Method for transforming T cells through phase separation and application thereof

Abstract

The invention provides an application and a method for promoting Lck activation and TCR cytoplasm domain phosphorylation by a CD3 epsilon cytoplasm domain or a CD3 epsilon cytoplasm domain variant through a liquid-liquid phase separation (LLPS) way, recruiting CD3 epsilon molecules in an inhibition state and the like. Also provided are CTR (Chimeric Trigger Receptor) molecules containing only the CD3 [epsilon] cytoplasmic domain as a TCR initiated switch, as well as corresponding engineered cells and methods of treatment.

Description

A method for modifying T cells through phase separation and its application

[0001] This application claims priority to a prior application, patent application number 202310164193.3, filed with the State Intellectual Property Office of China on February 24, 2023, entitled “A Method for Modifying T Cells by Phase Separation and Its Application.” The entire text of this prior application is incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of T cell functional transformation, and specifically relates to a new method for initiating T cell activation, a recombinant gene designed based on the method, a vector and cells containing the gene, and their applications, the results of which can be used for immunotherapy.

[0003] Background of the Invention

[0004] 1. About CD3 subunits

[0005] T cells can specifically recognize foreign antigens and trigger adaptive immune responses against invading pathogens or tumor cells. T cells primarily recognize antigens through their T cell receptors (TCRs). For αβ T cells, the TCR is primarily composed of a TCRαβ dimer responsible for antigen recognition and three signaling dimers: CD3εδ, CD3εγ, and CD3ζζ.

[0006] All CD3 subunits contain immunoreceptor tyrosine-based activation motifs (ITAMs; YxxL / Ix6-8YxxL / I). Tyrosine phosphorylation in ITAMs can activate downstream signaling pathways (Courtney, AH, WLLo, and A. Weiss, TCR Signaling: Mechanisms of Initiation and Propagation. Trends Biochem Sci, 2018. 43(2): p. 108-123.). Each of the CD3ε, δ, and γ subunits contains one ITAM motif, while CD3ζ contains three ITAMs. Therefore, a complete TCR-CD3 complex has 10 ITAMs with a total of 20 tyrosine phosphorylation sites to respond to different antigenic stimuli.

[0007] Other domains at the intracellular end of CD3 also play an important role in TCR signal transduction, including the BRS (Basic Residue Rich Sequence) that directly interacts between CD3ε and CD3ζ and the phospholipid membrane, and the PRS (Proline-Rich Sequence) in CD3ε that interacts with Lck.

[0008] CD3 ITAMs are primarily phosphorylated by Lck and dephosphorylated by CD45. Dual-phosphorylated CD3 ITAMs can bind to the Zap70 tandem SH2 domain, relieving Zap70's self-inhibition and activating downstream signaling pathways (Courtney, AH, WLLo, and A. Weiss, TCR Signaling: Mechanisms of Initiation and Propagation. Trends Biochem Sci, 2018. 43(2): p. 108-123.). Although CD3 ITAMs all have the YxxL / Ix6-8YxxL / I sequence, each CD3 has a different sequence, indicating that different CD3 subunits may have different functions. In addition, there is considerable controversy as to whether the type, number, and number of ITAMs in the CD3 subunits will have a qualitative and quantitative impact on TCR signaling.

[0009] For information on the multiple signaling roles of CD3ε and its application in CAR-T cell therapy, please refer to (Wu W, Zhou Q, Masubuchi T, Shi X, Li H, Xu X, Huang M, Meng L, He X, Zhu H, Gao S, Zhang N, Jing R, Sun J, Wang H, Hui E, Wong CC, Xu C. Multiple Signaling Roles of CD3ε and Its Application in CAR-T Cell Therapy. Cell. 2020 Aug 20; 182(4): 855-871.e23. doi: 10.1016 / j.cell.2020.07.018. Epub 2020 Jul 29. PMID: 32730808.), in which the authors simultaneously quantified the phosphorylation levels of the immunoreceptor tyrosine motifs (ITAMs) of all CD3 chains under TCR stimulation. Due to the substrate selectivity of Lck kinase, CD3ε's ITAM is mostly monophosphorylated and specifically recruits the inhibitory Csk kinase to attenuate TCR signaling, suggesting that the TCR possesses a self-constrained signaling mechanism with both activating and inhibitory motifs. Furthermore, incorporating the CD3ε cytoplasmic domain into second-generation chimeric antigen receptors (CARs) has been found to enhance the anti-tumor activity of CAR-T cells. Mechanistically, CD3ε recruits Csk via its ITAM, reducing CAR-T cytokine production, while the CD3ε basic residue-rich sequence (BRS) promotes CAR-T persistence through p85 recruitment. The article argues that CD3ε is a built-in multifunctional signal tuner, and increasing CD3 diversity represents a strategy for designing next-generation CARs. Furthermore, published patents (202010535848.X, 202010733636.2, and 202010734872.6) relate to the use of CD3ε, a subunit of the TCR complex, and its mutants in CAR-T therapy.

[0010] 2. About Lck enzyme

[0011] Lck belongs to the Src family of kinases (SFKs), a class of non-receptor tyrosine kinases. In immune cells, Src family kinases (SFKs) have been recognized as key regulators of numerous intracellular signaling pathways. Src family kinases (SFKs) occupy proximal positions in numerous signal transduction cascades, including those originating from T and B cell antigen receptors, Fc receptors, growth factor receptors, cytokine receptors, and integrins. In addition to these positive regulatory roles, Src family kinases (SFKs) can also act as negative regulators of cell signaling by phosphorylating immunoreceptor tyrosine-based inhibitory motifs (ITIMs) on inhibitory receptors, leading to the recruitment and activation of inhibitory molecules such as the 5′-inositol phosphatase (SHIP-1)-containing phosphatases SHP-1 and SHP-2.

[0012] Researchers (Li L, Guo X, Shi X, Li C, Wu W, Yan C, Wang H, Li H, Xu C. Ionic CD3-Lck interaction regulates the initiation of T-cell receptor signaling. Proc Natl Acad Sci US A. 2017 Jul 18; 114(29): E5891-E5899. doi: 10.1073 / pnas.1701990114. Epub 2017 Jun 28. PMID: 28659468; PMCID: PMC5530670.) found that the TCR kinase Lck exhibits high selectivity among the four CD3 signaling proteins of the TCR. CD3ε is the CD3 chain that can effectively interact with Lck, mainly through ionic interactions between the CD3ε basic residue rich sequence (BRS) and acidic residues in the unique domain of Lck. Furthermore, the initiation of TCR phosphorylation was clearly investigated using a TCR reconstitution system. Ionic CD3ε-Lck interactions control the phosphorylation level of the entire TCR upon antigen stimulation. In the resting state, the BRS sequence of CD3ε is tethered to the phospholipid membrane and is in an inhibitory state. Antigen stimulation can release this BRS inhibition. The dynamic activation of the CD3ε BRS and the subsequent recruitment of Lck may serve as a key switch for initiating TCR phosphorylation.

[0013] 3. About Liquid-Liquid Phase Separation (LLPS)

[0014] Liquid-liquid phase separation (LLPS) of biomacromolecules (proteins and RNA) has been an emerging hot topic in the international life sciences in recent years (Zheng C, Xu X, Zhang L, Lu D. Liquid-Liquid Phase Separation Phenomenon on Protein Sorting Within Chloroplasts. Front Physiol. 2021; 12: 801212. Published 2021 Dec 24. doi: 10.3389 / fphys.2021.801212). Cells are separated by numerous membrane-enclosed organelles and membrane-free compartments to ensure that a variety of cellular activities occur in a spatiotemporally controlled manner. The molecular mechanisms of membrane-bound organelle dynamics, such as their fusion and fission, vesicle-mediated transport, and membrane contact-mediated cell-cell interactions have been extensively characterized. However, the molecular details of the assembly and function of membrane-free compartments remain elusive. Recent evidence indicates that liquid-liquid phase separation (LLPS) plays a crucial role in the assembly of numerous membraneless compartments, collectively referred to as biomacromolecule aggregates. Phase-separated aggregates participate in diverse biological activities, including higher-order chromatin organization, gene expression, and the sorting of misfolded or unwanted proteins for autophagic degradation. Biomacromolecules, such as proteins and nucleic acids, can condense into liquid-like, membraneless aggregates via LLPS, providing an alternative means of aggregating and separating cellular components in a spatiotemporally defined manner for various functional processes.

[0015] Summary of the Invention

[0016] The activation of T cells is characterized by high sensitivity. Studies have shown that the binding of a single or several pMHCs is sufficient to activate the entire T cell. When a T cell comes into contact with an APC cell, the pMHC molecules on the APC cell will bind to the TCR molecules on the surface of the T cell. The TCR will move to the center of the T-APC cell contact surface in the form of a cluster, and then assemble into the central area of ​​the immune synapse (IS). In the resting state, the TCR on the surface of the T cell exists in the form of a monomer; and the interaction between TCR and pMHC will form an inverse lock key (under the condition of external force loading, the bond life increases), so a single pMHC does not have the ability to bind to multiple TCRs at the same time; therefore, the formation mechanism of the TCR cluster is not clear.

[0017] In the present invention, it was discovered that Lck kinase can phase separate from the intracellular domain of CD3ε or other intracellular domains of signaling molecules, thereby initiating and amplifying T cell activation signals. By constructing and expressing CTR (Chimeric trigger receptor) molecules containing only the intracellular domain of CD3ε, T cells can be specifically activated. Specific aspects of the present invention include:

[0018] In one aspect, the present invention provides the use of CD3ε, NKG2D, KIR3DS1, NCR1, NCR3, CD16α or FcεR1 cytoplasmic domains or variants thereof in the preparation of a liquid-liquid phase separation (LLPS) reagent that promotes Lck or its variants, wherein the Lck variants include phosphorylated variants, open conformational variants or regulatory domain constructs, wherein the liquid-liquid phase separation refers to liquid-liquid phase separation in immune cells, liquid-liquid phase separation in a loaded bilayer lipid, or liquid-liquid phase separation in a solution.

[0019] On the other hand, the present invention provides non-therapeutic uses of CD3ε, NKG2D, KIR3DS1, NCR1, NCR3, CD16α or FcεR1 cytoplasmic domains or their variants, wherein the non-therapeutic uses include experimental uses or research uses, characterized in that they promote liquid-liquid phase separation (LLPS) of Lck or its variants, Lck variants include phosphorylated variants, open conformational variants or regulatory domain constructs, wherein the liquid-liquid phase separation refers to liquid-liquid phase separation in immune cells, liquid-liquid phase separation in loaded bilayer lipids, or liquid-liquid phase separation in solution.

[0020] On the other hand, the present invention provides the use of CD3ε, NKG2D, KIR3DS1, NCR1, NCR3, CD16α or FcεR1 cytoplasmic domains or their variants in the preparation of reagents for promoting Lck activation and TCR cytoplasmic domain phosphorylation through a liquid-liquid phase separation (LLPS) pathway, wherein the Lck variants include phosphorylation variants, open conformational variants or regulatory domain constructs, wherein the liquid-liquid phase separation refers to liquid-liquid phase separation in immune cells, liquid-liquid phase separation in a loaded bilayer lipid, or liquid-liquid phase separation in a solution.

