID NO: 18).

EXAMPLE 1.2 construction of BaF3 cell lines stably expressing different M7 Rs

BaF3 (available from Nanjac, bai Biotechnology Co., ltd.) is murine pre-B lymphocytes that depend on exogenously added mouse IL-3 (mIL-3) cytokine (R & D system) for survival. When the exogenous gene is transferred into the cell line to enable the BaF3 cells to obtain the growth characteristic independent of IL-3, the transferred exogenous gene is indicated to be capable of transmitting the constitutive survival promoting signal, so that the BaF3 cell line is a common tool cell line for screening the cell line with the constitutive survival promoting signal gene.

To identify whether the tCD19-M7CR gene constructed in example 1.1 was able to continuously activate STAT5 signals after being introduced into the BaF3 cell line, different tCD19-M7CR genes were transferred into BaF3 cells by lentiviruses, and M7R genes having a function of continuously activating STAT5 were selected based on whether the BaF3 cells were able to produce IL-3 independent growth. The experimental procedure was as follows, lenti-X-293T cells (Takara Co., ltd.) in logarithmic growth phase (3X 10) ⁵ Individual cells) were inoculated into 6-well plates, after cell attachment, 27 expression plasmids pRK (Jin Weizhi construction) cloned with different tCD19-M7CR genes and cloned with the control IL7R-tCD19 gene, respectively, packaging plasmid pMDLg/pRRE (adedge, 12251, purchased from biofuels), regulatory plasmid pRSV-rev (adedge, 12253, purchased from biofuels) and envelope plasmid pMD2G (adedge, 12259, purchased from biofuels) were transfected with the PEI transfection method in a mass ratio of 3:3:2:2 into DMEM medium containing 10% FBS (Takara corporation), fresh DEME medium containing 2% Fetal Bovine Serum (FBS) was replaced after 16 hours of transfection, cell culture supernatants were collected, centrifuged to remove cell debris, and lentiviral-containing supernatants were obtained after further 48 hours of culture.

BaF3 cells were infected with lentiviral-containing supernatant for 24 hours and then routinely cultured for 48 hours with mIL-3 (R & D systems, 403-ML) in RPMI 1640 complete medium.

EXAMPLE 1.3 detection of M7R expression in cells

After infection of BaF3 cells by the lentivirus prepared in example 1.2, whether or not M7R was expressed successfully was determined by detecting whether or not tCD19 was expressed on the surface of BaF3 cells using flow cytometry.

Specifically, baF3 cells infected with lentivirus prepared in example 1.2 were taken, washed once with FACS buffer, and then the BaF3 cells were resuspended in FACS buffer, LIVE/DEAD Fixable Dead Cell Stain (Thermo, L34963) was added, and PE-CD19 antibody (BD Co., 555413) was incubated at 4℃for 30 to 45min. The cells were then washed once with FACS buffer and, after resuspension with FACS buffer, the expression of tCD19 on the cell surface was detected by flow cytometry.

As a result, as shown in FIG. 3A, on day 4 after infection of BaF3 cells with 27 lentiviruses containing different tCD19-M7CR genes, tCD19 was expressed on the surface of BaF3 cells, indicating successful expression of M7R gene in BaF3 cells.

Example 1.4. Screening for M7R with activating STAT5 function after 27 lentiviruses containing different M7R genes infest BaF3 cells and 27 different M7R genes were each expressed in the infected BaF3 cells, baF3 cells (each group of BaF3 cells expressing different M7R genes) prepared in example 1.2 were cultured in mIL-3-free RPMI-1640 (10% FBS) complete medium, and each M7R was judged for the mIL-3 independent growth promoting effect by observing whether BaF3 cells were able to grow in mIL-3 independent manner. Subsequently, each group of BaF3 cells selected to have mIL-3 independent growth was grown in the same cell number (i.e., 5X 10 ⁵ Individual cells/well) were added to 24-well plates, each group of cells was counted by a cell counter, the proliferation of the cells was recorded, and a growth curve was drawn.

As shown in FIG. 3B, the infected BaF3 cells were cultured without the addition of exogenous mIL-3, and then expressed IL7Rm1.1-tCD19, IL7Rm1.3-tCD19, IL7Rm3.1-tCD19, IL7Rm4-tCD19, IL7Rm5-tCD19, IL7Rm6-tCD19, IL7Rm7-tCD19, IL7Rm8-tCD19, IL7Rm9-tCD19, IL7Rm10-tCD19, IL7Rm11-tCD19, IL7Rm12-tCD19, IL7Rm13-tCD19, IL7Rm14-tCD19, IL7Rm15-tCD19, IL7 16-tCD19, IL7Rm 17-tCD19, IL7Rm 18-tCD19, IL7Rm 19-tCD19 in BaF3 cells ⁺ The proportion of cells increased and the remaining group of BaF3 cells failed to remain viable after culture without the addition of exogenous mIL-3, indicating IL7Rm1.1, IL7Rm1.3, IL7Rm3.1,IL7Rm4, IL7Rm5, IL7Rm6, IL7Rm7, IL7Rm8, IL7Rm9, IL7Rm10, IL7Rm11, IL7Rm12, IL7Rm13, IL7Rm14, IL7Rm15, IL7Rm 16,IL7Rm 17,IL7Rm 18,IL7Rm 19 are capable of providing a continuously activated IL7rα signal, promoting BaF3 cell growth in a mll-3 independent manner.

After infection of cells with the virus, the cells were screened without the addition of exogenous mIL-3 from day 3, and as shown in FIG. 3C, CD19 was counted before and after screening BaF3 cells without the addition of exogenous mIL-3 ⁺ The ratio of cells was varied, and the results showed that CD19 was detected on day 11 after culturing BaF3 cells without the addition of exogenous mIL-3 ⁺ CD19 in the group with cell survival ⁺ The ratio of (2) is significantly increased.

As shown in FIG. 3D, after cell counting, M7R genes such as IL7Rm1.1, IL7Rm1.3, IL7Rm3.1, IL7Rm 4-12, IL7Rm 14-18 and the like all can promote the proliferation of BaF3 cells in a mIL-3 independent manner.

Example 1.5 intracellular p-STAT5 detection of baf3 cells:

BaF3 cells not infected with lentivirus (as control) and BaF3 cells from each group capable of stably expressing tCD19-M7CR after the screening of example 1.4 were taken, washed twice with PBS, and resuspended in serum-free RPMI-1640 medium overnight. The following day, cells were collected by centrifugation, fixed with 3% paraformaldehyde, and washed 1 pass with FACS buffer; permeabilizing the cells with 0.1% Triton-X and washing 1 pass with FACS buffer; placing the cells in 95% precooled methanol, and standing at-20deg.C overnight; the following day, cells were collected by centrifugation, washed 1 time with FACS buffer, then incubated with AF647-p-STAT5 (BD, 562076) antibody at room temperature for 45min, and finally the expression level of p-STAT5 in BaF3 cells was detected by flow cytometry.

In fig. 4, ISO refers to staining with isotype control antibodies against STAT5, +il3 refers to the addition of IL3 to uninfected BaF3 cells to stimulate activation of STAT5 by the cells, as a positive control; without IL3 was used to detect basal levels of STAT5 phosphorylation in cells without IL3 stimulation of uninfected BaF3 cells, stained with anti-pSTAT 5 antibody.

As shown in fig. 4, baF3 cells not infected with lentivirus (ISO as control) only detected STAT5 phosphorylation signal upon stimulation with the addition of exogenous mll-3; whereas BaF3 cells expressing il7rm1.1, il7rm1.3, il7rm3.1, IL7Rm4, IL7Rm5, IL7Rm6, IL7Rm7, IL7Rm8, IL7Rm9, IL7Rm10, IL7Rm11, IL7Rm12, IL7Rm14, IL7Rm15, IL7Rm16, IL7Rm17, IL7Rm18 genes detected STAT5 phosphorylation signals without the addition of exogenous mll-3 stimulus, indicating that these M7R genes were able to constitutively activate STAT5 signaling pathways.

Example 2 influence of M7R on human T cell growth

According to the results of screening the constructed tCD19-M7CR gene using BaF3 cells in example 1, M7R sequences (IL7Rm1.1, 1.3, 3.1, 4 to 12, 14 to 18, respectively) having a constitutive activation function were selected.

To identify whether the M7R sequence was able to continuously activate the STAT5 signaling pathway in T cells after the tCD19-M7CR gene was introduced into a T cell line, different tCD19-M7CR genes (tCD 19-M7CR genes containing IL7Rm1.1, 1.3, 3.1, 4-12, 14-18, respectively) were transferred into T cells by lentivirus, and M7R genes having a continuous activation of STAT5 function in T cells were selected based on whether or not the T cells were able to produce IL-2 independent growth.

Lentiviral packaging procedure lentiviral supernatants of the tCD19-M7CR genes containing different M7R sequences (IL 7Rm1.1, 1.3, 3.1, 4-12, 14-18, respectively) were obtained as described in example 1.2. Activated human T cells were infected with lentiviruses expressing the different tCD19-M7CR genes (PBMC information see Table 1) to obtain T cells stably expressing the different tCD19-M7CR genes. The specific steps are as follows.

T cell sorting and activation steps: recombinant human interleukin-2 (national standard S20040020) for injection was added to TexMACS GMP Medium (Miltenyi Biotec, 170-076-309) to prepare a T cell culture medium having an IL-2 concentration of 200 IU/ml.

