Signal transduction receptors and their uses

Abstract

This invention provides signal transduction receptors and their applications. Signal transduction receptors comprise an extracellular domain of a receptor for immunosuppressive cytokines, or a fragment or mutant thereof retaining the function of specifically binding immunosuppressive cytokines; a transmembrane region or a mutant thereof; and an intracellular domain of a co-stimulatory signaling molecule, or a fragment or mutant thereof retaining the biological function of the co-stimulatory signaling molecule in transmitting co-stimulatory signals and activating immune cells. Immune cells modified with this signal transduction receptor, upon contact with immunosuppressive molecules on the surface of tumor cells and / or tumor stromal cells, are not negatively affected by these molecules. Instead, they exhibit immune activation, enhancing the proliferation and cytokine secretion of immune cells, and prolonging the survival time of activated immune cells. This overcomes the side effects of the tumor immunosuppressive microenvironment on effector cells in adoptive cell therapy for tumors.

Description

[0001] This application claims priority to Chinese Patent Application No. 202010485006.8, filed on June 1, 2020, entitled “Signal Conversion Receptor and Its Use Therein”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of biotechnology, and more specifically to a signal transduction receptor and its uses. Background Technology

[0003] In adoptive cell therapy (ACT) for tumors, the stability of the immune system is crucial. An underreaction can lead to severe infection, while an overreaction can cause allergic reactions. The human immune system has evolved a sophisticated and complex bidirectional immunomodulatory mechanism that regulates the immune response in both positive and negative ways. It activates and strengthens the immune response when eliminating foreign antigens; conversely, it weakens or terminates the immune response after the elimination of foreign antigens. In tumor ACT, positive regulatory molecules for effector T cells include the CD3 / TCR complex, CD28, CD134 (OX40), and CD137 (4-1BB), while negative regulatory molecules include PD-1 (PDCD1, CD279), CTLA-4 (CD152), LAG3 (CD223), and TIM3 (HAVCR2). However, the tumor microenvironment (TME) contains many factors that negatively regulate the T cell immune response, allowing tumor cells to evade the body's immune system's surveillance and clearance, and to continuously proliferate, invade, and metastasize.

[0004] TGF-β has a suppressive effect on the systemic immune system and inhibits the host's immune surveillance. TGF-β plays an important role in tumor initiation and progression. It has tumor-promoting effects, is typically produced in large quantities in many types of tumors and is known to have oncogenic effects, and it negatively impacts tumor immunity and significantly suppresses the host's tumor immune surveillance. TGF-β significantly and directly inhibits the cytotoxic program of cytotoxic T lymphocytes (CTLs) by inhibiting the transcription of genes encoding several key proteins, including at least perforin, granzyme A, granzyme B, Fas ligand, and γ-interferon. Simultaneously, TGF-β also significantly affects the differentiation and function of CD4+ T cells.

[0005] In recent years, several reports have described constructing novel fusion receptors by linking the extracellular regions of corresponding receptors with the transmembrane and intracellular regions of positive co-stimulatory signaling molecules to negatively regulate inhibitory signaling pathways in immune effector cells. These fusion receptors, when expressed in immune effector cells, function as signal transducers, converting immunosuppressive signals within the tumor microenvironment into immune-activating signals. Specifically, the extracellular polypeptide of the signal transducer receives immunosuppressive molecular signals from the surface of tumor cells, tumor stromal cells, or the tumor microenvironment and transmits them intracellularly. The intracellular region of the co-stimulatory signaling molecule activates the second signal of immune cells, thereby enhancing the proliferation and cytokine secretion of immune effector cells and prolonging their survival time. CN103965361B discloses a chimeric molecular transducer for T cell signals, comprising the extracellular region of PD1 (or a fusion protein of the extracellular region of PD1 and HERIN), the transmembrane region of CD28, and the intracellular co-stimulatory domain of CD28 (or 4-1BB). Tumor-infiltrating lymphocytes (TILs) expressing this T-cell signaling chimeric transducer exhibit significantly enhanced proliferation and killing effects on tumor target cells. CN105452287A discloses an immunosuppressive TGF-β signal transducer, which is a chimera comprising an extracellular domain of the TGF-β receptor and an intracellular domain from another molecule, such that the binding of TGF-β to the extracellular domain leads to the intracellular domain stimulating T-cell activity.

[0006] To stimulate the proliferation of immune effector cells and enhance their tumor immunity, more methods, including signal transducer molecules, are still needed to modify and activate immune effector cells. Summary of the Invention

[0007] The present invention provides an isolated fusion protein comprising: an extracellular domain of a receptor for an immunosuppressive cytokine or a functional fragment thereof or a mutant thereof that retains the specific binding of immunosuppressive cytokines; a transmembrane region thereof or a mutant thereof; and an intracellular domain of a costimulatory signaling molecule thereof or a functional fragment thereof or a mutant thereof that retains the biological function of the costimulatory signaling molecule in transmitting costimulatory signals and activating immune cells.

[0008] In one or more embodiments, the immunosuppressive cytokines include tumor cell or tumor stromal cell membrane proteins and tumor cell or tumor stromal cell secreted proteins.

[0009] In one or more embodiments, the tumor cell or tumor stromal cell membrane proteins include PDL1 / PDL2, FasL, B7-H4, Crry, and HLA-G.

[0010] In one or more embodiments, the tumor cell or tumor stromal cell secreted proteins include IL13, IL4, TGF-β, IL6, IL8, IL-10, CCL21, IDO, and VEGF.

[0011] In one or more embodiments, the receptors for TGF-β include TGF-β receptor 1, TGF-β receptor 2 and TGF-β receptor 3, preferably TGF-β receptor 1 and TGF-β receptor 2.

[0012] In one or more embodiments, the co-stimulatory signaling molecules include one or more of CD28, CD134 (OX40), CD137 (4-1BB), LCK, ICOS, DAP10, siglec-9, siglec-10, siglec-15, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-R2, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD27, and CD40.

[0013] In one or more embodiments, the transmembrane region includes any one or more transmembrane regions from CD28, CD8, CD134 (OX40), CD137 (4-1BB), LCK, ICOS, DAP10, siglec-9, siglec-10, siglec-15, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2Rbeta, IL-2Rgamma, IL-4Ralpha, IL-7Ralpha, IL-10R, IL-12R, IL-15R, IL-21R, CD27, and CD40.

[0014] In one or more embodiments, the transmembrane region is derived from the IL-7Ralpha transmembrane region or a mutant thereof.

[0015] In one or more embodiments, the amino acid sequence of the mutant of the IL-7Ralpha transmembrane region is as shown in any of SEQ ID NO:29-60.

[0016] In one or more embodiments, the coding sequence of the mutant of the IL-7Ralpha transmembrane region is shown in any of SEQ ID NO: 61-94.

[0017] In one or more embodiments, the extracellular region of the fusion protein is the extracellular region of TGF-β receptor 1, the transmembrane region is derived from the IL-7Ralpha transmembrane region or a mutant thereof, and the intracellular domain of the co-stimulatory molecule of the fusion protein is selected from any one of the following: IL-7Ralpha intracellular region, CD28 intracellular region tandem with 4-1BB intracellular region, CD28 intracellular region, 4-1BB intracellular region, CD28 intracellular region tandem with OX40 intracellular region, 28-IL2RB-z(YXXQ) intracellular region, 28-DeltaIL2RB-z(YXXQ) intracellular region, IL2RB-z(YXXQ) intracellular region, and DeltaIL2RB-z(YXXQ) intracellular region.

[0018] In one or more embodiments, the amino acid sequence of the fusion protein is any one of SEQ ID NO:95-350.

[0019] In one or more embodiments, the extracellular region of the fusion protein is the extracellular region of TGF-β receptor 2, the transmembrane region is derived from the IL-7Ralpha transmembrane region or a mutant thereof, and the intracellular domain of the co-stimulatory molecule of the fusion protein is selected from any one of the following: IL-7Ralpha intracellular region, CD28 intracellular region tandem with 4-1BB intracellular region, CD28 intracellular region, 4-1BB intracellular region, CD28 intracellular region tandem with OX40 intracellular region, 28-IL2RB-z(YXXQ) intracellular region, 28-DeltaIL2RB-z(YXXQ) intracellular region, IL2RB-z(YXXQ) intracellular region, and DeltaIL2RB-z(YXXQ) intracellular region.

[0020] In one or more embodiments, the amino acid sequence of the fusion protein is any one of SEQ ID NO:351-606, SEQ ID NO:1647, SEQ ID NO:1649, SEQ ID NO:1651, SEQ ID NO:1653, SEQ ID NO:1655, SEQ ID NO:1657, SEQ ID NO:1659, SEQ ID NO:1661, SEQ ID NO:1663, SEQ ID NO:1665, SEQ ID NO:1667, SEQ ID NO:1669, SEQ ID NO:1671, SEQ ID NO:1673, and SEQ ID NO:1675.

[0021] In one or more embodiments, the extracellular region of the fusion protein is the extracellular region of TGF-β receptor 3, the transmembrane region is derived from the IL-7Ralpha transmembrane region or a mutant thereof, and the intracellular domain of the co-stimulatory molecule of the fusion protein is selected from any one of the following: IL-7Ralpha intracellular region, CD28 intracellular region tandem with 4-1BB intracellular region, CD28 intracellular region, 4-1BB intracellular region, CD28 intracellular region tandem with OX40 intracellular region, 28-IL2RB-z(YXXQ) intracellular region, 28-DeltaIL2RB-z(YXXQ) intracellular region, IL2RB-z(YXXQ) intracellular region, and DeltaIL2RB-z(YXXQ) intracellular region.

[0022] In one or more embodiments, the amino acid sequence of the fusion protein is any one of SEQ ID NO:607-862.

[0023] The present invention also provides a polynucleotide molecule selected from: a polynucleotide molecule or complementary sequence encoding the fusion protein described in any embodiment of the present invention.