[0021] On the other hand, the present invention provides non-therapeutic uses of CD3ε, NKG2D, KIR3DS1, NCR1, NCR3, CD16α or FcεR1 cytoplasmic domains or their variants, wherein the non-therapeutic uses include experimental uses or research uses, and the uses include CD3ε cytoplasmic domains or CD3ε cytoplasmic domain variants promoting Lck activation and TCR cytoplasmic domain phosphorylation through a liquid-liquid phase separation (LLPS) pathway, wherein the CD3ε variants include phosphorylated variants, and the Lck variants include phosphorylated variants, open conformational variants or regulatory domain constructs, wherein the liquid-liquid phase separation refers to liquid-liquid phase separation in immune cells, liquid-liquid phase separation in loaded bilayer lipids, or liquid-liquid phase separation in solution.

[0022] In another aspect of the invention, the use of CD3ε / Lck phase-separated microclusters to recruit CD3ε molecular reagents in an inhibitory state (membrane-attached state) is provided, wherein the phase separation refers to liquid-liquid phase separation in immune cells, liquid-liquid phase separation in loaded bilayer lipids, or liquid-liquid phase separation in solution.

[0023] In another aspect of the invention, non-therapeutic uses of CD3ε / Lck phase-separated microclusters are provided, and the non-therapeutic uses include experimental uses or research uses, which include the use of CD3ε / Lck phase-separated microclusters to recruit CD3ε molecular reagents in an inhibitory state (membrane state), wherein the phase separation refers to liquid-liquid phase separation in immune cells, liquid-liquid phase separation in loaded bilayer lipids, or liquid-liquid phase separation in solutions.

[0024] The CD3ε cytoplasmic domain variants mentioned in the above invention are phosphorylated variants, specifically referring to the variant CD3ε-pY1 in which the first ITAM tyrosine is monophosphorylated, or the variant pCD3ε in which two ITAM tyrosines are doubly phosphorylated.

[0025] The Lck variant mentioned in the above invention is a regulatory domain construct LckUD-SH3-SH2, which refers to a construct formed by connecting the UD, SH3, and SH2 domains.

[0026] The present invention also provides a method for regulating CD3ε / Lck phase separation by mutating the CD3ε cytoplasmic domain, which is for non-therapeutic purposes, wherein the phase separation refers to liquid-liquid phase separation in immune cells, liquid-liquid phase separation in a loaded bilayer lipid, or liquid-liquid phase separation in a solution, and the mutation is a phosphorylation mutation or a mutation in the BRS region.

[0027] The immune cells described in the above invention include immune cells in living animals or immune cells cultured in vitro.

[0028] The immune cells in the above invention include T cells produced by the immune system, commercial model cells such as Jurkat cells, or artificially constructed simulation cells expressing TCR-CD3 complexes.

[0029] The present invention also provides non-therapeutic uses of Csk, which include experimental uses or research uses, and the use is to use Csk to dissolve CD3ε / Lck phase-separated microclusters, wherein the CD3ε / Lck phase-separated microclusters are aggregates formed by liquid-liquid phase separation of CD3ε / Lck in immune cells, liquid-liquid phase separation in loaded bilayer lipids, or liquid-liquid phase separation in solution, wherein CD3ε is a CD3ε cytoplasmic domain or a variant that can form phase separation, such as a phosphorylated variant, and Lck comprises a wild type or a variant that can form phase separation, such as a phosphorylated variant, an open conformational variant, or a regulatory domain construct.

[0030] The present invention also provides a CTR (Chimeric trigger receptor) molecule, whose intracellular domain contains CD3ε, NKG2D, KIR3DS1, NCR1, NCR3, CD16α or FcεR1 cytoplasmic domain or its variants as a switch for TCR activation or immune cell activation.

[0031] Preferably, the CTR (Chimeric trigger receptor) molecule comprises an extracellular domain, an optional hinge region, a transmembrane domain and an intracellular domain connected in sequence;

[0032] The extracellular domain includes an optional signal peptide and an antigen recognition region;

[0033] The intracellular domain contains only the CD3ε cytoplasmic domain or a variant thereof as a switch for TCR activation or immune cell activation.

[0034] In certain embodiments of the present invention, the CTR molecule does not comprise the extracellular domain of the CD3ε subunit or the transmembrane domain of the TCR complex subunit, especially the transmembrane domain of the CD3ε subunit.

[0035] In a specific embodiment of the present invention, the antigen recognition region in the CTR (Chimeric trigger receptor) molecule is selected from a single-chain antibody against a tumor surface antigen, wherein the tumor surface antigen is selected from one or more of CD19, mesothelin, CD20, CD22, CD123, CD30, CD33, CD38, CD138, BCMA, Fibroblast activation protein, Glypican-3, CEA, EGFRvIII, PSMA, Her2, IL13Rα2, CD171, claudin18.2 and GD2; the single-chain antibody is selected from a single-chain antibody fragment, a single-chain Fv (scFv), a single-chain Fab, a single-chain Fab′, a single-domain antibody fragment, a single-domain multispecific antibody, an intracellular antibody, a nanobody or a single-chain immune factor;

[0036] Preferably, the single-chain antibody is based on the following monoclonal antibodies: Inebilizumab, Rituximab, Ofatumumab, Glofitamab, Trastuzumab (trade name Herceptin), Pertuzumab (also known as 2C4, trade name Perjeta), Nimotuzumab (trade name Taixinsheng), Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, Retifanlimab, cBR96 and Glematumamab.

[0037] Most preferably, the antigen recognition region is a single-chain antibody fragment targeting CD19 (SEQ NO: 3), a myeloma antigen BCMA recognition domain sequence (SEQ NO: 5) or a single-chain antibody fragment targeting EGFR (SEQ NO: 7).

[0038] In another specific embodiment of the present invention, the antigen recognition region in the CTR (Chimeric trigger receptor) molecule also includes a protein domain based on natural ligand-receptor interaction, such as the extracellular domain of PD-1 protein (which can recognize PD-L1 / PD-L2 protein), the extracellular domain of CD2 (recognizing CD58 / CD59), the extracellular domain of CD28 / CD152 (recognizing CD80 / CD86), etc.

[0039] In another specific embodiment of the present invention, the transmembrane domain is selected from the transmembrane region of CD4, CD8, CD28, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, EGFR (epidermal growth factor receptor) or GITR; preferably, the transmembrane region of a type I single transmembrane molecule or a homologous multimer thereof; further preferably, the CD8 transmembrane domain;

[0040] The CTR molecules specifically provided by the present invention include but are not limited to:

[0041] αCD19-CD8-CD8-CD3ε

[0042] αCD19-CD28-CD28-CD3ε

[0043] PD1-PD1-PD1-CD3ε

[0044] PD1-PD1-PD1-CD3ε

[0045] PD1-PD1-PD1-NCR1

[0046] PD1-PD1-PD1-KIR3DS1

[0047] PD1-PD1-PD1-NKG2D

[0048] PD1-PD1-PD1-CD16α

[0049] αCD19-CD8-CD8-NCR1

[0050] αCD19-CD8-CD8-KIR3DS1

[0051] αCD19-CD8-CD8-NKG2D

[0052] αCD19-CD8-CD8-CD16α

[0053] αBCMA-CD8-CD8-CD3ε

[0054] αBCMA-CD28-CD28-CD3ε

[0055] αBCMA-CD8-CD8-NCR1

[0056] αBCMA-CD8-CD8-KIR3DS1

[0057] αBCMA-CD8-CD8-NKG2D

[0058] αBCMA-CD8-CD8-CD16α

[0059] αEGFR-CD8-CD8-CD3ε

[0060] αEGFR-CD28-CD28-CD3ε

[0061] αEGFR-CD8-CD8-NCR1

[0062] αEGFR-CD8-CD8-KIR3DS1

[0063] αEGFR-CD8-CD8-NKG2D

[0064] αEGFR-CD8-CD8-CD16α.

[0065] The present invention also provides a nucleic acid sequence selected from:

[0066] i) encoding the CTR (Chimeric trigger receptor) molecule of claim 1; or ii) a nucleic acid sequence complementary to i).

[0067] The present invention also provides a nucleic acid construct, which contains the above-mentioned nucleic acid sequence;

[0068] Preferably, the nucleic acid construct is a vector;

[0069] More preferably, the nucleic acid construct is a lentiviral vector, a retroviral vector, an adenoviral vector or an adeno-associated viral vector, containing the above-mentioned nucleic acid sequence.

[0070] The present invention also provides a lentiviral vector system, which contains the above-mentioned nucleic acid sequence and lentiviral vector auxiliary components.

[0071] The present invention also provides a genetically modified cell, characterized in that the cell expresses the above-mentioned CTR (Chimeric trigger receptor) molecule, or contains the above-mentioned nucleic acid sequence, or contains the above-mentioned nucleic acid construct, or is infected with the above-mentioned lentiviral vector system; preferably, the cell is selected from autologous or allogeneic T cells, B cells, NK cells, macrophages, monocytes, dendritic cells, neutrophils, basophils, eosinophils, mast cells, NK-T cells, MAIT cells, hematopoietic stem cells, embryonic stem cells, induced pluripotent stem cells, and red blood cells, and the T cells include αβT cells, γδT cells, and regulatory T cells.

[0072] The present invention also provides a pharmaceutical composition or kit comprising the aforementioned CTR (Chimeric trigger receptor) molecule, the aforementioned nucleic acid sequence, or the aforementioned nucleic acid construct, or the aforementioned lentiviral vector system, or the aforementioned genetically modified cells.

[0073] The present invention also provides the use of the above-mentioned CTR (chimeric trigger receptor) molecule, the above-mentioned nucleic acid sequence, or the above-mentioned nucleic acid construct, or the above-mentioned lentiviral vector system in preparing any one or more of the following use products: (1) preparing T cells or NK cells; (2) enhancing the proliferation ability of T cells or NK cells; (3) improving the killing ability of T cells or NK cells.

[0074] The present invention also provides the use of the aforementioned CTR (Chimeric trigger receptor) molecule, the aforementioned nucleic acid sequence, or the aforementioned nucleic acid construct, or the aforementioned lentiviral vector system, or the aforementioned genetically modified cell in the preparation of any one or more of the following products: (1) treating cancer; (2) inhibiting the cytokine storm generated during cancer treatment;

[0075] Preferably, the cancer is selected from adrenocortical carcinoma, bladder cancer, breast cancer, cervical cancer, bile duct carcinoma, colorectal cancer, esophageal cancer, glioblastoma, glioma, hepatocellular carcinoma, head and neck cancer, kidney cancer, leukemia, lymphoma, lung cancer, melanoma, mesothelioma, multiple myeloma, pancreatic cancer, pheochromocytoma, plasmacytoma, neuroblastoma, ovarian cancer, prostate cancer, sarcoma, gastric cancer, uterine cancer and thyroid cancer, and / or optionally wherein the cancer is a blood cancer or a solid tumor cancer.

[0076] The present invention also provides use of the CD3ε cytoplasmic domain or a variant thereof in preparing a T cell or NK cell activation reagent.

[0077] Explanation of relevant technical terms in the present invention:

[0078] Lck or its variants

[0079] Lck belongs to the Src family of kinases (SFKs), a type of non-receptor tyrosine kinase that contains UD, SH3, SH2 and other domains. Its activity is mainly regulated by the SH2 and SH3 domains and two tyrosine phosphorylation sites, Y505 and Y394.

[0080] In the present invention, Lck-wt refers to the wild type of Lck kinase.

[0081] In the present invention, Lck phosphorylation variants refer to variants in which the corresponding tyrosines are phosphorylated, such as Y394 and Y505, or expressed as pY394 or pY505, wherein pY394 is the activated form of Lck.

[0082] In the present invention, Lck mutants refer to mutants in which the corresponding amino acids are mutated, such as Y505F (open conformation) and K273R (null kinase activity) mutants.