Multiple donor PBMC cells were obtained from ORiCELLS, and specific information is shown in table 1 below:

TABLE 1 relevant Source information of donor PBMC cells

PBMC cell numbering	Catalog number	Batch number	Supplier ID number
				Donor 3PBMC	FPB004F-C	PCH2020110004	Z0052
Donor 5PBMC	FPB004F-C	PCH20210100004	Z0086
				Donor 13PBMC	FPB004F-C	LP211027009	Z0301
Donor 15PBMC	FPB004F-C	LP211013009	Z0290
				Donor 17PBMC	FPB004F-C	LP211214008	Z0349

Day 0The recovered donor PBMC were sorted using Pan T Cell Isolation Kit (human) (Miltenyi, 130-096-535) to obtain T cells, and the T cells were resuspended to a density (e.g., cell density 1X 10) using T cell culture medium ⁶ Individual cells/mL) and adding tranAct (Miltenyi, 130-111-160) for activation; on day 1, a certain amount of cells are isolated, the cells are not transduced cells (UNT cells, un-transduced T cells), the rest cells are added with the supernatants of the lentiviruses respectively containing different tCD19-M7CR genes, and T cells are blown uniformly; the lentiviral-containing supernatant was removed by centrifugation on day 2 and T cells were resuspended in fresh IL-2 containing T cell medium. No manipulation of UNT cells was performed. 37 ℃,5% CO ₂ After 48h incubation in the cell culture incubator, the expression level of the M7R gene in T cells and the phosphorylation level of STAT5 in T cells were detected using LIVE/DEAD Fixable Dead Cell Stain (Thermo, L34963), PE-CD19 (BD, 555413), AF647-p-STAT5 (BD, 562076) antibody combinations. Then the same number of tcd19+ T cells (5×10 ⁶ Cells/well) were added to 24-well plates and incubated with RPMI1640 complete medium (without hll-2) with liquid changes every 2 to 3 days. Total T cells were counted weekly by a cytometer and tCD19 was detected by flow cytometry ⁺ Cell ratio and cell growth curve was plotted.

T cells were assayed for tCD19 expression using PBMC cells of donor 3 48h after virus infection of T cells. As a result, as shown in FIG. 5, different ratios of tCD19 expression were detected in human T cells infected with different tCD19-M7CR genes, IL7R-tCD19 genes comprising a wild-type IL7Rα transmembrane region and intracellular segment, while tCD19 expression was not detected in T cells not transfected (UNT) and T cells expressing wild-type IL7Rα (IL 7R-WT). The M7R gene was demonstrated to be successfully expressed in T cells.

Using PBMC cells of donor 3, the level of phosphorylation of STAT5 in T cells was detected after 5 days of culture (5 days of co-culture starting from day 0 up to selection activation up to detection) after infection of T cells by the virus. As shown in fig. 6, untransfected T cells (UNT) only detected STAT5 phosphorylation signals upon stimulation with exogenous IL-2; whereas STAT5 phosphorylation signals were detected in T cells stably expressing different M7R, but not wild-type IL7R transmembrane and intracellular segments (control IL7R-tCD 19) without the addition of exogenous IL-2 stimulation, indicating that these M7R genes are also capable of constitutively activating STAT5 signaling pathways in human T cells.

Using PBMC cells of donor 3, cells were expanded in vitro for 2 weeks under the addition of exogenous IL-2 stimulus after which the cells stopped expanding and maintained for about 1 week, after which the cell numbers were significantly reduced, as shown in fig. 7A and 7B, after infection of T cells with virus, and cell growth curves were drawn. In the absence of added exogenous IL-2 stimulation, the number of T cells expressing IL7R-tCD19 decreased continuously and all cells died after 2 weeks.

In comparison to T cells expressing IL7R-tCD19, T cells expressing the IL7Rm4, 5, 7 and 8 constructs were able to sustain a slow decrease in number for 1 week without the addition of exogenous IL-2, starting after 1 week until all cells died for 4 weeks, and these in vitro experiments showed that expression of the M7R gene promoted T cell survival and had the ability to sustain T cell survival.

Example 3 construction of M7 CR-modified CAR

As shown in FIG. 8, different M7CR genes were designed in which the extracellular domain (ECD) and M7R (fused IL-7R mutant consisting of IL-7Rα mutant transmembrane and intracellular signaling domain) were directly linked to form a constitutive chimeric cytokine receptor M7CR; the N-terminus of M7CR was then linked to the C-terminus of a different CAR polypeptide by P2A, thereby constituting an M7CR modified CAR.

In the M7CR, ECDs located at the N-terminus of M7R include, but are not limited to, IL-15 (SEQ ID NO: 47), IL-15/IL-15Rα (SEQ ID NO: 140), IL-15/IL-15Rα (Sushi) (SEQ ID NO: 48), IL-15Rα (Sushi)/IL-15 (SEQ ID NO: 141), IL-12-P70 (SEQ ID NO: 49), IL-12-P40 (SEQ ID NO: 50), IL-21 (SEQ ID NO: 51), IL-9 (SEQ ID NO: 52), IL-18 (SEQ ID NO: 53), IL-23 (SEQ ID NO: 54), IL-36 γ (SEQ ID NO: 55), IFNα2b (SEQ ID NO: 56), 4-1BBL (SEQ ID NO: 57), CD40L (SEQ ID NO: 58), FLT3L (SEQ ID NO: 59), ICOS (SEQ ID NO: 60), GIOS (SEQ ID NO: 61), ICAM-62, CD2 (SEQ ID NO: 64), anti-CD-1 (SEQ ID NO: 64), anti-CD 1 (SEQ ID NO: 65)SEQ ID NO:67 anti-PD-L1) _VHH (SEQ ID NO: 68), anti-CD 47 (SEQ ID NO: 69), anti-IL-4 (SEQ ID NO: 70), anti-PD-1 (SEQ ID NO: 71), anti-CTLA-4 (SEQ ID NO: 72), anti-LAG-3 (SEQ ID NO: 73), anti-TIGIT (SEQ ID NO: 74), anti-CD 73 (SEQ ID NO: 75), anti-NKG 2D (SEQ ID NO: 76), anti-NKG 2C (SEQ ID NO: 77), anti-NKp 30 (SEQ ID NO: 78) and anti-NKp 46 (SEQ ID NO: 79). Different ECDs are linked to M7R to form respective M7CR molecules: IL-15-M7CR, IL-15/IL 15Rα -M7CR, IL15/IL15Rα (Sushi) -M7CR, IL15Rα (Sushi)/IL 15-M7CR IL-12-P70-M7CR, IL-12-P40-M7CR, IL-21-M7CR, IL-9-M7CR, IL-18-M7CR, IL-23-M7CR, IL-36 γ -M7CR, IFNα 2b-M7CR, 4-1BBL-M7CR, CD40L-M7CR, FLT3L-M7CR, ICOS-M7CR, GITR-M7CR, ICAM-1-M7CR, CD2-M7CR, anti-4-1 BB-M7CR, anti-CD 40-M7CR, anti-CD 83-M7CR, anti- β -M7CR, anti-PD-L1 _VHH -M7CR, anti-CD 47-M7CR, anti-IL-4-M7 CR, anti-PD-1-M7 CR, anti-CTLA-4-M7 CR, anti-LAG-3-M7 CR, anti-TIGIT-M7 CR, anti-CD 73-M7CR, anti-NKG 2D-M7CR, anti-NKG 2C-M7CR, anti-NKp 30-M7CR and anti-NKp 46-M7CR.

FIG. 1 shows the mechanism of action of T cells expressing constructed M7CR after T cell transduction by constructed M7CR.

Example 4 preparation of M7 CR-modified CAR-T cells

EXAMPLE 4.1 sequence Synthesis

A DNA sequence encoding a HuR968B CAR (SEQ ID NO: 15) (hereinafter/in the drawings also sometimes abbreviated as "8B"), H9.1.2-BB-L CAR (SEQ ID NO: 16) (sometimes referred to herein as "H9.1.2"), H9.2.1-BB-L-CAR (SEQ ID NO: 100) or H9.2.1-BB-L-CAR' (SEQ ID NO: 144) (sometimes referred to herein as "H9.2.1 or H9"), H9.2.1-28-L-CAR (SEQ ID NO: 142) (sometimes referred to herein as "H9.2.1 (CD 28)"), H9.1.2-P2A-tCD19-M7CR (hereinafter also referred to as "H9.1.2-tCD 19-M7 CR) (SEQ ID NO: 80), H9.1.2-P2A-tCD19-M7CR (CPT) (hereinafter also referred to as" H9.1.2-tCD19-M7CR (CPT)) (SEQ ID NO: 81), H9.1.2-P2A-IL-12-P70-M7CR (hereinafter also referred to as "H9.1.2-IL-12-M7 CR) (SEQ ID NO: 82), H9.1.2-P2A-IL-15/IL-15-M7 CR (hereinafter also referred to as" IL-15-M7 CR) (SEQ ID NO: 83), H9.1.2-P2A-IL-21-M7CR (hereinafter also referred to as "IL-15-M7 CR) (hereinafter also referred to as" IL-21-IL-12-M7 CR) (hereinafter also referred to as "IL-2-IL-15) (hereinafter also referred to as" IL-and Also referred to herein as H9.1.2-CD40L-M7 CR) (SEQ ID NO: 85), H9.1.2-P2A-4-1BBL-M7CR (hereinafter also referred to as H9.1.2-4-1BBL-M7 CR) (SEQ ID NO: 86), H9.1.2-P2A-anti-PD-L1 _VHH M7CR (hereinafter also referred to as H9.1.2-anti-PD-L1 _VHH -M7 CR) (SEQ ID NO: 87), 8B-P2A-tCD19-M7CR (hereinafter also referred to as 8B-tCD19-M7 CR) (SEQ ID NO: 88), 8B-P2A-tCD19-M7CR (CPT) (hereinafter also referred to as 8B-tCD19-M7CR (CPT)) (SEQ ID NO: 89), 8B-P2A-IL-15/IL-15Rα -M7CR (hereinafter also referred to as 8B-IL-15-M7 CR) (SEQ ID NO: 90), 8B-P2A-IL-12-M7 CR (hereinafter also referred to as 8B-IL-12-M7 CR) (SEQ ID NO: 91), 8B-P2A-IL-21-M7CR (hereinafter also referred to as 8B-IL-21-M7 CR) (SEQ ID NO: 92), 8B-P2A-M40 CR (hereinafter also referred to as 8B-CD40L-M7 CR) (SEQ ID NO: 90), 8B-P2A-IL-12-M7 CR (hereinafter also referred to as 8B-IL-12-M7 CR) (hereinafter also referred to as 8B-IL-21-M7 CR) (SEQ ID NO: 94), and 4B-P2A-L-7 CR (hereinafter also referred to as 8B-IL-21-M7 CR) (SEQ ID NO: 94) _VHH M7CR (hereinafter also referred to as 8B-anti-PD-L1 _VHH -M7 CR) (SEQ ID NO: 95), H9.2.1-P2A-tCD19-M7CR (hereinafter also referred to as H9.2.1-tCD19-M7 CR) (SEQ ID NO: 101), H9.2.1-P2A-IL-12-P70-M7CRin (hereinafter also called H9.2.1-IL-12-M7CRin or H9.2.1-IL12-M7 CRin) (SEQ ID NO: 102), H9.2.1in-P2A-IL-12-P70-M7CR (hereinafter also called H9.2.1in-IL-12-M7CR or H9.2.1in-IL12-M7 CR) (SEQ ID NO: 103), H9.2.1-P2A-IL-12-P70-M7CR (hereinafter also called H9.2.1-IL-12-M7CR or H9.2.1-IL12-M7 CR) (SEQ ID NO: 104), H9.2.1-P2A-sIL-12-P70 (hereinafter also called H9.2.1-sIL-12 or H9.2.1-sIL 12) (SEQ ID NO: 105), H9.2.1-P2A-IL-15/IL-15Rα (Sushi) -M7CR (hereinafter also called 37-IL-12-M7 CR) (hereinafter also called H9.2.1-IL-12-M7CR (hereinafter also called as "H9.2.1-IL-12-M7 CR) (SEQ ID NO: 104), and H9.2.1-P2A-sIL-12-P70 (hereinafter also called" IL-15-IL-37-IL-M7 CR (hereinafter also called 37-IL-37), H9.2.1in-P2A-IL-15/IL-15Rα (Sushi) -M7CR (hereinafter also referred to as H9.2.1in-IL-15-M7 CR) (SEQ ID NO: 134), H9.2.1-P2A-sIL-15 (hereinafter also referred to as H9.2.1-sIL-15) (SEQ ID NO: 139), H9.2.1 (CD 28) -P2A-IL-12-P70-M7CR (SEQ ID NO: 133), H9.2.1 (CD 28) -P2A-IL-15/IL15Rα (Sushi) -M7CR (SEQ ID NO: 137), H9.2.1-P2A-IL15Rα (Sushi)/IL-15-M7 CR (SEQ ID NO: 138) proteins.