[0024] In one or more embodiments, the polynucleotide molecule is selected from any one of SEQ ID NO:863-1630, SEQ ID NO:1648, SEQ ID NO:1650, SEQ ID NO:1652, SEQ ID NO:1654, SEQ ID NO:1656, SEQ ID NO:1658, SEQ ID NO:1660, SEQ ID NO:1662, SEQ ID NO:1664, SEQ ID NO:1666, SEQ ID NO:1668, SEQ ID NO:1670, SEQ ID NO:1672, SEQ ID NO:1674 and SEQ ID NO:1676, or is a complementary sequence of any of the polynucleotide molecules shown.

[0025] The present invention also provides a nucleic acid construct containing the polynucleotide molecule described in any embodiment of the present invention.

[0026] In one or more embodiments, the nucleic acid construct is a vector.

[0027] In one or more embodiments, the vector is an expression vector; preferably a viral vector.

[0028] The present invention also provides a genetically engineered cell that expresses the fusion protein described in any embodiment of the present invention and / or carries the coding sequence of the fusion protein.

[0029] In one or more embodiments, the cells are immune cells.

[0030] In one or more embodiments, the immune cells are T cells.

[0031] In one or more embodiments, the cell also expresses CAR or carries the coding sequence of CAR.

[0032] In one or more embodiments, the cell also expresses an exogenous TCR or carries a coding sequence for an exogenous TCR.

[0033] The present invention also provides the use of the fusion proteins, polynucleotide molecules, nucleic acid constructs and genetically engineered cells described in any embodiment of the present invention in the preparation of medicaments for the treatment or prevention of cancer. Attached Figure Description

[0034] Figure 1 Fluorescence micrographs of TCR-T cells electroporated with the TCR-EGFP fusion gene and SCR1-5 TCR-T cells co-transfected with the TCR-EGFP fusion gene + TGF-β signal transduction receptor gene.

[0035] Figure 2 In vitro killing effect of SCR1-5 TCR-T, RIID 2 TCR-T and TCR-T on A375 cells with an effector-to-target ratio of 1.5:1.

[0036] Figure 3 In vitro killing effect of SCR1-5 TCR-T, RIID 4 TCR-T and TCR-T on A375 cells with an effector-to-target ratio of 1.5:1.

[0037] Figure 4 In vitro killing effect of TCR-T, SCR5 TCR-T, and RIID2 TCR-T on A375 cells with an effector-to-target ratio of 1:1.

[0038] Figure 5 In vitro killing effect of TCR-T, SCR5 TCR-T, and RIID4 TCR-T on A375 cells with an effector-to-target ratio of 1:1.

[0039] Figure 6 In vitro killing activity of TCR-T, SCR6 TCR-T, SCR7 TCR-T, SCR8 TCR-T, SCR9 TCR-T and SCR10 TCR-T against A375 cells with an effector-to-target ratio of 1.5:1.

[0040] Figure 7In vitro killing activity of TCR-T, SCR10 TCR-T and RIID2 TCR-T against A375 cells with an effector-to-target ratio of 0.5:1.

[0041] Figure 8 In vitro killing activity of TCR-T, SCR11 TCR-T, SCR12 TCR-T, SCR13 TCR-T, SCR14 TCR-T and SCR15 TCR-T against A375 cells with an effector-to-target ratio of 1.5:1.

[0042] Figure 9 In vitro killing activity of TCR-T, SCR15 TCR-T and RIID4 TCR-T against A375 cells with an effector-to-target ratio of 0.75:1.

[0043] Figure 10 : IFN-γ secretion level of TCR-T, SCR1-5, and TCR-T.

[0044] Figure 11 Graph showing the inhibitory effects of TCR-T, RIID2 TCR-T, RIID4 TCR-T and SCR5 TCR-T on tumor growth after tumor formation in A375 mouse cell line. Detailed Implementation

[0045] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form preferred technical solutions.

[0046] This invention provides immune cells for adoptive cell therapy of tumors, which, after modification, can convert inhibitory signals that inhibit immune cell proliferation into stimulatory signals that enhance immune cell proliferation. In some embodiments, the stimulatory signal further includes a stimulatory signal that enhances antitumor activity.

[0047] In this invention, immune cells have the meaning known in the art, referring to cells that participate in or are related to the immune response, including lymphocytes, dendritic cells, monocytes / macrophages, granulocytes, mast cells, etc. Lymphocytes include T lymphocytes, tumor-infiltrating lymphocytes, B lymphocytes, K lymphocytes, and NK lymphocytes. Immune cells suitable for use in this invention particularly include those commonly used in adoptive cell therapy for tumors.

[0048] The immune cells of this invention express the signal transduction receptor described herein and / or contain the coding sequence of the signal transduction receptor. The signal transduction receptor of this invention is designed to bind inhibitory molecules but deliver positive signals rather than inhibitory signals. That is, these cells convert "braking" signals into "accelerating" signals to improve the antitumor activity of individual immune cells.

[0049] definition

[0050] The following terms are used in this invention. Terms not specifically defined herein have meanings commonly known in the art.

[0051] The term "expression frame" refers to the complete set of elements required to express a gene, including the promoter, the gene coding sequence, and the PolyA tailing signal sequence.

[0052] The term "coding sequence" is defined in this text as the portion of a nucleic acid sequence that directly identifies its protein product (e.g., signal transduction receptor, CAR). The boundaries of a coding sequence are typically defined by the ribosome-binding site (for prokaryotic cells) immediately upstream of the 5' open reading frame of the mRNA and the transcription termination sequence immediately downstream of the 3' open reading frame of the mRNA. Coding sequences can include, but are not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

[0053] The term "co-stimulatory signaling molecule" refers to a molecule present on the surface of antigen-presenting cells that binds to co-stimulatory signaling molecule receptors on Th cells, generating a co-stimulatory signal. These molecules activate the second signaling pathway of immune cells, enhancing their proliferative capacity and cytokine secretion, and prolonging the survival time of activated immune cells. Lymphocyte proliferation requires not only antigen binding but also the reception of signals from co-stimulatory molecules. Co-stimulatory signals are primarily transmitted to T cells through the binding of co-stimulatory molecules CD80 and CD86, expressed on the surface of antigen-presenting cells, to CD28 molecules on the surface of T cells. B cells receive co-stimulatory signals through common pathogen components such as LPS, complement components, or CD40L on the surface of activated antigen-specific Th cells.

[0054] The term "connector" or hinge is a polypeptide fragment that links different proteins or peptides, with the purpose of maintaining the spatial conformation of the linked proteins or peptides to preserve their function or activity. Exemplary connectors include those containing G and / or S, and, for example, the Furin 2A peptide.

[0055] The term "pharmaceuticalally acceptable excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0056] The term "effective dose" refers to a dose that can achieve therapeutic, preventive, alleviating and / or relieving disease or condition as described in this invention in a subject.

[0057] The term "disease and / or symptom" refers to a physical condition of the subject that is related to the disease and / or symptom described in this invention.

[0058] The term "subject" can refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, cow, horse, etc., that receives the pharmaceutical composition of the present invention to treat, prevent, reduce and / or alleviate the disease or condition described in the present invention.

[0059] The term "tumor stromal cells" refers to cells within the tumor microenvironment that support the malignant proliferation, anti-apoptosis, invasion, metastasis, and escape of immune surveillance of tumor cells. These cells mainly include fibroblasts, tumor-associated macrophages (TAMs), regulatory T cells (Tregs), undifferentiated bone marrow cells, endothelial cells, pericytes, platelets, and endothelial cells.

[0060] The term "immunosuppressive cytokines" refers to molecules produced by tumor cells or tumor stromal cells that suppress the immune system, allowing tumor cells to escape the body's immune surveillance and inducing immune tolerance. Examples include tumor cell or tumor stromal cell membrane proteins PDL1 / PDL2 (PD-1 ligand), FasL (Fas ligand), B7-H4 (immunostimulatory protein B7-H4, also known as VTCN1), Crry (membrane-associated complement regulatory protein, also known as Cr1l), HLA-G (non-classical MHC class I molecules), as well as tumor cell or tumor stromal cell secreted proteins IL13, IL4, TGF-β, IL6, IL8, IL-10, CCL21, IDO (indoleamine 2,3-dioxygenase), and VEGF.

[0061] The term "extracellular region" refers to the segment of membrane proteins located outside the cell.

[0062] The term "domain" refers to a region in a protein that has a specific structure and independent function. Common domains have between 100 and 400 amino acid residues, with the smallest domains having only 40 to 50 amino acid residues and the largest domains having more than 400 amino acid residues.

[0063] Signal transduction receptors

[0064] The signal transduction receptor of the present invention is a polypeptide, a fusion protein comprising an extracellular domain (also called an extracellular region) of a receptor for an immunosuppressive cytokine fused to an intracellular domain (also called an "intracellular region") of an immunostimulatory molecule (also called a "co-stimulatory signaling molecule"). More specifically, the signal transduction receptor of the present invention comprises an extracellular domain of a receptor for an immunosuppressive cytokine, a transmembrane region, and an intracellular domain of a co-stimulatory signaling molecule. The signal transduction receptor of the present invention binds to Th2 or immunosuppressive cytokines but induces immunostimulatory signals rather than immunosuppression, resulting in maintenance of the Th1 phenotype, proliferation, and cytotoxic characteristics.

