Chimeric polypeptides that regulate the physiological activity of cells

Abstract

A chimeric polypeptide comprising a binding peptide capable of specifically binding to a target molecule, a receptor modulating domain comprising one or more cleavage sites, and an intracellular domain, wherein the receptor modulating domain comprises an extracellular region and a transmembrane region, wherein the extracellular region and the transmembrane region are not both derived from a Notch protein, and binding of the binding peptide to the target molecule can induce cleavage of the receptor modulating domain, thereby releasing the intracellular domain.

Description

Priority information

[0001] This application claims priority to Chinese patent application CN202110730083.X, filed on June 29, 2021, Chinese patent application CN202111056553.5, filed on September 9, 2021, and Chinese patent application CN202210111505.X, filed on January 29, 2022, the entire contents of which are incorporated herein by reference in their entirety. Concurrent submission of sequence listing

[0002] The following sequence listing in computer-readable form (CRF) in ASCII text format (file name: FF00617PCT-sequence listing-20220629-yzg.txt, date: June 29, 2022, size: 232 KB) is incorporated herein by reference in its entirety. [Technical field]

[0003] The present application relates to chimeric polypeptides capable of modulating the physiological activity of cells, and cells expressing the chimeric polypeptides. [Background technology]

[0004] Although there are many methods to control physiological activities such as gene expression and cell differentiation in vitro, it remains technically difficult to achieve spatially specific control of cells in vivo. For example, some immune cells can activate cells and exert strong antitumor effects when exogenously expressed with antitumor molecules (CAR, cytokines, etc.), but it also brings about potential risks of off-target toxicity and functional impairment due to long-term activation of cells.

[0005] Currently, there exists a first-generation synthetic derivative of the Notch receptor, synNotch, which is composed of a ligand-binding domain, a Notch core regulatory region, and an intracellular signaling region. The principle is to replace the intracellular region of the natural Notch molecule with an artificial signaling molecule (such as a specific transcription factor) by utilizing the characteristic that shear occurs extracellularly and intracellularly after the natural Notch molecule binds to a target molecule (ligand), thereby basically realizing ligand-dependent gene expression control.

[0006] However, this technology still suffers from many problems, including background leakage of synNotch, i.e., weak gene expression even in the absence of ligand activation; low levels of synNotch-induced gene expression; and the long sequence and large molecule of synNotch, which make it difficult to modify by cell engineering. DISCLOSURE OF THEINVENTION

[0007] The present application relates to the following aspects.

[0008] a) a binding peptide capable of specifically binding to a target molecule; b) a receptor modulating domain comprising one or more cleavage sites; c) an intracellular domain; A chimeric polypeptide comprising: the receptor modulating domain comprises an extracellular region and a transmembrane region, the extracellular region and the transmembrane region not both derived from a Notch protein; A chimeric polypeptide, wherein binding of said binding peptide to said target molecule can induce cleavage of said receptor modulating domain, thereby releasing said intracellular domain.

[0009] In one embodiment said chimeric polypeptide is characterized in that said cleavage site is an enzymatic hydrolysis site, preferably a protease hydrolysis site.

[0010] In one embodiment, the chimeric polypeptide is characterized in that it contains a cleavage site in the transmembrane region of the receptor modulating domain, such that binding of the binding peptide to the target molecule causes cleavage and release of the intracellular domain.

[0011] In one embodiment, the chimeric polypeptide is characterized in that the transmembrane region comprises an I-CLiP (intramembranously cleaving proteases) enzyme cleavage site, preferably, the I-CLiP comprises a γ-secretase cleavage site, more preferably, the γ-secretase cleavage site comprises a Gly-Val dipeptide sequence.

[0012] In one embodiment, the extracellular region of the receptor modulating domain of the chimeric polypeptide comprises a cleavage site, and binding of the peptide that specifically binds to a target molecule causes cleavage of the extracellular region, which in turn causes cleavage and release of the intracellular domain.

[0013] In one embodiment, the cleavage site in the extracellular domain of the chimeric polypeptide is a sheddase protease cleavage site, preferably said sheddase protease is selected from BACE1, ADAM8, ADAM9, ADAM10, ADAM12, ADAM17, MT1-MMP.

[0014] In one embodiment, the extracellular region of the chimeric polypeptide is derived from the extracellular domain of Jagged2, EphrinB2, APLP1, APLP2, APP, CD44, CSF1R, CXCL16, CX3CL1, Delta1, E-cadherin, EphB2, EphrinB1, growth hormone receptor, HLA-A2, IFNaR2, IL1R2, L1, LRP, LRP2, LRP6, N-cadherin, Nectin1α, NRADD, p75-NTR, Pcdh α4, Pcdh γ-C3, PTPκ, PTP-LAR, SorCS1b, SorLA, Sortilin, ApoER2, PKHD1, ErbB4, IFNaR2, VEGF-R1, or VLDLR, or a fragment of the extracellular region of any of the above proteins, or a variant of the extracellular region of any of the above proteins.

[0015] In one embodiment, the extracellular region of the chimeric polypeptide comprises the extracellular region of Jagged2 or the extracellular region of EphrinB2, or a fragment of the extracellular region of Jagged2 or a fragment of the extracellular region of EphrinB2.

[0016] In one embodiment, the extracellular region of the chimeric polypeptide comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 3, 5, 7, 9, and 11.

[0017] In one embodiment, the intracellular domain of the chimeric polypeptide is selected from the group consisting of the following proteins: Transcriptional activator proteins;transcriptional repressor proteins;transcriptional co-activator proteins;transcriptional co-repressor proteins;DNA-binding polypeptides;RNA-binding polypeptides;translation regulatory polypeptides;hormones;cytokines;toxins;antibodies;chromatin regulators;suicide proteins;organelle-specific polypeptides (e.g., nuclear pore regulators, mitochondrial regulators, endoplasmic reticulum regulators, etc.);pro-apoptotic polypeptides;anti-apoptotic polypeptides;other polypeptides that promote cell death through other mechanisms;pro-proliferative polypeptides;anti-proliferative polypeptides;immune co-stimulatory polypeptides;site-specific nucleases;recombinases;inhibitory immune receptors;activating immune receptors;Cas9 and RNA-targeted nuclease mutants;DNA recognition polypeptides;signal transduction polypeptides;receptor tyrosine kinases;non-receptor tyrosine kinases;differentiation-promoting polypeptides and Preferably, the intracellular domain comprises a protein fragment selected from any of the following proteins: transcriptional activator proteins, transcriptional repressor proteins, site-specific nucleases, recombinases, inhibitory immunoreceptors, and activating immunoreceptors.

[0018] In one embodiment, the transcriptional activator protein is GLA4-VP64 or a fragment thereof, preferably said transcriptional activator protein comprises the sequence shown in SEQ ID NO:27.

[0019] In one embodiment, the intracellular domain of the chimeric polypeptide is a site-specific nuclease, preferably, said site-specific nuclease is a Cas9 polypeptide.

[0020] In one embodiment, the receptor modulating domain of the chimeric protein comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 85.

[0021] In one embodiment, the binding peptide is an antibody, an antigen, a ligand, a receptor, a cell adhesion molecule, or a non-antibody molecular scaffold.

[0022] In one embodiment, the binding peptide is an antibody, wherein the antibody is a single domain antibody, a single chain antibody, a diabody, a triabody, a minibody.

[0023] In one embodiment, said non-antibody molecular scaffold is an avimer, a DARPin, an adnectin, an avimer, an affinity body, an anticalin, or an affilin.

[0024] In one embodiment, the target molecule is present in a ligand in human pathological tissue and / or human normal tissue.

[0025] In one embodiment, the pathological tissue is a tumor tissue, an infectious disease tissue, or a genetically mutated tissue.

[0026] In one embodiment, the tumor tissue includes tumor cells, organs containing tumor cells, and the tumor microenvironment.

[0027] In one embodiment, the target molecule is specifically expressed or highly expressed in a particular human pathological tissue and / or human normal tissue.

[0028] In one embodiment, the transmembrane region is a transmembrane region from notch, preferably the transmembrane region comprises an amino acid sequence set forth in sequence 13, 14, 15, 16, 17, 18, 19, 20, 21, 102, 103, 104 or having at least 80% sequence identity to any one of SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 102, 103, 104.

[0029] In one embodiment, the receptor modulating domain has between 50 and 300 amino acids.

[0030] In one embodiment, a connecting peptide is included between the receptor modulating domain and the recognition domain.

[0031] In one embodiment, the chimeric polypeptide comprises, from N-terminus to C-terminus, a binding peptide, a receptor modulating domain, and an intracellular domain.

[0032] The present application also relates to cells expressing the above-mentioned chimeric polypeptides.

[0033] In one embodiment, the cell is an immune effector cell.

[0034] In one embodiment, the cells are T cells, NK cells, NKT cells, macrophages, CIK cells, and stem cell derived immune effector cells.

[0035] In one embodiment, the cell also contains a nucleic acid expressing another chimeric polypeptide or a T cell receptor.

[0036] In one embodiment, the additional chimeric polypeptide is a chimeric antigen receptor (CAR), a chimeric T cell receptor, or a T cell antigen coupler (TAC).

[0037] In one embodiment, the intracellular domain of the chimeric polypeptide can be released and then activate the expression of another chimeric polypeptide or a T cell receptor.

[0038] The present application relates to the nucleic acids, expression vectors, and viruses used to prepare the cells of the present application.

[0039] In one embodiment, the nucleic acid is constructed in an expression vector.

[0040] The present application relates to a method for activating a cell, the method comprising: The method includes contacting a cell of the present application described above with an immobilized antigen, wherein the binding peptide comprising the chimeric polypeptide comprises an antibody specific for a first antigen, and the contact releases the transcriptional activation protein comprised in the chimeric polypeptide, which regulates expression of the CAR and / or TCR in the cell, resulting in activation of the cell after the CAR and / or TCR binds to a second antigen.

[0041] In one embodiment, there is antigen expression heterogeneity for said first antigen and / or second antigen.

[0042] In one embodiment, the first antigen and / or the second antigen is a tumor antigen.

[0043] In one embodiment, the method is used to treat a tumor with heterogeneous expression of a first antigen.

[0044] The present application relates to a method for activating a cell, the method comprising: The method includes contacting a cell of the present application described above with the target molecule, wherein the binding peptide comprising the chimeric polypeptide comprises an antibody specific to a first target molecule, and the contact releases the transcriptional activation protein comprised in the chimeric polypeptide, which regulates expression of the CAR and / or TCR in the cell, resulting in activation of the cell after the CAR and / or TCR bind to a second target molecule.

[0045] In one embodiment, there is expression heterogeneity in said first target molecule and / or said second target molecule.

[0046] In one embodiment, the first target molecule and / or the second target molecule is a tumor antigen.

[0047] In one embodiment, the method is used to treat a tumor with heterogeneous expression of the first target molecule.

[0048] In one embodiment, the target molecule is an insoluble molecule.

[0049] In one embodiment, the first target molecule comprises a cell membrane surface antigen.

[0050] In one embodiment, the cells comprise T cells, NK cells, NKT cells, macrophages, CIK cells, stem cell-derived immune effector cells, or a combination thereof.

[0051] The present application relates to a method for modulating secretion of the cytokine IL-12, the method comprising: In one embodiment, the method comprises contacting a cell of the present application with an immobilized antigen, wherein the binding peptide comprising the chimeric polypeptide comprises an antibody specific for the antigen, and wherein the contact releases the transcriptional activation protein comprised in the chimeric polypeptide and modulates the expression of the IL-12 in the cell.

[0052] In one embodiment, the cell is a T cell.

[0053] In one embodiment, the antigen comprises a tumor antigen.

[0054] The present application relates to a method for modulating the secretion of cytokines IL-12, IL-7, CCL21 or a combination thereof, the method comprising: The method includes contacting a cell of the present application with the target molecule, wherein the binding peptide comprising the chimeric polypeptide comprises an antibody specific to the target molecule, and the contact releases the transcriptional activation protein comprised in the chimeric polypeptide to regulate expression of the IL-12, IL-7, CCL21, or a combination thereof in the cell.

[0055] In one embodiment, the cells comprise T cells, NK cells, NKT cells, macrophages, CIK cells, stem cell-derived immune effector cells, or a combination thereof.

[0056] In one embodiment, the target molecule comprises a tumor antigen.

[0057] In one embodiment, the target molecule is an insoluble molecule.

[0058] In one embodiment, the target molecule comprises a cell membrane surface antigen.

[0059] It should be understood that within the scope of this application, the above-mentioned technical features of this application and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions, which will not be described here one by one due to space limitations. [Brief description of the drawings]

[0060] [Figure 1] Figures 1A and 1B show that synJagged2EC, synEphrinB2EC, and synEphrinB2EC-APLP2(TM), which target GPC3, induced BFP expression levels comparable to or significantly higher than synNotch after co-incubation with GPC3-expressing hepatoma cells, respectively. [Diagram 2] FIG. 1 shows that chimeric polypeptides expressing synNotch, synEphrinB2EC can trigger transcriptional activation after co-incubation with coated antigen. [Diagram 3] FIG. 1 shows that chimeric polypeptides comprising the full-length EphrinB2 extracellular domain or truncations thereof that target GPC3 regulate gene expression. [Figure 4] FIG. 1 shows that when the antigen concentration is low (below 0.78125 μg / mL), cells containing the chimeric polypeptide remain quiescent, whereas when stimulated with high concentrations of antigen (above 0.3125 μg / mL), cells containing the chimeric polypeptide become activated. [Diagram 5] FIG. 1 shows that synJagged2EC and synEphrinB2EC induce equivalent or increased expression of IL12 compared to synNotch after co-incubation with GPC3-expressing hepatocarcinoma cells. [Figure 6]FIG. 1 shows GPC3 expression levels in different hepatic cancer cells. [Figure 7] FIG. 1 shows that the single vector system expresses synEphrinB2EC and synEphrinB2EC-APLP2(TM) at higher levels than synNotch. [Figure 8] FIG. 1 shows that synEphrinB2EC or synEphrinB2EC-APLP2(TM) induces higher transcription than synNotch following incubation with GPC3 low expressing cells. [Figure 9] FIG. 13 shows that the induced expression level of synEphrinB2EC-del3 is low after incubation with GPC3 low expressing cells, whereas its induced expression level after incubation with GPC3 high expressing cells is comparable to that of synNotch. [Figure 10] FIG. 13 shows that synEphrinB2EC, which regulates IL12 expression, exerts a synergistic anti-tumor effect with GPC3-CAR-T cells. [Figure 11] FIG. 13 shows that after co-incubation with EGFRvIII-expressing glioma cells U87MG-EGFRvIII and U251-EGFRvIII, respectively, cells expressing synEphrinB2EC targeting the tumor antigen EGFRvIII induce BFP expression at levels similar to cells expressing synNotch chimeric polypeptides, but cells expressing synNotch chimeric polypeptides have antigen-independent activation. [Figure 12] This figure shows that after human primary T cells were prepared into synEphrinB2EC-CAR and synNotch-CAR T cells expressing the tumor antigen EGFRvIII, the positive rate and expression level of synEphrinB2EC were higher than those of synNotch. [Figure 13] FIG. 1 shows that in the absence of antigen stimulation, synEphrinB2EC-CAR-T cells do not express CAR and have no spontaneous CAR signal. [Figure 14]FIG. 1 shows that in the absence of antigenic stimulation, synEphrinB2EC-CAR-T cells are in a less activated and differentiated state. [Figure 15] FIG. 1 shows that synEphrinB2EC-CAR-T cells are in a hypo-exhausted state in the absence of antigen stimulation. [Figure 16] FIG. 1 shows that synEphrinB2EC-CAR-T expressing cells targeting the tumor antigen EGFRvIII can be activated by EGFRvIII and kill IL13Ra2-positive glioma cells, and compared with synNotch-CAR T cells, synEphrinB2EC-CAR-T cells are more effective at killing tumors with strong heterogeneity in antigen expression. [Figure 17] FIG. 1 shows the in vivo antitumor effect of IL13Ra2-CAR-T expressing EGFRvIII-synEphrinB2EC. [Figure 18] 1 shows that 376.96-28Z-T cells that recognize only human B7H3 have a significant antitumor effect, whereas B7H3-28Z-T and B7H3-BBZ-T cells that recognize human and mouse B7H3 are less effective. By placing the less specific B7H3-28Z under the control of EGFRvIII-synEphrinB2EC, its specific killing effect can be improved. [Figure 19] FIG. 1 shows that both second and fourth generation Claudin18.2-CAR-T cells expressing mesothelin-synEphrinB2EC can be specifically activated by antigen and kill tumor cells. [Figure 20] FIG. 1 shows that both second and fourth generation Claudin18.2-CAR-T cells expressing mesothelin-synEphrinB2EC can be specifically activated by antigen and kill tumor cells. [Figure 21] FIG. 1 shows that Claudin18.2-CAR-T cells expressing FAP-synEphrinB2EC are antitumor and do not significantly reduce mouse body weight. [Figure 22]Figures 22A and 22B show that mesothelin-CAR-T cells containing Claudin18.2-synEphrinB2EC can be specifically activated by antigen and kill tumor cells. [Figure 23] FIG. 23A shows the expression levels of CLL1 and NKG2D in AML cells, and FIG. 23B shows the transcriptional activity induced by binding of a chimeric polypeptide targeting CLL1. [Figure 24] FIG. 13 shows that NKG2D-CAR-T cells kill THP1 and HL-60 cells expressing NKG2D ligands, and CD3Z-NKG2D-CAR-T cells induced by CLL1-synEphrinB2EC have a weak killing effect on THP-1 cells, which have low CLL1 expression, and can only kill HL-60 cells, which have high expression of both CLL1 and NKG2D ligands. [Diagram 25] FIG. 1 shows that T cells containing NKG2D-CAR regulated by CLL1-synEphrinB2EC proliferated well. [Figure 26] This figure shows that the mortality rate of T cells bearing NKG2D-CAR regulated by CLL1-synEphrinB2EC was basically the same as that of UTD, while the mortality rate of NKG2D-CAR-T cells was significantly increased, and T cells bearing NKG2D-CAR regulated by CLL1-synEphrinB2EC can effectively ensure the proliferation and activity of T cells in the culture system. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0061] The present application has discovered that a new type of chimeric polypeptide has a transcriptional regulatory activity triggered by specific binding to a target molecule (also known as a ligand or target antigen). The extracellular region and the transmembrane region of the receptor regulatory domain of the chimeric polypeptide are not derived from Notch at the same time, and the transmembrane region includes one or more cleavage sites. The transcriptional regulatory activity of the chimeric polypeptide of the present application triggered by specific binding to a target molecule (also known as a ligand or target antigen) is equal to or higher than the transcriptional regulatory activity of synNotch triggered by specific binding to a target molecule (also known as a ligand or target antigen). The present application provides a chimeric polypeptide having a binding-triggered transcriptional switch, a nucleic acid encoding the chimeric polypeptide, and a host cell genetically modified with the nucleic acid. The present application provides a transgenic organism comprising a nucleic acid encoding a chimeric polypeptide having binding-triggered transcriptional regulatory activity. Also provided are a method of locally regulating a cell's activity using one or more chimeric polypeptides having binding-triggered transcriptional regulatory activity, and a localized cell activation system using one or more chimeric polypeptides having binding-triggered transcriptional regulatory activity.