In the construct, the HuR968B CAR polypeptide (SEQ ID NO: 15) comprises from N-terminus to C-terminus CD8-SP (SEQ ID NO: 1), anti-PG antibody VH (SEQ ID NO: 9), G4S linker (SEQ ID NO: 5), VL (SEQ ID NO: 10), GGGGS hinge, CD8 TMD (SEQ ID NO: 8), 4-1BB CSD (SEQ ID NO: 11) and CD3 ζSSD (SEQ ID NO: 12). The CAR polypeptide is linked to the M7CR molecule by P2A (SEQ ID NO: 3).

In the construct, the H9.1.2-BB-L CAR molecule (SEQ ID NO: 16) comprises from N-terminus to C-terminus a CD8-SP (SEQ ID NO: 1), H9.1.2-VL (SEQ ID NO: 13), (G4S) 3 linker (SEQ ID NO: 4), H9.1.2-VH (SEQ ID NO: 14), CD8 hinge (SEQ ID NO: 7), CD8 TMD (SEQ ID NO: 8), 4-1BB CSD (SEQ ID NO: 11) and CD3 zeta SSD (SEQ ID NO: 12). The CAR polypeptide is linked to the M7CR molecule by P2A (SEQ ID NO: 3).

In the construct, the H9.2.1-BB-L CAR molecule (SEQ ID NO: 100) comprises from N-terminus to C-terminus a CD8-SP (SEQ ID NO: 1), H9.2.1-VL (SEQ ID NO: 98), (G4S) 3 linker (SEQ ID NO: 4), H9.2.1-VH (SEQ ID NO: 99), CD8 hinge (SEQ ID NO: 7), CD8 TMD (SEQ ID NO: 8), 4-1BB CSD (SEQ ID NO: 11) and CD3 zeta SSD (SEQ ID NO: 12). H9.2.1-BB-L CAR' molecules (SEQ ID NO: 144) comprise, from N-terminus to C-terminus, CD8-SP (SEQ ID NO: 1), H9.2.1-VL (SEQ ID NO: 98), linker sequence (SEQ ID NO: 145), H9.2.1-VH (SEQ ID NO: 99), CD8 hinge (SEQ ID NO: 146), CD8 TMD (SEQ ID NO: 147), 4-1BB CSD (SEQ ID NO: 11) and CD3 zeta SSD (SEQ ID NO: 12). The CAR polypeptide is linked to the M7CR molecule by P2A (SEQ ID NO: 3).

In the constructs, the H9.2.1-28-L CAR molecule (SEQ ID NO: 142) comprises, from N-terminus to C-terminus, CD8-SP (SEQ ID NO: 1), H9.2.1-VL (SEQ ID NO: 98), linker sequence (SEQ ID NO: 145), H9.2.1-VH (SEQ ID NO: 99), CD8 hinge (SEQ ID NO: 146), CD8 TMD (SEQ ID NO: 147), CD28 CSD (SEQ ID NO: 143) and CD3 zeta SSD (SEQ ID NO: 12). The CAR polypeptide is linked to the M7CR molecule by P2A (SEQ ID NO: 3).

The H9.1.2-P2A-tCD19-M7CR molecule (SEQ ID NO: 80) comprises H9.1.2-BB-L CAR (SEQ ID NO: 16), P2A (SEQ ID NO: 3) and tCD19-M7CR from N-terminus to C-terminus. The H9.1.2-P2A-tCD19-M7CR (CPT) molecule (SEQ ID NO: 81) comprises H9.1.2-BB-L CAR (SEQ ID NO: 16), P2A (SEQ ID NO: 3) and tCD19-M7CR (CPT) from N-terminus to C-terminus. The H9.1.2-P2A-IL-12-M7CR molecule (SEQ ID NO: 82) comprises H9.1.2-BB-L CAR (SEQ ID NO: 16), P2A (SEQ ID NO: 3) and IL-12-P70-M7CR from N-terminus to C-terminus. The H9.1.2-P2A-IL-15-M7CR molecule (SEQ ID NO: 83) comprises H9.1.2-BB-L CAR (SEQ ID NO: 16), P2A (SEQ ID NO: 3) and IL-15/IL from N-terminus to C-terminus15Rα -M7CR. The H9.1.2-P2A-IL-21-M7CR molecule (SEQ ID NO: 84) comprises H9.1.2-BB-L CAR (SEQ ID NO: 16), P2A (SEQ ID NO: 3) and IL-21-M7CR from N-terminus to C-terminus. The H9.1.2-P2A-CD40L-M7CR molecule (SEQ ID NO: 85) comprises H9.1.2-BB-L CAR (SEQ ID NO: 16), P2A (SEQ ID NO: 3) and CD40L-M7CR from N-terminus to C-terminus. The H9.1.2-P2A-4-1BBL-M7CR molecule (SEQ ID NO: 86) comprises H9.1.2-BB-L CAR (SEQ ID NO: 16), P2A (SEQ ID NO: 3) and 4-1BBL-M7CR from N-terminus to C-terminus. The H9.1.2-P2A-anti-PD-L1 _VHH The M7CR molecule (SEQ ID NO: 87) comprises, from N-terminus to C-terminus, H9.1.2-BB-L CAR (SEQ ID NO: 16), P2A (SEQ ID NO: 3) and anti-PD-L1 _VHH -M7CR。

The 8B-P2A-tCD19-M7CR molecule (SEQ ID NO: 88) comprises from N-terminus to C-terminus a HuR968B CAR (SEQ ID NO: 15), P2A (SEQ ID NO: 3) and tCD19-M7CR. The 8B-P2A-tCD19-M7CR (CPT) molecule (SEQ ID NO: 89) comprises from N-terminus to C-terminus a HuR968B CAR (SEQ ID NO: 15), P2A (SEQ ID NO: 3) and tCD19-M7CR (CPT). The 8B-P2A-IL-15-M7CR molecule (SEQ ID NO: 90) comprises from N-terminus to C-terminus a HuR968B CAR (SEQ ID NO: 15), P2A (SEQ ID NO: 3) and IL-15/IL-15Rα -M7CR. The 8B-P2A-IL-12-M7CR molecule (SEQ ID NO: 91) comprises from N-terminus to C-terminus a HuR968B CAR (SEQ ID NO: 15), P2A (SEQ ID NO: 3) and IL-12-P70-M7CR. The 8B-P2A-IL-21-M7CR molecule (SEQ ID NO: 92) comprises from N-terminus to C-terminus a HuR968B CAR (SEQ ID NO: 15), P2A (SEQ ID NO: 3) and IL-21-M7CR. The 8B-P2A-CD40L-M7CR molecule (SEQ ID NO: 93) comprises from N-terminus to C-terminus a HuR968B CAR (SEQ ID NO: 15), P2A (SEQ ID NO: 3) and CD40L-M7CR. The 8B-P2A-4-1BBL-M7CR molecule (SEQ ID NO: 94) comprises from N-terminus to C-terminus a HuR968B CAR (SEQ ID NO: 15), P2A (SEQ ID NO: 3) and 4-1BBL-M7CR. The 8B-P2A-anti-PD-L1 _VHH The M7CR molecule (SEQ ID NO: 95) comprises from N-terminus to C-terminus a HuR968B CAR (SEQ ID NO: 15), P2A (SEQ ID NO: 3) and an anti-PD-L1 _VHH -M7CR。

In the above-described "H9.1.2" and "8B" related constructs, all M7CR molecules comprise GM-CSFR alpha-SP (SEQ ID NO: 2) from N-terminus to C-terminus, ECD and M7R, optionally with Flag Tag attached between the C-terminus of ECD and N-terminus of M7R, (ILRm 8 shown as SEQ ID NO:34 is exemplified as M7R in this part of the examples below), ECD is selected from tCD19 (SEQ ID NO:17 IL-12-P70 (SEQ ID NO:49 IL-15/IL-15 ra (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). Wherein IL-12-P70-M7CR, IL-15/IL-15Rα -M7CR, IL-21-M7CR further comprises a Flag Tag (SEQ ID NO: 6) between the ECD sequence and the M7R sequence. tCD19-M7CR, IL-12-P70-M7CR (hereinafter also referred to as IL-12-M7 CR), IL-15/IL-15Rα -M7CR (hereinafter also referred to as IL-15-M7 CR), IL-21-M7CR, 4-1BBL-M7CR, CD40L-M7CR, anti-PD-L1 _VHH The M7R portion of the M7CR molecule is IL7Rm8 (SEQ ID NO: 34), tCD19-M7CR (CPT) as control, and the M7R portion is IL7Rm (CPT) (SEQ ID NO: 97) (IL 7Rm (CPT) is the M7R molecule as control). In addition, in the case of anti-PD-L1 _VHH The N-segment of the IL7Rm8 sequence of the M7R portion of the M7CR molecule also includes the "ESKYGPPCPPCP" sequence.