[0065] In this invention, immunosuppressive cytokines can be immunosuppressive cytokines secreted by cancer cells or surrounding tumor stroma, including tumor cell or tumor stromal cell membrane proteins and tumor cell or tumor stromal cell secreted proteins. The tumor cell or tumor stromal cell membrane proteins include, but are not limited to, PDL1 / PDL2 (PD-1 ligand), FasL (Fas ligand), B7-H4 (immunostimulatory protein B7-H4, also known as VTCN1), Crry (membrane-associated complement regulatory protein, also known as Cr1l), and HLA-G (non-classical MHC class I molecules); the tumor cell or tumor stromal cell secreted proteins include, but are not limited to, IL13, IL4, TGF-β, IL6, IL8, IL-10, CCL21, IDO (indoleamine 2,3-dioxygenase), and VEGF. This invention relates to constructing the signal transduction receptor of the present invention using the extracellular domains or functional fragments of receptors using these immunosuppressive cytokines, or mutants that retain the ability to bind the corresponding immunosuppressive cytokines.

[0066] In a particularly preferred embodiment, the receptor for the immunosuppressive cytokine in the signal transduction receptor of the present invention is a TGF-β receptor. The extracellular domain of the TGF-β receptor of the present invention may contain only the extracellular domain of the TGF-β receptor, or it may be a chimeric TGF-β receptor containing a TGF-β binding fragment of the TGF-β receptor. Any isotype of the natural TGF-β receptor's extracellular domain may be used, including TGF-β receptor 1, TGF-β receptor 2, and TGF-β receptor 3, preferably TGF-β receptor 1 and TGF-β receptor 2. The amino acid sequence and polynucleotide sequence of the extracellular region of an exemplary TGF-β receptor 1 may be as shown in SEQ ID NO: 1 and 2, respectively. An exemplary nucleotide sequence encoding TGF-β receptor 2 includes the sequence shown in Genbank accession numbers NM_001024847.2 or NM_003242.5, whose amino acid sequences have Genbank accession numbers NP_001020018 or NM_003242, respectively. In some embodiments, the extracellular domain of the present invention comprises amino acids 23-166 from TGF-β receptor 2, as shown in SEQ ID NO:3, and its coding sequence is shown in SEQ ID NO:4. The amino acid and polynucleotide sequences of the extracellular region of exemplary TGF-β receptor 3 may be shown in SEQ ID NO:5 and 6, respectively.

[0067] In some other embodiments, the receptor for the immunosuppressive factor in the signal transduction receptor of the present invention is an IL13 receptor, and the extracellular domain is the extracellular domain of the IL13 receptor, or a chimeric IL13 receptor containing an IL13-binding fragment of the IL13 receptor.

[0068] In this invention, the co-stimulatory signaling molecules include CD28, CD134 (OX40), CD137 (4-1BB), LCK, ICOS, DAP10, siglec-9, siglec-10, siglec-15, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD27, and CD40. The signal transduction receptor of this invention can be constructed using one or more of these co-stimulatory signaling molecules' intracellular domains (intracellular regions) or functional fragments thereof, or mutants that retain the biological functions of the co-stimulatory signaling molecules in transmitting co-stimulatory signals and activating immune cells. An exemplary IL-7R may be IL-7Ralpha, whose exemplary amino acid sequence and corresponding coding sequence are shown in SEQ ID NO:7 and 8, respectively. The amino acid sequence and corresponding coding sequence of the intracellular region of exemplary CD28 are shown in SEQ ID NO: 9 and 10, respectively. The amino acid sequence and corresponding coding sequence of the intracellular region of exemplary 4-1BB are shown in SEQ ID NO: 11 and 12, respectively. The amino acid sequence of the intracellular region of OX40 is shown in SEQ ID NO: 1645, and its coding sequence is shown in SEQ ID NO: 1646.

[0069] In this invention, the extracellular domain of the receptor for immunosuppressive cytokines is connected to the intracellular domain of a co-stimulatory signaling molecule via a transmembrane domain. The transmembrane domain can be of any origin. Transmembrane domains suitable for this invention include, but are not limited to, transmembrane domains of CD28, CD8, CD134 (OX40), CD137 (4-1BB), LCK, ICOS, DAP10, siglec-9, siglec-10, siglec-15, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2Rgamma, IL-2Rbeta, IL-4Ralpha, IL-7Ralpha, IL-10R, IL-12R, IL-15R, IL-21R, CD27, and CD40, or mutants thereof that retain transmembrane function. The transmembrane domain may originate from the same molecule as the intracellular domain, or from a different molecule. In this invention, the preferred transmembrane region is derived from the IL-7Ralpha transmembrane region and its mutants. Exemplary amino acid sequences and their coding sequences of the IL-7Ralpha transmembrane region are shown in SEQ ID NO:27 and 28, respectively. Exemplary amino acid sequences of mutants of the IL-7Ralpha transmembrane region may be selected from any one of SEQ ID NO:29-60; preferably, their coding nucleic acid sequences are selected from any one of SEQ ID NO:61-94.

[0070] It should be understood that the term "functional fragment" as used herein refers to a fragment that retains the desired biological function. For example, the functional fragment of the extracellular domain referred to herein refers to a fragment that retains the ability to bind to the corresponding immunosuppressive cytokine; the functional fragment of the intracellular domain refers to a fragment that retains the biological function of the co-stimulatory signaling molecule in transmitting co-stimulatory signals and activating immune cells. For instance, the functional fragment of the extracellular domain of TGF-β receptor 2 is a functional fragment from that extracellular domain that can bind to TGF-β receptor 2. The functional fragments of each extracellular domain and each intracellular domain applicable to this invention can be readily determined by those skilled in the art in conjunction with existing technical means.

[0071] The term "mutant" as used herein includes mutants of each extracellular domain, transmembrane region, and intracellular domain, provided that the mutant retains the corresponding biological function of each of the extracellular domain, transmembrane region, and intracellular domain. For example, mutants suitable for the extracellular domains of this invention include mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the extracellular domain as a comparison; mutants suitable for the transmembrane regions of this invention include mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the transmembrane region as a comparison; and mutants suitable for the intracellular domains of this invention include mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the intracellular domain as a comparison. Alternatively, compared to the sequence used for comparison, the mutants described in this invention have one or more (e.g., up to 20, 15, 10, 8, 5, or 3, such as 1-20, 1-10, etc.) amino acid residues inserted, substituted, or deleted.

[0072] This invention also includes mutants of the signal transduction receptors described above, such as mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the aforementioned signal transduction receptors. More specifically, this invention includes mutants that have one or more (e.g., up to 20, 15, 10, 8, 5, or 3, such as 1-20, 1-10, etc.) amino acid residues inserted, substituted, or deleted compared to the aforementioned signal transduction receptors. Such mutants retain the biological function of the signal transduction receptors described in this invention, including but not limited to converting inhibitory signals that inhibit immune cell proliferation into stimulatory signals that enhance immune cell proliferation. Mutations may occur in any one, any two, or all three of the extracellular domains, transmembrane domains, and intracellular domains described herein.

[0073] Exemplary signal transduction receptors of the present invention include, but are not limited to, signal transduction receptors containing, or composed of, the extracellular domains, transmembrane domains, and intracellular domains shown in each row of Table 1 below: Table 1

[0074] This invention may also include a mutated transmembrane region sequence of the activating receptor, preferably a mutant of the IL-7Ralpha transmembrane region. Mutants in the activating receptor transmembrane region can better aggregate the activating receptor, further activating and amplifying intracellular signals based on the wild-type receptor. The sequences of IL-7Ralpha transmembrane region mutants 1-32 (IL-7RalphaTM mut1-32) are shown in Tables 2 and 3 below: Table 2: Amino acid sequence of IL-7Ralpha transmembrane region mutant, 1-32

[0075] Table 3: Nucleotide sequences 1-32 of the IL-7Ralpha transmembrane region mutant

[0076] The present invention includes exemplary signal transduction receptors comprising IL-7Ralpha transmembrane mutants 1-32, including but not limited to the signal transduction receptors shown in any row of Table 4 below: Table 4

[0077] This invention also includes signal transduction receptors containing a CD28 transmembrane region and a 28-DeltaIL2RB-z(YXXQ) intracellular region or a 28-IL2RB-z(YXXQ) intracellular region. Exemplary signal transduction receptors include, but are not limited to, those shown in Table 5. Table 5

[0078] IL-7Ralpha TM mut1-32 in Shochat C et al., "Gain-of-function mutations in interleukin-7 receptor-α (IL7R) in childhood acute lymphoblastic leukemias", J Exp Med. The full text of the paper, published on May 9, 2011, in 208(5):901-8, is incorporated herein by reference.

[0079] The intracellular regions of 28-IL2RB-z(YXXQ), 28-DeltaIL2RB-z(YXXQ), and 28-IL2RB-z(YXXQ) are compared with those of 28-DeltaIL2RB-z(YXXQ) in Yuki Kagoya et al., “A Novel Chimeric Antigen Receptor Containing a JAK-STAT Signaling Domain Mediates Superior Antitumor Effects”. Nat Med The information was published in March 2018, 24(3):352-359, and is incorporated herein by reference in its entirety. Exemplary amino acid sequences and corresponding coding sequences are shown in SEQ ID NO:13 and 14, SEQ ID NO:15 and 16, SEQ ID NO:17 and 18, and SEQ ID NO:19 and 20, respectively. The intracellular region of IL2RB-z(YXXQ) (exemplary amino acid sequences and coding sequences are shown in SEQ ID NO:21 and 22, respectively) and the intracellular region of DeltaIL2RB-z(YXXQ) (exemplary amino acid sequences and coding sequences are shown in SEQ ID NO:23 and 24, respectively) are portions of the 28-IL2RB-z(YXXQ) intracellular region and the 28-DeltaIL2RB-z(YXXQ) intracellular region that do not include the CD28 intracellular region portion.

[0080] Preferably, the signal transduction receptor further comprises a signal peptide. Preferably, the signal peptide is located at the N-terminus of the signal transduction receptor. The signal peptide can be any signal peptide conventional in the art capable of guiding a polypeptide out of the nucleus, including but not limited to CD8, CD4, CD28, CD137, EGFR, TGFBRI, TGFBRII, TGFBRIII, and light chain signal peptides.