[0083] In the present invention, Lck regulatory domain constructs refer to various combinations of UD, SH3 and SH2 domains. For example, LckUD-SH3-SH2 refers to a construct formed by connecting UD, SH3 and SH2 domains.

[0084] In the present invention, the Lck kinase domain refers to the Lck kinase domain unit (245-501 a.a.) expressed alone.

[0085] CD3ε and its variants

[0086] The cytoplasmic domain of CD3ε or the intracellular domain of CD3ε refers to the region of the CD3ε subunit located in the cytoplasm (amino acid sequence positions 153-207), including domains such as BRS, PRS, RK and ITAM. Unless otherwise specified, when this application mentions CD3ε, it refers to its cytoplasmic domain.

[0087] The CD3ε cytoplasmic domain variants of the present invention include:

[0088] 1) Truncated or extended variants, based on the CD3ε cytoplasmic domain (153-207) sequence, truncated or extended by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at the C or N terminus, comprising complete BRS, PRS, RK, and ITAM domains, and having or not having the ability to form liquid-liquid phase separation with Lck. Variants that add tags or amino acids such as cysteine ​​for labeling or separation at the C or N terminus also belong to this type of variant. Those skilled in the art will appreciate that the above tags or amino acids can be removed when designing or preparing CTR molecules;

[0089] 2) Combination variants, comprising any combination of BRS, PRS, RK and ITAM domains or domain variants defined below, with or without the ability to form liquid-liquid phase separation with Lck;

[0090] For example, CD3ε-dBRS refers to a variant that combines the PRS, RK, and ITAM domains.

[0091] 3) Domain variants

[0092] 3-1) BRS domain mutants,

[0093] The BRS domain mutant refers to a mutant that retains the positively charged amino acids in the BRS motif KNRKAKAK and has the ability to form liquid-liquid phase separation with Lck;

[0094] Or it refers to a mutant in which the BRS motif is mutated from the positively charged amino acid in KNRKAKAK to other non-positively charged amino acids and does not have the ability to form liquid-liquid phase separation with Lck, for example, from KNRKAKAK to SNSSASAS;

[0095] 3-2) ITAM domain mutants;

[0096] The ITAM domain mutant is a mutant in which any one or two tyrosine residues in the domain are mutated to phenylalanine or to other amino acids;

[0097] Alternatively, the ITAM domain mutant is a phosphorylation variant, specifically a variant CD3ε-pY1 in which the first ITAM tyrosine is monophosphorylated, or a variant CD3ε-pY2 in which the second ITAM tyrosine is monophosphorylated, or a variant pCD3ε in which two ITAM tyrosines are dually phosphorylated;

[0098] 3-3) PRS domain mutants

[0099] For example, mutations such as PPPVPNP to SSSVSNS still possess phase separation activity;

[0100] 3-4) RK domain mutants

[0101] For example, mutations such as RKGQ to SSGQ still possess phase separation activity;

[0102] or a combination of the above domain variants;

[0103] 4) Conservative variants refer to variants in which one amino acid is substituted by another amino acid of the same class and still has the ability to form liquid-liquid phase separation with Lck, for example, one acidic amino acid is substituted by another acidic amino acid, one basic amino acid is substituted by another basic amino acid, or one neutral amino acid is substituted by another neutral amino acid;

[0104] 5) any combination of the above variants;

[0105] Optionally, the variant has greater than 95%, 96%, 97%, 98% or 99% sequence identity to the CD3ε cytoplasmic domain (153-207) sequence.

[0106] The following variations are included in the specific embodiments of the present invention:

[0107] The cysteine ​​residues in the above variants were used for in vitro labeling and were removed during the design and preparation of the CTR molecule.

[0108] Those skilled in the art can predict the phase separation activity of any variant based on the examples and analyses disclosed in the present invention, and use it to construct CTR chimeric molecules or to verify and study the phase separation mechanism.

[0109] CD3ε / Lck phase separation microclusters refer to aggregates formed by liquid-liquid phase separation in immune cells, liquid-liquid phase separation in loaded bilayer lipids, or liquid-liquid phase separation in solution, wherein CD3ε is the CD3ε cytoplasmic domain or a variant that can form phase separation, such as a phosphorylated variant, and Lck comprises a wild type or a variant that can form phase separation, such as a phosphorylated variant, an open conformation variant, or a regulatory domain construct.

[0110] Csk (C-terminal Src kinase Csk) is a kinase of the Src homologous family and one of the most important negative regulatory factors. It is a phosphorylation kinase of Lck kinase Y505 (negatively regulating Lck activity).

[0111] The "Chimeric trigger receptor (CTR) molecule" in the present invention is an artificially engineered molecule that can anchor specific molecules (such as antibodies) that recognize tumor cell surface antigens to immune cells (such as T cells), enabling the immune cells to recognize tumor antigens or viral antigens and activate the immune cells by using the intracellular domain of CD3ε or other receptor molecules as a switch for TCR activation. The CTR (Chimeric trigger receptor) molecule includes:

[0112] The extracellular domain, hinge region, transmembrane domain and intracellular domain (ICD) are connected in sequence;

[0113] The extracellular domain includes an optional signal peptide and an antigen recognition region;

[0114] The intracellular domain contains only the cytoplasmic domain of CD3ε or other receptor molecules as a switch for TCR activation.

[0115] The signal peptide is located at the extracellular end of the CTR molecule. Its function is to guide the nascent CAR protein into the endoplasmic reticulum of the cell. The CTR protein is glycosylated within the endoplasmic reticulum, and the glycosylated CTR can then appear on the T cell membrane. Any signal peptide sequence from animal cells can be used on the CTR molecule. In the present invention, the signal peptide is optional and can be used or not.

[0116] Among them, the antigen recognition region recognizes tumor surface antigens, and the antigen recognition region is preferably a single-chain antibody against a tumor surface antigen, and the tumor surface antigen is selected from one or more of CD19, mesothelin, CD20, CD22, CD123, CD30, CD33, CD38, CD138, BCMA, Fibroblast activation protein, Glypican-3, CEA, EGFRvIII, PSMA, Her2, IL13Rα2, CD171, claudin18.2 and GD2; the single-chain antibody is selected from a single-chain antibody fragment, a single-chain Fv (scFv), a single-chain Fab, a single-chain Fab′, a single-domain antibody fragment, a single-domain multispecific antibody, an intracellular antibody, a nanobody or a single-chain immune factor.

[0117] Preferably, the single-chain antibody is derived from the following monoclonal antibodies: Inebilizumab, Rituximab, Ofatumumab, Glofitamab, Trastuzumab (trade name Herceptin), Pertuzumab (also known as 2C4, trade name Perjeta), Nimotuzumab (trade name Taixinsheng), Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, Retifanlimab, cBR96 and Glematumamab.

[0118] Most preferably, the antigen recognition region is a single-chain antibody fragment targeting CD19 (SEQ NO: 3), a myeloma antigen BCMA recognition domain sequence (SEQ NO: 5), or a single-chain antibody fragment targeting EGFR (SEQ NO: 7).

[0119] Among them, the above-mentioned antigen recognition region also includes protein domains based on natural ligand-receptor interactions, such as the extracellular domain of PD-1 protein (which can recognize PD-L1 / PD-L2 protein), the extracellular domain of CD2 (recognizing CD58 / CD59), the extracellular domain of CD28 / CD152 (recognizing CD80 / CD86), etc.

[0120] The "hinge region" herein refers to the hydrophilic region between the antigen recognition domain and the transmembrane domain. In the present invention, the hinge region is optional and may or may not be used. The hinge region can be derived from a variety of different antibodies or antigen receptors, particularly from CD4 molecules. In a specific embodiment, the hinge region can be selected from, for example, CD4, CD8α, CD28, IgG1, IgG4, and CD279 (PD-1).

[0121] Here, the "transmembrane region" only needs to include a peptide that can penetrate the cell membrane. The transmembrane region preferably used is the transmembrane region of a CD molecule. In one embodiment, the transmembrane region can be selected from, for example, the transmembrane region of CD4, CD8, CD28, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, EGFR (epidermal growth factor receptor), or GITR. The transmembrane region of the present invention is preferably the transmembrane region of a type I single-pass transmembrane molecule or a homologous multimer thereof; in a preferred embodiment of the present invention, the CD8 transmembrane domain is used.

[0122] As used herein, the terms "identity" or "homology" generally refer to the proportion of nucleotide bases or amino acid residues in a candidate sequence that are identical to those in a corresponding sequence, after alignment and, if necessary, introduction of gaps to achieve the maximum percent identity over the entire sequence, without considering any conservative substitutions as part of the sequence identity. N-terminal or C-terminal extensions or insertions should not be construed as reducing identity or homology. Methods and computer programs for alignment are available and well known in the art. For example, sequence identity can be determined using sequence analysis software.

[0123] Nucleotide sequence and vector

[0124] The polynucleotide sequences of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand. The present invention also encompasses degenerate variants of polynucleotide sequences encoding fusion proteins, i.e., nucleotide sequences that encode the same amino acid sequence but differ in nucleotide sequence.

[0125] The polynucleotide sequences described herein can generally be obtained by PCR amplification. Specifically, primers can be designed based on the nucleotide sequences disclosed herein, particularly the open reading frame sequences, and amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates to obtain the relevant sequences. Long sequences often require two or more PCR amplifications, followed by splicing the fragments amplified from each amplification into the correct order.

[0126] The nucleic acid constructs provided herein also include one or more regulatory sequences operably linked to the aforementioned polynucleotide sequences. The coding sequence of the CTR (chimeric trigger receptor) molecule described herein can be manipulated in a variety of ways to ensure expression of the protein. Prior to insertion into a vector, the nucleic acid construct can be manipulated according to the specific expression vector or requirements. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0127] The regulatory sequence may be a suitable promoter sequence. The promoter sequence is generally operably linked to the coding sequence of the protein to be expressed. The promoter may be any nucleotide sequence that exhibits transcriptional activity in the selected host cell, including mutant, truncated, and hybrid promoters, and may be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell.

[0128] The regulatory sequence may also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operably linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell may be used in the present invention.

[0129] The regulatory sequence may also be a suitable leader sequence, a non-translated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell may be used in the present invention.

[0130] Preferably, the nucleic acid construct is a vector.

[0131] Typically, the expression of the polynucleotide sequence encoding the CTR (Chimeric trigger receptor) molecule is achieved by operably connecting the polynucleotide sequence encoding the CAR to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and integration of eukaryotic cells. Typical cloning vectors include transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

[0132] The polynucleotide sequence encoding the CTR (Chimeric trigger receptor) molecule of the present invention can be cloned into many types of vectors. For example, it can be cloned into a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Furthermore, the vector is an expression vector. The expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Typically, a suitable vector contains a replication origin that functions in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers.

[0133] More preferably, the nucleic acid construct is a lentiviral vector containing a replication origin, a 3'LTR, a 5'LTR and the aforementioned polynucleotide sequence.

[0134] The promoter can use a constitutive promoter sequence, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, heme promoter and creatine kinase promoter. Further, the use of inducible promoters can also be considered. The use of inducible promoters provides a molecular switch that can turn on the expression of a polynucleotide sequence operably connected to an inducible promoter when the expression period is expressed, and turn off the expression when the expression is not expected. Examples of inducible promoters include but are not limited to metallothionein promoters, glucocorticoid promoters, progesterone promoters and tetracycline promoters.