[0062] The chimeric polypeptides provided by the present application comprise, from N-terminus to C-terminus: (a) a binding domain capable of specifically binding to a target molecule, (b) a receptor modulating domain comprising one or more cleavage sites, and c) an intracellular domain, wherein the extracellular and transmembrane regions of the receptor modulating domain are distinct from Notch receptor polypeptides, and binding of the binding domain to the target molecule can induce cleavage of the receptor modulating domain, thereby releasing the intracellular domain.

[0063] Illustratively, the present application provides chimeric polypeptides synJagged2EC, synEphrinB2EC, synEphrinB2EC-APLP2(TM), and nucleic acids encoding the chimeric polypeptides of the present application, as well as host cells genetically modified with the nucleic acids. The present application provides transgenic organisms comprising nucleic acids encoding the chimeric polypeptides synJagged2EC, synEphrinB2EC, synEphrinB2EC-APLP2(TM).

[0064] Engineered cells expressing the chimeric polypeptides designed in the present application (e.g., synJagged2EC, synEphrinB2EC, synEphrinB2EC-APLP2(TM), etc.) can induce transcriptional regulatory activity upon binding to tumor cells expressing tumor antigens (e.g., GPC3, EGFRvIII, mesothelin, FAP, Claudin18.2, CLL1, CD123, etc.). In one example, transcription regulates BFP expression, expression of the cytokine IL12, or CAR. In one example, cells expressing synEphrinB2EC, synJagged2EC, or synEphrinB2EC-APLP2(TM) induce expression of BFP, IL12, and CAR at levels approaching or significantly higher than the expression levels of cells expressing synNotch chimeric polypeptides.

[0065] Proinflammatory cytokines such as IL12 have been proven to have a strong function in promoting T cell tumor immunity, but the risk of clinical application is high due to the possibility of toxic side effects. The problem of side effects can be effectively solved by regulating the specific expression of proinflammatory cytokines at tumor sites using the chimeric polypeptides provided in the present application. In certain embodiments, cells expressing synEphrinB2EC, synJagged2EC, or synEphrinB2EC-APLP2(TM) induce IL12 expression at levels close to or significantly higher than those of cells expressing synNotch chimeric polypeptides. In certain embodiments, synEphrinB2EC is more sensitive to target molecules (also known as ligands, target antigens) expressed at low levels and has a stronger ability to induce gene expression. Illustratively, the ability of synEphrinB2EC to induce IL12 expression after specific binding to tumor antigens expressed at low levels is significantly higher than synNotch, reaching approximately 4 to 6 times. For example, the level of BFP induced by synEphrinB2EC activated by tumor antigen EGFRvIII is equivalent to that of synNotch, but synNotch is nonspecifically activated even after co-incubation with U87 or U251 cells lacking tumor antigen EGFRvIII. This suggests that the chimeric polypeptide synEphrinB2EC not only has strong gene induction ability, but also has high antigen specificity and high safety.

[0066] The chimeric polypeptide provided in the present application is not activated by soluble antigen. Illustratively, soluble antigen cannot activate cells expressing synEphrinB2EC, synJagged2EC or synEphrinB2EC-APLP2(TM) chimeric polypeptide. In certain embodiments, soluble antigen refers to antigen that is dissolved and free in the extracellular environment.

[0067] The receptor regulatory domains of the chimeric polypeptides provided in the present application (e.g., synEphrinB2EC, synJagged2EC, synEphrinB2EC-APLP2(TM), etc.) are shorter than the corresponding domains of synNotch. When incorporated into the same lentiviral vector as the gene whose expression is regulated, the gene regulated by synEphrinB2EC can have a larger capacity in terms of sequence length. In a specific embodiment, the infection positive rate of T cells finally prepared by the chimeric polypeptide synEphrinB2EC, which includes a binding domain that specifically binds to a target molecule (also known as a ligand or target antigen) and an intracellular domain, is higher than that of synNotch, about 1.5 times, which reduces the difficulty of industrial production and preparation.

[0068] The chimeric polypeptides provided in the present application (e.g., synEphrinB2EC, synJagged2EC, synEphrinB2EC-APLP2(TM)) induce and regulate CAR expression by binding, thereby improving the antitumor effect of CAR-T cells against tumors with heterogeneity or strong antigen heterogeneity in antigen expression. Illustratively, cells expressing the chimeric polypeptides of the present application (e.g., synEphrinB2EC, synJagged2EC, synEphrinB2EC-APLP2(TM)) can induce transcriptional regulation of a chimeric antigen receptor (CAR) targeting a second target molecule (illustratively, IL13Ra2) after contacting a target cell expressing a first target molecule (illustratively, tumor antigen EGFRvIII). This achieves targeted killing of tumor cells expressing the second target molecule only when the first target molecule is present in the tumor microenvironment, enhancing the tumor specificity of CAR-T cells. Moreover, since a single target cell expressing the first and second target molecules simultaneously is not a necessary condition for initiating target killing, the present application can reduce the phenomenon of target cells escaping from effector cell killing by causing target cells expressing the second target molecule to lose or reduce the expression of the first target molecule. In certain embodiments, when the positive rate of the first target molecule is low, synNotch targeting the first target molecule cannot activate CAR-T cells to effectively kill tumor cells, but synEphrinB2EC, synJagged2EC, and synEphrinB2EC-APLP2(TM) targeting the first target molecule provided in the present application can still activate CAR-T cells to kill tumor cells.

[0069] The chimeric polypeptides provided by the present application that induce and regulate CAR expression upon binding (such as synEphrinB2EC, synJagged2EC, synEphrinB2EC-APLP2(TM)) do not express CAR in the absence of target molecule stimulation, and CAR phosphorylation does not occur. In the unactivated state, low levels of CD25 and CD69 expression are maintained, in the low depletion state, PD1, LAG3, TIM3, and CD39 are maintained at low levels of expression, and in the underdifferentiated state, most of the cells are Tscm cells.

[0070] The chimeric polypeptide provided by the present application, which induces and regulates CAR expression through binding, can achieve spatially specific regulation of target cell killing by CAR-T cells, reduce off-target toxicity, reduce dysfunction caused by long-term activation of cells, reduce activation of non-specific target molecules, and reduce the difficulties of industrial preparation through highly efficient virus packaging.

[0071] The present application also provides compositions and methods for producing such receptors, the nucleic acids encoding them, host cells genetically modified with these nucleic acids, and methods for modulating cellular activity and / or treating various health conditions, such as diseases (e.g., tumors).

[0072] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art of cell biology, cell culture, gene therapy, biochemistry, microbiology, recombinant DNA, immunology, genetics, and molecular biology, which techniques are fully explained in the literature. For example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wileyand son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrooketal, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (MJ Gaited., 1984); Mullis et al. al.USPat.No.4,683,195;Nucleic Acid Hybridization(BDHarries & SJHigginseds.1984);Transcription And Translation(BDHames & SJHigginseds.1984);Culture Of Animal Cells(RIFreshney,Alan R.Liss,Inc.,1987);Immobilized Cells And Enzymes(IRL Press,1986);B.Perbal,A Practical Guide To Molecular Cloning(1984);the series,Methods In ENZYMOLOGY(J.Abelson & M.Simon,eds.-in-chief,Academic Press,Inc.,New York), especially Vols.154, 155(Wuetal.eds.) and Vol.185, “Gene Expression Technology”(D.Goeddel,ed.);Gene Transfer Vectors For Mammalian Cells(JHMiller & MP Caloseds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer & Walker, eds., Academic Press, London, 1987); Hand book Of Experimental Immunology, Vols. I-IV (DM Weir & CC Blackwell, eds., 1986) and Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986). All methods and materials similar or equivalent to those described herein can be used in the practice or testing of this application, and suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. Furthermore, unless otherwise specified, the materials, methods, and examples in the present application are illustrative only and are not intended to be limiting. From the contents of this application, those skilled in the art will understand that many changes or modifications can be made in the specific embodiments disclosed without departing from the spirit and scope of this application and still obtain the same or similar results. The scope of this application is not limited to the specific embodiments described herein, which are intended merely as examples of aspects of this application, and functionally equivalent methods and components are within the scope of this application. This application covers variations and modifications of the subject matter of this application for various applications and conditions.

[0073] 1.Definition: As used herein, "about" can refer to a variation known or recognizable to one of ordinary skill in the art depending on the particular circumstances, or within a range of up to about ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, ±10%, ±11%, ±12%, ±13%, ±14%, ±15%, ±16%, ±17%, ±18%, ±19%, ±20%, ±25%, ±30% of a given value. That is, a range expressed as "about" covers ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, ±10%, ±11%, ±12%, ±13%, ±14%, ±15%, ±16%, ±17%, ±18%, ±19%, ±20%, ±25%, ±30% of a given value. Alternatively, particularly with respect to biological systems or methods, the term may refer to within an order of magnitude of a value, such as within about 5-fold or within about 2-fold of a value.

[0074] Ranges: Descriptions in range format are merely for convenience and brevity and should not be construed as inflexible limitations on the scope of the present application. Thus, the description of a range should be considered as specifically disclosing all possible subranges and individual values ​​within that range. For example, a description of a range from 1 to 6 should be considered as specifically disclosing subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and specific numerical points within these ranges, such as 1, 2, 3, 4, 5, 6, etc. The above principles apply equally regardless of the range of values ​​described. When a range description is used, the range includes the endpoints of the range.

[0075] The term "receptor" refers to a type of specialized protein or polypeptide that exists on the cell membrane or inside the cell and binds to a target molecule to activate a series of biochemical reactions within the cell, enabling the cell to produce a corresponding effect in response to an external stimulus. Target molecules (also called bioactive substances) that bind to receptors are collectively called ligands, or target antigens.

[0076] The term "chimeric antigen receptor" or "CAR" refers to an engineered molecule that can be expressed by immune cells, including but not limited to T cells. CARs are expressed in T cells and can redirect T cells to induce target cell killing with specificity determined by the chimeric receptor. CARs include an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The intracellular signaling domain includes a primary signaling domain and / or a costimulatory signaling domain. The extracellular binding domain of a CAR can be derived from a mouse monoclonal antibody, a humanized monoclonal antibody, or a fully human monoclonal antibody. The term CAR is not specifically limited to a CAR molecule, but also includes CAR variants. CAR variants include split CARs in which the extracellular portion (e.g., ligand-binding portion) and the intracellular portion (e.g., intracellular signaling portion) of the CAR are present on two separate molecules. CAR variants also include ON-switch CARs, which are CARs that can be conditionally activated, such as split CARs in which the conditional heterodimerization of the two portions of the split CAR is controlled by a drug. CAR variants also include bispecific CARs that contain a secondary CAR binding domain that can amplify or inhibit the activity of the primary CAR. CAR variants also include inhibitory chimeric antigen receptors (iCARs) that can be used, for example, as components of bispecific CAR systems, where binding of the secondary CAR binding domain inhibits primary CAR activation.

[0077] The term "engineered" refers to the application of principles and methods of cell and molecular biology to change the genetic material in a cell or to obtain a cellular product at the whole cell or organelle level through some engineering means. Engineered cells can also refer to cells that contain added, deleted, and / or altered genes.

[0078] The term "cell" or "engineered cell" may refer to a cell of human or non-human animal origin.

[0079] The terms "individual" and "test subject" may be used interchangeably and include humans or other species of animals, including but not limited to humans, mice, rats, hamsters and guinea pigs, rabbits, dogs, cats, sheep, pigs, goats, cows, horses, apes, and monkeys.

[0080] The term "transfection" refers to the introduction of exogenous nucleic acid into a eukaryotic cell. Transfection can be accomplished by a variety of means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran mediated transfection, polybrene mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.

[0081] The term "nucleic acid" or "polynucleotide" or "nucleic acid molecule" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form, including any nucleic acid molecule that encodes a polypeptide of interest or a fragment thereof. The nucleic acid molecule need only maintain substantial identity with the endogenous nucleic acid sequence, and need not be 100% homologous or identical to the endogenous nucleic acid sequence. A polynucleotide having "substantial identity" with an endogenous sequence can generally hybridize to at least one strand of a double-stranded nucleic acid molecule. "Hybridization" refers to the pairing of double-stranded molecules between complementary polynucleotide sequences or portions thereof under various stringent conditions. The term "homology" or "identity" refers to the identity of subunit sequences between two polymer molecules, for example between two nucleic acid molecules, such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. The term "substantial identity" or "substantial homology" refers to a polypeptide or nucleic acid molecule that exhibits at least about 50% homology or identity with a reference amino acid or nucleic acid sequence. In one example, such a sequence has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% homology or identity with the amino acid or nucleic acid sequence as a comparison. Sequence identity can be measured by using sequence analysis software (e.g., BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions generally include substitutions within the group of glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine, lysine, arginine; and phenylalanine, tyrosine. An exemplary method for determining the degree of identity can use the BLAST program, where a probability score between e-3 and e-100 indicates a closely related sequence.

[0082] The term "isolated" means changed or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the materials with which it naturally occurs is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form or can exist in a non-native environment, such as a host cell.

[0083] The term "operably linked" refers to a physical or functional link between two or more sequences (e.g., polypeptide or polynucleotide sequences) that allows them to operate in a desired manner. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Usually, operably linked DNA sequences are contiguous and, where necessary, join two protein coding regions in the same reading frame.

[0084] The terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds.

[0085] The term "immune cell activation" refers to changes in intracellular protein expression caused by signaling pathways that lead to the initiation of an immune response. In one example, the immune synapse formed after a CAR binds to an antigen includes the aggregation of many molecules near the binding receptor (e.g., CD4 or CD8, CD3γ / CDδ / CDε / CDζ, etc.). This aggregation of membrane-bound signaling molecules phosphorylates ITAM motifs contained in the CD3 molecule. This phosphorylation initiates the T cell activation pathway, ultimately activating transcription factors such as NF-κB and AP-1. These transcription factors induce global gene expression in the T cell, such as upregulating IL-2 production, promoting T cell proliferation, and thereby initiating a T cell-mediated immune response. "T cell activation" or "T cells are activated" refers to the state of T cells that have been stimulated to induce detectable cell proliferation, cytokine production, and / or detectable effector functions. In one example, the immune cells are activated after co-incubation with cells containing a specific antigen, or the immune cells are activated after infection with a virus.

[0086] The term "antigen" refers to a molecule that elicits an immune response, including, but not limited to, tumor antigens and pathogen antigens. Any tumor antigen may be used in the tumor-related embodiments described in this application ("examples" and "embodiments" may be used interchangeably herein). Tumor antigens in this application include thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD22; CD30; CD70; CD123; CD138; CD33; CD44; CD44v7 / 8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); interleukin-13 receptor subunit alpha (IL-13Rα); interleukin-11 receptor alpha (IL-11Rα); prostate stem cell antigen (PSCA); prostate specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1 Gag;MART-1;gp100;tyrosinase;mesothelin;EpCAM;protease serine 21 (PRSS21);vascular endothelial growth factor receptor, vascular endothelial growth factor receptor 2 (VEGFR2);Lewis (Y) antigen;CD24;platelet-derived growth factor receptor beta (PDGFR-β);stage-specific embryonic antigen-4 (SSEA-4);cell surface-associated mucin 1 (MUC1), MUC6;epidermal growth factor receptor family and its variants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII);neural cell adhesion molecule (NCAM);carbonic anhydrase IX (CAIX);LMP2;Ephrin type A receptor 2 (EphA2);fucosyl-GM1;sialic acid LeLewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer; TGS5; high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); folate receptor; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); Claudin 6, Claudin 18.2, Claudin 18.1; ASGPR1; CDH16; 5T4; 8H9; αvβ6 integrin; B-cell maturation antigen (BCMA); CA9; kappa light chain; CSPG4; EGP2, EGP40; FAP; FAR; FBP; fetal AchR;HLA-A1, HLA-A2; MAGEA1, MAGE3; KDR; MCSP; NKG2D ligand; PSC1; ROR1; Sp17; SURVIVIN; TAG72; TEM1; fibronectin; tenascin; oncofetal variants of the tumor necrotic zone; G protein-coupled receptor class C group 5 member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); policy alginate;placenta-specific 1 (PLAC1);hexose moiety of globoH glycoceramide (GloboH);mammary differentiation antigen (NY-BR-1);uroplakin 2 (UPK2);hepatitis A virus cell receptor 1 (HAVCR1);adrenergic receptor beta 3 (ADRB3);pannexin 3 (PANX3);G protein-coupled receptor 20 (GPR20);lymphocyte antigen 6 complex locus K9 (LY6K);olfactory receptor 51E2 (OR51E2);TCR gamma alternative receptor loading frame protein (TARP); Wilms tumor protein (WT1); ETS translocation mutated gene 6 (ETV6-AML); sperm protein 17 (SPA17); X antigen family member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen 2 (MAD-CT-2); Fos-related antigen 1; p53 mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; V-myc avian myelocytoma viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS); squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OYTES1); lymphocyte-specific protein tyrosine kinase (LCK);A kinase anchoring protein 4 (AKAP-4); synovial sarcoma X breakpoint 2 (SSX2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR); leukocyte immunoglobulin-like receptor subfamily member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); immunoglobulin lambda-like polypeptide 1 (IGLL1). Pathogen antigens include, but are not limited to, viral, bacterial, fungal, protozoan, or parasitic antigens. Viral antigens include, but are not limited to, cytomegalovirus (CMV) antigens, Epstein-Barr virus (EBV) antigens, human immunodeficiency virus (HIV) antigens, or influenza virus antigens;

[0087] The term "antigen-binding domain" refers to a molecule that specifically binds to an antigenic determinant, including immunologically active portions of immunoglobulin molecules and immune molecules, i.e., a molecule that contains an antigen-binding site that specifically binds to an antigen ("immunoreactive"). The term "antibody" includes intact antibody molecules as well as fragments of antibody molecules that retain antigen-binding ability. The term "antibody" can be used interchangeably with the terms "immunoglobulin" and "antigen domain" in this application. Antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, natural antibodies, bispecific antibodies, chimeric antibodies, Fv, Fab, Fab', Fab'-SH, F(ab')2, linear antibodies, single-chain antibodies (such as scFv), and single domain antibodies. In one example, an antibody comprises at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The CH is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The CL is composed of one structural domain. VH and VL can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of an antibody mediates the binding of immunoglobulins to various cells of the immune system (e.g., immune cells) and to host tissues or factors, including the first component (C1q) of the classical complement system. An antigen domain "specifically binds" to an antigen or is "immunoreactive" with an antigen if it binds to the antigen with a higher affinity (also called avidity) than it binds to other reference antigens (including polypeptides or other substances).