[0081] It should be understood that, as needed, the extracellular domains and transmembrane regions, and / or the transmembrane regions and intracellular domains described herein, can be connected by adapter sequences. Adapter sequences well-known in the art, such as adapter sequences containing G and S, such as (GSSS), can be used. n Or (GSSSS) n , where n is an integer from 1 to 8.

[0082] In some embodiments, the signal transduction receptor of the present invention may further include a signal peptide sequence. Typically, the signal peptide sequence is linked to an extracellular domain. Exemplary signal peptide sequences include the signal peptide of the TGF-β receptor. In some embodiments, the signal peptide of the TGF-β receptor is amino acids 1-22 of TGF-β receptor 2 (SEQ ID NO:1631), encoding the sequence shown in SEQ ID NO:1632.

[0083] In some embodiments, the signal transduction receptor of the present invention may further contain a single-chain antibody or natural peptide that recognizes and binds to tumor-specific antigens or tumor-associated antigens, thereby further enhancing immune activation after T cells expressing this molecular transducer come into contact with tumor cells. The tumor-specific antigens or tumor-associated antigens include CD19, CD20, CEA, GD2, FR, PSMA, gp100, CA9, CD171 / L1-CAM, IL-13Rα2, MART-1, ERBB2, NY-ESO-1, MAGE family proteins, BAGE family proteins, GAGE ​​family proteins, AFP, MUC1, CD22, CD23, CD30, CD33, CD44v7 / 8, CD70, VEGFR1, VEGFR2, IL-11Rα, ... EGP-2, EGP-40, FBP, GD3, PSCA, FSA, PSA, TAG-72, h5T4, fetal acetylcholine receptor, LeY, EpCAM, MSLN, IGFR1, EGFR, EGFRvIII, ERBB3, ERBB4, CA125, CA15-3, CA19-9, CA72-4, CA242, CA50, CYFRA21-1, SCC, AFU, EBV-VCA, TSGF, SF, POA, β2-MG, and PROGRP.

[0084] Chimeric antigen receptor (CAR)

[0085] The immune cells of this invention can further express CAR, or contain the coding sequence of CAR. The CAR described in this invention can be any CAR known in the art.

[0086] A CAR may sequentially comprise a polypeptide (such as scFv) that binds to tumor cell membrane antigens, a hinge region, a transmembrane region, and an intracellular signaling region. The CAR of this invention can be constructed using hinge regions, transmembrane regions, and intracellular signaling regions known in the art for constructing CARs. Typically, the polypeptide that binds to tumor cell membrane antigens can bind to membrane antigens widely expressed by tumor cells with moderate affinity. This polypeptide usually has an antigenic epitope inserted at any one, two, or three of the following three locations: the N-terminus of the polypeptide, between the polypeptide and the hinge region, and within the polypeptide. The polypeptide that binds to tumor cell membrane antigens is a natural polypeptide or a synthetic polypeptide; preferably, the synthetic polypeptide is a single-chain antibody or a Fab fragment.

[0087] The chimeric antigen receptor of the present invention can target one or more of the following antigens: CD19, CD20, CEA, GD2 (also known as B4GALNT1, ββGAL acetyl-galactosyltransferase 1), FR (Flavin reductase), PSMA (prostate-specific membrane antigen), PMEL promelanosome protein, CA9 (carbonic anhydrase IX), CD171 / L1-CAM, IL-13RL1, MART-1 (also known as mucin-A), ERBB2, NY-ESO-1 (also known as CTAG1B, cancer / testis antigen 1B), MAGE (melanoma-associated antigen E1) family proteins, BAGE (B melanoma antigen family) family proteins, GAGE ​​(growth hormone-releasing factor) family proteins, AFP (alpha fetal protein), MUC1 (mucin 1. Cell surface related), CD22, CD23, CD30, CD33, CD44v7 / 8, CD70, VEGFR1, VEGFR2, IL-11R / , EGP-2, EGP-40, FBP, GD3 (also known as ST8SIA1, ST8SIA1 acetyl-ceramide α-aminoamide α-sialyltransferase 1), PSCA (prostate stem cell antigen), FSA (also known as KIAA1109), PSA (also known as KLK3, kallikrein-associated peptidase 3), HMGA2, fetal acetylcholine receptor, LeY (also known as FUT3), EpCAM, MSLN (mesothelin), IGFR1, EGFR, EGFRvIII, ERBB3, ERBB4, CA125 (also known as MUC16, mucin 16, cell surface-related), CA15-3, CA19-9, CA72-4, CA242, CA50, CYFRA21-1, SCC (also known as SERPINB3), AFU (also known as FUCA1), EBV-VCA, POA (also known as VDR, vitamin D (1,25-dihydrovitamin D3) receptor), β body), G (ββ), microglobulin) and PROGRP (GRP gastrin-releasing peptide).

[0088] A single cell can express multiple CARs, including CARs that target different tumor antigens.

[0089] T cell receptor (TCR) - T

[0090] The immune cells of this invention can further express exogenous TCRs or contain coding sequences for genes expressing exogenous TCRs. The TCRs described in this invention can be various TCRs known in the art, such as HLA-matched TCRs with known sequences and structures, and whose binding antigen peptide sequences are also known.

[0091] The exogenous TCR described in this invention comprises an αβ double strand, which can form a complete TCR complex with the double-stranded structures of γε, δε, and ξξ endogenously expressed by immune effector cells such as T cells. The exogenous gene encoding the exogenous TCR described in this invention includes a gene encoding an αβ double strand. The coding sequences of the α and β strands are covalently linked by a DNA sequence encoding a cleavable adapter sequence, such as P2A, T2A, or F2A, or by a DNA fragment encoding an IRES sequence. In addition to the αβ double strand encoding the exogenous TCR, the gene encoding the exogenous TCR described in this invention may also include a tag protein gene fused with the αβ gene, such as EGFP, RFP, or YFP genes. The tag protein gene can be covalently linked to the gene encoding the αβ double strand by a cleavable adapter sequence, such as a 2A sequence, such as P2A, T2A, or F2A, or by a DNA sequence encoding an IRES sequence. The tag proteins, such as EGFP, RFP, and YFP genes, are co-expressed with the TCRαβ double strand and can serve as an identification indicator for detecting exogenous TCR expression.

[0092] The TCR-T of this invention can target one or more of the following antigens: CD19, CD20, CEA, GD2 (also known as B4GALNT1, ββGAL acetyl-galactosyltransferase 1), FR (Flavin reductase), PSMA (prostate-specific membrane antigen), PMEL promelanosome protein, CA9 (carbonic anhydrase IX), CD171 / L1-CAM, IL-13RL1, MART-1 (also known as mucin-A), ERBB2, NY-ESO-1 (also known as CTAG1B, cancer / testis antigen 1B), MAGE (melanoma-associated antigen E1) family proteins, BAGE (B melanoma antigen family) family proteins, GAGE ​​(growth hormone-releasing factor) family proteins, AFP (alpha fetal protein), and MUC1 (mucin). 1. Cell surface related), CD22, CD23, CD30, CD33, CD44v7 / 8, CD70, VEGFR1, VEGFR2, IL-11R / , EGP-2, EGP-40, FBP, GD3 (also known as ST8SIA1, ST8SIA1 acetyl-ceramide α-aminoamide α-sialyltransferase 1), PSCA (prostate stem cell antigen), FSA (also known as KIAA1109), PSA (also known as KLK3, kallikrein-associated peptidase 3), HMGA2, fetal acetylcholine receptor, LeY (also known as FUT3), EpCAM, MSLN (mesothelin), IGFR1, EGFR, EGFRvIII, ERBB3, ERBB4, CA125 (also known as MUC16, mucin 16, cell surface-related), CA15-3, CA19-9, CA72-4, CA242, CA50, CYFRA21-1, SCC (also known as SERPINB3), AFU (also known as FUCA1), EBV-VCA, POA (also known as VDR, vitamin D (1,25-dihydrovitamin D3) receptor), β body), G (ββ), microglobulin) and PROGRP (GRP gastrin-releasing peptide).

[0093] A single cell can express multiple exogenous TCRs, including exogenous TCRs that target different tumor antigens.

[0094] Polynucleotide molecules

[0095] This invention provides a polynucleotide molecule encoding the signal transduction receptor described herein. The invention also provides a complementary sequence to the coding sequence of the signal transduction receptor. The polynucleotide molecule may be a recombinant nucleic acid molecule or a synthetic one; it may contain DNA, RNA, and PNA (peptide nucleic acid) and may be a hybrid of these.

[0096] The invention also provides an expression cassette for the signal transduction receptor, which is a nucleic acid construct containing a promoter, a signal transduction receptor coding sequence, and a PolyA tailing signal sequence. The nucleic acid construct may also contain other elements required for expression, including but not limited to enhancers.

[0097] A vector containing the polynucleotide molecule, expression cassette, or nucleic acid construct described herein is also provided. The vector can be a plasmid, granule, virus, or bacteriophage. The vector can be a cloning vector or an expression vector. The expression vector can be a transposon vector. In some embodiments, the expression vector is one or more transposon vectors selected from: piggybac, sleeping beauty, frog prince, Tn5, and Ty. In addition to the polynucleotide molecule described herein, the expression vector typically contains other elements commonly found in vectors, such as multiple cloning sites, resistance genes, replication initiation sites, etc. In some embodiments, the recombinant expression vector uses pUC18, pUC19, pMD18-T, pMD19-T, pGM-T vectors, pUC57, pMAX, or pDC315 series vectors as a backbone. In other embodiments, the recombinant expression vector uses pCDNA3 series vectors, pCDNA4 series vectors, pCDNA5 series vectors, pCDNA6 series vectors, pRL series vectors, pUC57 vectors, pMAX vectors, or pDC315 series vectors as the backbone. In some embodiments, the present invention uses the pSN vector constructed according to CN201510638974.7.