[0135] In order to evaluate the expression of CTR (Chimeric trigger receptor) molecule polypeptides or portions thereof, the expression vector introduced into the cell may also contain either or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a cell population sought to be transfected or infected by a viral vector. In other aspects, the selectable marker can be carried on a single DNA segment and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neomycin, puromycin, and the like.

[0136] The reporter gene is used to identify the cells of potential transfection and to evaluate the functionality of the regulatory sequence. After DNA has been introduced into the recipient cells, the expression of the reporter gene is measured at the appropriate time. Suitable reporter genes can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein genes. Suitable expression systems are known and can utilize known technology to prepare or commercially obtain.

[0137] Methods for introducing genes into cells and expressing genes in cells are known in the art. Vectors can be easily introduced into host cells, for example, mammalian, bacterial, yeast, or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0138] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0139] Biological methods for introducing polynucleotides into host cells include the use of viral vectors, particularly lentiviral vectors, which have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, etc. Many virus-based systems have been developed for transferring genes into mammalian cells. For example, lentiviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into lentiviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject's cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0140] The present invention provides a lentiviral vector system comprising the aforementioned nucleic acid construct and lentiviral vector auxiliary components. The lentiviral auxiliary components include a lentiviral packaging plasmid and a cell line. The lentiviral vector system is constructed by viral packaging of the aforementioned nucleic acid construct with the assistance of the lentiviral packaging plasmid and cell line. The method for constructing the lentiviral vector system is commonly used in the art.

[0141] Cell therapy

[0142] The present invention also includes a type of cell therapy in which T cells are genetically modified to express the chimeric trigger receptor (CTR) molecule described herein (hereinafter referred to as CTR-T cells), and the CTR-T cells are infused into a recipient in need thereof. The infused cells are capable of killing tumor cells in the recipient.

[0143] The anti-tumor immune response elicited by CTR-T cells can be an active or passive immune response. In addition, the CTR-mediated immune response can be part of an adoptive immunotherapy procedure, in which CTR-T cells induce an immune response specific for the antigen-binding portion of the CTR molecule.

[0144] Treatable cancers may be non-solid tumors, such as hematological tumors, eg, leukemias and lymphomas.

[0145] The present invention provides gene-modified T cells or pharmaceutical compositions containing the gene-modified T cells, wherein the cells contain the aforementioned polynucleotide sequence, or contain the aforementioned nucleic acid construct, or are infected with the aforementioned lentiviral vector system.

[0146] The CTR molecule-modified T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or other components such as related cytokines or cell populations. Briefly, the pharmaceutical compositions of the present invention may include CTR cells as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0147] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease.

[0148] When an "immunologically effective amount," "anti-tumor effective amount," "tumor-inhibitory effective amount," or "therapeutic amount" is indicated, the precise amount of the composition of the present invention to be administered can be determined by a physician, who takes into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition. It can be generally stated that a pharmaceutical composition comprising the T cells described herein can be administered in an amount of 10 4 to 10 9 The dose of cells / kg body weight is preferably 10 5 to 10 6The T cell composition can be administered at a dose of 10 cells / kg body weight. The T cell composition can also be administered multiple times at these doses. The cells can be administered using infusion techniques well known in immunotherapy. The optimal dosage and treatment regimen for a particular patient can be readily determined by those skilled in the medical field by monitoring the patient's disease signs and adjusting treatment accordingly.

[0149] Administration of the subject compositions can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous injection, or intraperitoneally. In one embodiment, the T cell compositions of the present invention are administered to the patient by intradermal or subcutaneous injection. In another embodiment, the T cell compositions of the present invention are preferably administered by intravenous injection. The T cell compositions can be injected directly into a tumor, lymph node, or site of infection.

[0150] In some embodiments of the present invention, the CTR-T cells or compositions thereof of the present invention may be combined with other therapies known in the art. Such therapies include, but are not limited to, chemotherapy, radiotherapy, and immunosuppressants. For example, treatment may be combined with various radiotherapy agents, including cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, FK506, fludarabine, rapamycin, and mycophenolic acid. In further embodiments, the cell compositions of the present invention are administered to a patient in combination with (e.g., before, concurrently with, or after) bone marrow transplantation, T cell ablation therapy using chemotherapeutic agents such as fludarabine, external beam radiotherapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH.

[0151] In the present invention, "anti-tumor ability" refers to a biological effect, which can be represented by a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with cancer.

[0152] "Patient," "subject," "individual," and the like are used interchangeably herein to refer to a living organism, such as a mammal, in which an immune response can be elicited. Examples include, but are not limited to, humans, dogs, cats, mice, rats, and transgenic species thereof.

[0153] Optionally, the tumor is selected from one or more of leukemia and solid tumors.

[0154] Optionally, the tumor is selected from B-cell lymphoma, mantle cell lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, and acute myeloid leukemia.

[0155] Technical effects of the present invention

[0156] There are currently three generations of CARs, and "first generation" CARs are typically single-chain polypeptides consisting of a transmembrane domain fused to a cytoplasm / intracellular domain as an antigen binding domain by scFv, which includes a primary immune cell signaling sequence such as an intracellular domain from a CD3 ζ chain (which is a primary transmitter of a signal from an endogenous TCR). "First generation" CARs can provide de novo antigen recognition and cause activation of both CD4+ and CD8+ T cells by the CD3 ζ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation. "Second generation" CARs add intracellular domains from various costimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40) to the primary immune cell signaling sequence of CAR to provide additional signals to T cells. Therefore, "second generation" CARs include fragments that provide costimulation (e.g., CD28 or 4-1BB) and activation (e.g., CD3 ζ). Preclinical studies have shown that "second generation" CARs can improve the anti-tumor activity of T cells. For example, the robust efficacy of "second generation" CAR modified T cells has been demonstrated in clinical trials targeting CD19 molecules in patients with chronic lymphoblastic leukemia (CLL) and acute lymphoblastic leukemia (ALL). "Third generation" CARs include those fragments that provide multiple costimulations (e.g., CD28 and 4-1BB) and activation (e.g., CD3 ζ). It can be seen that the "CTR (Chimeric trigger receptor) molecule" of the present invention is different in structure from the above-mentioned three-generation CARs (the chimeric antigen receptor disclosed in CN 112500492 A contains the CD3ε intracellular region, but it is connected between the intracellular domain and the transmembrane domain, which is a typical second-generation CAR). Compared with the traditional CAR-T design, the CTR molecule based on the CD3ε intracellular domain has the following advantages: First, due to the membrane-attaching autoinhibitory nature of CD3ε, the CTR molecule has a higher specificity and ligand dependence (the excessively long intracellular domain of the traditional CAR molecule may cause the instability of its membrane-attaching autoinhibitory state, thereby leading to a stronger endogenous signal); second, because the CTR molecule itself does not participate in the transmission of T cell downstream signals, it mainly recruits endogenous TCR to initiate T cell activation through phase separation. Therefore, compared with traditional CAR-T, it has richer intracellular signals and is closer to native T cells, TCR-T and STAR-T. Compared with TCR-T and STAR-T, CTR-T is simpler to prepare and has the same flexibility in extracellular target and transmembrane domain selection as CAR-T. BRIEF DESCRIPTION OF THE DRAWINGS

[0157] Figure 1: CD3ε phase separates from Lck (left) and recruits other subunits of the TCR complex (right)

[0158] Left: The ability of LckUD-SH3-SH2 to form aggregates in solution with the indicated intracellular portions of CD3 molecules. CD3-Alexa647 and LckUD-SH3-SH2-Alexa488 were mixed in PBS containing 0.1% BSA for 1 hour. Scale bar, 20 μm.

[0159] Right: Recruitment of CD3 chains to CD3ε / LckUD-SH3-SH2 aggregates. CD3-Alexa647 and CD3ε-Alexa546 were simultaneously mixed with LckUD-SH3-SH2-Alexa488 in PBS containing 0.1% BSA and incubated at room temperature for 1 hour. Scale bar, 20 μm.

[0160] Figure 2: CD3ε / Lck phase separation promotes Lck activation and phosphorylation of CD3ε and CD3ζ

[0161] A: Schematic diagram of the experimental design. CD3ε-wt and CD3ζ were mixed with LckUD-SH3-SH2 to preform aggregates. As a control, CD3ε-mBRS was used in parallel to generate aggregation-free conditions. Subsequently, Lck-wt was added and recruited to preformed CD3ε / LckUD-SH3-SH2 aggregates, or in the case of CD3ε-mBRS, remained in solution. ATP-Mg was then provided. 2+ to initiate the phosphorylation reaction.

[0162] B: Phosphorylation levels of CD3ε, CD3ζ, and Lck-pY394 were measured by Western blotting.

[0163] Figure 3. Phosphorylation of CD3ε promotes phase separation of CD3ε / Lck

[0164] Figure 4. Phase separation of CD3ε and Lck on in vitro reconstituted two-dimensional phospholipid membranes

[0165] Figure 5. CD3ε / Lck phase separation can recruit CD3ε molecules in the inhibitory state (membrane state)

[0166] Figure 6. Csk dissolves CD3ε / Lck aggregates

[0167] Figure 7. CD3ε domain mutations affect CD3ε / Lck phase separation (CD3ε-null, referring to simultaneous mutations in the BRS / PRS / RK domains)

[0168] Figure 8. Comparison of four CD3 intracellular domains in mediating T cell IL-2 secretion

[0169] Figure 9. CD3ε intracellular domain-mediated T cell activation depends on endogenous TCR engagement

[0170] Wild-type Jurkat cells (A, B) and TCR-deficient Jurkat cells (C, D) containing chimeric molecules of the CD3ε intracellular domain (A, C) and CD3ζ intracellular domain (B, D), respectively, were co-cultured with Raji cells, and the cell culture supernatant was collected to detect the secretion of IL-2.

[0171] Figure 10. Effects of different hinge regions and transmembrane domains on CD3ε intracellular domain-mediated T cell activation

[0172] Chimeric molecules containing the intracellular domain of CD3ε were constructed using the hinge region and transmembrane domain of CD8 (A) and CD28 (B), respectively. Jurkat cells were used to express the two chimeric receptors and co-cultured with Raji cells. The culture supernatant was then collected for IL-2 detection.

[0173] Figure 11. Functional validation of engineered T cells expressing PD-1 and CD3ε chimeric receptor (CTR)

[0174] A: Antibody activation of Jurkat cells engineered with the CD3ε cytoplasmic domain. Jurkat cells overexpressing a chimeric molecule composed of the PD-1 extracellular domain, transmembrane domain, and CD3ε cytoplasmic domain were co-cultured with beads linked to the corresponding antibody. The PD-1 antibody-linked beads effectively activated the Jurkat cells expressing the chimeric molecule.

[0175] B: Raji cells stimulate IL-2 secretion from Jurkat cells engineered with the CD3ε cytoplasmic domain based on ligand-receptor interactions. The corresponding number of Raji cells overexpressing PD-L1 were co-cultured with Jurkat cells expressing a chimeric molecule (PD-1 extracellular domain, CD8 transmembrane domain, and CD3ε cytoplasmic domain).

[0176] Figure 12. CTR-T cells that recognize different antigens mediate primary T cell activation

[0177] A CTR containing a single-chain antibody, CD8 hinge region, CD8 transmembrane domain, and CD3ε intracellular domain was constructed and introduced into primary human T cells. CTR-T that recognizes CD19 (A) and BCMA (B), respectively, can recognize the corresponding antigens and mediate T cell activation, thereby secreting IL-2.

[0178] Figure 13. CTR-T treatment of mouse tumor model

[0179] A: Luciferase imaging of the mouse model inoculated with Nalm6 cells treated with CTR-T; B: Survival curves of mice in the PBS group and the CTR-T treatment group.