The H9.2.1-P2A-tCD19-M7CR molecule (SEQ ID NO: 101) comprises H9.2.1-BB-L CAR (SEQ ID NO: 100), P2A (SEQ ID NO: 3) and tCD19-M7CR from N-terminus to C-terminus. The H9.2.1-P2A-IL-12-P70-M7CR molecule (SEQ ID NO: 104) comprises H9.2.1-BB-L CAR (SEQ ID NO: 100), P2A (SEQ ID NO: 3) and IL-12-P70-M7CR from N-terminus to C-terminus. The H9.2.1-P2A-IL-12-M7CRin molecule (SEQ ID NO: 102) comprises H9.2.1-BB-L CAR (SEQ ID NO: 100), P2A (SEQ ID NO: 3) and IL-12-P70-M7CRin from N-terminal to C-terminal, wherein IL-12-P70-M7CRin represents the deletion of the intracellular Box1 domain of the IL-7 receptor (amino acids 1060-1071, PIVWPSLPDHKK shown as SEQ ID NO: 132) on the basis of IL-12-P70-M7CR and the simultaneous design of Y1239F, Y1246F (with IL7Rα (P16871-1) as reference) point mutation to inactivate the intracellular M7R signal. The H9.2.1in-P2A-IL-12-P70-M7CR molecule (SEQ ID NO: 103) comprises H9.2.1-BB-L CARin, P2A (SEQ ID NO: 3) and IL-12-P70-M7CR from N-terminus to C-terminus, wherein H9.2.1-BB-LCARin indicates that both domains of the H9.2.1-BB-L CAR (SEQ ID NO: 100) molecule intracellular 4-1BB and CD3 are deleted (to inactivate the CAR molecule intracellular signal) and a "KRGR" sequence is added at the C-terminus. The H9.2.1-P2A-sIL-12 molecule (SEQ ID NO: 105) comprises H9.2.1-BB-L CAR (SEQ ID NO: 100), P2A (SEQ ID NO: 3) and sIL-12 from N-terminus to C-terminus, wherein sIL-12 represents a gene consisting of GM-CSFR alpha-SP (SEQ ID NO: 2), IL-12-P70 (SEQ ID NO: 49) capable of expressing exocrine soluble IL-12.

The H9.2.1-P2A-IL-15/IL-15Rα (Sushi) -M7CR molecule (SEQ ID NO: 136) comprises H9.2.1-BB-L CAR' (SEQ ID NO: 144), P2A (SEQ ID NO: 3) and IL-15/IL-15Rα (Sushi) -M7CR from N-terminus to C-terminus. The H9.2.1-P2A-IL-15/IL-15Rα (Sushi) -M7CRin molecule (SEQ ID NO: 135) comprises H9.2.1-BB-L CAR' (SEQ ID NO: 144), P2A (SEQ ID NO: 3) and IL-15/IL-15Rα (Sushi) -M7CRin from N-terminus to C-terminus, wherein IL-15/IL-15Rα (Sushi) -M7CRin represents the deletion and design of Y1239F, Y1246F point mutation to inactivate the signal of intracellular M7R by deleting the IL-7 receptor intracellular Box1 domain (amino acids 1060-1071 (272-280 (with IL7Rα as reference (16871-1))) in sequence of PIVWPSLPDHKK shown in SEQ ID NO: 132) on the basis of IL-15/IL-15Rα (Sushi) -M7CR. The H9.2.1in-P2A-IL-15/IL-15Rα (Sushi) -M7CR molecule (SEQ ID NO: 134) comprises H9.2.1-BB-L CArin, P2A (SEQ ID NO: 3) and IL-15/IL-15Rα (Sushi) -M7CR from N-terminus to C-terminus, wherein H9.2.1-BB-LCARin indicates deletion of both domains within the H9.2.1-BB-L CAR' (SEQ ID NO: 144) molecule, 4-1BB and CD3, to inactivate the CAR molecule intracellular signal. The H9.2.1-P2A-sIL-15 molecule (SEQ ID NO: 139) comprises H9.2.1-BB-L CAR' (SEQ ID NO: 144), P2A (SEQ ID NO: 3) and sIL-15 from N-terminus to C-terminus, wherein sIL-15 represents a gene consisting of GM-CSFR alpha-SP (SEQ ID NO: 2), IL-15 (SEQ ID NO: 47) capable of expressing exocrine soluble IL-15. The H9.2.1-P2A-IL15Rα (Sushi)/IL-15-M7 CR molecule (SEQ ID NO: 138) comprises H9.2.1-BB-L CAR' (SEQ ID NO: 144), P2A (SEQ ID NO: 3) and IL15Rα (Sushi)/IL-15-M7 CR from N-terminus to C-terminus.

The H9.2.1 (CD 28) -P2A-IL-12-P70-M7CR molecule (SEQ ID NO: 133) comprises H9.2.1-28-L CAR (SEQ ID NO: 142), the sequence "RAKR", P2A (SEQ ID NO: 3) and IL-12-P70-M7CR from N-terminus to C-terminus. The H9.2.1 (CD 28) -P2A-IL-15/IL15Rα (Sushi) -M7CR molecule (SEQ ID NO: 137) comprises H9.2.1-28-L CAR (SEQ ID NO: 142), the sequence "RAKR", P2A (SEQ ID NO: 3) and IL-15/IL15Rα (Sushi) -M7CR from N-terminus to C-terminus.

In the constructs described above in connection with "H9.2.1" and "H9.2.1 (CD 28)", all M7CR molecules comprise GM-CSFR alpha-SP (SEQ ID NO: 2), ECD selected from tCD19 (SEQ ID NO: 17), IL-12-P70 (SEQ ID NO: 49), IL-15/IL-15R alpha (Sushi) (SEQ ID NO: 48), IL-15R alpha (Sushi)/IL-15 (SEQ ID NO: 141) and M7R (ILRm 8 shown as SEQ ID NO:34 is exemplified as M7R in this part of the examples below). the M7R portion of tCD19-M7CR, IL-12-P70-M7CR (hereinafter also referred to as IL-12-M7 CR), IL-15/IL-15Rα (Sushi) -M7CR and IL-15Rα (Sushi)/IL-15-M7 CR (hereinafter sometimes also referred to as IL-15-M7 CR) molecules was IL7Rm8 (SEQ ID NO: 34), tCD19-M7CR (CPT) was used as a control, and the M7R portion was IL7Rm (CPT) (SEQ ID NO: 97) (IL 7Rm (CPT) was the M7R molecule used as a control.

The DNA fragment synthesized above was inserted into the downstream of the EF 1. Alpha. Promoter of pRKN lentiviral expression vector (Jin Weizhi company) to replace EGFR sequence in the original vector, thereby obtaining the corresponding expression plasmid (synthesized by Jin Wei intelligent company).

EXAMPLE 4.2 preparation of lentiviruses

The expression plasmid obtained in example 4.1 was transfected with the structural plasmid pMDLg/pRRE (Addgene, 12251, purchased from biological wind), the regulatory plasmid pRSV-rev (Addgene, 12253, purchased from biological wind) and the envelope plasmid pMD2G (Addgene, 12259, purchased from biological wind) in a mass ratio of 3:3:2:2 by PEI transfection method, and after 16 hours of transfection, fresh DEME medium containing 2% Fetal Bovine Serum (FBS) was replaced, after further 48 hours of culture, cell supernatants were collected, centrifuged to remove cell debris, added PEG8000℃for 16-64 hours of incubation for virus concentration, centrifuged again to remove supernatant, and the virus pellet was resuspended with T cell medium (TexMACs) to obtain lentiviral concentrate, which was frozen at-80℃after sub-packaging. Lenti-X-293T cells (Takara) were digested, resuspended in DMEM medium containing 8. Mu.g/ml Polybrene (Sigma, H9268-5G) and added to 24-well plates, and different volumes of the lentiviral concentrate obtained above were added for 72 hours to conduct transduction of 293T cells. Transduced 293T cells were digested, stained with Biotin-SP-conjugated anti-Human IgG, F (ab') 2-specific (Jackson ImmunoResearch, 109-066-006) and APC-strepitadvidin (BioLegend, 405207), and the proportion of APC positive cells was detected using cytometry. Viral titer (TU/ml) was calculated from the starting cell amount, viral volume and positive cell ratio.