[0098] The CAR of this invention can also be expressed in the immune cells described in this invention using conventional vectors. The vector can be a conventional CAR expression vector, including but not limited to the various transposon vectors and recombinant expression vectors described above.

[0099] In some embodiments, the same vector simultaneously encodes the signal transduction receptor and CAR of the present invention. This vector may be bicistronic. The coding sequence of the CAR may be located at the 5' or 3' end of the coding sequence of the signal transduction receptor. The expression of the CAR and the signal transduction receptor may be guided by the same or different regulatory sequences.

[0100] When the polynucleotide sequence is known, the polynucleotide molecules can be prepared using methods conventional in the art, and the corresponding vectors can be constructed. Recombinant vectors can be constructed using methods well known to those skilled in the art, see, for example, techniques described in Sambrook et al., Ausubel (1989), or other standard textbooks. Alternatively, the nucleic acid molecules and vectors can be reconstituted into liposomes for delivery to target cells. Vectors containing the nucleic acid molecules of the present invention can be transferred into host cells using well-known methods, which vary depending on the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cell hosts, see Sambrook.

[0101] host cells

[0102] In this article, when expressing a heterologous nucleic acid sequence, "host cell" refers to a eukaryotic cell capable of replicating the vector and / or expressing the heterologous gene encoded by the vector. The host cell can serve as the recipient of the vector. The host cell can be "transfected" or "transformed," referring to the process of transfecting or transducing exogenous nucleic acids into the host cell. Transformed cells include primary target cells and their progeny. The terms "engineered" and "recombinant" cells or host cells used herein often refer to cells in which a exogenous nucleic acid sequence, such as a vector, has been introduced. Therefore, recombinant cells can be distinguished from naturally occurring cells that do not contain the introduced recombinant nucleic acid.

[0103] In this document, host cells include cells carrying the polynucleotide molecules and / or polypeptides described herein. Specifically, this invention provides cells carrying the signal transduction receptors and / or their coding sequences described herein. The cells of this invention are preferably immune cells and can be used for adoptive cell therapy of tumors. These cells of this invention are also referred to as signal transduction receptor-modified cells of this invention.

[0104] More specifically, the cells of the present invention are preferably immune effector cells, including T cells, such as cytotoxic T cells (also known as TC, cytotoxic T lymphocytes, CTL, T killer cells, cytolytic T cells, CD8+ T cells or killer T cells); NK cells; NKT cells; and other immune cells capable of inducing effector functions.

[0105] In this article, the cells can be autologous cells, homologous cells, allogeneic cells, and even xenotransplanted cells in some cases, in relation to the individuals that receive them.

[0106] The nucleic acid constructs / recombinant expression vectors of the present invention can be transferred into cells of interest. The transfer methods are conventional in the art, including but not limited to: viral transduction, microinjection, particle bombardment, gene gun transformation, and electroporation. In some embodiments, electroporation is used to transfer the nucleic acid constructs or recombinant expression vectors.

[0107] In addition to carrying the signal transduction receptors and / or their coding sequences as described in this invention, the cells of this invention may also possess one or more other properties that can be used for cell immunotherapy (such as adoptive cell therapy for tumors). These other properties may be inherent to the cell or may be part of the cell after genetic manipulation in humans. For example, the cells of this invention may carry chimeric antigen receptors, αβT cell receptors, and / or antigen-specific receptors, such as tumor-specific receptors, or their coding sequences.

[0108] Pharmaceutical Composition

[0109] In this document, "pharmaceutical composition" refers to a composition intended for administration to an individual and encompassing a cell-based composition for immunotherapy. The pharmaceutical compositions of this invention may also contain pharmaceutically acceptable carriers. Examples of suitable drug carriers are those known in the art and include phosphate-buffered saline solutions, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, etc. Compositions containing such carriers can be formulated using well-known conventional methods. These pharmaceutical compositions can be administered to the subject in appropriate doses.

[0110] Dosage regimens can be determined by the attending physician and clinical factors. As is well known in the medical field, the dosage for any given patient depends on a variety of factors, including the patient's body size, body surface area, age, the specific compound to be administered, sex, timing and route of administration, overall health status, and any other medications administered concurrently.

[0111] The compositions of the present invention can be administered locally or systemically. In some embodiments, the compositions provided by the present invention (e.g., cells expressing the signal transduction receptors described herein) can be administered parenterally, such as intravenously, intra-arterially, intrathecally, subdermally, or intramuscularly. In some other embodiments, DNA encoding the constructs provided by the present invention can be directly delivered to the target site, for example, via gene gun delivery to internal or external target sites or via catheter delivery to intra-arterial sites. In preferred embodiments, the pharmaceutical composition is administered subcutaneously, and in more preferred embodiments, intravenously. Parenteral formulations include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic solutions / aqueous solutions, emulsions, or suspensions, including saline and buffer media. Parenteral carriers include sodium chloride solution, Ringer's dextran, dextran and sodium chloride, lactated Ringer's solution, or fixed oil. Intravenous carriers include fluids and nutritional supplements, electrolyte supplements (e.g., those based on ringer's dextran), etc. Preservatives and other additives may also be present, such as antimicrobial agents, antioxidants, chelating agents, and inert gases. Furthermore, the pharmaceutical compositions of the present invention may contain protein carriers, such as serum albumin or immunoglobulins, preferably human. In addition to protein-based chimeric cytokine receptor constructs or nucleic acid molecules or carriers encoding them, the pharmaceutical compositions of the present invention are envisioned to also contain bioactive agents, depending on the intended use of the pharmaceutical composition.

[0112] Methods and Applications

[0113] The signal transduction receptors, polynucleotide molecules, carriers, host cells, and pharmaceutical compositions containing these substances described in this invention can be used to prevent, treat, or alleviate cancer, especially cancers in which cancer cells express corresponding tumor antigens on their surface, or to prepare drugs for the prevention, treatment, or alleviation of cancer.

[0114] As used herein, “treatment” or “treatment” includes any beneficial or necessary effect on the symptoms or lesions of a disease or pathological condition, and may include even a small reduction in one or more measurable markers of the disease or condition (e.g., cancer). Treatment may optionally include a reduction or relief of symptoms of the disease or condition, or a delay in the progression of the disease or condition. “Treatment” does not necessarily mean the complete eradication or cure of the disease or condition or its associated symptoms.

[0115] As used in this article, “prevention” refers to methods used to prevent, suppress, or reduce the likelihood of the occurrence or recurrence of a disease or condition (e.g., cancer). It also refers to delaying the onset or recurrence of a disease or condition, or delaying the appearance or recurrence of its symptoms. As used in this article, “prevention” also includes reducing the intensity, impact, symptoms, and / or burden of a disease or condition before it occurs or recurs.

[0116] This invention includes the administration of cells, polynucleotide molecules, and vectors, alone or in any combination, using standard vectors and / or gene delivery systems, optionally in conjunction with pharmaceutically acceptable carriers or excipients. In some embodiments, after administration, the polynucleotide molecule or vector can be stably integrated into the genome of the subject.

[0117] In specific embodiments, viral vectors that specifically target certain cells or tissues and persist within said cells may be used. Suitable drug carriers and excipients are well known in the art. The compositions prepared according to the present invention can be used to prevent, treat, or delay the aforementioned identified diseases.

[0118] Furthermore, the present invention provides a method for preventing, treating, or alleviating cancer, comprising the steps of: administering an effective amount of cells to a subject in need, said cells carrying signal transduction receptors, polynucleotide molecules, and / or carriers as described in and / or generated by the methods described in the present invention.

[0119] In this document, cancer includes, but is not limited to, breast, prostate, lung, and colon cancer or epithelial cancer, such as breast cancer, colon cancer, prostate cancer, head and neck cancer, and skin cancer; genitourinary cancers, such as ovarian cancer, endometrial cancer, and cervical cancer; kidney cancer, lung cancer, stomach cancer, small intestine cancer, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, esophageal cancer, salivary gland cancer, and thyroid cancer, etc. The compositions of this invention can be used for all stages and types of cancer, including for, for example, minimal residual disease, early-stage cancer, advanced cancer, and / or metastatic cancer and / or difficult-to-treat cancer.

[0120] By way of example, cancer patients, cancer-prone patients, or suspected cancer patients are treated as follows. The modified cells described herein can be administered to the individual and remain in the body for an extended period. The individual may receive one or more doses of the cells, with intervals between doses ranging from days to weeks to months to years. In specific embodiments, multiple doses may occur over weeks or months, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks or months. In some embodiments, the genetically modified cells are encapsulated to suppress immune recognition and are located at the tumor site. In cases where cells are provided to an individual after tumor recurrence following initial treatment with the cells of the present invention, these cells may be modified to recognize different target tumor antigens. For example, when the initial round includes cells carrying the signal transduction receptor of the present invention and another receptor specifically targeting a particular antigen, subsequent rounds (including after tumor recurrence) may use receptors targeting different specific antigens.

[0121] In some embodiments, an effective amount of therapeutic cells carrying or expressing the signal transduction receptors described in any embodiment of the invention and optionally a CAR or exogenous transgenic TCR is provided to the individual in need. These cells may be delivered simultaneously or separately from one or more other cancer treatments. These cells and other cancer therapeutics may be delivered in the same or separate formulations. Cells and other cancer therapeutics may be delivered to the individual via separate delivery routes. Cells and / or other cancer therapeutics may be delivered, for example, by injection at the tumor site, intravenous administration, or oral administration. Conventional delivery routes for such compositions are known in the art.

[0122] The number of cells used will depend on a variety of factors, such as the purpose of introduction, cell lifespan, the protocol to be used, the number of administrations, cell proliferation capacity, and the stability of the recombinant construct.

[0123] Cells can be administered as needed. In some embodiments, multiple protocols can be used to adjust protocol parameters. In specific embodiments, the route or frequency or timing of administration, cell lifespan, and / or the number of cells present can vary. The frequency of administration may, for example, depend at least in part on the factors described above.