[0088] The terms "therapeutically effective amount", "therapeutically effective", "effective amount", or "in an effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, substance or composition, pharmaceutical composition effective to achieve a particular biological result described herein, such as, but not limited to, an amount or dose sufficient to promote a T cell response. An effective amount of immune cells refers to, but is not limited to, the number of immune cells that can increase, enhance, or prolong anti-tumor activity; increase the number of anti-tumor immune cells or the number of activated immune cells; the number of immune cells that promote IFN-γ secretion, tumor regression, tumor shrinkage, and tumor necrosis.

[0089] The term "promoter" as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell or introduced synthetic machinery necessary to initiate the specific transcription of a polynucleotide sequence.

[0090] The term "endogenous" refers to a nucleic acid molecule or polypeptide, etc., that originates from the organism itself.

[0091] The term "exogenous" refers to a nucleic acid molecule or polypeptide that is not endogenously present in a cell or whose expression levels are insufficient to achieve a function when overexpressed, and covers any recombinant nucleic acid molecule or polypeptide expressed in a cell, for example, exogenous, heterologous and overexpressed nucleic acid molecules and peptides.

[0092] The term "recognition" refers to selective binding to a target antigen. Immune cells expressing an exogenous receptor in this application are able to recognize cells expressing the antigen to which said exogenous receptor specifically binds.

[0093] The term "specifically binds" refers to an antibody or ligand that recognizes and binds to a binding partner (e.g., a tumor antigen) protein present in a sample, but does not substantially recognize or bind to other molecules in the sample.

[0094] The term "vector" as used herein refers to a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the inside of a cell. Many vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides related to ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses, and also includes non-plasmid and non-viral compounds that facilitate the introduction of nucleic acids into cells, such as transposon vectors, polylysine compounds, liposomes, etc. Viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, etc.

[0095] The term "disease" refers to any condition that damages or interferes with the normal function of cells, tissues, or organs, such as a tumor (cancer) or a pathogen infection. Refractory cancers include, but are not limited to, cancers that are insensitive to radiation therapy, cancers that recur after radiation therapy, cancers that are insensitive to chemotherapy, cancers that recur after chemotherapy, cancers that are insensitive to CAR-T therapy, or cancers that recur after treatment.

[0096] The term "tumor" refers to a disease characterized by the pathological proliferation of cells or tissues and their subsequent metastasis or invasion into other tissues or organs. The proliferation of tumor cells is usually uncontrolled and progressive, and is characterized by loss of migration and contact inhibition. Tumors include cancers and their precancerous lesions. Tumors include hematological tumors, solid tumors, or their metastatic lesions.

[0097] The term "antigen expression heterogeneity or antigenic heterogeneity" means that cells within a cell population express different antigens, or that the same antigen is expressed by only some cells of a cell population.

[0098] 2. Chimeric Polypeptides The chimeric polypeptide of the present application is a receptor that regulates transcriptional activity in a target molecule (also known as a ligand or target antigen)-dependent manner. The chimeric polypeptide of the present application is a recombinant, non-natural receptor that includes a binding domain, a receptor regulatory domain, and an intracellular domain.

[0099] In one example, after the chimeric polypeptide binds to a target molecule, the hydrolysis of the chimeric polypeptide is triggered, releasing the intracellular domain.In one example, after the chimeric polypeptide binds to a target molecule (e.g., tumor antigen) displayed on the surface of a target cell, the hydrolysis of the chimeric polypeptide is triggered, releasing the intracellular domain.Exemplarily, after the chimeric polypeptide binds to a tumor antigen on the surface of a tumor cell, it regulates the transcription factor that regulates the custom transcription program in the cell.

[0100] The chimeric polypeptide of the present application comprises, from N-terminus to C-terminus: (a) a binding domain capable of specifically binding to a target molecule, (b) a receptor modulating domain comprising one or more cleavage sites, and c) an intracellular domain, wherein the extracellular region and the transmembrane region of the receptor modulating domain are not simultaneously derived from Notch, and binding of the binding domain to the target molecule can induce cleavage of the receptor modulating domain to release the intracellular domain. The binding domain capable of specifically binding to a target molecule and the intracellular domain are heterologous to the Notch receptor polypeptide.

[0101] The chimeric polypeptides of the present application have superior transcriptional regulatory activity over existing SynNotch receptors and provide a more modular platform for engineering. Existing SynNotch receptors can be engineered using ligand-binding domains (such as single-chain antibodies and nanobodies), but it is difficult to use the native extracellular domains of the receptors / ligands on SynNotch receptors. The chimeric polypeptides of the present application are suitable for use with other types of ligand-binding domains, thereby expanding the possibilities for targeting diseases and tissues.

[0102] 2.1 Receptor Modulation Domain The receptor modulating domain of the chimeric polypeptide comprises an extracellular region and a transmembrane region (also known as a transmembrane domain). The receptor modulating domain comprises one or more ligand-inducible proteolytic cleavage sites selected from an I-CLiP (intramembrane-cleaving protease) enzymatic cleavage site or a sheddase protease cleavage site. I-CLiPs are transmembrane-cleaving proteases that can catalyze the hydrolysis of a specific site on the transmembrane region of a transmembrane protein. In one example, the I-CLiP comprises a γ-secretase cleavage site. In one example, the γ-secretase cleavage site comprises a γ-secretase cleavage site of the Gly-Val dipeptide sequence. In one example, the sheddase protease is selected from BACE1, ADAM8, ADAM9, ADAM10, ADAM12, ADAM17, MT1-MMP, or a combination thereof.

[0103] In one example, the receptor modulating domain comprises one or more ligand-inducible proteolytic cleavage sites located in the transmembrane region, said cleavage sites being selected from I-CLiP enzymatic cleavage sites. In one example, the extracellular region comprises one or more ligand-inducible sheddase proteolytic cleavage sites, and the transmembrane region comprises an I-CLiPs enzymatic cleavage site. In one example, the cleavage site in the extracellular region is a sheddase protease cleavage site. In one example, the transmembrane region comprises an I-CLiPs enzymatic cleavage site.

[0104] In one example, the extracellular region comprises one or more cleavage sites. In one example, the transmembrane region comprises one or more cleavage sites. In one example, the extracellular region comprises one or more cleavage sites and the transmembrane region comprises one or more cleavage sites. In one example, the extracellular region does not include a cleavage site. In one example, the transmembrane region comprises one or more cleavage sites. In one example, the extracellular region does not include a cleavage site and the transmembrane region comprises one or more cleavage sites.

[0105] 2.1.1 Transmembrane region In one example, the transmembrane region is a single-pass transmembrane receptor transmembrane region that contains at least one gamma-secretase cleavage site. In one example, the transmembrane region includes, but is not limited to, the transmembrane regions of CLSTN1, CLSTN2, APLP1, APLP2, LRP8, APP, BTC, TGBR3, SPN, CD44, CSF1R, CXCL16, CX3CL1, DCC, DLL1, DSG2, DAG1, CDH1, EPCAM, EPHA4, EPHB2, EFNB1, EFNB2, ErbB4, GHR, HLA-A, and IFNAR2, where the transmembrane region contains at least one gamma-secretase cleavage site. In one example, transmembrane regions include, but are not limited to, IL1R1, IL1R2, IL6R, INSR, ERN1, ERN2, JAG2, KCNE1, KCNE2, KCNE3, KCNE4, KL, CHL1, PTPRF, SCN1B, SCN3B, NPR3, NGFR, PLXDC2, PAM, AGR, ROBO1, SORCS3, SORCS1, SORL1, SDC1, SDC2, SPN, TYR, TYRP1, DCT, VASN, FLT1, CDH5, PKHD1, NECTIN1, PCDHGC3, NRG1, LRP1B, CDH2, NRG2, PTPRK, SCN2B, Nradd, and PTPRM transmembrane regions.

[0106] In one example, the transmembrane region comprises a Notch1 transmembrane region, a Notch2 transmembrane region, a Notch3 transmembrane region, or a Notch4 transmembrane region from a human or non-human animal (e.g., mouse, zebrafish, Drosophila, Xenopus, or Gallus). In one example, the transmembrane region comprises a fragment of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology or identity to the amino acid sequence set forth in SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 102, 103 or 104, and / or up to 1, 2, 3, 4, 5 or more amino acid residues, optionally substituted with different amino acid residues. In one example, an amino acid residue other than "GV" in SEQ ID NO: 13, 14, 15, 17, 18, 19, 102, 103 or 104 is replaced with a different amino acid residue.

[0107] In one example, the transmembrane region comprises an APLP1 transmembrane region or an APLP2 transmembrane region derived from a human or non-human animal. In one example, the transmembrane region comprises a fragment having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology or identity to the amino acid sequence set forth in SEQ ID NO:21, and / or up to 1, 2, 3, 4, 5 or more amino acid residues, optionally substituted with different amino acid residues.

[0108] In one example, the carboxyl terminus of the transmembrane region contains a stop transfer sequence (STS). The STS links the intracellular domains of the chimeric polypeptide and prevents entry into the lumen of the endoplasmic reticulum. In one example, the STS contains about 4-10 residues, e.g., 4, 5, 6, 7, 8, 9, or 10 amino acid residues. In one example, the STS comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the STS sequence of Notch1, Notch2, Notch3, Notch4, CLSTN1, CLSTN2, CSF1R, CXCL16, DAG1, GHR, PTPRF, AGR, KL, NRG1, LRP1B, Jag2, EPCAM, KCNE3, CDH2, NRG2, PTPRK, BTC, EPHA3, IL1R2, or PTPRM. In one example, the STS comprises a sequence that includes only Lys (K) or Arg (R) in the first four residues. In one example, an STS comprises a fragment having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology or identity to the amino acid sequence set forth in SEQ ID NO: 22, 23, 24, 25 or 26, and / or optionally up to 1, 2, 3, 4, 5 or more amino acid residues substituted with different amino acid residues.

[0109] 2.1.2 Extracellular domain In one example, the extracellular region comprises the full length of the extracellular domain of Jagged2, EphrinB2, APLP1, APLP2, APP, CD44, CSF1R, CXCL16, CX3CL1, Delta1, E-cadherin, EphB2, EphrinB1, growth hormone receptor, HLA-A2, IFNaR2, IL1R2, L1, LRP, LRP2, LRP6, N-cadherin, Nectin1α, NRADD, p75-NTR, Pcdh α4, Pcdh γ-C3, PTPκ, PTP-LAR, SorCS1b, SorLA, Sortilin, ApoER2, PKHD1, ErbB4, IFNaR2, VEGF-R1, or VLDLR, or a fragment or truncated or truncated structure of the extracellular domain of any of the above proteins, or a variant of the extracellular domain of any of the above proteins. In one example, the extracellular domain of the above proteins is full length. In one example, the extracellular region of the receptor regulatory domain of the chimeric polypeptide comprises the entire length of the above-mentioned EphrinB2EC or Jagged2 extracellular region, respectively, and the chimeric polypeptide can induce cleavage of the chimeric polypeptide after specific binding to a target molecule, releasing the intracellular domain.

[0110] The fragment of the extracellular region of a protein according to the present application does not include the entire extracellular region, but includes only a part of the extracellular region of a protein. In one example, a chimeric polypeptide including an extracellular region fragment of EphrinB2 specifically binds to a target molecule, and then causes cleavage of the chimeric polypeptide to release the intracellular domain of the chimeric polypeptide. In one example, the extracellular region fragment of EphrinB2 includes a fragment obtained by removing a potential ADAM10 cleavage site in the extracellular region of EphrinB2, such as EphrinB2EC-del23 and EphrinB2EC-del3. In one example, the extracellular region fragment of EphrinB2 includes a fragment obtained by removing the N-terminal vicinity of the extracellular region of EphrinB2, such as EphrinB2EC-del1. In one example, the extracellular region fragment of EphrinB2 includes a fragment obtained by removing the C-terminal vicinity of the extracellular region of EphrinB2, such as EphrinB2EC-del3. In one example, the extracellular domain fragment of EphrinB2 includes a fragment in which the intermediate sequence of the extracellular domain of EphrinB2 has been deleted, such as EphrinB2EC-del2. In one example, any of the chimeric polypeptides comprising the extracellular domain of the receptor regulatory domain of EphrinB2EC-del1, EphrinB2EC-del2, EphrinB2EC-del3, or EphrinB2EC-del23 described above can be cleaved to release the intracellular domain after specifically binding to a target molecule.

[0111] A mutant of the extracellular domain of a protein according to the present application means a protein having a mutation, deletion, or addition in a part of the amino acid sequence of the entire amino acids of the extracellular domain. In one example, a chimeric polypeptide comprising an extracellular domain of a receptor modulating domain composed of a mutant of the extracellular domain of EphrinB2 specifically binds to a target molecule, and then causes cleavage of the chimeric polypeptide to release the intracellular domain. In one example, a chimeric polypeptide comprising an extracellular domain of a receptor modulating domain composed of a mutant of the extracellular domain of Jagged2 specifically binds to a target molecule, and then causes cleavage of the chimeric polypeptide to release the intracellular domain.

[0112] The truncated structure or truncated form of the extracellular domain of the protein of the present application refers to a fragment of the extracellular domain of the protein that is truncated from the entire amino acid fragment of the extracellular domain. In one example, any of the chimeric polypeptides comprising the extracellular domain of the receptor regulatory domain of truncated EphrinB2EC-del1, EphrinB2EC-del2, EphrinB2EC-del3, or EphrinB2EC-del23 can be cleaved to release the intracellular domain after specifically binding to a target molecule.

[0113] In the present application, a fragment, variant, or truncated structure (also known as a truncated form or a truncation) of an extracellular domain of a protein may be used interchangeably, and a fragment, variant, or truncated structure (also known as a truncated form or a truncation) of an extracellular domain of a protein has at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity compared to the sequence of the extracellular domain of the protein. In one example, the amino acid sequence of the truncated structure is shown in SEQ ID NO: 5, 7, 9, and 11.

[0114] In certain embodiments of the present application, the fragment, mutant or truncated structure (also referred to as truncated form) of the extracellular domain of the protein is a potential site of cleavage by ADAM10 in which the extracellular domain of EphrinB2 has been removed or mutated.

[0115] In one example, the extracellular region comprises the full length or a truncated version of the extracellular region of EphrinB2 of a human or non-human animal (e.g., white-fronted gibbon, bonobo, Sumatran orangutan, chimpanzee, gorilla, rabbit, Peruvian night monkey, marmoset, crested lemur, small-eared greater galago, mouse, rat, cow, Xenopus laevis). In one example, the extracellular region comprises the full length or a truncated version of the extracellular region of Jagged2. In one example, the extracellular region comprises the extracellular region of EphrinB2 that includes the cleavage site. In one example, the extracellular region comprises the extracellular region of EphrinB2 that does not include the cleavage site. In one example, the extracellular region comprises a fragment that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:1, 3, 5, 7, 9 or 11, and / or optionally up to 1, 2, 3, 4, 5 or more amino acid residues substituted with different amino acid residues.

[0116] In one example, the receptor regulatory domain is EphrinB2 extracellular domain / Notch1 transmembrane domain (Notch1STS), EphrinB2 extracellular domain / Notch1 transmembrane domain (Notch2STS), EphrinB2 extracellular domain / Notch1 transmembrane domain (Notch3STS), EphrinB2 extracellular domain / Notch1 transmembrane domain (Notch4STS), EphrinB2 extracellular domain / Notch2 transmembrane domain (Notch1STS), EphrinB2 extracellular domain / Notch2 transmembrane domain (Notch3STS), EphrinB2 extracellular domain / Notch1 transmembrane domain (Notch4STS), EphrinB2 extracellular domain / Notch2 transmembrane domain (Notch1STS), EphrinB2 extracellular domain / Notch2 transmembrane domain (Notch2 ... Notch2 transmembrane domain (Notch2STS), EphrinB2 extracellular domain / Notch2 transmembrane domain (Notch3STS), EphrinB2 extracellular domain / Notch2 transmembrane domain (Notch4STS), EphrinB2 extracellular domain / Notch3 transmembrane domain (Notch1STS), EphrinB2 extracellular domain / Notch3 transmembrane domain (Notch2STS), EphrinB2 extracellular domain / Notch3 transmembrane domain (Notch3STS), Eph rinB2 extracellular region / Notch3 transmembrane region (Notch4STS), EphrinB2 extracellular region / Notch4 transmembrane region (Notch1STS), EphrinB2 extracellular region / Notch4 transmembrane region (Notch2STS), EphrinB2 extracellular region / Notch4 transmembrane region (Notch2STS), Extracellular region / Notch4 transmembrane region (Notch3STS), EphrinB2 extracellular region / Notch4 transmembrane region (Notch4STS), EphrinB2 extracellular region / APLP2 transmembrane region (APLP2STS), EphrinB2 extracellular region / A PLP2 transmembrane domain (Notch1STS), EphrinB2 extracellular domain / APLP2 transmembrane domain (Notch2STS), Jagged2 extracellular domain / Notch1 transmembrane domain (Notch1STS), Jagged2 extracellular domain / Notch1 transmembrane domain ( Notch2STS), Jagged2 extracellular region / APLP2 transmembrane region (APLP2STS), Jagged2 extracellular region / APLP2 transmembrane region (Notch1STS), and Jagged2 extracellular region / APLP2 transmembrane region (Notch2STS).In this specification, when a transmembrane region is the Notch1 transmembrane region (Notch1STS), it means that this transmembrane region contains the Notch1 transmembrane region and the STS portion of Notch1; when a transmembrane region is the Notch1 transmembrane region (Notch2STS), it means that this transmembrane region contains the Notch1 transmembrane region but the STS portion uses Notch2STS; when a transmembrane region is the Notch4 transmembrane region (Notch1STS), it means that the transmembrane region contains the Notch4 transmembrane region and Notch1STS, and the remaining explanation is inferred.