Example 4.3 acquisition of T cells and lentiviral transduction

Recombinant human interleukin-2 (national standard S20040020) for injection was added to TexMACS GMP Medium (Miltenyi Biotec, 170-076-309) to prepare a T cell culture medium having an IL-2 concentration of 200IU/ml. Sorting the resuscitated PBMC using Pan T Cell Isolation Kit (human) (Miltenyi, 130-096-535) on day 0 to obtain T cells, resuspending the cells to a certain density using the T cell medium described above and adding TransAct (Miltenyi, 130-111-160) for activation; on day 1, a certain amount of cells, which are untransduced cells (UNT cells, un-transduced T cells), were isolated, cultured continuously without adding lentivirus, and the remaining cells were added with different kinds of lentivirus concentrates obtained in example 4.2 above at moi=1 to 5 and the cells were blown up uniformly; the virus supernatant was removed by centrifugation on day 2 and the cells were resuspended in fresh T cell medium. UNT cells do not have any manipulation; all cells were transferred to G-Rex (WILSONWOL, cat# 80040S) on day 3, and appropriate amount of fresh T cell culture medium was added and placed at 37℃CO ₂ Standing and culturing in an incubator; half of the cell culture supernatant was discarded every 2-3 days, and an equal volume of fresh IL-2-containing T cell culture medium or an equal volume of fresh T cell culture medium was supplemented. The cells were replaced with fresh medium at half the medium or were directly supplemented with IL-2, wherein IL-2 was added to a concentration of 200IU/ml in the cell culture medium. When the cell number is amplified by about 20-80 times, harvesting the cells after meeting the requirement, centrifuging to remove the culture medium, and adopting CAR-T cells CS10 (Stemcell, 07930) was resuspended and then sub-packaged, and the temperature was programmed to-80 ℃ for cryopreservation.

Example 4.4 detection of CAR expression and phenotypic detection

An appropriate amount of CAR-T cells obtained from example 4.3 above were taken, washed once with FACS buffer (PBS+2% FBS), resuspended, added with LIVE/DEAD Fixable Dead Cell Stain (Thermo, L34963) containing FACS buffer, stained 10-15min at room temperature, washed twice, added with PE-Cy7-CD45RA (Biolegend, 304126), FITC-CCR7 (Biolegend, 353216), BV605-CD4 (BD, 562658), BV421-CD8 (BD, 749366), APC-Antibodies to Strep (Biolegend, 405207) were combined with PE-Flag (Biolegend, 637310), PE-CD40L (Biolegend, 310806), PE-41BBL (Biolegend, 311504), PE-IL15 (R)&D, MAB 247-SP), PE-CD19 (BD, 555413), his-PD-L1 protein and PE-anti-his detect different ECD molecules, wherein anti-PD-L1 _VHH ECD was detected using His-tagged PD-L1 purified protein as a first staining reagent and then PE-anti-His antibody as a second staining reagent. Dyeing for 30-45 min at 4 ℃; after two washes, the cells were resuspended in FACS buffer and examined by flow cytometry.

Using PBMC cells of donor 5, the expression levels of CAR and M7CR at day 9 after CAR-T preparation are shown in figure 9A. In the figure, "H9.1.2" is H9.1.2-BB-L CAR-T cells, the remainder being tCD19-M7CR, tCD19-M7CR (CPT), IL-15/IL-15Rα -M7CR (labeled 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-BB-L CAR-T cells.

As can be seen from fig. 9A, the expression of CAR polypeptide and M7CR was detected by FACS at day 9, the CAR polypeptide was able to be expressed in all CAR-T cells, CAR in H9.1.2 ⁺ The proportion of cells was about 26%, while tCD19-M7CR, tCD19-M7CR (CPT), IL-15-M7CR, IL-12-M7CR, IL-21-M7CR, CD40L-M7CR, 4-1BBL-M7CR and anti-PD-L1 _VHH CAR in the M7CR group ⁺ The ratio of cells was: 12.14%, 11.87%, 6.81%, 6.37%, 18.75%, 12.97%, 11.13%, 10.62%. tCD19-M7CR, tCD19-M7CR (CPT), IL-15-M7CR, IL-12-M7CR, IL-21-M7CR, CD40L-M7CR, 4-1BBL-M7CR and anti-PD-L1 _VHH The M7CR molecules in the M7CR group are all expressed with the expression efficiencies of respectively: 6.6%, 6.81%, 1.43%, 3.59%, 4.95%, 2.97%, 1.03%, 7.45%. It is demonstrated that both CAR polypeptide and M7CR molecules can be expressed, and that the expression of CAR and each M7CR molecule has a certain correlation.

As can be seen in fig. 9B, the proportion of CD4 and CD8 positive cells in the cells expressing H9.1.2CAR was 59.9%,36.3%, respectively; 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 CD4 in the M7CR group ⁺ CellsThe proportions are respectively as follows: 55.9%, 62.5%, 60.2%, 56.1%, 62%, 65.7%, 60.5%, and 46.5%; CD8 ⁺ The cell ratios are respectively as follows: 36.3%, 33.5%, 26.9%, 38%, 31.5%, 29.8%, 32.7% and 48.2%.

Using donor 13 PBMC cells, when the virus infects T cells, as shown in FIG. 9C, the expression levels of CAR and M7CR at day 9 after CAR-T preparation are shown, where "8B" is HuR968B CAR-T cells, the remainder are tCD19-M7CR, tCD19-M7CR (CPT), IL-15/IL-15Rα -M7CR (labeled IL-15-M7CR in the figure), IL-12-M7CR, IL-21-M7CR, CD40L-M7CR, 4-1BBL-M7CR, anti-PD-L1 BBL-T cells _VHH -M7CR modified HuR968B CAR-T cells.

As can be seen from fig. 9C, the expression of CAR polypeptide and M7CR was detected by FACS at day 9, the CAR polypeptide was able to be expressed in all CAR-T cells, CAR in 8B ⁺ The proportion of cells was about 39%, other groups tCD19-M7CR, tCD19-M7CR (CPT), IL-15-M7CR, IL-12-M7CR, IL-21-M7CR, CD40L-M7CR, 4-1BBL-M7CR and anti-PD-L1 _VHH CAR of-M7 CR ⁺ The ratio of cells was: 20.41%, 17.58%, 11.29%, 12.01%, 26.12%, 21.86%, 20.12%, 21.68%. tCD19-M7CR, tCD19-M7CR (CPT), IL-15-M7CR, IL-12-M7CR, IL-21-M7CR, CD40L-M7CR, 4-1BBL-M7CR and anti-PD-L1 in each group _VHH The M7CR molecules are expressed with the following expression efficiencies: 6.6%, 6.81%, 1.43%, 3.59%, 4.95%, 2.97%, 1.03%, 7.45%. It is illustrated that both CAR polypeptide and M7CR molecules are expressed, and that there is a correlation between CAR and expression of each M7CR molecule.

As can be seen from fig. 9D, the proportion of CD4 and CD8 positive cells in 8B CAR expressing cells was 33.2%,61.6%, respectively; 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 CD4 in the M7CR group ⁺ The cell ratios are respectively as follows: 31.1%, 32.4%, 29.8%, 38.2%, 32.4%, 32.2%, and 32.2%; CD8 ⁺ The cell ratios are respectively as follows: 64.3%, 63.2%, 65.1%, 55.7%, 63.5%, 63.3%, 63.5% and 61.1%.

H9.1.2-BB-L CAR-T and M7CR repair were testedTotal T cells, CD4 in decorated H9.1.2-BB-L CAR-T samples ⁺ And CD8 ⁺ T phenotype, the results are shown in FIG. 9E. Representative flow cytometry shows the expression of different CAR-T cells CD45RA, CCR7, CD45RA ⁺ CCR7 ⁺ Representing primary T cells or stem memory T cells (TN/TSCM), CD45RA-CCR7 ⁺ Representing central memory T Cells (TCM), CD45RA ^- CCR7 ^- Representing effector memory T cells (TEM), CD45RA ⁺ CCR7 ^- Representing a subset of effector T cells (Teff), the majority of CAR-T cells are TN/TSCM, TCM cells. The phenotype of H9.1.2 was not significantly altered compared to UNT.

IL12-M7CR modified H9.1.2-BB-L CAR-T cell sample Total T cells, CD4, compared to unmodified H9.1.2-BB-L CAR-T ⁺ And CD8 ⁺ T cells differentiated significantly, the proportion of TCM and TN subpopulations decreased, the proportion of TEM and Teff subpopulations increased, and the T cell phenotype did not differentiate significantly in the other groups.

Detection of total T cells, CD4, in unmodified HuR968B CAR-T and M7CR modified HuR968B CAR-T samples ⁺ And CD8 ⁺ T phenotype, the results are shown in FIG. 9F. In comparison to unmodified HuR968B CAR, IL12-M7CR modified HuR968B CAR-T cell samples total T cells, CD4 ⁺ And CD8 ⁺ T cells differentiated significantly, the proportion of TCM and TN subpopulations decreased, the proportion of TEM and Teff subpopulations increased, and the T cell phenotype did not differentiate significantly in the other groups. Further detection of total T cells, CD4, in H9.2.1-BB-L CAR ' -T (wherein H9.2.1-BB-L CAR ' has the sequence shown in SEQ ID NO: 144) and M7CR modified H9.2.1-BB-L CAR ' -T samples ⁺ And CD8 ⁺ T phenotype, the results are shown in FIGS. 9G and 9H. In both donors, total T cells, CD4, in IL-15-M7CR, sIL-15 or IL-15-M7CRin modified H9.2.1 CAR '-T cell samples compared to unmodified H9.2.1 CAR' -T were found ⁺ And CD8 ⁺ The proportion of T cell TCM and TN subpopulations increases; whereas IL-12-M7CR modified CAR-T cell samples in total T cells, CD4 ⁺ And CD8 ⁺ T cells partially differentiated, the proportion of TCM and TN sub-populations decreased, and the proportion of TEM and Teff sub-populations increased. Elucidation of different ECD structures on CAR-T cellsThe phenotype can have different effects.

Example 4.5CAR-T cell STAT5 Signal activation

The activation of STAT5 was studied by FACS detection of intracellular p-STAT5 expression levels. The experimental procedure was as follows, T cells of 1E6 were taken, washed once with PBS and resuspended in serum-free RPMI1640 medium overnight. The next day, UNT cells will be stimulated with IL-2 (200 UI/mL) for 20min before FACS buffer wash once. Then, an AF647-p-STAT5 (BD, 562076) antibody was added to conduct intracellular staining, and the procedure was the same as in example 1.5. As shown in fig. 9I and 9J, UNT increased p-STAT5 levels under stimulation with IL-2, and neither car+ nor CAR-, p-STAT5 was increased in H9.2.1CAR-T cells. Whereas in the car+ cells expressing M7R, the average expression level of p-STAT5 was increased, whereas in H9.2.1-IL-12-M7CRin, in which the inactivating mutation was performed on M7R, the expression level of p-STAT5 was not increased. The above results indicate that M7R is able to continue to provide an activation signal in CAR-T cells, activating downstream signaling pathways.