[0124] Reagent test kit

[0125] Any of the compositions described herein may be included in the kit. In one non-limiting embodiment, the kit may include cells expressing the signal transduction receptors described in any embodiment of the invention for cell therapy and / or reagents for generating one or more cells for cell therapy containing a recombinant expression vector. The kit components are provided in suitable containers.

[0126] Some components of these kits may be packaged in an aqueous matrix or in lyophilized form. The containers for these kits typically include at least one vial, test tube, flask, bottle, syringe, or other container in which the component can be placed, and preferably appropriately aliquoted. In kits containing more than one component, the kit typically also includes a second, third, or other container in which other components can be placed separately. However, various combinations of components can be contained in vials. The kits of the present invention typically also include a device containing the component in a commercially available, closed-form constraint. Such containers may include injection-molded or blow-molded plastic containers in which the desired vials are held.

[0127] When the kit components are provided in one or more liquid solutions, the liquid solutions are aqueous solutions, and sterile aqueous solutions are particularly preferred. In some cases, the container itself may be a syringe, pipette, and / or other such devices.

[0128] The kit components can also be provided in dry powder form. When reagents and / or components are provided as dry powder, the powder can be reconstituted by adding a suitable solvent. Therefore, the kit may also include a second container containing sterile, pharmaceutically acceptable buffers and / or other diluents.

[0129] In specific embodiments of the invention, cells intended for use in the cell therapies described herein are provided in the kit. In some embodiments, the cells are essentially the only component of the kit. The kit may contain reagents and materials for preparing the desired cells. In specific embodiments, the reagents and materials contain primers, nucleotides, suitable buffers or buffering agents, salts, etc., for amplifying the desired sequence, and in some cases, the reagents include DNA and / or vectors encoding the signal transduction receptor and / or its regulatory elements as described in any embodiment herein.

[0130] Effects of the present invention

[0131] This invention creatively links a polypeptide, a transmembrane peptide, and an intracellular peptide of a co-stimulatory signaling molecule that efficiently binds to immunosuppressive molecules on the surface of tumor cells and / or tumor stromal cells, forming a fusion protein with T signaling molecule converter function. T cells modified with this fusion protein, upon contact with immunosuppressive molecules on the surface of tumor cells and / or tumor stromal cells, are not negatively affected by the immunosuppressive molecules but instead exhibit immune activation, enhancing T cell proliferation and cytokine secretion, and prolonging the survival time of activated immune cells. This overcomes the side effects of the tumor immunosuppressive microenvironment on effector cells in adoptive cell therapy for tumors, effectively improving the inhibitory and killing effects of immune effector cells on tumors both in vivo and in vitro.

[0132] The embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the examples, they should be performed according to the techniques or conditions described in the literature in the art (e.g., refer to J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Science Press), relevant references, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0133] Example 1: Construction of a signal transduction receptor expression vector

[0134] The pNB vector was constructed according to the method described in Example 1 on page 15 of the specification of Chinese Patent CN105154473B. The pNB vector containing the expression cassette of TGF-β signal transducer 2-1-TGF-β signal transducer 2-15 (TGFBR2-SCR1-TGFBR2-SCR15) was constructed according to the method described in Example 2 on page 15 of the specification of CN105154473B.

[0135] Chinese patent application CN105452287A discloses a signal transduction receptor containing the extracellular region of TGF-β receptor 2. Example 1 on page 21 of the specification describes two signal transduction receptor structures, RIID2 and RIID4, which respectively contain a TGF-β signal transduction receptor 2 signal peptide, a TGF-β signal transduction receptor 2 extracellular region, a TLR2 transmembrane region, and a TLR2 intracellular region, and a TGF-β signal transduction receptor 2 signal peptide, a TGF-β signal transduction receptor 2 extracellular region, a TLR4 transmembrane region, and a TLR4 intracellular region. Based on the structures described above, signal transduction receptors SCR-RIID2 and SCR-RIID4, including the aforementioned elements, were synthesized. Neither of these contains IRES or mOrange or GFP fluorescent protein.

[0136] The structures corresponding to TGFBR2-SCR1-TGFBR2-SCR15, SCR-RIID2, and SCR-RIID4 are shown in Table 6 below: Table 6

[0137] Specifically, the company was commissioned to synthesize SEQ ID NO:1648, SEQ ID NO:1650, SEQ ID NO:1652, SEQ ID NO:1654, SEQ ID NO:1656, SEQ ID NO:1658, SEQ ID NO:1660, SEQ ID NO:1662, SEQ ID NO:1664, SEQ ID NO:1666, SEQ ID NO:1668, SEQ ID NO:1670, SEQ ID NO:1672, SEQ ID NO:1674, SEQ ID NO:1676, SEQ ID NO:1678, SEQ ID The sequence shown in NO:1680 was cloned into the prepared pNB vector according to the method described in Example 2 on page 15 of the specification CN105154473B, and named pNB328-1648, pNB328-1650, pNB328-1652, pNB328-1654, pNB328-1656, pNB328-1658, pNB328-1660, pNB328-1662, pNB328-1664, pNB328-1666, pNB328-1668, pNB328-1670, pNB328-1672, pNB328-1674, pNB328-1676, pNB328-1678, and pNB328-1680, respectively.

[0138] The recombinant plasmid obtained above was transformed into... E. coli (DH5c), after being correctly sequenced, plasmids were extracted and purified using a plasmid purification kit from Qiagen to obtain high-quality plasmids for each recombinant expression vector.

[0139] Example 2: Preparation of NY-ESO-1 TCR expression vector

[0140] Synthesized and encoded the NY-ESO-1 antigen peptide SLLMWITQC (HLA- The α and β strands of the TCR gene (02:01) were linked together using a DNA sequence encoding the P2A peptide, resulting in the sequence shown in SEQ ID NO:1681. Then, a DNA sequence encoding EGFP was linked to the 3' end of SEQ ID NO:1681 using the DNA sequence encoding the P2A peptide, resulting in the sequence shown in SEQ ID NO:1682. SEQ ID NO:1682 was synthesized by a company and cloned into the prepared pNB vector according to the method described in Example 2 on page 15 of the CN105154473B specification, named pNB328-1682. The PB transposase coding region and CMV promoter in the pNB vector were deleted to obtain the pNC vector without the PB transposase expression cassette. SEQ ID NO:1682 was cloned into the prepared pNC vector according to the method described in Example 2 on page 15 of the CN105154473B specification, named pNC328-1682. The obtained recombinant plasmids were transformed into E. coli (DH5c). After correct sequencing, the plasmids were extracted and purified using a plasmid purification kit from Qiagen to obtain high-quality plasmids for each recombinant expression vector.

[0141] Example 3 Preparation of TCR-T

[0142] Preparation of human activated T cells: Coat six-well plates with a coating solution containing 5 μg / ml anti-CD3 antibody and 5 μg / ml anti-CD28 antibody at room temperature for 2-4 hours. After removing the coating solution, wash the plates 1-3 times with physiological saline and add AIM-V medium containing 2% FBS for later use. Human peripheral blood PBMCs (HLA- At 02:01, PBMCs (purchased from ALLCELLS) were revived in a 37°C water bath and cultured for 2-4 hours to ensure adherence. The unadhered suspension cells were identified as naïve T cells. These suspension cells were collected into 15ml centrifuge tubes, centrifuged at 1200 rpm for 3 minutes, and the supernatant was discarded. Physiological saline was added, and the cells were centrifuged at 1200 rpm for 3 minutes. The saline was discarded, and this step was repeated. The washed naïve T cells were then transferred to antibody-coated wells containing culture medium and cultured at 37°C with 5% CO2 for 3-4 days before proceeding with subsequent experiments.

[0143] Electroporation preparation of TCR-T cells expressing NY-ESO-1 TCR and NY-ESO-1 TCR+TGF-β signal transduction receptor: 1) Add 2 mL of AIM-V medium to each of the 19 wells of a 12-well plate beforehand, and then transfer the plate to a cell culture incubator and preheat at 37°C with 5% CO2 for 1 hour. 2) Prepare the electro-spreading solution for each well according to the dosage in Table 7, for a total of 19 wells: Table 7

[0144] 3) Take the obtained activated T cells into 19 EP tubes, add 5×106 cells to each EP tube, centrifuge at 1200 rpm for 5 min, discard the supernatant, then resuspend the cells with 500 μL of physiological saline, and repeat the centrifugation steps to wash the cell pellet. 4) Add the following plasmids to the prepared electroporation solution in step 2): pNB328-1682 6 μg, pNC328-1682 3 μg + pNB328-1648 3 μg, pNC328-1682 3 μg + pNB328-1650 3 μg, pNC328-1682 3 μg + pNB328-1652 3 μg, pNC328-1682 3 μg + pNB328-1654 3 μg, pNC328-1682 3 μg + pNB328-1656 3 μg, pNC328-1682 3 μg + pNB328-1658 3 μg, pNC328-1682 3 μg + pNB328-1660 3 μg, respectively. 3μg, pNC328-16823μg+ pNB328-1662 3μg, pNC328-1682 3μg+ pNB328-1664 3μg, pNC328-1682 3μg+ pNB328-1666 3μg, pNC328-1682 3μg+ pNB328-1668 3μg, pNC328-1682 3μg+ pNB328-16703μg, pNC328-1682 3μg+ pNB328-1672 3μg, pNC328-1682 3μg+ pNB328-1674 3μg, pNC328-1682 3μg+ pNB328-1676 3 μg of pNC328-1682 + 3 μg of pNB328-1678, 3 μg of pNC328-1682 + 3 μg of pNB328-1680, 3 μg of pNC empty vector + 3 μg of pNB empty vector, and then let stand at room temperature for no more than 30 min; 5) Resuspend 19 tubes of activated T cells in the plasmid-containing electroporation buffer prepared in step 4), 100 μL per tube. Carefully transfer the cell resuspended buffer into a LONZA 100 μL electroporation cuvette. Place the cuvette into the LONZA Nucleofector™ 2b electroporation tank and start the electroporation program. Select the T-020 electroporation program. 6) After electroporation, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to EP tubes. Add 200 μL of preheated AIM-V medium to each tube, and then transfer it to the wells of the 12-well plate containing preheated AIM-V medium from step 1). Incubate at 37°C and 5% CO2. After 1 hour of incubation, add compound G150 (purchased from MedChemExpress) to a final concentration of 5 μM. Continue incubation for 13 days, during which time the cells are passaged according to their proliferation. After 13 days, the cell count and cell viability of each electroporated sample are measured.