[0117] Illustratively, the chimeric polypeptide synJagged2EC comprises a receptor modulating domain comprised of a Jagged2 extracellular domain fragment (SEQ ID NO: 1) and a Notch1 transmembrane domain (SEQ ID NO: 13). In one example, the receptor modulating domain of the chimeric polypeptide synJagged2EC comprises the sequence shown in SEQ ID NO: 28.

[0118] Exemplary chimeric polypeptides include a receptor regulatory domain composed of an EphrinB2 extracellular domain (SEQ ID NO: 1, 3, 5, 7, 9, or 11) and a Notch1 transmembrane domain (SEQ ID NO: 13), or an EphrinB2 extracellular domain (SEQ ID NO: 3, 4) and a Notch1 transmembrane domain (Notch2STS) (SEQ ID NO: 102), or an EphrinB2 extracellular domain (SEQ ID NO: 1, 3, 5, 7, 9, or 11) and a Notch1 transmembrane domain (Notch2STS) (SEQ ID NO: 103). ch1 transmembrane domain (Notch3STS) (SEQ ID NO: 103), or EphrinB2 extracellular domain (SEQ ID NO: 1, 3, 5, 7, 9 or 11) and Notch1 transmembrane domain (Notch4STS) (SEQ ID NO: 104), or EphrinB2 extracellular domain (SEQ ID NO: 1, 3, 5, 7, 9 or 11) and Notch2 transmembrane domain (SEQ ID NO: 14), or EphrinB2 extracellular domain (SEQ ID NO: 1, 3, 5, 7, 9 or 11) and No or the EphrinB2 extracellular region (SEQ ID NO: 1, 3, 5, 7, 9 or 11) and a Notch4 transmembrane domain (SEQ ID NO: 16), or the EphrinB2 extracellular region (SEQ ID NO: 1, 3, 5, 7, 9 or 11) and a human Notch1 transmembrane domain (SEQ ID NO: 17), or the EphrinB2 extracellular region (SEQ ID NO: 1, 3, 5, 7, 9 or 11) and a human Notch2 transmembrane domain (SEQ ID NO: 18), or the EphrinB2 extracellular region (SEQ ID NO: 1, 3, 5, 7, 9 or 11) and a human Notch3 transmembrane domain (SEQ ID NO: 19), or the EphrinB2 extracellular region (SEQ ID NO: 1, 3, 5, 7, 9 or 11) and a human Notch4 transmembrane domain (SEQ ID NO: 20), or the EphrinB2 extracellular region (SEQ ID NO: 1, 3, 5, 7, 9 or 11) and a human APLP2 transmembrane domain (SEQ ID NO: 21). In one example, the receptor modulating domain of the chimeric polypeptide synEphrinB2EC comprises the sequence set forth in SEQ ID NO:29, 30, 31, 32, 33, 34, 35, 37, 38, 39, 40, 41 or 85.

[0119] The chimeric polypeptide synEphrinB2EC-APLP2(TM) comprises a receptor modulating domain composed of an EphrinB2 extracellular domain fragment (SEQ ID NO:3) and an APLP2 transmembrane domain (SEQ ID NO:21). In one example, the receptor modulating domain of the chimeric polypeptide synEphrinB2EC-APLP2(TM) comprises the sequence set forth in SEQ ID NO:36.

[0120] In one example, the receptor modulating domain contained in the chimeric polypeptide has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity or homology to any one of the sequences set forth in SEQ ID NOs: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 85.

[0121] The receptor regulatory domain of the chimeric polypeptide is short, which is beneficial for viral packaging and expression, and reduces the difficulty of industrial production and preparation. The full length of the coding sequence of the chimeric polypeptide synEphrinB2EC, synEphrinB2EC-del, synJagged2EC, or synEphrinB2EC-APLP2(TM) is shorter than the full length of the synNotch coding sequence, and the final prepared vector containing the complete chimeric polypeptide has high cell transduction efficiency, which reduces the difficulty of industrial production and preparation.

[0122] In one example, the chimeric polypeptides synEphrinB2EC, synEphrinB2EC-del, synJagged2EC, or synEphrinB2EC-APLP2(TM) are not located in the same vector as the gene they bind to and regulate, i.e., a dual vector system. In one example, the chimeric polypeptides synEphrinB2EC, synEphrinB2EC-del, synJagged2EC, synEphrinB2EC-APLP2(TM) and the gene they bind to and regulate are located in the same vector, i.e., a single vector system. In one example, the infection efficiency of the synEphrinB2EC, synEphrinB2EC-del, synJagged2EC, and synEphrinB2EC-APLP2(TM) single vector system is higher than that of synNotch, reducing the difficulty of industrial production and preparation. In one example, the infection efficiency of the synEphrinB2EC single vector system is about 1.5-fold higher than that of synNotch, which reduces the difficulties of industrial production and preparation.

[0123] Compared with synNotch, the level of leaky expression of transcriptional regulation induced by the chimeric polypeptide synEphrinB2EC-del is significantly lower. Compared with synNotch, the chimeric polypeptide synEphinB2EC-Del3 has both stringency of inducible expression and strong induction ability. The chimeric polypeptide synEphinB2EC-Del3 can better distinguish different expression levels of the same target molecule, is suitable for identifying target molecules with low expression in normal tissues but high expression in tumor tissues, and is less likely to cause off-targeting in normal tissues. Compared with synNotch, the chimeric polypeptide synEphinB2EC-Del3 regulates gene expression more strictly and has low sensitivity to low-expressing target molecules, and when targeting target molecules that are highly expressed in tumors and low-expressed in normal tissues, synEphinB2EC-Del3 has higher selectivity for tumor tissues and is safer.

[0124] 2.2 Intracellular domain The chimeric polypeptides of the present application include an intracellular domain that is released by hydrolysis after the chimeric polypeptide binds to a target molecule.

[0125] The intracellular domain of the chimeric polypeptide of the present application may be selected from the group consisting of the following proteins: The protein fragments include those selected from any of the following proteins or combinations: transcription factors (including transcription activating proteins and transcription repressing proteins), transcription co-activating proteins, transcription co-repressing proteins, DNA-binding polypeptides, RNA-binding polypeptides, translation regulatory polypeptides, hormones, cytokines, toxins, antibodies, chromatin regulators, suicide proteins, organelle-specific polypeptides (e.g., nuclear pore regulators, mitochondrial regulators, endoplasmic reticulum regulators, etc.), apoptosis-promoting polypeptides, anti-apoptotic polypeptides, other polypeptides that promote cell death through other mechanisms, proliferation-promoting polypeptides, anti-proliferative polypeptides, immune co-stimulatory polypeptides, site-specific nucleases, recombinases, inhibitory immune receptors, activating immune receptors, mutants of Cas9 and RNA-targeted nucleases, DNA recognition polypeptides, signal transduction polypeptides, receptor tyrosine kinases, non-receptor tyrosine kinases, and polypeptides that promote differentiation.

[0126] The intracellular domain of the chimeric polypeptide of the present application comprises a transcription activator protein that promotes or inhibits transcription of a promoter-driven DNA sequence. In one example, the transcription factor directly regulates cell differentiation. In one example, the transcription factor indirectly regulates cell differentiation by regulating the expression of a second transcription factor. The transcription factor is a transcription activator protein or a transcription repressor protein. In one example, the transcription factor is a transcription repressor protein. In one example, the transcription factor is a transcription activator protein. In one example, the transcription factor also comprises a nuclear localization signal. In one example, the transcription factor is selected from Gal4-VP16, Gal4-VP64, tetR-VP64, ZFHD1-VP64, Gal4-KRAB, and HAP1-VP16. In one example, the transcription factor is Gal4. In one example, the transcription factor is Gal4-VP64.

[0127] In some cases, the antibodies induced by the intracellular domain of the chimeric polypeptide of the present application are therapeutic antibodies used to treat diseases (including immune diseases, tumors).

[0128] In one example, the intracellular domain comprises a protein fragment or combination thereof selected from any of the following proteins: transcriptional activator protein, transcriptional repressor protein, site-specific nuclease, recombinase, inhibitory immunoreceptor, activating immunoreceptor. In one example, the transcriptional activator protein comprises GLA4, GLA4-VP64, or a fragment thereof. In one example, the transcriptional activator protein comprises an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity or homology to that set forth in SEQ ID NO:27.

[0129] In one example, the site-specific nuclease is a Cas9 polypeptide. In one example, the intracellular domain is a recombinase. In one example, the intracellular domain is an inhibitory immunoreceptor. In one example, the intracellular domain is an activating immunoreceptor.

[0130] In one example, the chimeric polypeptide comprises an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity or homology to any one of the amino acid sequences of SEQ ID NOs: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 85 each linked in order to SEQ ID NO: 27.

[0131] 2.3 Binding domains (also called binding peptides) The chimeric polypeptide comprises a binding domain that specifically binds to a target molecule. In one example, the binding domain of the chimeric receptor disclosed herein specifically binds to one or more target molecules. In one example, the chimeric polypeptide comprises a linker interposed between the binding domain and the receptor structure-modulating domain. The binding domain comprises an antibody, an antigen, a ligand, a receptor, a target (e.g., tag FLAG), an Fc receptor, an extracellular matrix component, a cell adhesion molecule, a non-antibody molecular scaffold, or a combination thereof.

[0132] In one example, the binding domain of the chimeric polypeptide comprises an antigen-binding domain. In one example, the antigen-binding domain is selected from an antibody, a receptor, a cell adhesion molecule, a non-antibody molecular scaffold, or a combination thereof. In one example, the antigen-binding domain comprises an antibody-based recognition scaffold. In one example, the antigen-binding domain comprises an antibody. In one example, the antibody contained in the antigen-binding domain specifically binds to a tumor antigen, a disease-associated antigen, or an extracellular matrix component. In one example, the antibody contained in the antigen-binding domain specifically binds to a cell surface antigen, a soluble antigen, or an antigen immobilized on an insoluble substrate. In one example, the antigen-binding domain comprises a single-chain antibody Fv (scFv). In one example, the antibody contained in the antigen-binding domain can specifically bind to multiple antigens. In one example, the antigen-binding domain comprises a nanobody, a single-domain antibody, a diabody, a triabody, a mini-antibody, or a combination thereof. In one example, the antigen-binding domain is a non-antibody-based recognition scaffold, such as an avimer, a DARPin, an adnectin, an avimer, an affinity body, an anticalin, or an affilin. In one example, the antibody is a single domain antibody, a single chain antibody, a diabody, a triabody, a minibody, a F(ab')2 fragment, a F(ab)v fragment, a scFv, a single domain antibody (sdAb), and functional fragments thereof or combinations thereof.

[0133] In one example, the binding domain comprises an antigen, such as an endogenous antigen and an exogenous antigen. In one example, the binding domain comprises a ligand for a receptor. In one example, the binding domain comprises a receptor. In one example, the binding domain comprises a cell adhesion molecule (e.g., all or a portion of the extracellular domain of a cell adhesion molecule). In one example, the binding domain comprises a portion of a polymerization domain.

[0134] In one example, the binding domain specifically binds to a tumor antigen and / or pathogen antigen as described in the 1. Definition term "Antigen". In one example, the binding domain comprises an antibody that specifically binds to a tumor antigen and / or pathogen antigen as described in the 1. Definition term "Antigen". In one example, the antibody comprised in the binding domain specifically binds to mesothelin, FAP, Claudin18.2, CLL1, CD19, GPC3, WT1, HER2, EGFR, EGFRvIII, BCMA, CD123, or a combination thereof. In one example, the binding domain comprises an antibody VH or VL or scFV that specifically binds to mesothelin, FAP, Claudin18.2, CLL1, CD19, GPC3, WT1, HER2, EGFR, EGFRvIII, BCMA, CD123, or a combination thereof. In one example, the binding domain comprises an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity or homology to that set forth in SEQ ID NO: 42, 43, 44, 45, 46, 47, 86, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or 101.

[0135] In one example, the chimeric polypeptide comprises an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity or homology to any one of the amino acid sequences set forth in SEQ ID NOs: 50, 51, 52, 53, 54, 55, 56, 57, 61, 73, 87, 105, 106, 108 or 110.

[0136] 2.4 Target molecules The target molecule that binds to the chimeric polypeptide of the present application is also referred to as a ligand or target antigen.

[0137] The target molecule may be membrane bound. The target molecule may be present on a cell surface. The target molecule may be immobilized on an insoluble substrate (e.g., polyethylene, polystyrene, polyvinylpyrrolidone, polycarbonate, nitrocellulose, etc.). The target molecule may be soluble. The target molecule may be present in an extracellular environment (e.g., extracellular matrix). The target molecule may be present in an artificial matrix. The target molecule may be present in a non-cellular environment. The target molecule may be present on an insoluble support in various forms, such as a plate, tissue culture dish, column, etc. The target molecule may be present in an extracellular matrix (ECM) (e.g., the antigen is an ECM component). The target molecule may be present in an artificial matrix. The target molecule may be present in a non-cellular environment. Target molecules include polypeptides, nucleic acids, glycoproteins, small molecules, carbohydrates, lipids, glycolipids, lipoproteins, and lipopolysaccharides. In one example, the target molecule is selected from differentiation marker clusters, cell surface receptors, adhesion proteins, integrins, mucins, lectins, and tumor antigens.

[0138] In one example, the target molecule is a cluster of differentiation (CD) marker. In some embodiments, the CD markers are CD1, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3d, CD3e, CD3g, CD4, CD5, CD7, CD8a, CD8b, CD19, CD20, CD21, CD22, CD23, CD25, CD27, CD28, CD33, CD34, CD40, CD45, CD48, CD52, CD59, CD66, CD70, CD71, CD72, CD73, CD79A, CD79B, CD80 (B7.1), CD86 (B7.2), CD94, CD95, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD178, CD181 (CXCR1), The antibody is selected from the group consisting of CD182 (CXCR2), CD183 (CXCR3), CD210, CD246, CD252, CD253, CD261, CD262, CD273 (PD-L2), CD274 (PD-L1), CD276 (B7H3), CD279, CD295, CD339 (JAG1), CD340 (HER2), EGFR, FGFR2, CEA, AFP, CA125, MUC-1, MAGE, alkaline phosphatase, placenta-like 2 (ALPPL2), B-cell maturation antigen (BCMA), blue fluorescent protein (BFP), green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), and signal regulatory protein alpha (SIRPα).

[0139] In one example, the target molecule is an antigen. In one example, the target molecule includes a tumor antigen and / or a pathogen antigen. In one example, the tumor antigen is Mesothelin, FAP, Claudin18.2, CLL1, CD19, GPC3, WT1, EGFR, BCMA, CD7, NKG2D-ligand, CD19, B7H3, ALPPL2, CD123, CD171, CD179a, CD20, CD213A2, CD22, CD24, CD246, CD272, CD30, CD33, CD38, CD44v6, CD46, CD71, CD97, CEA, CLDN6, CLECL1, CS-1, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, FLT3, GD2, GD3, GM3, GPRC5D, HER2(ERBB2), IGLL1, IL 11Ra, IL13Ra2, CD 117, MUC1, NCAM, PAP, PDGFR-b, PRSS21, PSCA, PSMA, ROR1, SIRPa, SSEA-4, TAG72, TEM1 / CD248, TEM7R, TSHR, VEGFR2, ALPI, cMet, and Axl.

[0140] In one example, the chimeric polypeptide and its transcriptionally regulated CAR each recognize a different tumor antigen.

[0141] In the treatment of tumors with antigen heterogeneity, the proportion of positive cells (i.e., positivity rate) of the first target molecule (e.g., EGFRvIII) is low, and the positivity rate of the second target molecule (e.g., IL13Ra2) is high. Such tumor cells avoid the treatment targeting the first target molecule, thereby reducing the efficacy of the treatment. Due to the widespread expression of the second target molecule, the treatment targeting the second target molecule may cause off-target toxicity. In one example, the present application constructs a chimeric polypeptide that binds to the first target molecule to cause transcriptional regulation and recognizes the expression of the exogenous receptor of the second target molecule, and realizes specific and widespread killing of tumor cells that express the second target molecule and partially express the first target molecule. In one example, the killing effect of the CAR targeting the second target molecule depends on the induction of the transcriptional regulation activity of the chimeric polypeptide that binds to the first target molecule. That is, the killing effect requires the presence of the first target molecule and the second target molecule in the environment. Using a chimeric polypeptide (synEphrinB2EC, synJagged2EC, synEphrinB2EC-APLP2(TM)) that binds to a first target molecule to induce and regulate the expression of an exogenous receptor (such as a CAR) that targets a second target molecule can significantly improve the anti-tumor effect of CAR-T cells transcriptionally regulated by the chimeric polypeptide against tumors with heterogeneous expression of the first target molecule, compared to synNotch.

[0142] In the treatment of tumors with heterogeneity of target molecules, where a first target molecule (e.g., mesothelin) is highly expressed but also expressed in normal tissues, and a second target molecule (e.g., Claudin18.2) is highly expressed but also expressed in other normal tissues, targeting the first target molecule or the second target molecule alone will result in off-target toxicity. In one example, using a chimeric polypeptide (synEphrinB2EC, synJagged2EC, synEphrinB2EC-APLP2(TM)) that specifically binds to a first target molecule to induce transcriptional regulation of the expression of an exogenous receptor that targets a second target molecule can significantly improve the anti-tumor effect of CAR-T cells that are transcriptionally regulated by binding of the chimeric polypeptide against tumors with heterogeneous expression of the first and second target molecules, compared to synNotch. In one example, using a chimeric polypeptide that specifically binds to a second target molecule (synEphrinB2EC, synJagged2EC, synEphrinB2EC-APLP2™) to induce and modulate expression of an exogenous receptor that targets a first target molecule can significantly improve the anti-tumor effect of CAR-T cells that are induced and modulated by binding of the chimeric polypeptide against tumors with heterogeneous expression of the first and second target molecules, compared to synNotch.