Example 5 quantitative analysis of cell surface antigen molecular number by Qufikit:

with saturated concentrations of mouse anti-human CLDN18 antibodies and cell lines (SNU-601 ^high ，SNU-601 ^low Incubation with the DAN-G18.2 cell line purchased from Nanjac Bai Biotech, followed by quantitative measurement of the number of antigens of CLDN18 on the cell surface by the qufikit kit (Agilent company).

Specifically, DAN-G18.2 and SNU-601 cells (1E 5) were taken in 96-well V-plates, and supernatants were discarded. The FACS buffer was then added for resuspension washing, and the supernatant was removed by centrifugation again. A saturated mouse anti-human CLDN18.2 mix was prepared using FACS buffer and 100. Mu.L of the mix was added to the wells. mu.L of FACS buffer was added to the wells. Incubate at 4℃for 30min in the dark. Centrifuge at 300g for 5min, discard supernatant. 40ul beads (Vial 1 and Vial 2) after shaking and mixing are added to the wells of the standard in the qufikit respectively, and the supernatant is discarded after centrifugation at 300g for 5 min. Preparation of secondary antibodies (Alexa) using FACS buffer488AffiniPure coat anti-Mouse IgG, F (ab') 2fragment specific, 1:100), 100 μl of secondary antibody dye was added to each well and incubated at 4deg.C for 30min in the absence of light. The supernatant was discarded by resuspension using FACS buffer, centrifugation at 300g for 5 min. The FACS buffer was added at 150 μl for resuspension and FACS analysis was performed on the machine.

As shown in FIG. 10A, SNU-601 ^high Cells, SNU-601 ^low The cells and DAN-G18.2 cell surface expressed amounts of CLDN18 were varied, with the expression level of DAN-G18.2 being highest.

TABLE 2

Ability to bind specific antibodies	DAN-G18.2 cells	SNU-601 cells
			High expression	655891	87427
Low expression	—	24360

As shown in Table 2, the cell surface molecular numbers, SNU-601high, SNU-601low, and DANG-18.2high surface CLDN18 molecular numbers were calculated by quantitative analysis of Qufikit, respectively: 87427. 24360 and 655891.

The CLDN18.2 on the surface of different target cells was labeled with antibodies, and then the expression level of CLDN18.2 on the surface of target cells was detected by flow cytometry. Specifically, A6 antibody was used to incubate target cells for 30min at 4 ℃; then after washing once with FACS buffer, the cells were incubated with APC-labeled anti-human Fc antibodies for 30min at 4 ℃; finally, after washing once with FACS buffer, the sample was checked on the machine. As shown in fig. 10B, the peak chart shows the expression amount of CLDN18.2 in each cell, and the expression amount was quantified by calculating MFI. Wherein, the expression level of CLDN18.2 in the DANG18.2 and NUGC-4 cells is higher, SNU-601 and Hup-T4 are in medium expression, SNU-620 and PANC-1 are in low expression, ISO is isotype antibody control, and K562 is CLDN18.2 negative control cell.

Example 6 in vitro dynamic killing experiments

The killing of the target cells by CAR-T cells was detected dynamically in real time using a xCELLigence RTCA MP instrument (Agilent company). After 50. Mu.L of medium was added to the E-Plates and the instrument read the baseline values, 50. Mu.L of tumor target cells were added and then placed in the machine for dynamic monitoring of cell growth. Resuscitates the UNT cells prepared in example 4.3 and CAR-T cells (T cells, PBMC cells from donor 5, donor 13) were placed in a 37 ℃ cell incubator overnight. The next day, adding CAR-T into corresponding E-Plates holes according to the ratio of E to T according to the experimental requirement, simultaneously adding a P329G mutation A6 antibody into the holes corresponding to HuR968B CAR-T cells and M7CR modified HuR968B CAR-T cells, combining a tumor target cell by utilizing the VH/VL domain of the P329G mutation A6 antibody and combining an Fc end P329G mutation with an extracellular binding region of the HuR968B CAR-T cells, thereby activating the targeted tumor killing function of the HuR968B CAR-T cells or the M7CR modified HuR968B CAR-T cells. xCELLigence RTCA MP the instrument system dynamically monitors the killing of the target cells by the CAR-T cells for 48-96 hours.

As shown in FIG. 11A, H9.1.2-BB-L CAR-T cells modified with H9.1.2-BB-L CAR-T cells or different M7 CRs, respectively, were incubated with tumor target cells DAN-G18.2, and killing by tCD19-M7CR modified H9.1.2-BB-L CAR-T cells was comparable and better than H9.1.2-BB-L CAR-T cells at E:1, 1:3, 1:10, respectively, and killing by CAR-T cells in the other groups was substantially comparable to H9.1.2-BB-L CAR-T cells.

As shown in FIG. 11B, 4-1BBL-M7CR modified H9.1.2-BB-L CAR-T cells killed more than tCD19-M7CR modified H9.1.2-BB-L CAR-T cells at E:T of 1:1, 1:3, 1:10, respectively.

As in figure 11C, when E:T is 1:1, 1:3, 1:10 respectively, anti-PD-L1 _VHH The killing effect of both M7CR modified H9.1.2-BB-L CAR-T cells was stronger than that of tCD19-M7CR modified H9.1.2-BB-L CAR-T cells and unmodified H9.1.2-BB-L CAR-T cells, and this advantage was more pronounced with decreasing E:T ratio. Description of anti-PD-L1 _VHH And M7R are combined to modify, so that the modified H9.1.2-BB-L CAR-T cells have synergistic effect on killing target cells.

As shown in FIG. 11D, huR968B CAR-T cells and A6 antibodies (2 nM) modified with HuR968B CAR-T cells and M7CR and target cell SUN-601 ^high Or SUN-601 ^low Co-incubation (E: t=1). IL-12-M7CR modified HuR968B CAR-T cell pair SNU-601 ^high And SNU-601 ^low Is better than unmodified HuR968B CAR-T, tCD19-M7CR modified HuR968B CAR-T cells and other M7CR modified HuR968B CAR-T cells, and the effect is in SNU-601 ^low More pronounced. In addition, IL-15/IL-15Rα -M7CR (labeled IL-15-M7CR in FIG. 11D) modified HuR968B CAR-T cell pair SNU-601 ^low Slightly better killing than unmodified HuR968B CAR-T cells and tCD19-M7CR modified HuR968B CAR-T cells.

Example 7 in vitro tumor cell repeated stimulation experiments

SNU-601 on day 0 ^high Cells (2.5E5) were added to 24-well plates and allowed to adhere overnight while resuscitating the different sets of HuR968B CAR-T cells prepared in example 4.3 (using PBMC cells of donor 13). The CAR positive rate of CAR-T cells was adjusted and made uniform on day 1, then different groups of HuR968B CAR-T cells were added to each well, respectively, at E: t=2, and P329G mutant A6 antibody was added to a final concentration of 2nM. On day 2, 100 μl of supernatant was collected for cytokine detection experiments. Half-volume changes were made to each group on day 3, day 5. SNU-601 will be on day 7 ^high Cells (2X 10) ⁵ ) A new 24-well plate (noted as day 0 of the next round of stimulation) was added, the proportion and phenotype of CAR-T cells in each group was examined by flow cytometry, and the T cell number was examined by a cytometer. 5E5 CAR-T cells were removed on day 8 and added to a new 24-well plate (noted as day 1 of the next round of stimulation). After each round of stimulation, increaseFold proliferation (CAR-T cells) =car-T cell number (day 7)/5E 5, cumulative fold proliferation=fold proliferation Round1 x fold proliferation Round2 x ….

As shown in FIG. 12A, huR968B CAR-T cells in all groups proliferated under stimulation of target cells for the first 7 days, and from day 7 to day 21, both IL-15/IL-15Rα -M7CR (labeled IL-15-M7CR in FIG. 12A) and HuR968B CAR-T cells in the IL-12-M7CR modified group continued to proliferate at a higher fold than the unmodified HuR968B CAR-T cell group and tCD19-M7CR modified HuR968BCAR-T cells. Wherein the CAR-T cells in the IL-12-M7CR modified group had the highest cumulative fold expansion (about 44 fold) after 21 days of stimulation. The above demonstrates that IL-12-M7CR modification can increase the proliferative capacity of CAR-T cells upon repeated stimulation of target cells.

As shown in fig. 12B, CARs in HuR968B CAR-T cells at day 14 after multiple rounds of target cell stimulation ⁺ The cell proportion was 53.3% and slightly increased as compared to day 1. Whereas modified HuR968B CAR-T cells CAR ⁺ The proportion of cells increased significantly, and CARs in the group modified by tCD19-M7CR, tCD19-M7CR (CPT), IL15-M7CR, IL-12-M7CR and IL-21-M7CR at day 21 ⁺ The cell ratios are respectively: 76.5%, 78.6%, 90.1%, 79.52% and 51.39%.

As shown in fig. 12C and 12D, CAR-T cells modified with 4-1BBL-M7CR, CD40L-M7CR, and tCD19-M7CR proliferated significantly higher than HuR968B CAR-T cells at day 7 after target cell stimulation. From day 7 to day 14, the proliferative capacity of CAR-T cells began to decline in all groups after target cell stimulation.