[0180] Figure 14. CTR-mediated specific activation of NK cells

[0181] A: NK92MI cells expressing CTR:αCD19-CD8-CD8-CD3ε molecules recognized IFN-γ secretion activated by Raji cells; B: CD19 antibody blocked the recognition of Raji cells by CTR-NK cells.

[0182] Figure 15. New switch molecules based on phase separation mechanism and functional verification

[0183] A: Representative images of phase separation between the intracellular domains of different receptors and Lck; B: Statistical diagram of phase separation between different receptors and Lck mediating Lck aggregation on the diastereomer membrane; C: CTR-mediated NK cell activation based on these new switch molecules. DETAILED DESCRIPTION

[0184] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0185] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0186] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0187] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in the art.

[0188] 1. Materials and Methods

[0189] Antibody

[0190] The antibodies used in this invention were purchased from various commercial suppliers:

[0191] Biotin-labeled CD3ε antibody (UCHT1, Abcam, ab191112), FITC-labeled CD3ε antibody (OKT-3, Biolegend, 317306), PE-labeled Lck antibody (pY505, BD, 558552), PE-labeled Src antibody (pY418, BD, 560094), Csk antibody (Sino Biological, 200710-T44), Anti-Phospho-tyrosine (4G10, Millipore, 05-321), Goat anti-Rabbit IgG-APC (Solarbio, K0034G-APC), pCD3ζ antibody (pY142, Abcam, ab245764), CD3ζ antibody (Santa Cruz Biotechnology, sc_1239), pZap70 antibody (Cell Signaling Technology, 2701), Zap70 antibody (Abcam, ab32410), pPLCγ1 antibody (Cell Signaling Technology, 14008), PLCγ1 antibody (Santa Cruz Biotechnology, sc_7290), pErk antibody (Cell Signaling Technology, 4370S), Erk antibody (Cell Signaling Technology, 4695S), CD3 antibody (eBioscience, 16-0037-85), APC-labeled CD3 antibody (eBioscience, 17-0038-42).

[0192] Chimeric molecule construction

[0193] All chimeric molecules are carried using lentiviral vectors. The entire chimeric molecule consists of an extracellular domain, a hinge region, a transmembrane domain, and an intracellular domain from the N-terminus to the C-terminus. The four domains are connected in sequence by enzyme ligation. The extracellular domain is a CD19 single-chain antibody (clone number FMC63) sequence using the CD8α signal peptide.

[0194] Jurkat cells

[0195] The chimeric molecules were transfected into Jurkat cells via lentiviral transfection for overexpression. Successfully transfected cells were sorted by flow cytometry and used for subsequent functional experiments. All Jurkat cells were cultured in RPMI1640 medium supplemented with 100 U / mL streptomycin, 100 μg / mL penicillin, and 10% fetal bovine serum at 37°C and 5% CO2.

[0196] Cell function experiments

[0197] 1×10 4 Jurkat cells were seeded in U-shaped 96-well culture plates 30 minutes in advance. The indicated number of antibody-linked beads or Raji cells were added to a total volume of 200 μl. The cells were incubated at 37°C for 16–24 hours. The culture supernatant was collected and assayed for IL-2 secretion by ELISA. Each experiment was performed in triplicate and three independent replicates.

[0198] Peptides

[0199] Unless otherwise specified, all peptides used in the present invention were synthesized by GL Biochemistry (Shanghai, China).

[0200] Recombinant protein expression and labeling

[0201] Lk

[0202] Full-length Lck, Lck regulatory domain constructs (various combinations of UD, SH3, and SH2 domains), or the Lck kinase domain were expressed in Expi293F cells, bacteria, and insect cells, respectively. To express full-length Lck, cDNAs encoding human wild-type and mutant Lck with an N-terminal 10× His tag (3-509 a.a., Lck-wt; K273R & Y505F, Lck-open) were subcloned into the pHAGE vector. pHAGE-Lck-wt or pHA GE-Lck-open plasmids were transiently transfected into Expi293F cells using polyethyleneimine (PEI) (Polysciences, 23966). After 48 hours of expression, cells were harvested and lysed in 50 mM Na2HPO4, 300 mM NaCl, 1 mM EDTA, 1 mM DTT, 1× protease inhibitor cocktail, 1 mM PMSF, 0.3% Triton X-100, pH=8.0. The lysate was centrifuged and filtered through a 0.22 μm membrane filter to obtain a clarified lysate containing the recombinant protein.

[0203] To express the Lck regulatory domain constructs, cDNA encoding the human Lck domain of UD (3-60a.a.), UD-SH3 (3-121a.a.), UD-SH3-SH2 (3-226a.a.), or SH3-SH2 (59-226a.a.) with an N-terminal 10×His tag was subcloned into the pET28a vector and transduced into BL21(DE3) competent E. coli strain. Recombinant BL21 was induced with 0.25mM IPTG at 16°C for 12 hours for recombinant protein expression. Bacteria were harvested and lysed by ultrasonic cell disruptor in 50mM Na2HPO4, 300mM NaCl, 1mM EDTA, 1mM DTT, 1X protease inhibitor cocktail, 1mM PMSF, pH=8.0. For UD recombinant proteins, the lysate was first boiled at 95°C for an additional 30 minutes. The lysate was centrifuged and filtered through a 0.22 μm membrane filter to obtain a clarified lysate containing the recombinant protein.

[0204] To express the Lck kinase domain (245-501a.a.), the encoding cDNA was inserted into the pFast Bac1-MBP vector, which contains an N-terminal MBP and HRV 3C sites and a 10×His sequence. Recombinant baculovirus containing the expression plasmid was generated using the Bac to Bac expression system. Baculovirus was produced in SF9 insect cells and used to infect BTI-Tn-5B1-4 insect cells for 48 hours. Cells were harvested and lysed using an ultrasonic cell disruptor in 50mM Na2HPO4, 300mM NaCl, 1mM EDTA, 1mM DTT, 1X protease inhibitor cocktail, 1mM PMSF, pH=8.0. The cleared lysate was incubated with dextrin beads (Smart-Lifesciences, SA026025). MBP was removed using 3C protease at 4°C for 12 hours. After centrifugation, cleaved recombinant Lck kinase was obtained.

[0205] Csk

[0206] The full-length and truncated Csk were constructed into pCold plasmids with N-terminal GST and 8×His tags. All constructs were transduced into Escherichia coli BL21 (DE3) and induced with 1mM IPTG at 15°C for 18 hours. The bacteria were collected and lysed by cell disruption (JN-3000plus, JNBIO) in lysis buffer (50mM Tris-HCl, pH=8.0, 500mM NaCl, 10% glycerol, 5mM DTT). The clarified lysate was loaded onto glutathione agarose resin (Genestar) and eluted with elution buffer (50mM Tris-HCl, pH=8.0, 100mM NaCl, 5% glycerol, 1mM DTT). The GST tag was then removed by precision protease (SAE0045, Sigma Aldrich), and the protein was further purified by gel filtration (Hiload 16 / 600, superdex 200, GE Healthcare).

[0207] CD3ε

[0208] The cytoplasmic domain of human CD3ε(153-207) was subcloned into a laboratory-adapted pET-28a vector with an N-terminal 10×His-tag for membrane anchoring, followed by a cystine for fluorescent labeling. The recombinant vector was expressed in E. coli BL21(DE3) for 24 hours at 16°C with 1 mM IPTG induction. Harvested bacteria were lysed using a cell disruption method (JN-3000plus, JNBIO), and the clarified lysate was purified using nickel ion affinity chromatography (Ni-IDA agarose) and gel filtration (Hiload 16 / 600, Superdex 200, GE Healthcare).

[0209] CD3ζ

[0210] The cytoplasmic domain of human CD3ζ was purified using known techniques. Residues 52 to 164 of human CD3ζ were expressed as a GST fusion protein in E. coli BL21(DE3) cells. The protein was purified using a GST affinity column, followed by removal of the GST tag by TEV cleavage. The protein was then purified by HPLC.

[0211] Fluorescent dye labeling

[0212] Proteins and peptides for fluorescent labeling were first reduced with TCEP supplement at a 10-fold protein molar ratio, followed by incubation with maleimides (C5-maleimide Alexa-488, C5-maleimide Alexa-546, and C2-maleimide Alexa-647, Thermo Scientific) at a 5-fold protein molar ratio and incubated at room temperature for 2 hours. Excess dye was removed by exchanging the buffer for PBS (Zeba spin desalting columns, Thermo Scientific).

[0213] Phase separation on loaded lipid bilayers

[0214] Small unilamellar vesicles (SUVs)

[0215] Phospholipids (95% DOPC, 5% DGS-NTA-Ni 2+ The 1% PEG-1000 (0.1% DSPE-PEG5000) was dried in a 37°C water bath under a stream of nitrogen. The dried lipid film was further dehydrated in a desiccator for 2 more hours and resuspended in PBS to a final concentration of 2 mg / ml. The lipid solution was sonicated for 5-10 minutes and then repeatedly frozen and thawed until the solution became clear. The solution was centrifuged at 21,380 g for 60 minutes at 4°C. The supernatant containing the SUVs was then collected.

[0216] Loaded lipid bilayer (SLB)

[0217] A glass-bottomed 96-well plate was washed with 5% Hellmanex III and rinsed thoroughly three times with MilliQ HO. The plates were then washed with 6M NaOH at 50°C for two hours, followed by a thorough rinse with MilliQ HO, and equilibrated with PBS for 30 minutes. 15 μl of freshly prepared SUVs were added and incubated at 37°C for one hour to induce SLB formation. The SLBs were then washed three times with aggregation buffer (PBS containing 1% BSA) and incubated at room temperature for 30 minutes.

[0218] On-membrane phase separation assay

[0219] Typically, 2 μM His-tagged wild-type or truncated Lck was incubated with preformed SLBs at room temperature for 1 hour. Unbound proteins were removed by washing three times with aggregation buffer. After an additional 30-minute incubation, 2 μM His-tagged CD3ε or 10 μM CD3 molecules (CD3ε, CD3δ, CD3γ, and CD3ζ without the His-tag) were added to the glass wells and mixed thoroughly to trigger cluster formation. The occupied area and Feret diameter of the formed clusters were measured using Image J.

[0220] For Lck recruitment and Csk dissolution assays, 2 μM Alexa-488-labeled LckUD-SH3-SH2 was attached to SLBs, and clusters were preformed by injecting 5 μM wild-type or phosphorylated CD3ε into the solution. 2 μM Alexa-647-labeled full-length Lck-wt or Lck-open was then added and mixed thoroughly to quantify Lck aggregation, and 5 μM full-length or truncated Csk was added to the wells and mixed thoroughly to assess cluster dissolution.

[0221] In the signal reconstitution assay, 2.5 μM wild-type His-tagged CD3ε was used to trigger the phase separation of SLB-bound LckUD-SH3-SH2 in signal buffer. 1 μM Alexa-647-labeled Lck-wt was then added to the solution and incubated for 30 minutes. Unbound Lck-wt was removed by washing with signal buffer. 1 mM ATP-Mg was injected. 2+ The whole system was incubated at room temperature for 1 hour, and then 5 μM Csk protein was added and mixed thoroughly.

[0222] Unless otherwise mentioned, all imaging experiments were performed on an Olympus FV1200 microscope equipped with a 60× oil immersion objective.

[0223] Phase separation determination in solution

[0224] Typically, 20 μM of truncated Lck was mixed with 50 μM of CD3 in PBS supplemented with 0.1% BSA. The solution was then transferred to a 96-well optical plate and incubated at room temperature for 1 hour.