[0143] In one example, the cells used for treatment express a second target molecule targeted by the CAR, and then the treatment targeting the second target molecule causes the cells to kill each other during the culture process, affecting the activity and production of the cells. In one example, a chimeric polypeptide that specifically binds to a first target molecule is used to cause the transcriptional regulation of the expression of a CAR that targets a second target molecule, and the cells, after specifically binding to the first target molecule on the tumor cells, induce the transcriptional regulation of the expression of an exogenous receptor that targets the second target molecule in the cells, killing the tumor cells expressing the second target molecule and / or attacking the host immune cells expressing the second target molecule, thereby increasing the survival and proliferation of the cells and further improving the anti-tumor activity. In one example, the second target molecule comprises an NK cell marker selected from the NKG2 receptor family, such as NKG2A, NKG2D, NKG2C; the killer immunoglobulin-like receptor (KIR) family, such as KIR2DL1, KIR2DL2 / 3, KIR2DL4, KIR2DL5, KIR3DL1, 15 KIR3DL2, KIR2DS1, KIR2DS2 / S3, KIR2DS4, KIR2DS5, KIR3DS1; the natural cytotoxicity receptor (NCR), such as NKP30, NKP44, NKP46, NKp80; and other NK cell-specific expressed antigens, such as CD159a, CD159c, CD94, CD158, CD56, LIR / ILT2, CD244, CD226, CD2, CD16, CD161, TIGIT, CS1. In one example, a chimeric polypeptide that specifically binds to a first target molecule on leukemia cells (e.g., CLL1, CD123) is used to induce transcriptional regulation of expression of an exogenous receptor that targets NKG2D, thereby killing the tumor cells.

[0144] In one example, the target molecule is an inflammatory disease-associated molecule, including AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basigin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (alpha chain of the IL-2 receptor), CD3, CD4, CD5, IFN-α, IFN-γ, IgE, IgE These include, but are not limited to, the Fc region, IL-1, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6 receptor, integrin alpha4, integrin alpha4beta7, LFA-1 (CD11a), myostatin, OX-40, sclerosin, SOST, TGFbeta1, TNF-alpha, and VEGF-A.

[0145] 2.5. Genes Inducibly Regulated by Chimeric Polypeptide Binding The gene induced by chimeric polypeptide binding is operably linked to the transcriptional control element that is activated or inhibited by the intracellular domain of the chimeric polypeptide.In one example, the gene regulated is expressed under the control of the promoter regulated by GAL-4, tetR, ZFHD1, HNF1A or HAP1.In one example, the gene expression product regulated is selected from non-coding RNA, cytokine, cytotoxin, chemokine, immunomodulator, pro-apoptotic factor, anti-apoptotic factor, hormone, differentiation factor, dedifferentiation factor, modified TCR, CAR, reporter gene or combination thereof.

[0146] In one example, genes induced and regulated by chimeric polypeptide binding include, but are not limited to, chemokines, chemokine receptors, cytokines, cytokine receptors, differentiation factors, growth factors, growth factor receptors, hormones, metabolic enzymes, proliferation inducers, receptors, small molecule second messenger synthesis enzymes, T cell receptors, transcriptional activator proteins, transcriptional repressor proteins, transcriptional activator proteins, transcriptional repressor proteins, translation regulators, translational activator proteins, translational repressor proteins, activating immunoreceptors, apoptosis inhibitors, apoptosis inducers, immune activators, immunosuppressants, and inhibitory immunoreceptors.

[0147] In one example, the gene induced to be regulated by chimeric polypeptide binding includes, but is not limited to, a transcription activator protein, a transcription repressor protein, a CAR, a second chimeric polypeptide, a translation regulator, a cytokine, a hormone, a chemokine, or an antibody molecule. In one example, the nucleic acid sequence encoding the transcription activator protein, the transcription repressor protein, the exogenous receptor, the second chimeric polypeptide, the translation regulator, the cytokine, the hormone, the chemokine, the antibody molecule, or a combination thereof is operably linked to a transcription control element, which is activated or inhibited by the intracellular domain of the chimeric polypeptide. In one example, the transcription control element comprises a UAS (upstream activating sequence), the sequence of which is shown in SEQ ID NO: 48. In one example, the intracellular domain of the chimeric polypeptide comprises GAL4-VP64, the sequence of which is shown in SEQ ID NO: 27, and the nucleic acid sequence of the gene induced to be regulated by chimeric polypeptide binding is operably linked to a UAS, the sequence of which is shown in SEQ ID NO: 48. In one example, the intracellular domain of the chimeric polypeptide comprises GAL4-VP64, the sequence of which is shown in SEQ ID NO: 27, and the nucleic acid sequence of a gene that is inducibly regulated by chimeric polypeptide binding is operably linked to a UAS-CMV promoter, the sequence of which is shown in SEQ ID NO: 49.

[0148] In one example, the gene induced and regulated by chimeric polypeptide binding includes an exogenous cytokine or CAR. In one example, the exogenous cytokine nucleic acid sequence is operably linked to a transcriptional control element that is activated or inhibited by the intracellular domain of the chimeric polypeptide. In one example, the chimeric polypeptide in the cell binds to a target molecule and then causes hydrolysis of the receptor, which is cleaved to release the intracellular domain, and the cell is stimulated to produce interferons (α-interferon, β-interferon, γ-interferon), interleukins (IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13, IL-14, IL-15, Induce expression of cytokines or chemokines including IL-16, IL-17, IL-17A, IL-18, IL-19, IL-20, IL-24), tumor necrosis factor (TNF-α), transforming growth factor-β, TRAIL, MIP-1, MIP-1β, MCP-1, RANTES, IP10, CCL2, CCL3, CCL5, CCL17, CCL19, CCL21, CCR7, CXCL9, CXCL10, CXCL11, CXCL16, MCSF. In one example, the cytokines include IL-2, IL-7, IL-9, IL-12, IL-15, IL-18, CCL21, or a combination thereof.

[0149] In one example, the cytokine IL12 nucleic acid sequence is operably linked to a transcriptional control element that is activated by the intracellular domain of the chimeric polypeptide. IL12 subunit P35 GENE ID: 3592, subunit P40 GENE ID: 3593. In one example, the IL12 comprises the sequence shown in SEQ ID NO: 58.

[0150] In one example, the chimeric polypeptide in the cell causes hydrolysis of the receptor after binding to the target molecule, the receptor is cleaved, releasing the intracellular domain, and inducing the expression of the CAR or modified TCR by the cell. The nucleic acid sequence of the CAR or modified TCR is operably linked to a transcriptional control element that is activated or inhibited by the intracellular domain of the chimeric polypeptide. In one example, the CAR or modified TCR specifically recognizes a tumor antigen, a cancer cell-associated antigen, a hematological malignancy antigen, a solid tumor antigen, a cell surface antigen, an intracellular antigen, etc.

[0151] In one example, the first target molecule and the second target molecule are different and are respectively: 1) Hematological tumor antigens: CD19 (expressed on B cells), CD20 (expressed on B cells), CD22 (expressed on B cells), CD30 (expressed on B cells), CD33 (expressed on myeloid cells), CD70 (expressed on B cells / T cells), CD123 (expressed on myeloid cells), κ (expressed on B cells), Lewis Y (expressed in myeloid cells), NKG2D ligand (expressed in myeloid cells), ROR1 (expressed in B cells), SLAMF7 / CS1 (expressed in myeloma cells, natural killer cells, T cells and most B cells), CD138 (expressed in malignant plasma cells of multiple myeloma), CD56 (expressed in myeloma cells, neuronal cells, natural killer cells, T cells and trabecular osteoblasts), CD38 (expressed in B cells / T cells) and CD160 (expressed in NK cells / T cells); 2) Solid tumor antigens: B7H3 (expressed in malignant tumors and gliomas), CAIX (expressed in kidney), CD44v6 / v7 (expressed in cervix), CD171 (expressed in neuroblastoma), CEA (expressed in colon), EGFRvIII (expressed in neuroblastoma), EGFRvIII (expressed in gli ... expressed in glioma), EGP2 (expressed in cancer), EGP40 (expressed in colon), EphA2 (expressed in glioma, lung), ErbB2 (HER2) (expressed in breast, lung, prostate, glioma), ErbB receptor family (expressed in breast, lung, prostate, glioma), ErbB3 / 4 (expressed in breast, ovary), HLA-A1 / MAGE1 (expressed in melanoma), HLA-A2 / NY-ESO-1 (expressed in malignant tumors, melanoma), FR-a (expressed in ovary), FAR (expressed in rhabdomyosarcoma), GD2 (expressed in neuroblastoma, malignant tumors, melanoma), GD3 (expressed in melanoma, lung cancer), HMW-MAA (expressed in melanoma), IL11Ra (expressed in osteosarcoma), IL13Ra2 (expressed in glioma), LewisY (expressed in breast / ovary / pancreas), mesothelin (expressed in mesothelioma, breast, pancreas), Muc1 (expressed in ovary, breast, prostate), NCAM (expressed in neuroblastoma, colorectal), NKG2D ligand (expressed in ovary, malignant tumors), PSCA (expressed in prostate, pancreas), PSMA (expressed in prostate), TAG72 (expressed in colon), VEGFR-2 (expressed in tumor vasculature), Axl (expressed in lung cancer), Met (expressed in lung cancer), α5β3 (expressed in tumor vasculature), α5β1 (expressed in tumor vasculature), TRAIL-R1 / TRAIL-R2 (expressed in solid tumors (colon, lung, pancreas) and blood) malignant tumors), RANKL (expressed in prostate cancer and bone metastases), tenascin (expressed in glioma, epithelial tumors (breast, prostate)), EpCAM (expressed in epithelial tumors (breast, colon, lung)), CEA (expressed in epithelial tumors (breast, colon, lung)), gpA33 (expressed in colorectal cancer), mucin (expressed in epithelial tumors (breast, colon, lung, ovary)), TAG-72 (expressed in epithelial tumors (breast, colon, lung)), EphA3 (expressed in lung, kidney, melanoma, glioma, hematological malignancies), and IGF1R (expressed in lung, breast, head and neck, prostate, thyroid, glioma). Examples of surface and intracellular antigens include, for example, Her2 (ERBB2), MAGE-A1 (MAGEA1), MART-1 (MLANA), NY-ESO (CTAG1), WT1, MUC17, and MUC13. In one example, the first and second target molecules are BCMA, B7H6, CAIX, CD123, CD138, CD171, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CEA, CS1, EGFRvIII, EGP2, EGP40, Erb family members (ERBB1, ERBB2, ERBB3, ERBB4), FAP, fetal acetylcholine receptor (AChR), folate receptor alpha (FOLR1), folate receptor alpha (FOLR2), and folate receptor alpha (FOLR3). and selected from the group consisting of IFN-gamma-gamma (IFN-gamma), IFN ...

[0152] In one example, the binding domain of the chimeric polypeptide specifically binds to a first target molecule and the CAR specifically binds to a second target molecule different from the first target molecule, and is selected from the group consisting of Mesothelin, FAP, Claudin18.2, CLL1, CD19, GPC3, WT1, EGFR, EGFRvIII, BCMA, CD7, NKG2D-ligand, MOG, CD19, B7H3 (CD276), BCMA (CD269), ALPPL2, CD123, CD171, CD179a, CD20, CD213A2, CD22, CD24, CD246, CD272, CD30, CD33, CD38, CD44v6, CD46, CD71, CD97, CEA, CLDN6, CLECL1, CS-1, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, Ephrin B2, FAP, FLT3, GD2, GD3, GM3, GPRC5D, HER2(ERBB2 / neu), IGLL1, IL 11Ra, IL13Ra2, CD117, MUC1, NCAM, PAP, PDGFR-b, PRSS21, PSCA, PSMA, ROR1, SIRPa, SSEA-4, TAG72, TEM1 / CD248, TEM7R, TSHR, VEGFR2, ALPI, citrulline vimentin, cMet, and Axl.

[0153] In one embodiment, the transcription factor induced by the chimeric polypeptide in response to the first target molecule drives expression of the CAR in response to the second target molecule, such that the CAR is active and activates the T cell only in the presence of the first and second target molecules. In one example, the first and second target molecules are ASGR1 and GPC3, EGFRvIII and IL13Ra2, EGFRvIII and B7H3, mesothelin and Claudin18.2, Claudin18.2 and mesothelin, FAP and Claudin18.2, CLL1 and NKG2D, CD123 and NKG2D, respectively. In one example, the chimeric polypeptide and CAR comprise the sequences set forth in SEQ ID NOs: 61 and 62, or the sequences set forth in SEQ ID NOs: 61 and 64, or the sequences set forth in SEQ ID NOs: 61 and 65, or the sequences set forth in SEQ ID NOs: 61 and 66, or the sequences set forth in SEQ ID NOs: 105 and 67, or the sequences set forth in SEQ ID NOs: 106 and 67, or the sequences set forth in SEQ ID NOs: 108 and 107, or the sequences set forth in SEQ ID NOs: 73 and 109, or the sequences set forth in SEQ ID NOs: 50 and 109, respectively.

[0154] U87 and U251 cells express endogenous IL13Ra2 but do not express EGFRvIII. Killing of cells expressing at least two or more target molecules at different positive rates by T cells containing a chimeric polypeptide that regulates CAR expression is detected. In one example, EGFRvIII-positive cells and EGFRvIII-negative cells are mixed in different ratios to simulate gliomas that are IL13Ra2-positive and only partially EGFRvIII-positive under natural conditions. Gastric cancer cells HGC-27 express endogenous mesothelin but do not express Claudin18.2. HGC-27-A2 is an HGC-27 cell that overexpresses human Claudin18.2.

[0155] In one example, the nucleic acid sequence encoding the CAR is operably linked to a transcriptional control element regulated by the intracellular domain of the chimeric polypeptide. In one example, the nucleic acid sequence encoding the CAR is operably linked to a UAS-CMV promoter, and the intracellular domain of the chimeric polypeptide includes GLA4-VP64. In one example, the extracellular antigen binding region of the CAR includes an antibody that recognizes mesothelin, FAP, Claudin18.2, CLL1, CD19, GPC3, WT1, HER2, EGFR, EGFRvIII, BCMA, CD7, CD123, NKG2D-ligand, or a combination thereof. In one example, the extracellular antigen binding region of the CAR includes an antibody scFV that recognizes mesothelin, FAP, Claudin18.2, CLL1, CD19, GPC3, WT1, HER2, EGFR, EGFRvIII, BCMA, CD7, CD123, NKG2D-ligand, or a combination thereof. In one example, the extracellular antigen binding region of the CAR comprises an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity or homology to that set forth in SEQ ID NO: 42, 43, 44, 45, 46, 47, 86, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or 101. In one example, the IL13Ra2-CAR, B7H3-CAR, Claudin18.2-CAR, mesothelin-CAR, or NKG2D-CAR whose expression is regulated by the chimeric polypeptide is operably linked to a transcriptional control element, and the transcriptional control element is regulated by the intracellular domain of the chimeric polypeptide. In one example, the IL13Ra2-CAR, B7H3-CAR, Claudin18.2-CAR, mesothelin-CAR, or NKG2D-CAR whose expression is regulated by the chimeric polypeptide is inserted downstream of a UAS-CMV promoter, and the intracellular region of the chimeric polypeptide comprises Gal4-VP64.In the examples of the present application, "Z-CAR-T expressing X-chimeric polypeptide Y" or "X-chimeric polypeptide YZ-CAR-T" is used to describe T cells comprising a chimeric polypeptide Y targeting antigen X that regulates the expression of a CAR that recognizes antigen Z, where the Z-CAR is inserted downstream of a UAS-CMV promoter, and the intracellular domain of the chimeric polypeptide Y comprises Gal4-VP64. In the above, the symbols X and Z represent different tumor antigens. That is, the chimeric polypeptide Y can target antigen X, and the CAR shown can recognize antigen Z, and when the chimeric polypeptide Y and the CAR are combined and used in a T cell, the expression of the CAR is regulated by the intracellular domain of the chimeric polypeptide Y.

[0156] Gastric cancer highly expresses both mesothelin and Claudin18.2, preventing Claudin18.2-CAR-T cells from killing normal tissues that express Claudin18.2, thus limiting the killing effect of Claudin18.2-CAR-T cells to the tumor area. In one example, mesothelin-synEphrinB2EC-expressing Claudin18.2-CAR-T cells.

[0157] In one example, the CAR extracellular antigen binding region is linked directly to the transmembrane region or linked via a hinge. In one example, the hinge comprises a sequence having 95-100% identity to a CD8 hinge, e.g., SEQ ID NO: 75. In one example, a polynucleotide encoding a signal peptide is included upstream of the nucleic acid molecule encoding the CAR. In one embodiment, the signal peptide comprises a sequence having 95-100% identity to a CD8 signal peptide, e.g., SEQ ID NO: 81.

[0158] The transmembrane domain of the CAR molecule of the present application comprises a CD28 or CD8 transmembrane domain. In one example, the CAR comprises a CD8 transmembrane domain, e.g., a sequence having 95-100% identity to SEQ ID NO: 76. In one example, the CAR comprises a CD28 transmembrane domain, e.g., a sequence having 95-100% identity to SEQ ID NO: 77.

[0159] In one example, the CAR comprises an intracellular signal domain, i.e., a primary signal domain and / or a costimulatory signal domain. In one example, the primary signal domain comprises a CD3ζ intracellular domain, e.g., a sequence having 95-100% identity to SEQ ID NO: 80. In one example, the costimulatory signal domain comprises a CD28 or 4-1BB intracellular domain.

[0160] In one example, the CAR comprises a 4-1BB intracellular domain, e.g., a sequence having 95-100% identity to SEQ ID NO: 79. In one example, the CAR comprises a CD28 intracellular domain, e.g., a sequence having 95-100% identity to SEQ ID NO: 78.

[0161] In one example, the intracellular signaling domain of the CAR comprises a human CD3 zeta intracellular domain. In one example, the intracellular signaling domain of the CAR comprises a human CD3 zeta intracellular domain and a CD28 intracellular domain. In one example, the intracellular signaling domain of the CAR comprises a human CD3 zeta intracellular domain and a 4-1BB intracellular domain. In one example, the intracellular signaling domain of the CAR comprises a CD3 zeta intracellular domain, a CD28 intracellular domain, and a 4-1BB intracellular domain. In one example, the cell expresses a CAR and a cytokine.

[0162] Exemplarily, a CAR whose expression is transcriptionally regulated by the chimeric polypeptide of the present application comprises a sequence as set forth in SEQ ID NO: 42, 43, 44, 45, 46, 47, 86, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or 101, sequentially linked to SEQ ID NO: 82, 83 or 84, respectively.

[0163] In one example, genes inducibly regulated by chimeric polypeptide binding include, but are not limited to, trastuzumab (Herceptin, available from Chugai Pharmaceutical Co., Ltd.); bevacizumab (Avastin, available from Genentech, Inc.); infliximab (Remicade); rituximab (Rituxan, available from Biogen Idec Inc.); adalimumab (Humira). In one example, the therapeutic antibodies described above are inserted behind the UAS-CMV promoter regulated by Gal4-VP64.

[0164] 2.6 Other sequences In one example, the chimeric polypeptide of the present application also contains one or more other structural domains, including a signal peptide, an epitope tag, an affinity domain, a nuclear localization signal (NLS), and a polypeptide that generates a detectable signal.