As shown in FIGS. 12E and 12F, anti-PD-L1 after multiple rounds of stimulation by target cells _VHH -M7CR modified HuR968B CAR-T cells proliferate slightly higher than tCD19-M7CR modified HuR968B CAR-T cells and unmodified HuR968B CAR-T cells. CAR in HuR968B CAR-T cells at day 14 ⁺ The ratio was 53.3%, which was slightly higher than that at day 1. And passes through tCD19-M7CR, CD40L-M7CR, 4-1BBl-M7CR and anti-PD-L1 _VHH -CAR in M7CR modified HuR968B CAR-T cells ⁺ The ratio rose significantly, CAR by day 14 ⁺ The proportions of (2) are respectively as follows: 77.4%, 71.8%, 66.3%, and,75.1％。

FIG. 13A shows CD4 in each group after the first and third rounds of stimulation of the target cells of FIGS. 12A-12F ⁺ And CD8 ⁺ Representative flow cytometry assay results for T cell numbers. As shown in fig. 13A, CD4 in each group at the start of the experiment ⁺ And CD8 ⁺ The ratio of T cells was maintained at substantially 1:2. After multiple experiments, IL-12-M7CR modification can improve CD4 ⁺ And CD8 ⁺ T cell ratio (about 1:1), while CD8 in the other groups ⁺ T cell ratio was significantly increased, CD4 ⁺ The T cell fraction was significantly reduced. It was shown that IL-12-M7CR modification promotes CD4 upon stimulation of target cells ⁺ Expansion of T cells.

FIG. 13B shows the comparison of CD4 in each group of FIG. 13A ⁺ And CD8 ⁺ Statistics of the proportion of T cells. As shown in the figure, IL-12-M7CR modification can increase CD4 after multiple rounds of stimulation ⁺ And CD8 ⁺ T cell ratio to 1:1 around, and CD8 in other groups ⁺ T cell ratio was significantly increased, CD4 ⁺ The T cell fraction was significantly reduced.

Example 8 cytokine detection assay

Using BD ^TM Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit II. Equal volumes of Capture Beads were mixed and plated at 25. Mu.L/well. Equal volumes of in vitro tumor cells are added to repeatedly stimulate the supernatant or supernatant dilutions or standards in the experiment. After mixing, 25. Mu.L of an equal volume of human Th1/Th2 PE detection reagent was added and incubated at room temperature for 3h in the dark. After washing twice with wash buffer, the cytokine concentration was calculated by flow cytometry PE channel MFI values.

As shown in FIG. 13C, the levels of IL-2, IFN-gamma and TNF cytokines in the supernatants 24h after effector cell addition at round 1 stimulation were detected by CBA. IL-2 levels were found to be minimal in supernatants of IL-15/IL-15Rα -M7CR (labeled IL-15-M7CR in FIG. 13C) modified group (< 2000 pg/mL), IL-12-M7CR modified and tCD19-M7CR (CPT) modified group (2000-4000 pg/mL) times, unmodified HuR968B CAR group, tCD19-M7CR, IL-21-M7CR, CD40L-M7CR, 4-1BBl-M7CR and αPD-L1VHH-M7CR modified HuR968B CAR group (> 6000 pg/mL) were higher. IFN-gamma levels were highest in supernatants of IL-12-M7CR modified groups (> 10000 pg/mL) and below 5000pg/mL in supernatants of other groups. IL-12-M7CR modified group (> 1000 pg/mL) supernatant levels of TNF were highest, with other groups of supernatants having lower levels of TNF.

Example 9 in vitro killing of IL-12-M7CR/IL-15-M7CR modified H9.2.1-BB-L CAR-T cells

Further, in vitro killing experiments were performed against target cells with different levels of Claudin18.2 expression, and the killing effect of IL-12-M7CR/IL-15-M7CR modified H9.2.1 CAR-T on target cells in vitro was investigated.

The killing of the target cells by CAR-T cells was detected dynamically in real time using a xCELLigence RTCA MP instrument (Agilent company). The experimental procedure is as described previously. Cell lines with different expression levels of CLDN18.2 were selected as target cells. Wherein PANC-1 is a CLDN18.2 low expression cell line, SNU-601 and Hup-T4 are medium and high expression cell lines, and DAN-G18.2 is a high expression cell line. CAR-T cells were prepared using PBMCs from donor 15 and donor 17. Wherein H9.2.1-IL12-M7CR represents IL-12-P70 modified H9.2.1-BB-L CAR-T cells; H9.2.1in-IL12-M7CR indicates that the 4-1BB costimulatory domain and the CD3 zeta signaling domain in H9.2.1-BB-L CAR-T are deleted, thereby achieving the purpose of CAR structure function deletion; H9.2.1-IL12-M7CRin shows that the domain of the intracellular Box1 of the M7CR is deleted, and mutations Y449F and Y456F (taking IL7 Ralpha (P16871-1) as a reference) are introduced to further achieve the aim of inactivating the intracellular structural function of the M7 CR; H9.2.1-sIL12 represents a combination of H9.2.1-BB-L CAR-T cells and soluble IL 12. As shown in fig. 14A-14D, killing of CAR-T cells in target cells with different expression levels of CLDN18.2 increased with increasing expression level of CLDN18.2, demonstrating that killing of H9.2.1-BB-L CAR-T cells was antigen-dependent. The killing capacity of the CAR-T cells is from low to high in different target ratios, namely the H9.2.1in-IL-12-M7CR CAR-T cells, the H9.2.1-BB-L CAR-T cells are sequentially arranged, the tCD19-M7CR modified H9.2.1-BB-L CAR-T cells are equivalent to H9.2.1-IL-12-M7CRin CAR-T cells and H9.2.1-sIL12 CAR-T cells, and the IL-12-M7CR modified H9.2.1-BB-L CAR-T cells have the strongest killing effect. It was demonstrated that IL-12-P70 and M7R have a combined effect in promoting killing of CAR-T cells.

Similarly, repeated killing experiments on target cells Hup-T4 were performed using H9.2.1-BB-L CAR-T cells.

The killing of target cells Hup-T4 by CAR-T cells was examined using an Xcelligence instrument. The specific experimental procedure is as follows, day0 targets Hup-T4 (4X 10) ⁴ Individual/well) was added to Eplate and placed in an xcelligent instrument overnight. After the Cell index increased to about 1, experiments were performed. The Cell index is the read out of the xcelligent instrument, and for killing experiments using this model instrument, the Cell index value is used to represent the amount of living cells as a universal standard. According to E: t=1: 1 and E: t=1: 5, different groups of CAR-T cells were added to the wells, lysis buffer was added to the PC group, and T cells from the same donor, which were not transfected with CAR, were added to the UNT group. When the cell index stability is no longer decreasing, the killing of this round is ended. Eplate is then removed, the supernatant removed by centrifugation, the cells resuspended in fresh RPMI 1640 complete medium, and added to the Eplate containing target cells plated the day before for the second round of experiments. Three experiments were performed in total. As shown in fig. 14E, after three continuous rounds of killing experiments, IL-12-M7CR modified CAR-T cells still had better killing effect, while unmodified H9.2.1 CAR-T cells gradually attenuated with increasing number of rounds of killing in multiple rounds of killing. The results demonstrate that IL-12-M7CR molecules are capable of increasing the killing capacity of H9.2.1 CAR-T cells, as well as the effect of sustained killing, in repeated killing experiments.

Example 10 anti-tumor effects of CAR-T cells expressing constitutive chimeric cytokine receptor in mice.

Through animal experiments, the anti-tumor effect of the CAR-T cells expressing the constitutive chimeric cytokine receptor in mice is studied, and whether the IL-12-M7CR molecules can promote the anti-tumor effect and proliferation capacity of the PG CAR-T cells in the mice is detected. The experimental method is specifically as follows, NOG mice (purchased from Vetong Liwa) are selected, and Day-7 is given intraperitoneal injection of NUGC-4-Gluc cells (1X 10 each) ⁶ ) Modeling, detecting the modeling condition of mice by a living animal imaging system, and determining the tumor load to be 1×10 when Day0 ⁹ p/s (for tumors in IVIS imaging systemsAnimals were grouped (5 per group) when producing calculated values and units of photon numbers. Each mouse was then given 5×10 by tail vein injection ⁵ The number of UNT cells given to the UNT group was consistent with the total number of T cells infused by the group of mice with the lowest CAR positive rate. Mouse load was imaged weekly, number of CAR-T cells in peripheral blood.

As shown in fig. 15 and 16, the M7 CR-expressing CAR-T cells constructed based on PG CAR-T cells (HuR 968B CAR-T cells) were injected into the model mice via tail vein injection while 0.3mg/kg of P329G containing mutant A6 antibody was injected, and as a result of in vivo imaging of small animals, it was found that the PG CAR-T cells modified by IL-12-M7CR and tCD19-M7CR had better antitumor effect in vivo with time, and as shown in fig. 17, the PG CAR-T cells had higher expansion level from day 7 to day 28, while the unmodified PG CAR-T cells had weaker antitumor effect and poorer expansion ability in vivo. The above in vivo results demonstrate that M7CR modification has the effects of promoting expansion of PG CAR-T cells and enhancing anti-tumor effect of CAR-T cells in vivo.

As shown in fig. 18, CAR-T cells expressing M7CR constructed based on conventional CAR-T cells (H9.2.1-BB-L CAR-T cells, hereinafter exemplified by H9.2.1-BB-L CAR sequences as shown in SEQ ID No. 100) were injected into the model mice via tail vein, and the IL-12-M7CR and tCD19-M7CR modified CAR-T cells were found to have better antitumor effect in vivo by in vivo imaging of small animals, and at Day13, the IL-12-M7CR modified CAR-T cells were able to completely eliminate tumors in the model mice. As shown in fig. 19, which is a quantitative statistic of IVIS images, H9.2.1 CAR-T cells, M7R H9.2.1 CAR-T cells, and IL-12-M7CR CAR-T cells were found to have better antitumor effects in model mice than the UNT group. Wherein, the M7R modification can improve the anti-tumor effect of H9.2.1 CAR-T cells, and the IL-12 modification can further improve the anti-tumor effect of M7R H9.2.1 CAR-T cells. As shown in fig. 20, the numbers of total human T cells and CAR-T cells in the peripheral blood of mice were detected by flow cytometry, and after one injection of CAR-T cells, the numbers of total T cells and CAR-T cells increased with time in the other groups except for the UNT group, wherein the number of CAR-T cells expanded was the greatest in the IL-12-M7CR group, the M7R group was the next, and the H9.2.1 group was the last. The in vivo results show that the M7CR modification has the effects of promoting the expansion of traditional CAR-T cells and improving the anti-tumor effect of the CAR T cells in vivo.