[0145] Cells expressing exogenous genes, prepared using the above method, were named TCR-T, SCR1 TCR-T, SCR2 TCR-T, SCR3 TCR-T, SCR4 TCR-T, SCR5 TCR-T, SCR6 TCR-T, SCR7 TCR-T, SCR8 TCR-T, SCR9 TCR-T, SCR10 TCR-T, SCR11 TCR-T, SCR12 TCR-T, SCR13 TCR-T, SCR14 TCR-T, SCR15 TCR-T, RIID2 TCR-T, RIID4 TCR-T, and Mock TCR-T, respectively. The percentage of EGFP-positive cells was detected using a Beckman Cytoflex flow cytometer. For TCR-T, EGFP-positive cells can be considered as cells expressing the NY-ESO-1 TCR gene. For cells co-transfected with TGF-β signaling receptor and TCR, since the pNC vector does not contain the PB transposase expression cassette, EGFP positivity indicates that the PB transposase, upon which successful TCR-EGFP integration expression depends, originates from the pNB vector expressing the TGF-β signaling receptor gene. Therefore, in cells co-transfected with TGF-β signaling receptor and TCR, EGFP-positive cells can be considered as exhibiting dual integration and expression of TGF-β signaling receptor and NY-ESO-1 TCR. Results are as follows... Figure 1 As shown in Table 8: Table 8

[0146] Figure 1Fluorescence micrographs of TCR-T cells electrotransformed with the TCR-EGFP fusion gene and SCR1-5 TCR-T cells co-transformed with the TCR-EGFP fusion gene + TGF-β signal transduction receptor gene are shown. The EGFP fluorescence intensity of TCR-T cells is brighter than that of SCR1-5 TCR-T cells. This may be because TCR-T cells are prepared using a single plasmid, which includes both the exogenous TCR-EGFP fusion gene and the PB transposase expression cassette; while SCR1-5 TCR-T cells require co-transformation with two plasmids, with the exogenous TCR-EGFP fusion gene and the PB transposase expression cassette requiring separate pNC and pNB vectors.

[0147] Example 4: Killing effect of TCR-T cells expressing TGF-β signaling receptor SCR1-5 on target cells

[0148] Select HLA-1 positive expression of NY-ESO-1 02:01 type melanoma cell line A375 (all purchased from the American Type Culture Collection, ATCC) was used as target cells. The in vitro killing activity of TCR-T, SCR1 TCR-T, SCR2 TCR-T, SCR3 TCR-T, SCR4 TCR-T, SCR5 TCR-T, RIID2 TCR-T, and RIID4 TCR-T cells prepared in Example 3 was detected using a Real-Time Label-Free Cell Function Analyzer (RTCA) from Essen. The specific steps are as follows: (1) Zeroing: Add 50 μl of DMEM or 1640 culture medium to each well, place it in the instrument, select step 1, and zero it; (2) Target cell plating: Melanoma A375 cells were plated at 104 cells / 50μl per well in a plate containing the detection electrode. After a few minutes of incubation to allow the cells to stabilize, the plate was placed in the instrument to begin step 2, cell culture. (3) Add effector cells: After culturing target cells for 8-48 hours until the cell index reaches 1.0, pause step 2, add effector cells, 50 μl per well, and add effector cells according to the positive cell ratio in Table 1 at an effector-target ratio of 1.5:1. Start step 3 and continue co-culturing for more than 120 hours, then observe the cell proliferation curve.

[0149] The results are as follows Figure 2 and Figure 3 As shown. Figure 2 and Figure 3 The cytotoxic effects of SCR1-5 TCR-T, RIID 2 TCR-T, and TCR-T on target cells were compared with those of SCR1-5 TCR-T, RIID 4 TCR-T, and TCR-T on target cells. The effector-to-target ratio was set to 1.5:1, and the cytotoxic effects were observed over a long period of time. Figure 2 and Figure 3 All studies showed that shortly after the addition of effector cells (24h), SCR4 TCR-T and SCR5 TCR-T exhibited significantly better target cell killing effects than TCR-T, SCR1 / 2 / 3 TCR-T, RIID2 TCR-T, and RIID4 TCR-T. After 120h of cytotoxicity, SCR1-5 TCR-T still showed more significant killing effects on A375 target cells compared to TCR-T. The killing effects of SCR1 / 2 / 3 TCR-T were similar, while SCR4 TCR-T was more effective than SCR1 / 2 / 3 TCR-T. SCR5-TCR-T, however, showed significantly better killing effects than SCR1-4 TCR-T and TCR-T, making it the best among the various effector cell types. Figure 2 The results showed that the RIID2 TCR-T had a similar killing effect to TCR-T, but was slightly lower than that of TCR-T. Figure 3 The results showed that RIID4 TCR-T had a slightly better killing effect than TCR-T, but lower than SCR1 / 2 / 3 TCR-T, and significantly lower than SCR4 TCR-T and SCR5 TCR-T. These results indicate that the signal transduction receptors SCR1-5 of this invention can still significantly enhance the killing level of effector T cells against tumor target cells even under low effector-to-target ratio conditions, and this enhancement effect persists even with longer killing times.

[0150] Example 5: The specific killing effect of TCR-T cells expressing the TGF-β signaling receptor SCR5 on target cells under inefficient target conditions.

[0151] Referring to Example 4, A375 cells were selected as the target cells, and TCR-T, SCR5 TCR-T, RIID2 TCR-T, and RIID4 TCR-T cells were selected as effector cells. The effector-to-target ratio was further reduced to 1:1, and the killing levels of several effector cells on A375 cells were compared. The procedure was the same as in Example 4. The long-term observation at the 1:1 low effector-to-target ratio was intended to simulate the scenario of T cell killing tumors in a real in vivo environment.

[0152] The results are as follows Figure 4 and Figure 5 As shown. Figure 4 The results showed that shortly after the effector cell addition time point (just over 40 hours), RIID2 TCR-T exhibited superior cytotoxicity compared to TCR-T, while SCR5 TCR-T showed significantly better cytotoxicity than the other two. Even after 120 hours of cytotoxicity testing, RIID2 TCR-T remained superior to TCR-T, while SCR5 TCR-T remained superior to both. Similarly, Figure 5The results showed that shortly after the effector cell addition time point (just over 40 hours), RIID4 TCR-T exhibited superior killing efficacy compared to TCR-T, while SCR5 TCR-T showed significantly better killing efficacy than the former two. Even after the killing assay duration exceeded 120 hours, the killing efficacy of RIID4 TCR-T remained superior to TCR-T, and the killing efficacy of SCR5 TCR-T was also superior to both. These results indicate that even under conditions of extremely low effector-to-target ratio and long killing time, the signal transduction receptor SCR5 of this invention can still effectively enhance the killing level of effector T cells against tumor target cells.

[0153] Example 6: Killing effect of TCR-T cells expressing TGF-β signaling receptor SCR6-10 on target cells.

[0154] The in vitro killing activity of the TCR-T, SCR6 TCR-T, SCR7 TCR-T, SCR8 TCR-T, SCR9 TCR-T, and SCR10 TCR-T prepared in Example 3 against A375 cells was detected. The effector-to-target ratio was set at 1.5:1 based on the proportion of EGFP-positive cells, and the specific detection steps were the same as in Example 4. The results are as follows: Figure 6 As shown, shortly after the addition of effector cells (24h), SCR10TCR-T exhibited significantly better target cell killing effects than TCR-T and SCR6 / 7 / 8 / 9 TCR-T. Even after 90h of induction, SCR6-10 TCR-T remained more effective at killing A375 target cells than TCR-T. The killing effects of SCR6 / 7 / 8TCR-T were similar, while SCR9 TCR-T and SCR10 TCR-T were more effective than SCR6 / 7 / 8TCR-T, with SCR10 TCR-T showing the best killing effect among all effector cell types. These results indicate that the signal transduction receptor SCR6-10 of this invention can significantly enhance the killing level of effector T cells against tumor target cells even under low effector-to-target ratio conditions, and this enhancement effect persists even at longer induction times.

[0155] Example 7: Killing effect of TCR-T cells expressing TGF-β signaling receptor SCR10 on target cells at low target-to-cell ratio.

[0156] Referring to Example 4, A375 cells were selected as target cells, and TCR-T, SCR10 TCR-T, and RIID2 TCR-T cells were selected as effector cells. The effector-to-target ratio was further reduced to 0.5:1. The killing levels of several effector cells on A375 cells were compared, and the operation was the same as in Example 4. The long-term observation at a low effector-to-target ratio of 0.5:1 was intended to simulate the scenario of T cell killing tumors in a real in vivo environment.

[0157] The results are as follows Figure 7 As shown. Figure 7The results showed that after 80 hours, RIID2 TCR-T exhibited superior killing efficacy compared to TCR-T, while SCR10 TCR-T showed significantly superior killing efficacy. Even after 140 hours of killing assay, RIID2 TCR-T still showed superior killing efficacy compared to TCR-T, while SCR10 TCR-T showed significantly superior killing efficacy compared to both. These results indicate that even under conditions of a low effector-to-target ratio of 0.5:1 and long killing time, the signal transduction receptor SCR10 of this invention can still effectively enhance the killing level of effector T cells against tumor target cells.