[0225] For the Lck recruitment assay, 20 μM LckUD-SH3-SH2 and 50 μM CD3ε were mixed to preform phase-separated droplets. Subsequently, 10 μM Lck-wt or Lck-open was added and incubated for 30 minutes. The solution and droplets were separated by centrifugation at 21,380 g for 30 minutes at 4°C. Fractions were analyzed by SDS-PAGE with Coomassie blue staining, and band intensities were quantified using Image J.

[0226] For Lck activation assay, 10 μM CDζ was premixed with 50 μM CD3ε-wt or CD3ε-mBRS, and the mixture was then transferred and preformed into phase-separated droplets with 20 μM LckUD-SH3-SH2 in signaling buffer (35 mM HEPES, 60 mM NaCl, 30 mM KCl, 1 mM MgCl2, pH 7.2). Subsequently, 10 μM Lck-wt was added and incubated for 30 minutes. 1 mM ATP-Mg was then added. 2+The phosphorylation reaction was triggered at room temperature. The reaction was stopped by adding 5× SDS-PAGE loading buffer at different time points. The phosphorylation levels of CD3 and Lck were then detected and quantified by Western blotting.

[0227] NK92MI functional experiments

[0228] NK92MI cells and target cells (Raji / Nalm6) were centrifuged, the culture medium discarded, and the cells resuspended in fresh medium (MEMα supplemented with 12.5% ​​FBS, 12.5% ​​HS, and 1% PS) to adjust the cell density. 10,000 NK92MI cells were plated per well of a 96-well spherical-bottom plate, followed by the corresponding number of target cells (Raji / Nalm6) in a final volume of 200 μL per well. After incubation at 37°C, 5% CO2 for 24 hours, the supernatant was collected and assayed for IFNγ secretion using an ELISA kit.

[0229] In the antibody blocking experiment, the culture medium of Raji cells was centrifuged and discarded, and the cells were resuspended in fresh culture medium (MEMα culture medium containing 12.5% ​​FBS, 12.5% ​​HS and 1% PS). The cell density was adjusted and 5000 cells were added to each well of a spherical bottom 96-well plate at 50 μL. Then, different gradients of FMC63 (anti-CD19) antibodies were added and incubated for half an hour (37°C, 5% CO2). The culture medium of NK92MI cells was centrifuged and discarded, and the cells were resuspended in fresh culture medium. The cell density was adjusted and 5000 cells were added to each well of the incubated Raji cells at 50 μL. After co-culture at 37°C, 5% CO2 for 24 hours, the supernatant was collected and the secretion of IFNγ was detected using an ELISA kit.

[0230] Mouse tumor model experiments

[0231] NSIG immunodeficient mice were purchased from Huafukang (Beijing) and housed at the Animal Experimental Center, Institute of Biophysics, Chinese Academy of Sciences. On Day 0, NSIG mice (6-8 weeks old, female) were injected with 0.5×10⁶ Nalm6-luciferase-mCherry tumor cells via tail vein injection. Three days after tumor injection, intraperitoneal injection of D-Luciferin was performed to observe and record tumor formation in the mice. On Day 4, 1×10⁶ CTR-T cells (human primary CD3+ T cells lentivirally transduced with the CTR: αCD19-CD8-CD8-CD3ε) were injected into the tail vein of the randomly assigned mice for treatment; one group received PBS as a control. Twice weekly, intravital imaging was performed to monitor treatment outcomes.

[0232] 2. Specific Examples

[0233] Example 1

[0234] Our results show that the CD3ε subunit in the TCR complex can undergo liquid-liquid phase separation (LLPS) with the kinase Lck molecule. While the other subunits (CD3ζ, CD3γ, and CD3δ) cannot phase separate with Lck, they can be recruited to the CD3ε / Lck phase-separated droplets (Figure 1). Furthermore, we found that CD3ε / Lck phase separation promotes phosphorylation of Lck at tyrosine 394, a condition sufficient for Lck to enter its activated conformation. Therefore, CD3ε / Lck phase separation enhances Lck's phosphokinase activity and promotes phosphorylation of the cytoplasmic domains of various TCR complex subunits (Figure 2). Next, by testing different Lck truncations and CD3ε in different phosphorylation states, we found that activated Lck and CD3ε phosphorylated at the first tyrosine of the ITAM motif undergo stronger phase separation (Figure 3). Therefore, the TCR complex forms a self-promoting phosphorylation reaction microcluster through the phase separation of CD3ε and Lck, so that a small amount of TCR activation can cause the phosphorylation of a large number of TCRs on T cells, thereby leading to the activation of the entire T cell.

[0235] In this section, we discovered that CD3ε enhances TCR signaling through phase separation with Lck and phosphorylation of its ITAM.

[0236] Example 2

[0237] We also validated full-length Lck on reconstituted two-dimensional phospholipid membranes in vitro. We first attached Lck to the membrane and then added CD3ε. Clear phase separation was observed using confocal microscopy (Figure 4). However, when CD3ε was first attached to the membrane and then Lck was added, no phase separation was observed. In this case, adding CD3ε to the system still effectively initiated phase separation. Furthermore, we found that the previously added membrane-attached CD3ε was also recruited into the microclusters generated by phase separation (Figure 5). Our results further confirm that CD3ε in resting T cells is in a membrane-attached, autoinhibitory state. Furthermore, we found that CD3ε / Lck phase-separated microclusters can recruit CD3ε in the membrane-attached (autoinhibitory) state. Based on this, we hypothesize that CD3ε acts as a "switch" within the entire TCR complex. CD3ε can recruit the endogenous TCR in T cells through phase separation with Lck, leading to Lck phosphorylation of the cytoplasmic domains of each subunit of the endogenous TCR, thereby activating the entire T cell.

[0238] Example 3

[0239] We next tested whether the CD3ε-Csk interaction would affect the liquid-liquid phase separation of CD3ε-Lck. In the absence of Csk, CD3ε-pY1 and pCD3ε formed larger clusters with Lck than unphosphorylated CD3ε. Addition of Csk led to rapid dissolution of pCD3ε-Lck aggregates, whereas this effect was not observed with CD3ε-Lck aggregates (Figure 6).

[0240] Example 4

[0241] To further investigate the molecular mechanism of CD3ε-Lck phase separation, we designed a series of mutants based on the amino acid sequence of CD3ε. The results showed that the BRS domain of CD3ε is crucial for CD3ε / Lck phase separation (Figure 7). Mutations in the PRS and RK domains did not significantly alter CD3ε / Lck phase separation (Figure 7). Furthermore, tyrosine mutations in the ITAM domain did not alter CD3ε / Lck phase separation (Figure 7). Furthermore, because tyrosine mutations prevent CD3ε from being phosphorylated, they do not recruit Csk, leading to dissolution of phase-separated microclusters.

[0242] Example 5

[0243] Based on the results of the separation of the intracellular domains of each CD3 subunit from Lck obtained in Example 1, we used the technical platform of the first generation CAR-T to preliminarily identify the functions of the intracellular domains of the four CD3 subunits (αCD19-CD8-CD8-CD3 / extracellular domain-hinge region-transmembrane region-intracellular region). The results showed that the secretion levels of IL-2 of T cells mediated by the intracellular domains of CD3ε and CD3ζ were comparable (Figure 8), while the secretion levels of IL-2 of T cells mediated by CD3δ and CD3γ were very low. Among them, the phosphorylation of CD3ζ recruits Zap70 and transmits activation signals to downstream, which is a known TCR activation mechanism. However, there is currently no research to prove that CD3ε can also recruit Zap70 after phosphorylation. Therefore, the mechanism by which the CD3ε intracellular domain mediates TCR downstream signal activation is not clear.

[0244] Example 6

[0245] Based on the in vitro experimental results in Example 2, we speculate that CD3ε is likely to directly recruit and activate endogenous TCR by phase separation and then transmit signals to downstream to cause the activation of T cells. In order to verify this hypothesis, we transferred two chimeric molecules (αCD19-CD8-CD8-CD3ε and αCD19-CD8-CD8-CD3ζ) into wild-type Jurkat cells and TCR-deficient Jurkat cells (TCRβ knockout), and simultaneously performed functional identification of the four cells (Fig. 9). The results show that in wild-type Jurkat cells, the IL-2 secretion levels caused by CD3ε and CD3ζ intracellular domains are comparable (Fig. 9A-B); however, in TCR-deficient Jurkat cells, only CD3ζ can effectively mediate the secretion of IL-2 (Fig. 9C-D). These results show that the T cell activation mediated by the CD3ε intracellular domain depends on the participation of endogenous TCR, and it itself can not directly lead to the activation of T cells.

[0246] Example 7

[0247] We also tested the effects of the hinge region and transmembrane domain on CD3ε-mediated T cell activation. The results showed that CTR molecules with two completely different hinge regions and transmembrane domains (αCD19-CD8-CD8-CD3ε and αCD19-CD28-CD28-CD3ε) can both effectively mediate T cell activation (Figure 10).

[0248] To further verify the role of the extracellular domain, we designed a chimeric molecule (PD1-PD1-PD1-CD3ε) that connects the intracellular domain of CD3ε to the extracellular domain and transmembrane domain of PD-1. By overexpressing this molecule in Jurkat cells, Jurkat cells can be activated by PD-1 antibodies (Figure 11A) and can also be activated by Raji cells expressing PD-L1 (Figure 11B).

[0249] Example 8

[0250] The inventors further constructed CTR-T based on human primary T cells. By co-culturing with Raji cells expressing CD19, we found that CTR-T can recognize Raji cells and secrete IL-2 (Figure 9A). At the same time, we also constructed another CTR-T targeting BCMA, which can also be successfully activated by target cells and secrete IL-2 (Figure 9B). The degree of activation of both CTR-Ts is proportional to the number of target cells. At the same time, in the group without target cells, CTR-T cannot be activated and has a lower endogenous activation signal. These results show that the design of CTR can be applied to recognize different targets and has lower off-target.

[0251] Example 9

[0252] To verify the tumor therapeutic efficacy of CTR-T, we inoculated Nalm6 cells into immunodeficient mice via tail vein injection. On the third day after inoculation, PBS and CTR-T cells were injected into the tail vein. Tumor growth was observed using small animal imaging (Figure 13A) and mouse survival curves were calculated (Figure 13B). The results showed that CTR-T treatment effectively inhibited Nalm6 cell growth in mice and prolonged their survival.

[0253] Example 10

[0254] Since many activating receptors of NK cells are also substrates of Lck, we speculate that receptors that can phase-separate with Lck can also serve as switches for NK cell activation. To verify the application prospects of CTR in NK cells, we constructed the CTR:αCD19-CD8-CD8-CD3ε molecule into NK92MI cells and co-cultured CTR-NK with Raji cells. The results showed that Raji cells can effectively activate CTR-NK (Figure 14A). At the same time, because NK cells themselves have the properties of innate immunity and can directly kill target cells, we used the CD19 antibody with the same clone number as CTR to block Raji cells. The results showed that as the concentration of CD19 antibody increased, the IFN-γ secretion of CTR-NK cells gradually decreased, indicating that CTR-NK has good antigen specificity (Figure 14B).

[0255] Example 11

[0256] We also screened a series of other potential switch receptors. By testing the intracellular domains of different receptors with membrane-bound Lck, we discovered many receptors that can directly phase-dissociate with Lck (Figures 15A-15B). By constructing the intracellular domains of these receptors into the CTR:αCD19-CD8-CD8-ICD and functionally validating them using NK92MI cells, we found that these receptors can mediate NK92MI cell recognition of Nalm6 cells and secretion of IFN-γ.