[0165] 3. Nucleic acids and vectors The present application provides nucleic acids and expression vectors that contain genes that encode the chimeric polypeptides of the present application and / or induce transcriptional regulation upon binding of the chimeric polypeptides.

[0166] In one example, the nucleotide sequence encoding the chimeric polypeptide of the present application is operably linked to a transcriptional control element (e.g., a promoter, an enhancer, etc.). In one example, the transcriptional control element is inducible. In one example, the transcriptional control element is constitutive. In one example, the promoter functions in a eukaryotic cell. In one example, the promoter is a cell type specific promoter. In one example, the promoter is a tissue specific promoter.

[0167] In one example, the expression vector is a viral vector, such as an adeno-associated virus (AAV) vector, an adenovirus vector, a lentivirus vector, or a retrovirus vector. In one example, a retrovirus vector (gamma-retrovirus or lentivirus) is used to introduce the nucleic acid molecule into the cell. Non-viral vectors can also be used. Any suitable viral vector or non-viral delivery system can be used for transduction. In a specific embodiment of the present application, a pRRLSIN vector is constructed to express the chimeric polypeptide. The chimeric polypeptide or CAR can be constructed using a single polycistronic expression cassette, multiple expression cassettes in a single vector, or accessory molecules (e.g., cytokines) in multiple vectors. Examples of elements generating polycistronic expression cassettes include, but are not limited to, various viral and non-viral internal ribosome entry sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-κB IRES, RUNX1 IRES, p53 IRES, Hepatitis A IRES, Hepatitis C IRES, pestivirus IRES, non-baculovirus IRES, picornavirus IRES, poliovirus IRES, and encephalomyocarditis virus IRES) and cleavable linkers (e.g., 2A peptides such as P2A, T2A, E2A, and F2A peptides).

[0168] Other viral vectors that may be used include, for example, adenoviruses, lentiviruses and adeno-associated viral vectors, vaccinia virus, bovine papilloma virus, or herpes viruses such as Epstein-Barr virus.

[0169] Non-viral methods can also be used for genetic modification of immune cells. For example, nucleic acid molecules can be introduced into immune cells by lipofection, asialomucoid-polylysine conjugation, or microinjection under surgical conditions. Other non-viral gene transfer methods include in vitro transfection using transposons, liposomes, calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Nucleic acid molecules can be first introduced into a cell type that can be cultured in vitro (e.g., its own or allogeneic primary cells or their progeny), and then the cells modified by said nucleic acid molecules (or their progeny) can be injected into the target tissue or whole body of the examinee.

[0170] 4. Engineered Cells The present application provides engineered cells genetically modified with a nucleic acid of the present application, a nucleic acid encoding a chimeric polypeptide of the present application, or a chimeric polypeptide of the present application, a gene and a nucleic acid whose transcriptional regulation is induced by chimeric polypeptide binding. The present application provides a method of modulating the activity of a cell expressing a chimeric polypeptide of the present application. The method generally involves contacting the engineered cell with a target molecule to induce cleavage of the chimeric polypeptide, thereby releasing the intracellular domain, and modulating the activity of the cell upon release of the intracellular domain.

[0171] In one example, engineered cells are genetically modified to express the chimeric polypeptide of the present application and further genetically modified to express a CAR. For example, engineered cells are genetically modified with a nucleic acid comprising a nucleotide sequence encoding a CAR, and the intracellular domain of the chimeric polypeptide is a transcriptional activation protein, and the nucleotide sequence encoding the CAR is operably linked to a transcriptional control element that is activated by the intracellular domain of the chimeric polypeptide. Many CAR polypeptides have been described in the art, any of which are suitable for use herein.

[0172] In one example, the engineered cell is an immune cell, a neuron, an epithelial cell, an endothelial cell, or a stem cell. Stem cells include human pluripotent stem cells, including human induced pluripotent stem cells (iPSCs) and human embryonic stem cells. In one example, the cell includes an immune cell. In one example, the cell is a primary cell. In one example, the immune cell is a B cell, a monocyte, a natural killer cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a regulatory T cell, a helper T cell, a cytotoxic T cell, another T cell, or a combination thereof. In one example, the engineered cell includes a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 59, 63, 68, 69, 70, 71, 72, 74, 88, or a translated amino acid sequence thereof.

[0173] The immune cells of the present application may be lymphoid cells. The lymphoid system, including B, T, and natural killer (NK) cells, provides antibody production, regulation of the cellular immune system, detection of exogenous reagents in blood, detection of host exogenous cells, etc. Non-limiting examples of immune cells of the lymphoid system include T cells, natural killer T (NKT) cells, and their precursors, including embryonic stem cells and pluripotent stem cells (e.g., stem cells or pluripotent stem cells that differentiate into lymphoid cells). T cells may be lymphocytes that mature in the thymus and are primarily responsible for cellular immunity. T cells participate in the adaptive immune system. T cells include, but are not limited to, helper T cells, cytotoxic T cells, memory T cells (including central memory T cells, stem cell-like memory T cells (or stem-like memory T cells), and two effector memory T cells (such as TEM cells and TEMRA cells)), regulatory T cells (also called suppressor T cells), natural killer T cells, mucosal-associated invariant T cells, γδ T cells, or αβ T cells. Cytotoxic T cells (CTL or killer T cells) are T lymphocytes that can induce the death of infected somatic or tumor cells. The examinee's own T cells can be engineered to express the chimeric polypeptide of the present application and bind to a CAR that causes transcriptional activity. In one example, the cells of the present application are selected from T cells, NK cells, cytotoxic T cells, NKT cells, macrophages, CIK cells, and stem cell-derived immune cells, or combinations thereof. In one example, the immune cells are T cells. In one example, the T cells can be CD4+ T cells and / or CD8+ T cells. In one example, the immune cells are CD3+ T cells. In one example, the cells in the composition of the present application include a cell population collected from PBMC cells after stimulation with CD3 magnetic beads.

[0174] The cells (e.g., T cells) of the present application can be autologous, non-autologous (e.g., allogeneic), or derived in vitro from engineered progenitor or stem cells, and can be obtained from many sources, such as peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, tumors, etc.

[0175] In certain aspects of the present application, T cells can be obtained from a blood sample taken from a test subject using any number of techniques known to those skilled in the art, such as Ficoll™ isolation techniques. In a preferred aspect, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, such as T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one aspect, the cells collected by apheresis can be washed to remove the plasma portion and place the cells in an appropriate buffer or medium for subsequent processing steps. Multiple rounds of selection can also be used in the context of the present application. In some aspects, it may be necessary to perform a selection procedure and use "unselected" cells during activation and expansion. The "unselected" cells can also undergo other rounds of selection.

[0176] The cells of the present application are capable of modulating the tumor microenvironment.

[0177] The source of the unpurified CTLs can be any source known in the art, such as bone marrow, fetal, neonatal or adult, or other hematopoietic cell sources such as fetal liver, peripheral blood or umbilical cord blood. A variety of techniques can be used to isolate the cells. For example, in negative selection, non-CTLs can be first removed. mAbs are particularly useful for identifying markers associated with specific cell lineages and / or differentiation stages of positive and negative selection.

[0178] Most of the terminally differentiated cells can be removed initially by a relatively crude isolation. For example, magnetic bead separation can be used initially to remove many unrelated cells. In certain embodiments, at least about 80%, typically at least about 70%, of the total hematopoietic cells are removed prior to isolating the cells.

[0179] Isolation procedures include, but are not limited to, density gradient centrifugation; resetting; coupling to particles that change cell density; magnetic separation with antibody-coated magnetic beads; affinity chromatography; cytotoxic agents combined or used in combination with mAbs, including but not limited to complement and cytotoxins; and antibody panning attached to a solid matrix (e.g., plate, chip, elutriation) or any other convenient technique.

[0180] Separation and analysis techniques include, but are not limited to, flow cytometry and can be of varying sophistication, including multiple color channels, low and oblique angle light scatter detection channels, impedance channels, etc.

[0181] Cells can be selected for dead cells using a dead cell associated dye such as propidium iodide (PI). In certain embodiments, cells are harvested, such as in medium containing 2% fetal calf serum (FCS) or 0.2% bovine serum albumin (BSA), or other suitable sterile isotonic medium.

[0182] 5. Genetically Modified Organisms The present application provides a non-human transgenic organism comprising a nucleic acid encoding a chimeric polypeptide of the present application. The transgenic non-human organism of the present application comprises a genome genetically modified to include a nucleic acid encoding a chimeric polypeptide of the present application.

[0183] 6. Method The present application provides a method of regulating the activity of an engineered cell expressing a chimeric polypeptide of the present application, the method comprising contacting an engineered cell comprising the chimeric polypeptide of the present application with a target molecule, whereupon binding between the target molecule and the domain of the chimeric polypeptide is induced by cleavage of a protease cleavage site of the receptor modulating domain of the chimeric polypeptide to release an intracellular domain, whereby the release of the intracellular domain regulates the activity of the engineered cell. In one example, the contacting is performed in vivo, ex vivo, or in vitro. In one example, the target molecule is present on the surface of the target cell, immobilized on an insoluble substrate, present in an extracellular matrix, present in an artificial matrix, or soluble. In one example, the release of the intracellular domain regulates the proliferation of the engineered cell. In one example, the release of the intracellular domain regulates apoptosis in the engineered cell. In one example, the release of the intracellular domain induces cell death by a mechanism other than apoptosis. In one example, the release of the intracellular domain regulates gene expression in the engineered cell through transcriptional regulation, chromatin regulation, translation, trafficking, or post-translational processing. In one example, release of the intracellular domain modulates differentiation of the engineered cell. In one example, release of the intracellular domain modulates migration of the engineered cell. In one example, release of the intracellular domain modulates expression and secretion of a molecule from the engineered cell. In one example, release of the intracellular domain modulates adhesion of the engineered cell to a target cell or to an extracellular matrix. In one example, release of the intracellular domain induces re-expression of a gene product in the engineered cell. Release of the intracellular domain induces re-expression of a gene product in the engineered cell. In one example, the gene product is a transcription activator protein, a transcription repressor protein, a chimeric antigen receptor, a translation regulator, a cytokine, a hormone, a chemokine, or an antibody. In one example, the released transcription factor modulates differentiation of the engineered cell that is an immune cell, a stem cell, a progenitor cell, or a precursor cell. In one example, the method is used to treat a tumor.

[0184] The present application provides a method of activating a T cell, the method comprising contacting a T cell described herein, wherein the T cell is genetically modified with one or more nucleic acids comprising i) a chimeric polypeptide according to the present application; and ii) a nucleotide sequence encoding a CAR, with an immobilized antigen, wherein a domain of the chimeric polypeptide comprises an antibody specific for a first target molecule, the contacting causes release of an intracellular domain of the chimeric polypeptide (e.g., a transcriptional activator protein) and expression of a CAR polypeptide in the T cell, which CAR polypeptide results in activation of the T cell upon binding of a second target molecule. In one example, the first target molecule and the second target molecule are different tumor antigens, or the first target molecule is a tissue-specific molecule and the second target molecule is a tumor antigen.

[0185] The present disclosure provides a method of modulating an activity of an engineered cell, the method comprising contacting an engineered cell with a surface-immobilized antigen, where expression of a chimeric polypeptide of the present disclosure by the engineered cell can result in release of an intracellular domain and an engineered cell activity. In some cases, the intracellular domain is a transcription factor that modulates cell differentiation.

[0186] The present disclosure provides a method of locally modulating an activity of an engineered cell, comprising expressing a chimeric polypeptide comprising the present application within a cell; and releasing the intracellular domain after the engineered cell contacts a target molecule to modulate an activity of the engineered cell (expression of a gene product of the cell, proliferation of the cell, apoptosis of the cell, non-apoptotic death of the cell, differentiation of the cell, dedifferentiation of the cell, migration of the cell, secretion of molecules from the cell, and cell adhesion of the cell).

[0187] In one example, the present application provides a method of treating a tumor in a subject suffering from a tumor, the method comprising: i) genetically modifying T lymphocytes or NKT cells obtained from an individual with a chimeric polypeptide of the present application, or a vector comprising the chimeric polypeptide and a CAR whose transcriptional activity is induced by binding thereof, wherein the chimeric polypeptide is specific for an antigen on the subject's tumor cells; ii) introducing the genetically modified T lymphocytes or NKT cells into the subject, wherein the genetically modified T lymphocytes or NKT recognize and kill the tumor cells, thereby treating the tumor.

[0188] In one example, the present application provides a method of inhibiting the activity of a target cell in a subject, the method comprising administering to the subject a therapeutically effective amount of an engineered cell expressing a chimeric polypeptide of the present application, the engineered cell inhibiting the activity of the target cell in the subject. In one example, the target cell is a tumor cell. In one example, the target cell is an acute myeloma leukemia cell, an anaplastic lymphoma cell, an astrocytoma cell, a B cell cancer cell, a breast cancer cell, a colon cancer cell, an ependymoma cell, an esophageal cancer cell, a glioblastoma cell, a glioma cell, a leiomyosarcoma cell, a liposarcoma cell, a hepatoma cell, a lung cancer cell, a mantle cell lymphoma cell, a melanoma cell, a neuroblastoma cell, a non-small cell lung cancer cell, an oligodendroglioma cell, an ovarian cancer cell, a pancreatic cancer cell, a peripheral T cell lymphoma cell, a renal cancer cell, a sarcoma cell, a gastric cancer cell, a liver cancer cell, a mesothelioma cell, or a sarcoma cell. In one example, the target cell expresses a low level of the target molecule. In one example, the engineered cell further comprises a CAR, a modified TCR, an exogenous cytokine, and / or a therapeutic monoclonal antibody that is activated by initiating transcription when the chimeric polypeptide binds to a target molecule. In one example, the chimeric polypeptide specifically binds to a first target molecule, and the CAR and / or the modified TCR specifically bind to a second target molecule, and the first target molecule and the second target molecule in the target cell express a heterogeneous tumor antigen, or the first target molecule is a tissue-specific molecule, and the second target molecule is a tumor antigen. In one example, the first target molecule is not a tumor antigen but has tissue-specific expression, and the second target molecule is a tumor antigen. In one example, the positive rate of the first target molecule is lower than the positive rate of the second target molecule. In one example, this method can improve the anti-tumor specificity of the engineered cell.

[0189] 7. Medication A composition comprising the chimeric polypeptide of the present application can be provided systemically or directly to a subject to induce and / or enhance an immune response to an antigen and / or to treat and / or prevent a tumor, a pathogenic infection or an infectious disease. In one example, the composition of the present application is directly injected into the organ of interest (e.g., an organ affected by a tumor). Alternatively, the composition of the present application may be provided indirectly to the organ of interest, such as by administration to the circulatory system (e.g., a vein, tumor vasculature). Proliferation and differentiation agents can be provided before, simultaneously with, or after administration of the composition to increase the production of T cells, NKT cells, or CTL cells in vitro or in vivo.

[0190] The immune cells in the compositions of the present application may comprise a purified cell population. One of skill in the art can easily determine the percentage of immune cells of the present application in a population using various well-known methods, such as fluorescence-activated cell sorting (FACS). In a population containing immune cells of the present application, suitable ranges of purity are about 50% to about 55%, about 5% to about 60%, and about 65% to about 70%. In certain embodiments, the purity is about 70% to about 75%, about 75% to about 80%, or about 80% to about 85%. In certain embodiments, the purity is about 85% to about 90%, about 90% to about 95%, and about 95% to about 100%. The dosage can be easily adjusted by one of skill in the art (e.g., as the purity decreases, the dosage may need to be increased). The cells can be introduced by injection, catheter, or the like.

[0191] The compositions of the present application may be pharmaceutical compositions comprising the immune cells or progenitor cells of the present application and a pharma- ceutically acceptable carrier. Administration may be autologous or allogeneic. For example, immune cells or progenitor cells may be obtained from one subject and administered to the same subject or to a different matched subject. Peripheral blood-derived immune cells or their progeny (e.g., from in vivo, ex vivo, or in vitro sources) may be administered by catheter administration, systemic injection, local injection, intravenous injection, or local injection, including parenteral administration. When administering the compositions of the present application, they may be formulated in a unit dose injectable form (such as a solution, suspension, emulsion, etc.).

[0192] 8. Dosage Form The compositions comprising the chimeric polypeptides of the present application may be conveniently provided in the form of a sterile liquid preparation, such as an isotonic aqueous solution, suspension, emulsion, dispersion or viscous composition, which may be buffered to a selected pH. Liquid preparations are generally easier to prepare than gels, other viscous compositions, and solid compositions. In addition, liquid compositions are somewhat more convenient to administer, particularly by injection. On the other hand, viscous compositions may be formulated within an appropriate viscosity range to allow for longer contact time with a particular tissue. The liquid or viscous composition may include a carrier, which may be a solvent or dispersion medium, including, for example, water, saline, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures.

[0193] Sterile injectable solutions can be prepared by incorporating the immune cells of the composition of the present application in a desired amount of an appropriate solvent, incorporating various amounts of other ingredients as needed. Such compositions can be mixed with a suitable carrier, diluent or excipient, such as sterile water, saline, glucose, dextrose, etc. The compositions can also be lyophilized. The compositions may contain auxiliary substances, such as wetting agents, dispersing or emulsifying agents (e.g., methylcellulose), pH buffers, gelling or thickening agents, preservatives, flavorings, pigments, etc., depending on the route of administration and the formulation required.

[0194] To enhance the stability and sterility of the composition, various additives including antimicrobial preservatives, antioxidants, chelating agents and buffers can be added. Protection against the action of microorganisms can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, such as aluminum monostearate and gelatin. However, any vehicle, diluent, or additive used must be compatible with the genetically modified immune cells or their precursor cells.

[0195] The compositions may be isotonic, i.e., they may have the same osmotic pressure as blood and / or tears.The desired isotonicity of the composition can be achieved using sodium chloride or other pharma- ceutically acceptable agents such as glucose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes.Sodium chloride is particularly suitable for buffers that contain sodium ions.

[0196] If necessary, a pharma- ceutically acceptable thickening agent can be used to maintain the viscosity of the composition at a selected level. For example, methylcellulose is easily and economically available and easy to use. Other suitable thickening agents include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, etc. The concentration of the thickening agent depends on the agent selected. It is important to use an amount that achieves the selected viscosity. Obviously, the selection of suitable carriers and other additives depends on the exact route of administration and the nature of the particular dosage form, for example, a liquid dosage form (for example, whether the composition is formulated as a solution, suspension, gel, or other liquid, such as a time-release form or liquid-filled form).