[0158] Example 8: Killing effect of TCR-T cells expressing TGF-β signaling receptor SCR11-15 on target cells

[0159] The in vitro killing activity of TCR-T and SCR11-15 TCR-T prepared in Example 3 against A375 cells was detected. The effector-to-target ratio was set at 1.5:1 based on the proportion of EGFP-positive cells. The specific detection steps were the same as in Example 4. The results are as follows: Figure 8 As shown, shortly after the addition of effector cells (24h), SCR11 / 12 / 14 / 15 TCR-T cells exhibited significantly superior target cell killing effects compared to TCR-T cells. After 90h of induction, SCR11-15 TCR-T cells showed more significant killing effects on A375 target cells compared to TCR-T cells. Specifically, the killing effects of SCR11 / 12 TCR-T cells were similar but superior to SCR13 TCR-T cells; the killing effects of SCR14 / 15 TCR-T cells were better than those of SCR11 / 12 TCR-T cells, with SCR15 TCR-T cells showing the best killing effect among all effector cell types. These results indicate that the signal transduction receptor SCR11-15 of this invention can significantly enhance the killing level of effector T cells against tumor target cells even under low effector-to-target ratio conditions, and this enhancement effect persists even at longer induction times.

[0160] Example 9: Killing effect of TCR-T cells expressing TGF-β signaling receptor SCR15 on target cells at low target-to-cell ratio.

[0161] Referring to Example 4, A375 cells were selected as target cells, and TCR-T, SCR15 TCR-T, and RIID4 TCR-T cells were selected as effector cells. The effector-to-target ratio was further reduced to 0.75:1. The killing levels of several effector cells on A375 cells were compared, and the operation was the same as in Example 4. The long-term observation at the low effector-to-target ratio of 0.75:1 was intended to simulate the scenario of T cell killing tumors in a real in vivo environment.

[0162] The results are as follows Figure 9 As shown. Figure 9The results showed that shortly after the addition of effector cells, RIID4 TCR-T exhibited superior killing efficacy compared to TCR-T, while SCR15 TCR-T began to show significantly superior killing efficacy compared to the former two starting 60 hours prior. Even after 100 hours of killing assays, RIID4 TCR-T still showed superior killing efficacy compared to TCR-T, while SCR15 TCR-T showed significantly superior killing efficacy compared to the former two. These results indicate that even under conditions of a low effector-to-target ratio of 0.75:1 and a long killing time, the signal transduction receptor SCR15 of this invention can still effectively enhance the killing level of effector T cells against tumor target cells.

[0163] Example 10: TGF-β signaling receptor enhances the secretion level of TCR-T cytokines

[0164] TGF-β1 (purchased from R&DSYSTEMS, catalog number: 240-B-002 / CF) was added to the TCR-T and SCR1-5 TCRT cell culture systems prepared in Example 3 to a final concentration of 2.0 ng / mL. Three parallel sample wells were set up for each cell group. An additional TCR-T group was not treated with TGF-β1. After 24 hours of treatment, IFN-γ secretion levels were measured using the HTRF IFN-γ detection kit (CisbioHuman IFN gamma kit, catalog number: 62HIFNGPET) according to the instructions. Results are as follows: Figure 10 As shown.

[0165] Figure 10 The results showed that, compared with the untreated TCR-T group, the IFN-γ secretion level of the TCR-T group treated with TGF-β1 was significantly reduced, indicating that the naturally occurring TGF-β receptor on the T cell surface inhibits T cell activation after binding to TGF-β1. After treatment with TGF-β1, the IFN-γ secretion level of SCR1-5 TCR-T cells was significantly increased compared with both the TGF-β1-treated and untreated TCR-T groups. This indicates that immune effector cells, such as TCR-T cells, after introducing the signal transduction receptor of this invention, can effectively enhance T cell activation levels when binding to corresponding ligands such as TGF-β1, and this enhancement effect can offset and surpass the inhibitory effect of the corresponding ligand, such as TGF-β1, binding to the naturally occurring inhibitory receptor on the cell surface.

[0166] Example 11: TGF-β signaling receptor enhances in vivo tumor suppression level of TCR-T.

[0167] The inhibitory effects of TCR-T, RIID2 TCR-T, RIID4 TCR-T, and SCR5 TCR-T on tumor growth after tumor formation in A375 cell line mice were examined in an immunodeficient mouse model. Immunodeficient NSG mice (purchased from The Jackson Laboratory) were used for in vivo tumor formation of the A375 cell line. NSG mice were housed in a standard SPF animal facility for two weeks to acclimatize before tumor cell inoculation, with a 12-hour circadian rhythm, an ambient temperature of 20-24℃, and a humidity of 45-65%. A375 cells were cultured in DMEM medium and 10% fetal bovine serum until 80-90% confluence, then cell counting and viability calculation were performed. Each mouse was anesthetized with isoflurane and subcutaneously injected with 100 μL of a solution containing 1×10⁻⁶ cells on the left side. 7 A375 live cells were suspended in PBS to minimize pain in mice. After tumor formation, tumor-bearing mice were randomly divided into 5 groups (n=6): control group (A375), TCR-T group, RIID2 group, RIID4 group, and SCR5 group. Cells prepared in Example 3 were used for reinfusion. After passing the administered cells through a 70 μm sieve, they were resuspended in PBS. According to the EGFP positivity rate recorded in Table 8, TCR-T had a rate of 44.22%, approximately twice the average proportion of EGFP-positive cells in RIID2 TCR-T, RIID4 TCR-T, and SCR5 TCR-T. The cell dosage for the TCR-T group was 1 × 10⁻⁶ cells / day. 7 Cells were resuspended in 200 μL PBS. The cell dosage for the RIID2 TCR-T, RIID4 TCR-T, and SCR5 TCR-T groups was 2 × 10⁻⁶ cells / mL. 7 Cells were resuspended in 200 μL PBS; the control group received 200 μL PBS per mouse. The drugs were administered via tail vein injection at three time points: D0, D1, and D2. The trend of tumor size change over time was observed in the mice.

[0168] The results are as follows Figure 11 As shown in the figure, compared with the control group that did not undergo cell reinfusion, tumor proliferation in the TCR-T group of tumor-bearing mice was slower. Tumor proliferation in the RIID2 and RIID4 groups was further inhibited compared to the TCR-T group, with the RIID4 group showing slightly slower tumor proliferation than the RIID2 group. The inhibition of tumor proliferation in the SCR5 group was the most significant among all mouse groups, with the tumor proliferation rate further suppressed compared to the RIID2 and RIID4 groups. These results indicate that immune effector cells expressing the TGF-β signaling receptor SCR5 of this invention, such as TCR-T cells, can significantly enhance the inhibitory effect on tumor proliferation in vivo.

[0169] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of this invention. The full scope of this invention is given by the appended claims and any equivalents thereof.

Claims

1. An isolated fusion protein, comprising the following components: (1) Extracellular domains of receptors for immunosuppressive cytokines; (2) Transmembrane region; and (3) Intracellular domains of co-stimulatory signaling molecules; in, (1) The extracellular domain sequence of the receptor for the immunosuppressive cytokine is shown in SEQ ID NO: 3; (2) The amino acid sequence of the intracellular domain of the co-stimulatory signal molecule is shown in any one of SEQ ID NO:7, 9, 11; (3) The amino acid sequence of the transmembrane region is shown in any one of SEQ ID NO:27, 29, 35, 36, 54.

2. The fusion protein as described in claim 1, characterized in that, The amino acid sequence of the fusion protein is shown in any one of SEQ ID NO:1647, SEQ ID NO:1649, SEQ ID NO:1651, SEQ ID NO:1653, SEQ ID NO:1655, SEQ ID NO:1657, SEQ ID NO:1659, SEQ ID NO:1661, SEQ ID NO:1663, SEQ ID NO:1665, SEQ ID NO:1667, SEQ ID NO:1669, SEQ ID NO:1671, SEQ ID NO:1673, and SEQ ID NO:1675.

3. A polynucleotide molecule, selected from: (1) A polynucleotide molecule encoding the fusion protein of claim 1 or 2; and (2) (1) The complementary sequence of the polynucleotide molecule.

4. The polynucleotide molecule as described in claim 3, characterized in that, The polynucleotide molecule includes a sequence selected from any one of SEQ ID NO:1648, SEQ ID NO:1650, SEQ ID NO:1652, SEQ ID NO:1654, SEQ ID NO:1656, SEQ ID NO:1658, SEQ ID NO:1660, SEQ ID NO:1662, SEQ ID NO:1664, SEQ ID NO:1666, SEQ ID NO:1668, SEQ ID NO:1670, SEQ ID NO:1672, SEQ ID NO:1674 and SEQ ID NO:1676, or a complementary sequence thereof.

5. A nucleic acid construct containing the polynucleotide molecule as described in claim 3 or 4.

6. The nucleic acid construct as described in claim 5, characterized in that, The nucleic acid construct is a vector.

7. The nucleic acid construct as described in claim 5, characterized in that, The nucleic acid construct is an expression vector.

8. Genetically engineered cells that express the fusion protein of claim 1 or 2, and / or carry the coding sequence of the fusion protein.

9. The cell as described in claim 8, characterized in that, The cells in question are immune cells.

10. The cell as claimed in claim 8, characterized in that, The cells in question are T cells.

11. The cell as claimed in claim 8, characterized in that, The cells also express CAR or carry the coding sequence for CAR.

12. The cell as claimed in claim 8, characterized in that, The cells also express exogenous TCRs or carry the coding sequence of exogenous TCRs.

13. Use of the fusion protein of claim 1 or 2, the polynucleotide molecule of claim 3 or 4, the nucleic acid construct of any one of claims 5-7, and the genetically engineered cell of any one of claims 8-12 in the preparation of a medicament for the treatment or prevention of melanoma.

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