While exemplary embodiments of the invention have been described above, it will be appreciated by those skilled in the art that these disclosures are exemplary only, and that various other substitutions, adaptations, and modifications may be made within the scope of the invention. Therefore, the present invention is not limited to the specific embodiments set forth herein.

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Claims (22)

1. A constitutive chimeric cytokine receptor comprising an extracellular domain consisting of an effector molecule having the ability to remodel the tumor microenvironment, e.g., selected from a cytokine, an immune effector molecule, an inhibitory molecular antagonist, or an effector molecule targeting an NK cell activating receptor, and a constitutively activated IL-7R mutant; the constitutively active IL-7R mutants comprise an IL-7R mutant transmembrane domain and an IL-7R intracellular domain.

2. The constitutive chimeric cytokine receptor according to claim 1, wherein the constitutive activated IL-7R mutant comprises any amino acid sequence selected from the group consisting of SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 28, SEQ ID No. 30 to SEQ ID No. 45, preferably the constitutive activated IL-7R mutant comprises any amino acid sequence selected from the group consisting of SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 33 and SEQ ID No. 34, most preferably the constitutive activated IL-7R mutant comprises the amino acid sequence shown in SEQ ID No. 34.

3. The constitutive chimeric cytokine receptor according to claim 1 or 2, wherein the cytokine that is an extracellular domain is selected from any of IL-12 (e.g., IL-12p40 or IL-12p 70), IL-15 (e.g., IL-15 or IL-15FP, wherein the IL-15FP is a fusion protein of IL-15 and IL-15 ra, whereby the IL-15FP comprises a fusion protein of IL-15/IL-15 ra and IL-15 ra/IL-15 two forms, the IL-15 ra being selected from IL-15 ra or IL-15 ra (Sushi)), IL-21, IL-18, IL-9, IL-23, IL-36 γ and ifnα2b; the immune effector molecule as an extracellular domain is selected from any of a 4-1BB targeting molecule moiety (e.g., 4-1BB ligand, anti-4-1 BB antibody), a CD40 targeting molecule moiety (e.g., CD40 ligand, anti-CD 40 antibody), a CD83 targeting molecule moiety (e.g., anti-CD 83 antibody), a FLT3 targeting molecule moiety (e.g., FLT3 ligand (FTL 3L), anti-FLT 3 antibody (αflt3)), GITR, ICOS, CD2, and ICAM-1; the inhibitory molecular antagonist as an extracellular domain is selected from any one of an anti-PD-L1 molecule, an anti-CD 47 molecule, an anti-IL-4 molecule, an anti-TGF-beta molecule, an anti-PD-1 molecule, an anti-CTLA-4 molecule, an anti-LAG-3 molecule, an anti-TIGIT molecule and an anti-CD 73 molecule; the effector molecule targeting the NK cell activating receptor as extracellular domain is selected from molecules targeting the NK cell activating receptors NKG2C, NKG2D, NKp, NKp44 and NKp46, e.g. anti-NKG 2C, anti-NKG 2D, anti-NKp 30, anti-NKp 44, anti-NKp 46, by activating endogenous NK cells, an enhanced anti-tumor immune effect is obtained.

4. The constitutive chimeric cytokine receptor according to claim 1 or 2, wherein the extracellular domain is selected from IL-12 (e.g., IL-12p40 or IL-12p 70), IL-15 (e.g., IL-15 or IL-15FP, wherein the IL-15FP is a fusion protein of IL-15 and IL-15 ra, whereby the IL-15FP comprises a fusion protein of IL-15/IL-15 ra and IL-15 ra/IL-15, the IL-15 ra being selected from IL-15 ra or IL-15 ra (Sushi)), IL-21, 4-1BB ligand, CD40 ligand or anti-PD-L1 nanobody.

5. A nucleic acid molecule encoding the constitutive chimeric cytokine receptor of any one of claims 1 to 4.

6. A vector comprising the nucleic acid molecule of claim 5, e.g., the vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector.

7. A cell comprising the constitutive chimeric cytokine receptor of any one of claims 1 to 4, the nucleic acid molecule of claim 5, or the vector of claim 6, said cell being, for example, an immune effector cell, e.g., a T cell, an NK cell, e.g., said T cell is an autologous T cell or an allogeneic T cell, e.g., said immune effector cell is prepared after isolation of T cells, NK cells from human PBMCs.

8. Pharmaceutical composition comprising

An immune effector cell (e.g., T cell, NK cell) selected from the group consisting of a constitutive chimeric cytokine receptor of any one of claims 1 to 4, a nucleic acid molecule encoding a constitutive chimeric cytokine receptor of any one of claims 1 to 4, a vector of claim 6, and any combination thereof; and

optionally pharmaceutically acceptable excipients;

for example, the immune effector cell is a T cell expressing the constitutive chimeric cytokine receptor of any one of claims 1 to 4 prepared from autologous T cells or allogeneic T cells, e.g., the immune effector cell is a T cell expressing the constitutive chimeric cytokine receptor of any one of claims 1 to 4 prepared from T cells isolated from human PBMCs.

9. Use of a pharmaceutical composition according to claim 8 for enhancing an anti-tumor immune effect, e.g. activating, proliferating, surviving and exerting immune effector functions, in a subject.

10. Use of a pharmaceutical composition according to claim 8 for the preparation of a medicament for the treatment of tumors.

11. A constitutive chimeric cytokine receptor-modified CAR polypeptide or TCR polypeptide of any one of claims 1-4, comprising the constitutive chimeric cytokine receptor of any one of claims 1-4 at the N-terminus or C-terminus of the CAR polypeptide or TCR polypeptide, and having a self-cleaving peptide or IRES sequence between the constitutive chimeric cytokine receptor and the CAR polypeptide or TCR polypeptide, e.g., the self-cleaving peptide is a 2A self-cleaving peptide from an oro-hoof virus or cardiovirus, e.g., P2A as set forth in SEQ ID NO: 3.

12. The constitutive chimeric cytokine receptor-modified CAR polypeptide according to claim 11, which CAR polypeptide targets one or more cancer-associated antigens directly or by "molecular switching", e.g. a CAR polypeptide that targets a cancer-associated antigen directly comprises a signal peptide, an antigen binding domain, a transmembrane domain, an intracellular signal domain from the N-terminus to the C-terminus; a CAR polypeptide that targets a cancer-associated antigen via a "molecular switch" comprises, from N-terminus to C-terminus, a signal peptide, a P329G mutation binding domain, a transmembrane domain, an intracellular signal domain, which CAR polypeptide binds to the P329G mutation of the "molecular switch" and then targets the cancer-associated antigen via the "molecular switch".

13. A constitutive chimeric cytokine receptor-modified CAR polypeptide according to claim 11 or 12 which is directly targeted to or targeted by "molecular switch" to the cancer-associated antigen CLDN18.2.

14. The constitutive chimeric cytokine receptor-modified CAR polypeptide according to claim 13, the constitutive chimeric cytokine receptor selected from IL-12 (e.g., IL-12p40 or IL-12p 70), IL-15 (e.g., IL-15 or IL-15FP, wherein the IL-15FP is a fusion protein of IL-15 and IL-15 ra, whereby the IL-15FP comprises IL-15/IL-15 ra and a fusion protein of two forms of IL-15 ra/IL-15, the IL-15 ra selected from IL-15 ra or IL-15 ra (Sushi)); the CAR polypeptide targets the cancer-associated antigen CLDN18.2 directly or through a "molecular switch" (e.g., a CAR polypeptide that targets CLDN18.2 directly comprises a signal peptide, CLDN18.2 antigen binding domain, transmembrane domain, intracellular signaling domain from N-terminus to C-terminus; a CAR polypeptide that targets CLDN18.2 through a "molecular switch" comprises a signal peptide, P329G mutation binding domain, transmembrane domain, intracellular signaling domain from N-terminus to C-terminus, that binds to the P329G mutation of a "molecular switch", which in turn targets the cancer-associated antigen CLDN18.2 through a "molecular switch").

15. A nucleic acid molecule encoding the constitutive chimeric cytokine receptor-modified CAR polypeptide or TCR polypeptide of any one of claims 11-14.

16. A vector comprising the nucleic acid molecule of claim 15, e.g., the vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector.

17. A cell that expresses (1) a CAR polypeptide or a TCR polypeptide; and (2) the constitutive chimeric cytokine receptor of any one of claims 1 to 4;

for example, comprising the constitutive chimeric cytokine receptor-modified CAR polypeptide or TCR polypeptide of any one of claims 11-14, the nucleic acid molecule of claim 15, or the vector of claim 16; alternatively, it comprises a nucleic acid construct of the nucleic acid molecule of claim 5 and a nucleic acid construct expressing a CAR polypeptide or TCR polypeptide;

the cells are, for example, immune effector cells, e.g., T cells, NK cells, e.g., autologous T cells or allogeneic T cells, e.g., prepared after isolation of T cells, NK cells from human PBMCs.

18. Pharmaceutical composition comprising

Selected from (1) the cell of claim 17; (2) A nucleic acid molecule encoding the constitutive chimeric cytokine receptor-modified CAR polypeptide of claim 15; (3) the vector of claim 16; (4) A nucleic acid construct of the nucleic acid molecule of claim 5 and a nucleic acid construct expressing a CAR polypeptide or TCR polypeptide; and (5) any combination of the (1) to (4); and optionally pharmaceutically acceptable excipients;

for example, the cells are prepared from autologous T cells or allogeneic T cells, e.g., the cells are prepared from T cells isolated from human PBMCs.

19. The pharmaceutical composition of claim 18, wherein when the CAR polypeptide is a molecular switch-regulated CAR polypeptide, the pharmaceutical composition further comprises a molecular switch.

20. Use of the pharmaceutical composition of claim 18 or 19 for treating a tumor in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 18 or 19.

21. Use of a pharmaceutical composition according to claim 18 or 19 in the manufacture of a medicament for the treatment of a tumor.

22. A method of treating a tumor, the method comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition of any one of claims 8, 18 and 19.