[0197] The number of cells in the composition administered will vary depending on the subject being treated. Fewer cells can be administered that are more effective. The exact determination of the effective amount can be determined based on each subject's individual factors, including the subject's size, age, sex, weight, and subject's condition. Dosages can be easily determined by those skilled in the art from this application and knowledge in the art.

[0198] One skilled in the art can readily determine the amount of cells and any additives, vehicles and / or carriers in the composition and administered in the method. Typically, any additives (other than the one or more active cells and / or one or more reagents) are present in phosphate buffered saline in an amount of 0.001% to 50% by weight of the solution, and the active ingredient is present in micrograms to milligrams, for example, in the order of about 0.0001% to about 5% by weight, about 0.0001% to about 1% by weight, about 0.0001% to about 0.05% by weight or about 0.001% to about 20% by weight, about 0.01% to about 10% by weight or about 0.05% to about 5% by weight. For any composition administered to animals or humans, the following results can be determined: toxicity by determining the lethal dose (LD) and LD50 in an appropriate animal model, such as a rodent, such as a mouse; the dose of the composition, where the concentration of the ingredients and the time of administration of the composition, will elicit the appropriate response.

[0199] 9. Treatment method The present application provides a method of inducing and / or increasing an immune response in a subject in need of a composition of the present application. A composition comprising the chimeric polypeptide of the present application can be used to treat and / or prevent a tumor in an elephant subject. The composition of the present application can be used to prolong the survival of a subject suffering from a tumor. The composition of the present application can also be used to treat and / or prevent a pathogenic or other infection, such as in an immunocompromised human subject. Such a method includes administering an effective amount of the composition of the present application to achieve the desired effect, whether amelioration of an existing condition or prevention of recurrence. In the case of treatment, the amount administered is an amount effective to produce the desired effect. An effective amount can be provided in one or more administrations. An effective amount can be provided by bolus administration or continuous infusion.

[0200] In one example, the compositions of the present application can be used to treat subjects who have tumor cells with low levels of surface antigen expression, such as due to disease recurrence, where the subject has undergone a treatment that results in the persistence of tumor cells, in certain embodiments, the tumor cells have a low density of target molecules on the tumor cell surface.

[0201] In one example, the compositions of the present application can be used to treat subjects suffering from disease recurrence who have been administered immune cells (e.g., T cells), including administration of CAR alone. In one example, tumor cells have low density of tumor-specific antigens on the tumor cell surface. Such methods include administering an effective amount of the compositions of the present application to achieve the desired effect, alleviate an existing condition, or prevent recurrence.

[0202] An "effective amount" (or "therapeutically effective amount") is an amount sufficient to produce beneficial or desired clinical results after treatment. An effective amount may be administered to a subject in one or more doses. For therapeutic purposes, an effective amount is an amount sufficient to palliate, ameliorate, stabilize, reverse, or slow the progression of a disease, or otherwise alleviate the pathological consequences of a disease. An effective amount is generally determined by a physician on a case-by-case basis and is within the capabilities of one of ordinary skill in the art. When determining the appropriate dosage to achieve an effective amount, several factors are usually taken into consideration. These factors include the age, sex, and weight of the subject, the disease being treated, the severity of the disease, the form of the composition of the present application being administered, and the effective concentration.

[0203] Adoptive immunotherapy using antigen-specific T cells typically involves approximately 10 6 ~10 10 A cell amount ranging from 0.1 to 100 mg / kg is injected. The immune cells of the present application are administered to the host and subsequently differentiated to induce T cells specific for a particular antigen. The compositions of the present application can be administered by any method known in the art, including but not limited to intravenous, subcutaneous, intranodal, intratumoral, intrathecal, intrapleural, intraperitoneal, and direct administration to the thymus.

[0204] The present application provides a method for treating and / or preventing a tumor in a subject, which may include administering to a subject suffering from a tumor an effective amount of a composition of the present application.

[0205] Non-limiting examples of tumors include blood cancers (e.g., leukemia, lymphoma, and myeloma), ovarian cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, gastric cancer, glioblastoma, laryngeal cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcoma, and miscellaneous cancers (including prostate cancer and small cell lung cancer). Non-limiting examples of tumors include astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, ependymoma, medulloblastoma, primitive neuroectodermal tumor (PNET), chondrosarcoma, osteosarcoma, pancreatic ductal adenocarcinoma, small cell lung adenocarcinoma and large cell lung adenocarcinoma, chordoma, angiosarcoma, endothelial sarcoma, squamous cell carcinoma, bronchioloalveolar carcinoma, epithelial adenocarcinoma and its liver metastases, lymphangiosarcoma, lymphangioendothelial sarcoma, hepatocellular carcinoma, cholangiocarcinoma, synovium, mesothelioma, Ewing's tumor, rhabdomyosarcoma, colon carcinoma, basal cell carcinoma, sweat gland carcinoma, papillary carcinoma, sebaceous gland carcinoma, adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, uterine carcinoma, uterine sarcoma ... These include, but are not limited to, leukemia, multiple myeloma, Waldenstrom's macroglobulinemia and severe interstitial disease, breast tumors such as ductal and lobular adenocarcinoma, squamous cell carcinoma and adenocarcinoma of the cervix, epithelial carcinoma of the uterus and ovaries, adenocarcinoma of the prostate, transitional squamous cell carcinoma of the bladder, B-cell and T-cell lymphomas (nodular and diffuse) plasmacytoma, acute and chronic leukemia, malignant melanoma, soft tissue sarcoma and leiomyosarcoma. In certain embodiments, the tumor is selected from hematological cancers (e.g., leukemia, lymphoma, and myeloma), ovarian cancer, prostate cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, gastric cancer, glioblastoma, and laryngeal cancer. In one example, the compositions of the present application can be used to treat and / or prevent solid tumors that are unsuitable for conventional treatment measures or that have relapsed and become refractory, such as liver cancer, lung cancer, breast cancer, ovarian cancer, kidney cancer, thyroid cancer, gastric cancer, and colorectal cancer. In one example, the tumor is a hematological tumor.

[0206] The therapeutic goal of the compositions of the present application may include slowing or reversing disease progression and / or alleviating side effects, or the therapeutic goal may include reducing or delaying the risk of recurrence.

[0207] The present application provides a method of treating and / or preventing a pathogenic infection (e.g., a viral infection, a bacterial infection, a fungal infection, a parasitic infection, or a protozoal infection), for example, in an immunocompromised subject. The method may include administering to a subject suffering from a pathogenic infection an effective amount of a composition of the present application. Examples of viral infections amenable to treatment include, but are not limited to, cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus, and influenza virus infections.

[0208] The term "enhancement" refers to the ability of a subject or tumor cells to respond to a treatment disclosed herein to be improved. For example, an enhancement of response can include an increase in responsiveness of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% or more. As used herein, "enhancement" can also refer to increasing the number of subjects who respond to a treatment, such as immune cell therapy. For example, enhanced response can refer to the total percentage of subjects who respond to treatment, where the percentage is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% or greater.

[0209] 10. Kit The present application provides a kit for inducing and / or enhancing an immune response in a subject and / or treating and / or preventing a tumor or pathogen infection. In one example, the kit includes an effective amount of a composition of chimeric polypeptides and a pharmaceutical composition of the present application. In one example, the kit includes a sterile container, which may be in the form of a box, an ampoule, a bottle, a vial, a tube, a bag, a sachet, a blister pack, or other suitable container known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing drugs. In one example, the kit includes a nucleic acid molecule encoding a CAR of the present application that recognizes an antigen of interest in an expressible form, which may be included in one or more vectors.

[0210] In one example, the compositions and / or nucleic acid molecules of the present application are provided with instructions (protocols) for administering the compositions or nucleic acid molecules to a subject suffering from or at risk of developing a tumor, pathogen, or immune disorder. The instructions typically include information regarding the use of the composition to treat and / or prevent a tumor or a pathogenic infection. In one example, the instructions include at least one of the following: description of the therapeutic agent; dosing schedule and administration for the treatment or prevention of a tumor, a pathogenic infection, or an immune disorder, or a symptom thereof; precautions; warnings; indications; contraindications; drug information; side effects; animal pharmacology; clinical studies; and / or references. These instructions can be printed directly on the container, printed as a label affixed to the container, or provided as a separate sheet, booklet, card, or folder in or with the container.

[0211] 11. Therapeutic or pharmaceutical uses In this application, the methods described in this application can also be interpreted as therapeutic applications. That is, the methods described in this application can be considered as therapeutic applications of the compositions, nucleic acid molecules or engineered cells of this application, or as applications for preparing a medicament corresponding to a therapeutic application. In one example, this application relates to the use of said engineered cells or nucleic acid molecules for regulating the activity of an engineered cell, or the use of said engineered cells for preparing a medicament for regulating the activity of an engineered cell. This application relates to the use of said engineered cells or nucleic acid molecules for activating an engineered cell, or the use of said engineered cells for preparing a medicament for activating an engineered cell. This application relates to the use of said engineered cells or nucleic acid molecules for inhibiting the activity of a target cell in a test subject, or for preparing a medicament for inhibiting the activity of a target cell in a test subject. This application relates to the use of said engineered cells or nucleic acid molecules for improving or treating a health condition of a test subject in need, or for preparing a medicament for improving or treating a health condition of a test subject in need. All of the above in this application can also be applied to therapeutic applications or medicament preparation applications.

[0212] This application is, for example, related to Chinese patent application publication numbers CN107058354A, CN107460201A, CN105194661A, CN105315375A, CN105713881A, CN106146666A, CN106519037A, CN106554414A, CN105331585A, CN106397593A, CN106467573A, CN104140974A, CN108884459A, C N107893052A, CN108866003A, CN108853144A, CN109385403A, CN109385400A, CN109468279A, CN109503715A, CN109908176A, CN109880803A, CN110055275A, CN110123837A, CN110438082A, CN110468105A, and for example, International Patent Application Publication No. WO201 7186121A1, WO2018006882A1, WO2015172339A8, WO2018 / 018958A1, WO2014180306A1, WO2015197016A1, WO201600 8405A1, WO2016086813A1, WO2016150400A1, WO2017032293A1, WO2017080377A1, WO2017186121A1, WO2018045811A 1, WO2018108106A1, WO2018 / 219299, WO2018 / 210279, WO2019 / 024933, WO2019 / 114751, WO2019 / 114762, WO2019 / 141270, WO2019 / 149279, WO2019 / 170147A1, WO2019 / 210863, and WO2019 / 219029, including CAR-T cells, methods for preparing the same, and antibodies therefor. EXAMPLES

[0213] Example 1: Construction of chimeric polypeptides synJagged2EC, synEphrinB2EC, synEphrinB2EC-APLP2(TM) In this example, various exemplary chimeric polypeptide compositions were constructed for use in the present application (Table 1). TIFF2024523636000001.tif208170

[0214] For example, the fragment sequences in Table 1 above are as follows: antiGPC3-scFv (SEQ ID NO: 42), antiEGFRvIII-scFv (SEQ ID NO: 43), antiMesothelin-scFv (SEQ ID NO: 44), antiFAP-scFv (SEQ ID NO: 45), antiClaudin18.2-scFv (SEQ ID NO: 46), antiCLL1-scFv (SEQ ID NO: 47), Jagged2 extracellular domain (SEQ ID NO: 1, 2), EphrinB2 extracellular domain (SEQ ID NO: 3, 4), EphrinB2-del1 extracellular domain (SEQ ID NO: 5, 6), EphrinB2-del2 extracellular domain (SEQ ID NO: 7, 8), EphrinB2-del3 extracellular domain (SEQ ID NO: 9, 10), EphrinB2-del23 extracellular domain (SEQ ID NO: 11, 12), mouse Notch1 (Notch1STS) transmembrane domain (SEQ ID NO: 13), mouse Notch2 transmembrane domain (Notch2STS) (sequence No. 14), mouse Notch3 transmembrane domain (Notch3STS) (sequence number 15), mouse Notch4 transmembrane domain (Notch4STS) (sequence number 16), human Notch1 transmembrane domain (Notch1STS) (sequence number 17), human Notch2 transmembrane domain (Notch2STS) (sequence number 18), human Notch3 transmembrane domain (Notch3STS) (sequence number 19), human Notch4 transmembrane domain (Notch4STS) (sequence number 20), APLP2 transmembrane domain (APLP2STS) (sequence number 21), Notch1STS (sequence number 22), Notch2STS (sequence number 23), Notch3STS (sequence number 24), Notch4STS (sequence number 25), APLP2 STS (sequence number 26), GAL4-VP64 (sequence number 27), where mouse Notch is represented by the English symbol Notch, and human Notch is represented by the English symbol huNotch.

[0215] The chimeric polypeptides were introduced into Jurkat cells (purchased from the Cell Bank of the Chinese Academy of Sciences), and the expression was detected by flow cytometry using biotin-labeled antigen polypeptide + PE-labeled streptavidin. The results showed that all of the above polypeptides were stably expressed on the cell membrane.

[0216] In this example, BFP (genebank ID: QJR97815.1) was inserted into the Gal4-VP64-controlled UAS-CMV promoter (SEQ ID NO: 49) in Table 1 to form a reporter gene activated by the chimeric polypeptide. In this example, green fluorescent protein (GFP) regulated by the pGK promoter (Addgene #79120, 7721-8220 bp) was inserted downstream of BFP as a positive transfer marker. The GFP protein sequence is shown in genebank ID: UDY80669.1. In this example, a UAS-BFP-PGK-GFP fragment consisting of the nucleic acid sequences UAS-CMV, BFP, pGK, and GFP in this order was introduced into Jurkat cells to construct Jurkat-responsive cells.

[0217] Example 2: synJagged2EC, synEphrinB2EC, synEphrinB2EC-APLP2(TM) regulate gene expression Jurkat responder cells transfected with each chimeric peptide targeting GPC3 were mixed with hepatoma cells at a 1:1 ratio (SK-Hep1 is GPC3 negative, SK-Hep1-GPC3 is exogenously overexpressed GPC3 protein, HuH7 and PLC / PRF / 5 are GPC3 positive), co-incubated for 24 hours, and BFP was detected by flow cytometry. Figures 1A and 1B (numbers represent the percentage of BFP-positive cells) show that after co-incubation with target cells, synJagged2EC (SEQ ID NO: 51), synEphrinB2EC (SEQ ID NO: 52), and synEphrinB2EC-APLP2(TM) (SEQ ID NO: 53) induce equivalent or significantly increased BFP expression levels compared to synNotch (modified from plasmid Addgene#79125, replacing the anti-CD19 scFv sequence with the anti-GPC3-scFv sequence).

[0218] Example 3: Antigen co-incubation experiments Coating group: GPC3 antigen (5 μg / mL, overnight at 4°C) was pre-coated on 96-well plates, and Jurkat responder cells expressing chimeric polypeptides targeting GPC3 were plated at 50,000 cells per well. Lysis group: Jurkat responder cells expressing chimeric peptides targeting GPC3 were plated at 50,000 cells per well, and GPC3 antigen (5 μg / mL) was added directly to the medium. BFP was detected by flow cytometry at 0, 4, 8, 24, and 32 hours after co-incubation of cells with antigen. Figure 2 shows that the expression of BFP in cells of each group was comparable after co-incubation with coated antigen. BFP was barely detectable in cells incubated with lysed antigen.

[0219] Example 4. SynEphrinB2EC-del, which contains a truncated form of the EphrinB2 extracellular domain, regulates gene expression With reference to Examples 1 and 2, Jurkat responder cells expressing GPC3-synEphrinB2EC-del1 (SEQ ID NO: 54), GPC3-synEphrinB2EC-del2 (SEQ ID NO: 55), GPC3-synEphrinB2EC-del3 (SEQ ID NO: 56), and GPC3-synEphrinB2EC-del23 (SEQ ID NO: 57) were constructed, and BFP expression was detected after GPC3 stimulation. Figure 3 shows that chimeric polypeptides targeting GPC3, including full-length EphrinB2 extracellular domain or its truncated form, all regulate gene expression. When expression was induced by synEphrinB2EC-del3 or synEphrinB2EC-del23, leakage was significantly reduced.

[0220] Example 5. Response of synEphrinB2EC and synEphrinB2EC-APLP2(TM) to antigen stimulation at different concentrations Biotin-labeled GPC3 antigen polypeptide was dissolved in PBS buffer (10 μg / mL) and diluted 2-fold with PBS to a concentration of 0.019531 μg / mL. 50 μL of each concentration was added to a streptavidin-coated 96-well plate (Thermo 15500) and incubated at room temperature for 2 hours for GPC3 antigen coating.

[0221] Forty thousand Jurkat responder cells expressing a chimeric peptide targeting GPC3 were added to wells coated with various concentrations of GPC3 antigen, cultured at 37°C for 24 hours, and BFP was detected by flow cytometry (Figure 4).

[0222] Example 6: synJagged2EC, synEphrinB2EC regulate IL12 expression IL12 was inserted downstream of the UAS-CMV promoter, and GFP controlled by the pGK promoter was linked downstream of IL12. The resulting fragment UAS-IL12-PGK-GFP was introduced into T cells simultaneously or consecutively with the chimeric polypeptide targeting GPC3, and then mixed separately with liver cancer cells at a 1:1 ratio and co-incubated for 24 hours, and IL12 in the culture supernatant was detected by ELISA. Figure 5 shows that after co-incubation with target cells, synJagged2EC and synEphrinB2EC induced equivalent or increased IL12 expression compared to synNotch. Combined with Figure 6, it can be seen that transcription induced by synEphrinB2EC was improved after incubation with GPC3 low-expressing cells.

[0223] Example 7. Single vector system of synEphrinB2EC, synEphrinB2EC-APLP2(TM) regulating IL12 expression In Example 6, the chimeric polypeptide and the regulated expression gene are placed in two different vectors, and double viral infection is required to achieve the regulation of the target gene by the chimeric polypeptide. To simplify the operation and improve the transduction efficiency, IL12 was inserted downstream of the UAS-CMV promoter and incorporated into the chimeric polypeptide expression vector to transduce T cells. Figure 7 shows that the single vector system expresses synEphrinB2EC and synEphrinB2EC-APLP2(TM) at higher levels than synNotch. Figure 8 combined with Figure 6 shows that after incubation with GPC3 low expressing cells, synEphrinB2EC or synEphrinB2EC-APLP2(TM) induced higher transcription than synNotch. FIG. 9 shows that after incubation with GPC3 low expressing cells, the induced expression level of synEphrinB2EC-del3 was low, whereas after incubation with GPC3 high expressing cells, its induced expression level was comparable to that of synNotch, indicating that synEphrinB2EC-del3 could more effectively discriminate between low and high antigen expression.