[0257] Sequence information

Claims

1. Use of CD3ε, NKG2D, KIR3DS1, NCR1, NCR3, CD16α or FcεR1 cytoplasmic domains or their variants in the preparation of liquid-liquid phase separation (LLPS) reagents that promote Lck or its variants, wherein Lck variants include phosphorylated variants, open conformational variants or regulatory domain constructs, wherein the liquid-liquid phase separation refers to liquid-liquid phase separation in immune cells, liquid-liquid phase separation in loaded bilayer lipids, or liquid-liquid phase separation in solutions.

2. Non-therapeutic uses of CD3ε, NKG2D, KIR3DS1, NCR1, NCR3, CD16α or FcεR1 cytoplasmic domains or variants thereof, wherein the non-therapeutic uses include experimental uses or research uses, characterized in that The invention promotes liquid-liquid phase separation (LLPS) of Lck or its variants, wherein the Lck variants include phosphorylation variants, open conformation variants or regulatory domain constructs, wherein the liquid-liquid phase separation refers to liquid-liquid phase separation in immune cells, liquid-liquid phase separation in loaded bilayer lipids, or liquid-liquid phase separation in solutions.

3. The use of CD3ε, NKG2D, KIR3DS1, NCR1, NCR3, CD16α or FcεR1 cytoplasmic domains or their variants in the preparation of reagents for promoting Lck activation and TCR cytoplasmic domain phosphorylation through a liquid-liquid phase separation (LLPS) pathway, wherein Lck variants include phosphorylation variants, open conformation variants or regulatory domain constructs, wherein the liquid-liquid phase separation refers to liquid-liquid phase separation in immune cells, liquid-liquid phase separation in a loaded bilayer lipid, or liquid-liquid phase separation in a solution.

4. Non-therapeutic uses of CD3ε, NKG2D, KIR3DS1, NCR1, NCR3, CD16α or FcεR1 cytoplasmic domains or their variants, including experimental uses or research uses, including the use of the above-mentioned cytoplasmic domains or their variants to promote Lck activation and TCR cytoplasmic domain phosphorylation reagents through a liquid-liquid phase separation (LLPS) pathway, Lck variants including phosphorylated variants, open conformational variants or regulatory domain constructs, wherein the liquid-liquid phase separation refers to liquid-liquid phase separation in immune cells, liquid-liquid phase separation in a loaded bilayer lipid, or liquid-liquid phase separation in a solution.

5. The use of CD3ε / Lck phase separation microclusters to prepare reagents for recruiting CD3ε molecules in an inhibited state (membrane state), wherein the CD3ε / Lck phase separation microclusters are aggregates formed by liquid-liquid phase separation of CD3ε and Lck in immune cells, liquid-liquid phase separation in loaded bilayer lipids, or liquid-liquid phase separation in solution, wherein CD3ε includes the CD3ε cytoplasmic domain or a variant that can form phase separation, and Lck includes the wild type or its variants that can form phase separation such as phosphorylated variants, open conformational variants or regulatory domain constructs.

6. The use of CD3ε / Lck phase-separated microclusters for non-therapeutic purposes, including experimental uses or research uses, and the uses include the step of recruiting CD3ε molecular reagents in an inhibited state (membrane state) with CD3ε / Lck phase-separated microclusters, wherein the CD3ε / Lck phase-separated microclusters are aggregates formed by liquid-liquid phase separation of CD3ε and Lck in immune cells, liquid-liquid phase separation in loaded bilayer lipids, or liquid-liquid phase separation in solution, wherein CD3ε includes the CD3ε cytoplasmic domain or a variant thereof that can form phase separation, and Lck includes the wild type or a variant thereof that can form phase separation such as a phosphorylated variant, an open conformational variant or a regulatory domain construct.

7. A method for regulating CD3ε / Lck phase separation by mutating the CD3ε cytoplasmic domain, wherein the method is for non-therapeutic purposes, wherein the phase separation is liquid-liquid phase separation in immune cells, liquid-liquid phase separation in a loaded bilayer lipid, or liquid-liquid phase separation in a solution.

8. The use according to any one of claims 1 to 6, wherein the CD3ε cytoplasmic domain variant is: 1) Truncated or extended variants, based on the CD3ε cytoplasmic domain (153-207) sequence, the C or N terminus is truncated or extended by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, comprising complete BRS, PRS, RK and ITAM domains, and having or not having the ability to form liquid-liquid phase separation with Lck. Variants that add tags or amino acids such as cysteine ​​for labeling or separation at the C or N terminus also belong to this type of variants. The above tags or amino acids are removed when designing or preparing CTR molecules; 2) Combination variants, any combination of BRS, PRS, RK and ITAM domains or domain variants defined below, with or without the ability to form liquid-liquid phase separation with Lck; For example, CD3ε-dBRS refers to a variant that combines the PRS, RK, and ITAM domains; 3) Domain variants 3-1) BRS domain mutants, The BRS domain mutant refers to the positively charged amino acids in the BRS motif KNRKAKAK. A mutant that retains the protein and has the ability to form a liquid-liquid phase separation with Lck; Or it refers to a mutant in which the BRS motif is mutated from the positively charged amino acid in KNRKAKAK to other non-positively charged amino acids and does not have the ability to form liquid-liquid phase separation with Lck, for example, from KNRKAKAK to SNSSASAS; 3-2) ITAM domain mutants; The ITAM domain mutant is a mutant in which any one or two tyrosines in the domain are mutated to phenylalanine, or mutated to other amino acids; Alternatively, the ITAM domain mutant is a phosphorylation variant, specifically a variant CD3ε-pY1 in which the first ITAM tyrosine is mono-phosphorylated, or a variant CD3ε-pY2 in which the second ITAM tyrosine is mono-phosphorylated, or a variant pCD3ε in which two ITAM tyrosines are dually phosphorylated; 3-3) PRS domain mutants For example, mutations such as mutating PPPVPNP to SSSVSNS still have phase separation activity; 3-4) RK domain mutants For example, mutations such as RKGQ mutated to SSGQ still have phase separation activity; or a combination of the above domain variants; 4) Conservative variants refer to variants in which one amino acid is replaced by another amino acid in the same class and still has the ability to form liquid-liquid phase separation with Lck, for example, one acidic amino acid is replaced by another acidic amino acid, one basic amino acid is replaced by another basic amino acid, or one neutral amino acid is replaced by another neutral amino acid; 5) any combination of the above variants; Optionally, the variant has greater than 95%, 96%, 97%, 98% or 99% sequence identity to the CD3ε cytoplasmic domain (153-207) sequence; Preferred are the following variants: CD3ε (SEQ NO: 35) CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLY SGLNQRRI; CD3ε-pY1 (SEQ NO: 36) CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPD-pY-EPIRKGQRD LYSGLNQRRI; CD3ε-pY2 (SEQ NO: 37) CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDL-p Y-SGLNQRRI; pCD3ε (SEQ NO: 38) CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPD-pY-EPIRKGQRD L-pY-SGLNQRRI; CD3ε-YYFF (SEQ NO: 39) CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDFEPIRKGQRDLF SGLNQRRI; CD3ε-mPRS (SEQ NO: 40) CKNRKAKAKPVTRGAGAGGRQRGQNKERS S SVSNSDYEPIRKGQRDLY SGLNQRRI; CD3ε-mBRS (SEQ NO: 41) CSNSSASASPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYS GLNQRRI; CD3ε-dBRS (SEQ NO: 42) CPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI CD3ε-mRK (SEQ NO: 43) CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPISSGQRDLY SGLNQRRI; CD3ε-null (SEQ NO: 44) CSNSSASASPVTRGAGAGGRQRGQNKERSSSVSNSDYEPISSGQRDLYS GLNQRRI; The cysteine ​​in the above variants was used for in vitro labeling and was removed during the design and preparation of the CTR molecule.

9. The use according to any one of claims 1 to 6, wherein the Lck phosphorylation variant is a variant in which the corresponding tyrosine is phosphorylated, such as Y394 and Y505, and the Lck regulatory domain construct is LckUD-SH3-SH2, which is a construct formed by connecting the UD, SH3, and SH2 domains; and the open conformation variant is a mutant comprising Y505F.

10. The use according to any one of claims 1 to 6 or the method according to claim 7, wherein the immune cells include immune cells in living animals, or immune cells cultured in vitro.

11. The use according to any one of claims 1 to 6 or the method according to claim 7, wherein the immune cells include T cells produced by the immune system, commercial model cells such as Jurkat cells, or artificially constructed simulation cells expressing the TCR-CD3 complex.

12. The method of claim 7, wherein the CD3ε cytoplasmic domain variant is: 1) Truncated or extended variants, based on the CD3ε cytoplasmic domain (153-207) sequence, the C or N terminus is truncated or extended by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, comprising complete BRS, PRS, RK and ITAM domains, and having or not having the ability to form liquid-liquid phase separation with Lck. Variants that add tags or amino acids such as cysteine ​​for labeling or separation at the C or N terminus also belong to this type of variants. The above tags or amino acids are removed when designing or preparing CTR molecules; 2) Combination variants, any combination of BRS, PRS, RK and ITAM domains or domain variants defined below, with or without the ability to form liquid-liquid phase separation with Lck; For example, CD3ε-dBRS refers to a variant that combines the PRS, RK, and ITAM domains; 3) Domain variants 3-1) BRS domain mutants, The BRS domain mutant refers to a mutant in which the positively charged amino acids in the BRS motif KNRKAKAK are retained and which has the ability to form a liquid-liquid phase separation with Lck; Or it refers to a mutant in which the BRS motif is mutated from the positively charged amino acid in KNRKAKAK to other non-positively charged amino acids and does not have the ability to form liquid-liquid phase separation with Lck, for example, from KNRKAKAK to SNSSASAS; 3-2) ITAM domain mutants; The ITAM domain mutant is a mutant in which any one or two tyrosines in the domain are mutated to phenylalanine, or mutated to other amino acids; Alternatively, the ITAM domain mutant is a phosphorylation variant, specifically a variant CD3ε-pY1 in which the first ITAM tyrosine is mono-phosphorylated, or a variant CD3ε-pY2 in which the second ITAM tyrosine is mono-phosphorylated, or a variant pCD3ε in which two ITAM tyrosines are dually phosphorylated; 3-3) PRS domain mutants For example, mutations such as mutating PPPVPNP to SSSVSNS still have phase separation activity; 3-4) RK domain mutants For example, mutations such as RKGQ mutated to SSGQ still have phase separation activity; or a combination of the above domain variants; 4) Conservative variants refer to variants in which one amino acid is replaced by another amino acid in the same class and still has the ability to form liquid-liquid phase separation with Lck, for example, one acidic amino acid is replaced by another acidic amino acid, one basic amino acid is replaced by another basic amino acid, or one neutral amino acid is replaced by another neutral amino acid; 5) any combination of the above variants; Optionally, the variant has greater than 95%, 96%, 97%, 98% or 99% sequence identity to the CD3ε cytoplasmic domain (153-207) sequence; Preferred are the following variants: CD3ε (SEQ NO: 35) CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLY SGLNQRRI; CD3ε-pY1 (SEQ NO: 36) CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPD-pY-EPIRKGQRD LYSGLNQRRI; CD3ε-pY2 (SEQ NO: 37) CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDL-p Y-SGLNQRRI; pCD3ε (SEQ NO: 38) CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPD-pY-EPIRKGQRD L-pY-SGLNQRRI; CD3ε-YYFF (SEQ NO: 39) CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDFEPIRKGQRDLF SGLNQRRI; CD3ε-mPRS (SEQ NO: 40) CKNRKAKAKPVTRGAGAGGRQRGQNKERSSSVSNSDYEPIRKGQRDLY SGLNQRRI; CD3ε-mBRS (SEQ NO: 41) CSNSSASASPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYS GLNQRRI; CD3ε-dBRS (SEQ NO: 42) CPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI CD3ε-mRK (SEQ NO: 43) CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPISSGQRDLY SGLNQRRI; CD3ε-null (SEQ NO: 44) CSNSSASASPVTRGAGAGGRQRGQNKERSSSVSNSDYEPISSGQRDLYS GLNQRRI; The cysteine ​​in the above variants was used for in vitro labeling and was removed during the design and preparation of the CTR molecule.