[0224] Example 8. GPC3-synEphrinB2ECs modulate IL12 expression and synergize with CAR-T cells to treat tumors This is an in vivo antitumor experiment using GPC3-synEphrinB2EC T cells that regulate IL12 expression and T cells expressing GPC3-CAR (sequence number 60) prepared in Example 7.

[0225] NPG mice were subcutaneously inoculated with PLC / PRF / 5 hepatoma cells, and after tumor formation, they were divided into groups according to the diagram, the corresponding number of cells were administered, and the tumor volume and body weight were measured. Figure 10 shows that synEphrinB2ECs regulating IL12 expression exert a synergistic antitumor effect with GPC3-CAR-T cells.

[0226] Example 9: EGFRvIII-synEphrinB2EC regulates gene expression Jurkat responder cells containing a chimeric polypeptide targeting EGFRvIII (SEQ ID NO: 61) were co-incubated with glioma cells at a 1:1 ratio for 24 hours and BFP was detected by flow cytometry (FIG. 11).

[0227] Example 10. CAR-T cells containing synEphrinB2EC that regulate CAR expression IL13Ra2-CAR (SEQ ID NO: 62) was inserted downstream of the UAS-CMV promoter and incorporated into a chimeric polypeptide expression vector, and transduced into T cells to generate T cells that regulate the expression of IL13Ra-CAR with a chimeric polypeptide targeting EGFRvIII (Figure 12) and were cultured in vitro for 14 days. CAR expression and phosphorylation levels were detected by Western blot (Figure 13); cells were labeled with CD25, CD69, PD1, LAG3, TIM3 and CD39, CD45RA, CD62L antibodies, respectively, and detected by flow cytometry (Figures 14, 15).

[0228] Example 11. In vivo and in vitro killing of T cells containing EGFRvIII-synEphrinB2EC regulating IL13Ra2-CAR expression Target cells: U87, U87-10%EGFRvIII(90%U87+10%U87-EGFRvIII), U87-50%EGFRvIII(50%U87+50%U87-EGFRvIII), U 251, U251-10%EGFRvIII(90%U251+10%U251-EGFRvIII), U251-50%EGFRvIII(50%U251+50%U251-EGFRvIII).

[0229] The effector-to-target ratios were 3:1, 1:1, and 1:3, and the killing of target cells was monitored in real time by detecting supernatant LDH or xCELLigence RTCA. Figure 16 shows that IL13Ra2-CAR-T cells expressing EGFRvIII-synEphrinB2EC can kill tumors with EGFRvIII antigen heterogeneity and also have a killing effect on tumor cell populations with low EGFRvIII positivity.

[0230] U251-EGFRvIII cells were subcutaneously inoculated into NPG mice, and after tumor formation, they were divided into groups according to the figure, administered the corresponding number of CAR-T cells, and measured the tumor volume and body weight. Figure 17 shows the in vivo antitumor effect of IL13Ra2-CAR-T expressing EGFRvIII-synEphrinB2EC.

[0231] Example 12. In vivo killing of B7H3-CAR-T cells expressing EGFRvIII-synEphrinB2EC chimeric polypeptides T cells expressing B7H3-28Z (SEQ ID NO: 64), B7H3-BBZ (SEQ ID NO: 65), and 376.96-28Z (SEQ ID NO: 66) were constructed, respectively. T cells containing EGFRvIII-synEphrinB2EC regulating B7H3-28Z were constructed with reference to Example 10.

[0232] U251-EGFRvIII cells were subcutaneously inoculated into NPG mice, and after tumor formation, the mice were divided into groups according to the diagram, the corresponding number of cells were administered, and the tumor volume and body weight were measured. Figure 18 shows that 376.96-28Z-T cells that only recognize human B7H3 have a significant antitumor effect, but the therapeutic efficacy of B7H3-28Z CAR-T and B7H3-BBZ CAR-T that recognize human and mouse B7H3 is low. By placing the low-specificity B7H3-28Z under the control of EGFRvIII-synEphrinB2EC, its specific killing effect can be improved.

[0233] Example 13. In vitro killing of Claudin18.2-CAR-T cells expressing mesothelin-synEphrinB2EC T cells expressing mesothelin-targeting chimeric polypeptide (SEQ ID NO: 105) to regulate the expression of Claudin18.2-CAR (SEQ ID NO: 67), or T cells regulating the co-expression of Claudin18.2-CAR with IL7 and CCL21 were prepared with reference to Examples 1 and 10. Here, the positive rates of mesothelin-synEphrinB2EC regulating the expression of Claudin18.2-CAR (SEQ ID NO: 68), Claudin18.2-CAR and IL7 (SEQ ID NO: 69), and Claudin18.2-CAR and IL7 and CCL21 (SEQ ID NO: 70) were 40.1%, 31.1%, and 33.8%, respectively. Co-cultured according to different effector-to-target ratios, and LDH was detected in the supernatant. Figures 19 and 20 show that both second and fourth generation Claudin18.2-CAR-T cells expressing mesothelin-synEphrinB2EC can be specifically activated by antigen and kill tumor cells.

[0234] Example 14. In vivo killing of Claudin18.2-CAR-T cells expressing FAP-synEphrinB2EC T cells containing FAP-synEphrinB2EC (SEQ ID NO: 106) regulating Claudin18.2-CAR (SEQ ID NO: 67) were constructed with reference to Examples 1 and 10. After tumor formation, pancreatic cancer PDX models were grouped according to the diagram, and the corresponding number of CAR-T cells were administered, and tumor volume and body weight were measured. Figure 21 shows that Claudin18.2-CAR-T cells expressing FAP-synEphrinB2EC are antitumor, and the body weight of mice is not significantly reduced.

[0235] Example 15. In vitro killing of mesothelin-CAR-T cells expressing Claudin18.2-synEphrinB2EC T cells containing Claudin18.2-synEphrinB2EC (SEQ ID NO: 108) for regulating mesothelin-CAR (SEQ ID NO: 107) were constructed with reference to Examples 1 and 10. The effector-to-target ratios were co-cultured at 3:1, 1:1, and 1:3, respectively, and LDH was detected in the supernatant. Figures 22A and 22B show that mesothelin-CAR-T cells containing Claudin18.2-synEphrinB2EC can be specifically activated by antigen and kill tumor cells.

[0236] Example 16. CLL1-synEphrinB2EC regulates gene expression Referring to Examples 1 and 2, Jurkat responder cells containing chimeric polypeptides targeting CLL1 (sequence numbers 73 and 110) were constructed and co-incubated 1:1 with AML cell lines (Figure 23A) for 24 hours, and BFP was detected by flow cytometry (Figure 23B).

[0237] Example 17. In vitro killing of NKG2D-CAR-T cells expressing CLL1-synEphrinB2EC With reference to Example 10, T cells containing CLL1-synEphrinB2EC (SEQ ID NO: 73) regulating NKG2D-CAR (SEQ ID NO: 109) were constructed. They were co-cultured according to different effector-to-target ratios, and LDH was detected in the supernatant. The results show that NKG2D-CAR-T cells kill THP1 and HL-60 cells expressing NKG2D ligand, and CD3Z-NKG2D-CAR-T cells induced by CLL1-synEphrinB2EC have a weak killing effect on THP-1 cells with low CLL1 expression, and can only kill HL-60 cells with high expression of both CLL1 and NKG2D ligand (Figures 23A and 24). This result is consistent with the experiment of BFP-induced expression, indicating that CLL1-synEphrinB2EC further enhances the specificity of NKG2D-CAR-T cells targeting AML cells.

[0238] Example 18. Proliferation and activity of NKG2D-CAR-T cells cultured in vitro In vitro cultured CAR-T cells were collected for cell counting and viability measurement. Figure 25 shows that T cells containing NKG2D-CAR regulated by CLL1-synEphrinB2EC proliferated well.

[0239] The CAR-T cells cultured in vitro were collected, labeled with antibodies, stained with 7-AAD, and detected by flow cytometry. Figure 26 shows that the mortality rate of T cells containing NKG2D-CAR regulated by CLL1-synEphrinB2EC is basically the same as that of UTD, while the mortality rate of NKG2D-CAR-T cells is significantly increased, indicating that T cells containing NKG2D-CAR regulated by CLL1-synEphrinB2EC can effectively ensure the proliferation and activity of T cells in the culture system.

[0240] Example 19. Chimeric polypeptides targeting other AML therapeutic targets induce NKG2D-CAR expression Referring to Examples 16-18, taking CD123 as an example, T cells were constructed in which NKG2D-CAR (SEQ ID NO: 109) was regulated by CD123-synEphrinB2EC chimeric polypeptide (SEQ ID NO: 50), which can enhance the specificity of NKG2D-CAR-T cells targeting AML cells and effectively ensure the proliferation and activity of T cells in the culture system.

[0241] Example 20. Chimeric polypeptides induce expression of therapeutic monoclonal antibodies The genes whose expression is regulated by the chimeric polypeptides GPC3-synJagged2EC, GPC3-synEphrinB2EC, and GPC3-synEphrinB2EC-APLP2(TM) of the present application were replaced with coding sequences for trastuzumab, bevacizumab, infliximab, rituximab, and adalimumab, and when co-incubated with GPC3-positive tumor cells, synJagged2EC, synEphrinB2EC, synEphrinB2EC-APLP2(TM) induced comparable or significantly increased expression levels of trastuzumab, bevacizumab, infliximab, rituximab, and adalimumab compared to synNotch.

[0242] The embodiments described herein include this embodiment as any single embodiment or in combination with any other embodiment or part thereof. Furthermore, after reading the above teachings of this application, a person skilled in the art may make various changes or modifications to this application, and these equivalents are also included in the scope defined by the appended claims of this application. TIFF2024523636000002.tif250170TIFF2024523636000003.tif246170TIFF2024523636000004.tif252170TIFF2024523636000005.tif250170TIFF2024523636000006.tif250170TIFF2024523636000007.tif250170TIFF2024523636000008.tif250170TIFF2024523636000009.tif250170TIFF2024523636000010.tif250170TIFF2024523636000011.tif250170TIFF2024523636000012.tif250170TIFF2024523636000013.tif250170TIFF2024523636000014.tif250170TIFF2024523636000015.tif250170TIFF2024523636000016.tif250170TIFF2024523636000017.tif251170TIFF2024523636000018.tif250170TIFF2024523636000019.tif250170TIFF2024523636000020.tif244170TIFF2024523636000021.tif148170

Claims

1. a) a binding domain capable of specifically binding to a target molecule; b) a receptor modulating domain comprising one or more cleavage sites; c) an intracellular domain; A chimeric polypeptide comprising: the receptor modulating domain comprises an extracellular region and a transmembrane region, and the extracellular region and the transmembrane region are not simultaneously derived from a Notch protein; Binding of the binding domain to the target molecule can induce cleavage of the receptor modulating domain, thereby releasing the intracellular domain; Preferably, the extracellular domain is selected from the group consisting of Jagged2, EphrinB2, APLP1, APLP2, APP, CD44, CSF1R, CXCL16, CX3CL1, Delta1, E-cadherin, EphB2, EphrinB1, growth hormone receptor, HLA-A2, IFNaR2, IL1R2, L1, LRP, LRP2, LRP6, N-cadherin, nectin1α, NRADD, p75-NTR, Pcdh α4, Pcdh The present invention is characterized in that it comprises an extracellular domain derived from gamma-C3, PTPκ, PTP-LAR, SorCS1b, SorLA, sortilin, ApoER2, PKHD1, ErbB4, IFNaR2, VEGF-R1, or VLDLR, or a combination thereof, or a fragment or a combination thereof of the extracellular domain of any of the above proteins, or a mutant or a combination thereof of the extracellular domain of any of the above proteins, or a truncated structure or a combination thereof of the extracellular domain of any of the above proteins, Chimeric polypeptides.

2. The transmembrane region further comprises a stop transfer sequence (STS); 2. The chimeric polypeptide of claim 1, characterized in that the STS comprises an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 22, 23, 24, 25, and 26.

3. the binding domain comprises an antigen binding domain capable of binding to a target molecule on the surface of a target cell, or the binding domain comprises a ligand portion capable of binding to a target molecule; The chimeric polypeptide according to claim 1, characterized in that the target molecule is selected from the group consisting of differentiation marker clusters, cell surface receptors, adhesion proteins, integrins, mucins, lectins and tumor antigens.

4. the target cell is a pathogen or the target cell is a human cell; The chimeric polypeptide according to claim 3, characterized in that the human cell is preferably a tumor cell.

5. the antigen-binding domain is selected from the group consisting of an antibody, a receptor, a cell adhesion molecule, a non-antibody molecular scaffold, or a combination thereof; Preferably, the antibody is a single domain antibody, a single chain antibody, a diabody, a triabody, a minibody, a F(ab') ２ Fragment, F(ab) ｖ The chimeric polypeptide according to claim 1, characterized in that it is a fragment, a scFv, a single domain antibody (sdAb) and functional fragments thereof or a combination thereof.

6. The antigen binding domain may be selected from the group consisting of mesothelin, FAP, Claudin18.2, CLL1, CD19, GPC3, WT1, EGFR, BCMA, CD7, NKG2D-ligand, CD19, B7H3, ALPPL2, CD123, CD171, CD179a, CD20, CD213A2, CD22, CD24, CD246, CD272, CD30, CD33, CD38, CD44v6, CD46, CD71, CD97, CEA, CLDN6, CLECL1, CS-1, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, FLT3, GD2, GD3, GM3, GPRC5D, HER2 (ERBB2), IGLL1, IL 4. The chimeric polypeptide of claim 3, which specifically binds to a tumor antigen selected from the group consisting of IL11Ra, IL13Ra2, CD 117, MUC1, NCAM, PAP, PDGFR-b, PRSS21, PSCA, PSMA, ROR1, SIRPa, SSEA-4, TAG72, TEM1 / CD248, TEM7R, TSHR, VEGFR2, ALPI, cMet, and Axl.

7. the transmembrane region of the receptor modulating domain comprises a γ-secretase cleavage site; Preferably, the transmembrane region of the receptor modulating domain comprises a Notch transmembrane region, More preferably, the chimeric polypeptide of claim 1, characterized in that the transmembrane region of the receptor modulating domain comprises a Notch1, Notch2, Notch3 or Notch4 transmembrane region.

8. the intracellular domain comprises a transcription factor, a site-specific nuclease, a recombinase, an inhibitory immunoreceptor, an activating immunoreceptor, or a combination thereof; 2. The chimeric polypeptide according to claim 1, characterized in that the transcription factor is selected from Gal4-VP16, Gal4-VP64, tetR-VP64, ZFHD1-VP64, Gal4-KRAB, HAP1-VP16, or a combination thereof.

9. the extracellular region of the receptor modulating domain comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 3, 5, 7, 9, and 11; and / or the transmembrane region of the receptor modulating domain comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 102, 103, and 104; Preferably, the receptor modulating domain comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 85; the chimeric polypeptide comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 50, 51, 52, 53, 54, 55, 56, 57, 61, 73, 87, 105, 106, 108, or 110; The chimeric polypeptide of claim 1.

10. 10. An engineered cell comprising the chimeric polypeptide of any one of claims 1 to 9, Preferably, the engineered cell is an immune cell, a neuron, an epithelial cell, an endothelial cell or a stem cell; Preferably, the immune cells are B cells, monocytes, natural killer cells, basophils, eosinophils, neutrophils, dendritic cells, macrophages, regulatory T cells, helper T cells, cytotoxic T cells, other T cells, or combinations thereof. Engineered cells.

11. further comprising an expression cassette encoding a foreign gene operably linked to the intracellular domain of the chimeric polypeptide, wherein the intracellular domain of the chimeric polypeptide regulates expression of the foreign gene; The engineered cell of claim 10, characterized in that the exogenous gene is preferably expressed under the control of a promoter regulated by GAL-4, tetR, ZFHD1, HNF1A, or HAP1.

12. the exogenous gene expression product is selected from a non-coding RNA, a cytokine, a cytotoxin, a chemokine, an immunomodulatory factor, a pro-apoptotic factor, an anti-apoptotic factor, a hormone, a differentiation factor, a de-differentiation factor, a modified TCR, a CAR, a reporter gene, or a combination thereof; Preferably, the binding domain of the chimeric polypeptide specifically binds to a first target molecule, the exogenous gene expression product is a CAR, the CAR specifically binds to a second target molecule different from the first target molecule, and the first target molecule and the second target molecule are mesothelin, FAP, Claudin18.2, CLL1, CD19, GPC3, WT1, EGFR, BCMA, CD7, NKG 2D-ligand, MOG, CD19, B7H3, ALPPL2, CD123, CD171, CD179a, CD20, CD213A2, CD22, CD24, CD246, CD272, CD30, CD 33, CD38, CD44v6, CD46, CD71, CD97, CEA, CLDN6, CLECL1, CS-1, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, Ephrin B2, FAP, FLT3, GD2, GD3, GM3, GPRC5D, HER2 (ERBB2), IGLL1, IL 11Ra, IL13Ra2, CD 117, MUC1, NCAM, PAP, PDGFR-b, PRSS21, PSCA, PSMA, ROR1, SIRPa, SSEA-4, TAG72, TEM1 / CD248, TEM7R, TSHR, VEGFR2, ALPI, cMet, and Axl; More preferably, the engineered cell of claim 11 is characterized in that the genes encoding the chimeric peptide and the CAR are present in the same expression vector.

13. The first target molecule and the second target molecule are each selected from the combinations of ASGR1 and GPC3, EGFRvIII and IL13Ra2, EGFRvIII and B7H3, mesotheli and Claudin18.2, Claudin18.2 and mesotheli, FAP and Claudin18.2, CLL1 and NKG2D, and CD123 and NKG2D; Preferably, the chimeric polypeptide and the CAR comprise the sequences shown in SEQ ID NOs: 61 and 62, or the sequences shown in SEQ ID NOs: 61 and 64, or the sequences shown in SEQ ID NOs: 61 and 65, or the sequences shown in SEQ ID NOs: 61 and 66, or the sequences shown in SEQ ID NOs: 105 and 67, or the sequences shown in SEQ ID NOs: 106 and 67, or the sequences shown in SEQ ID NOs: 108 and 107, or the sequences shown in SEQ ID NOs: 73 and 109, or the sequences shown in SEQ ID NOs: 50 and 109, respectively.

16. The engineered cell of claim 15.

14. 13. The engineered cell of claim 12, wherein the cytokine is IL-2, IL-7, IL-9, IL-12, IL-15, IL-18, CCL21, or a combination of the above cytokines.

15. The engineered cell of claim 12, characterized in that the engineered cell comprises a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 59, 63, 68, 69, 70, 71, 72, 74, and 88, or its translated amino acid sequence.

Images