13. The method according to claim 12, wherein the CD3ε cytoplasmic domain variant is a variant having phase separation activity or a variant not having phase separation activity.

14. Non-therapeutic uses of Csk, including experimental uses or research uses, wherein the uses are to use Csk to dissolve CD3ε / Lck phase-separated microclusters, wherein the CD3ε / Lck phase-separated microclusters are aggregates formed by liquid-liquid phase separation of CD3ε and Lck in immune cells, liquid-liquid phase separation in loaded bilayer lipids, or liquid-liquid phase separation in solution, wherein CD3ε is the CD3ε cytoplasmic domain or a variant that can form phase separation such as a phosphorylated variant, and Lck comprises a wild type or a variant that can form phase separation such as a phosphorylated variant, an open conformational variant or a regulatory domain construct.

15. A CTR (Chimeric trigger receptor) molecule, whose intracellular domain contains CD3ε, NKG2D, KIR3DS1, NCR1, NCR3, CD16α or FcεR1 cytoplasmic domain or its variants as a switch for TCR activation or immune cell activation; Preferably, the CTR (Chimeric trigger receptor) molecule comprises an extracellular domain, an optional hinge region, a transmembrane domain and an intracellular domain connected in sequence; The extracellular domain includes an optional signal peptide and an antigen recognition region; The intracellular domain contains only the above-mentioned cytoplasmic domain or its variant as a switch for TCR activation or immune cell activation; Preferably, the intracellular domain comprises the CD3ε, NKG2D, KIR3DS1, NCR1 or NCR3 cytoplasmic domain.

16. The CTR (Chimeric trigger receptor) molecule of claim 15, wherein the antigen recognition region is selected from a single-chain antibody against a tumor surface antigen, wherein the tumor surface antigen is selected from one or more of CD19, mesothelin, CD20, CD22, CD123, CD30, CD33, CD38, CD138, BCMA, Fibroblast activation protein, Glypican-3, CEA, EGFRvIII, PSMA, Her2, IL13Rα2, CD171, claudin18.2 and GD2; the single-chain antibody is selected from a single-chain antibody fragment, a single-chain Fv (scFv), a single-chain Fab, a single-chain Fab′, a single-domain antibody fragment, a single-domain multispecific antibody, an intracellular antibody, a nanobody or a single-chain immune factor; Preferably, the single-chain antibody is based on the following monoclonal antibodies: Inebilizumab, Rituximab, Ofatumumab, Glofitamab, Trastuzumab (trade name Herceptin), Pertuzumab (also known as 2C4, trade name Perjeta), Nimotuzumab (trade name Taixinsheng), Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, Retifanlimab, cBR96 and Glematumamab; Most preferably, the antigen recognition region is a single-chain antibody fragment targeting CD19 (SEQ NO: 3), Myeloma antigen BCMA recognition domain sequence (SEQ NO: 5) or single-chain antibody fragment targeting EGFR (SEQ NO: 7).

17. The CTR (Chimeric trigger receptor) molecule as described in claim 15, wherein the antigen recognition region also includes a protein domain based on natural ligand-receptor interaction, such as the extracellular domain of PD-1 protein (which can recognize PD-L1 / PD-L2 protein), the extracellular domain of CD2 (recognizing CD58 / CD59), the extracellular domain of CD28 / CD152 (recognizing CD80 / CD86), etc.

18. The CTR (Chimeric trigger receptor) molecule of claim 15, wherein the transmembrane domain is selected from the transmembrane region of CD4, CD8, CD28, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, EGFR (epidermal growth factor receptor) or GITR; preferably, the transmembrane region of a type I single transmembrane molecule or a homologous multimer thereof; further preferably, it is the CD8 transmembrane domain.

19. A nucleic acid sequence selected from: i) encoding the CTR (Chimeric trigger receptor) molecule according to any one of claims 15 to 18; or ii) a nucleic acid sequence complementary to i).

20. A nucleic acid construct comprising the nucleic acid sequence of claim 19; Preferably, the nucleic acid construct is a vector; More preferably, the nucleic acid construct is a lentiviral vector, a retroviral vector, an adenoviral vector or an adeno-associated viral vector, containing the nucleic acid sequence of claim 19.

21. A lentiviral vector system, comprising the nucleic acid sequence of claim 19 and lentiviral vector auxiliary components.

22. A genetically modified cell, characterized in that The cell expresses the CTR (Chimeric trigger receptor) molecule described in claims 15-18, or contains the nucleic acid sequence described in claim 19, or contains the nucleic acid construct described in claim 20, or is infected with the lentiviral vector system described in claim 21; preferably, the cell is selected from autologous or allogeneic T cells, B cells, NK cells, macrophages, monocytes, dendritic cells, neutrophils, basophils, eosinophils, mast cells, NK-T cells, MAIT cells, hematopoietic stem cells, embryonic stem cells, induced pluripotent stem cells, and red blood cells, and the T cells include αβT cells, γδT cells, and regulatory T cells.

23. A pharmaceutical composition or kit comprising the CTR (Chimeric trigger receptor) molecule according to any one of claims 15 to 18, the nucleic acid sequence according to claim 19, or the nucleic acid construct according to claim 20, or the lentiviral vector system according to claim 21, or the genetically modified cell according to claim 22.

24. Use of the CTR (chimeric trigger receptor) molecule of any one of claims 15-18, the nucleic acid sequence of claim 19, the nucleic acid construct of claim 20, or the lentiviral vector system of claim 21 in the preparation of any one or more of the following products: (1) preparing T cells or NK cells; (2) enhancing the proliferation capacity of T cells or NK cells; (3) improving the killing ability of T cells or NK cells.

25. Use of the CTR (chimeric trigger receptor) molecule according to any one of claims 15 to 18, the nucleic acid sequence according to claim 19, or the nucleic acid construct according to claim 20, or the lentiviral vector system according to claim 21, or the genetically modified cell according to claim 22 in the preparation of any one or more of the following products: (1) treating cancer; (2) inhibiting cytokine storm generated during cancer treatment; Preferably, the cancer is selected from adrenocortical carcinoma, bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, esophageal cancer, glioblastoma, glioma, hepatocellular carcinoma, head and neck cancer, kidney cancer, leukemia, lymphoma, lung cancer, melanoma, mesothelioma, multiple myeloma, pancreatic cancer, pheochromocytoma, plasmacytoma, neuroblastoma, ovarian cancer, prostate cancer, sarcoma, gastric cancer, uterine cancer and thyroid cancer, and / or optionally wherein the cancer is a blood cancer or a solid tumor cancer.

26. Use of CD3ε, NKG2D, KIR3DS1, NCR1, NCR3, CD16α, FcεR1α cytoplasmic domain or variants thereof in preparing modified T cells or NK cells.

27. The CTR (chimeric trigger receptor) molecule of any one of claims 15 to 18, the nucleic acid sequence of claim 19, or the nucleic acid construct of claim 20, the lentiviral vector system of claim 21, or the genetically modified cell of claim 22, or the CD3ε cytoplasmic domain variant for use of claim 26, comprising: 1) Truncated or extended variants, based on the CD3ε cytoplasmic domain (153-207) sequence, the C or N terminal is truncated or extended by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, containing complete BRS, PRS, RK and ITAM domains, and having or not having the ability to form liquid-liquid phase separation with Lck, and adding tags or amino acids such as cysteine ​​for labeling or separation at the C or N terminal also belong to this category Variants of this type, wherein the above tags or amino acids are removed when designing or preparing CTR molecules; 2) Combination variants, any combination of BRS, PRS, RK and ITAM domains or domain variants defined below, with or without the ability to form liquid-liquid phase separation with Lck; For example, CD3ε-dBRS refers to a variant that combines the PRS, RK, and ITAM domains; 3) Domain variants 3-1) BRS domain mutants The BRS domain mutant refers to a mutant in which the positively charged amino acids in the BRS motif KNRKAKAK are retained and which has the ability to form a liquid-liquid phase separation with Lck; Or it refers to a mutant in which the BRS motif is mutated from the positively charged amino acid in KNRKAKAK to other non-positively charged amino acids and does not have the ability to form liquid-liquid phase separation with Lck, for example, from KNRKAKAK to SNSSASAS; 3-2) ITAM domain mutants; The ITAM domain mutant is a mutant in which any one or two tyrosines in the domain are mutated to phenylalanine, or mutated to other amino acids; Alternatively, the ITAM domain mutant is a phosphorylation variant, specifically a variant CD3ε-pY1 in which the first ITAM tyrosine is mono-phosphorylated, or a variant CD3ε-pY2 in which the second ITAM tyrosine is mono-phosphorylated, or a variant pCD3ε in which two ITAM tyrosines are dually phosphorylated; 3-3) PRS domain mutants For example, mutations such as mutating PPPVPNP to SSSVSNS still have phase separation activity; 3-4) RK domain mutants For example, mutations such as RKGQ mutated to SSGQ still have phase separation activity; or a combination of the above domain variants; 4) Conservative variants refer to variants in which one amino acid is replaced by another amino acid in the same class and still has the ability to form liquid-liquid phase separation with Lck, for example, one acidic amino acid is replaced by another acidic amino acid, one basic amino acid is replaced by another basic amino acid, or one neutral amino acid is replaced by another neutral amino acid. amino acid substitution; 5) any combination of the above variants; Optionally, the variant has greater than 95%, 96%, 97%, 98% or 99% sequence identity to the CD3ε cytoplasmic domain (153-207) sequence; Preferred are the following variants: CD3ε1 (SEQ NO: 35) CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLY SGLNQRRI; CD3ε-pY1 (SEQ NO: 36) CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPD-pY-EPIRKGQRD LYSGLNQRRI; CD3ε-pY2 (SEQ NO: 37) CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDL-p Y-SGLNQRRI; pCD3ε (SEQ NO: 38) CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPD-pY-EPIRKGQRD L-pY-SGLNQRRI; CD3ε-YYFF (SEQ NO: 39) CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDFEPIRKGQRDLF SGLNQRRI; CD3ε-mPRS (SEQ NO: 40) CKNRKAKAKPVTRGAGAGGRQRGQNKERS S SVSNSDYEPIRKGQRDLY SGLNQRRI; CD3ε-mBRS (SEQ NO: 41) CSNSSASASPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYS GLNQRRI; CD3ε-dBRS (SEQ NO: 42) CPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI CD3ε-mRK (SEQ NO: 43) CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPISSGQRDLY SGLNQRRI; CD3ε-null (SEQ NO: 44) CSNSSASASPVTRGAGAGGRQRGQNKERSSSVSNSDYEPISSGQRDLYS GLNQRRI; The cysteine ​​in the above variants was used for in vitro labeling and was removed during the design and preparation of the CTR molecule.

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