CN118546879A - Method for transforming T cells through phase separation and application thereof - Google Patents

Abstract

The present invention provides uses and methods of a CD3ε cytoplasmic domain or a CD3ε cytoplasmic domain variant for promoting Lck activation and TCR cytoplasmic domain phosphorylation through a liquid-liquid phase separation (LLPS) pathway, and recruiting CD3ε molecules in an inhibitory state; the present invention also provides a CTR (Chimeric trigger receptor) molecule containing only the CD3ε cytoplasmic domain as a switch for TCR activation, as well as corresponding engineered cells and treatment methods.

Description

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

Technical Field

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 a cell containing the gene, and their application, and the result can be used for immunotherapy.

Background of the Invention

1. About CD3 subunits

T cells can specifically recognize foreign antigens and trigger adaptive immune responses to invading pathogens or tumor cells. T cells mainly recognize antigens through T cell receptors (TCR). For αβ-type T cells, their TCR is mainly composed of TCRαβ dimer units responsible for antigen recognition and three CD3εδ, CD3εγ, and CD3ζζ dimer units that transmit signals.

CD3 subunits all contain immunoreceptor tyrosine-based activation motifs (ITAMs; YxxL/Ix6-8YxxL/I). Tyrosine phosphorylation in ITAMs can lead to activation of downstream signaling pathways (Courtney, A.H., W.L.Lo, and A.Weiss, TCR Signaling: Mechanisms of Initiation and Propagation. Trends Biochem Sci, 2018. 43(2): p.108-123.). Each of CD3ε, δ, and γ subunits contains an 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.

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.

CD3 ITAMs are mainly phosphorylated by Lck and dephosphorylated by CD45. Dual phosphorylated CD3 ITAMs can bind to the Zap70 tandem SH2 domain, relieve the self-inhibition of Zap70, and activate downstream signaling pathways (Courtney, A.H., W.L.Lo, 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 controversy as to whether the types, number, and number of phosphorylation sites of ITAMs in CD3 subunits will have a qualitative and quantitative effect on TCR signal transduction.

For 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 CD3εand 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 immune receptor tyrosine motifs (ITAMs) of all CD3 chains under TCR stimulation. Due to the substrate selectivity of Lck kinase, the ITAM of CD3ε is mostly in a monophosphorylated state, and specifically recruits the inhibitory Csk kinase to attenuate TCR signaling, indicating that TCR has a self-restrained signaling mechanism with both activation and inhibition motifs. In addition, it was found that incorporating the CD3ε cytoplasmic domain into the second-generation chimeric antigen receptor (CAR) can improve the anti-tumor activity of CAR-T cells. Mechanistically, CD3ε reduces the production of CAR-T cytokines through ITAM recruitment of Csk, while the basic residue-rich sequence (BRS) of CD3ε promotes the persistence of CAR-T through p85 recruitment. The article believes that: CD3ε is a built-in multifunctional signal tuner, and increasing CD3 diversity represents a strategy for designing the next generation of CAR. In addition, the published patents (202010535848.X, 202010733636.2, 202010734872.6) involve the use of subunit CD3ε and its mutants in the TCR complex in CAR-T therapy.

2. About Lck enzyme

Lck belongs to the Src family kinases (SFKs), a class of non-receptor tyrosine kinases that have been implicated as key regulators of a large number of intracellular signaling pathways in immune cells. Src family kinases (SFKs) occupy proximal positions in many signal transduction cascades, including those derived 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 inhibitory motifs (ITIMs) on inhibitory receptors, leading to the recruitment and activation of inhibitory molecules such as the phosphatases SHP-1 and SHP-2 containing 5' inositol phosphatase (SHIP-1).

The 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 U S 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 on 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 the acidic residues in the unique domain of Lck. At the same time, the initiation of TCR phosphorylation was clearly studied using the TCR reconstitution system. The ionic CD3ε-Lck interaction controls the phosphorylation level of the entire TCR during antigen stimulation. In the resting state, the BRS sequence of CD3ε is bound by the phospholipid membrane and is in an inhibitory state. Antigen stimulation can release the inhibitory state of the BRS sequence. The dynamic opening of CD3ε BRS and its subsequent Lck recruitment can serve as an important switch for the initiation of TCR phosphorylation.

3. About Liquid-Liquid Phase Separation (LLPS)

Liquid-liquid phase separation (LLPS) of biomacromolecules (proteins and RNA) has been an emerging hot topic in the international life science field 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 2021Dec 24. doi: 10.3389/fphys.2021.801212). Cells are separated by many 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. There is increasing evidence that liquid-liquid phase separation (LLPS) plays an important role in the assembly of many membraneless compartments, collectively referred to as biomacromolecule aggregates. Phase-separated aggregates participate in various biological activities, including higher-order chromatin organization, gene expression, and sorting of misfolded or unwanted proteins for autophagic degradation. Biomacromolecules, such as proteins and nucleic acids, can condense into liquid-like membraneless aggregates through liquid-liquid phase separation (LLPS), providing another means to aggregate and separate cellular components in a spatiotemporally defined manner for various functional processes.

Summary of the invention

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, and 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.

In the present invention, it is found that Lck kinase and the intracellular region of CD3ε can be phase-separated, thereby initiating and amplifying the activation signal of T cells. By constructing and expressing a CTR (Chimeric trigger receptor) molecule containing only the intracellular domain of CD3ε, T cells can be specifically activated. Specific aspects of the present invention include:

In one aspect, the present invention provides the use of a CD3ε cytoplasmic domain or a CD3ε cytoplasmic domain variant in the preparation of a liquid-liquid phase separation (LLPS) reagent that promotes the production of 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 a loaded bilayer lipid, or liquid-liquid phase separation in a solution.

On the other hand, the present invention provides non-therapeutic uses of the CD3ε cytoplasmic domain or a CD3ε cytoplasmic domain variant, wherein the non-therapeutic uses include experimental uses or research uses, characterized in that the liquid-liquid phase separation (LLPS) of Lck or its variants is promoted, 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 loaded bilayer lipids, or liquid-liquid phase separation in solutions.

On the other hand, the present invention provides the use of a CD3ε cytoplasmic domain or a CD3ε cytoplasmic domain variant in the preparation of a reagent for promoting Lck activation and TCR cytoplasmic domain phosphorylation through a liquid-liquid phase separation (LLPS) pathway, wherein 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.

On the other hand, the present invention provides non-therapeutic uses of the CD3ε cytoplasmic domain or CD3ε cytoplasmic domain variants, which non-therapeutic uses include experimental uses or research uses, and the uses include CD3ε cytoplasmic domain 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, Lck variants include phosphorylated 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 solution.

In another aspect of the invention, the use of CD3ε/Lck phase-separated microclusters to recruit CD3ε molecular reagents in an inhibited state (membrane 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 solutions.

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.

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

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.

The present invention also provides 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 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.

The immune cells described in the above invention include immune cells in living animals, or immune cells under in vitro culture conditions.

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.

The present invention also provides non-therapeutic uses of Csk, which include experimental uses or research uses, and 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/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 contains 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.

The present invention also provides a CTR (Chimeric trigger receptor) molecule, whose intracellular domain contains only the CD3ε cytoplasmic domain or its variant 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 CD3ε cytoplasmic domain or a variant thereof as a switch for TCR activation or immune cell activation.

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

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, 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;

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 the myeloma antigen BCMA recognition domain sequence (SEQ NO: 5) or a single-chain antibody fragment targeting EGFR (SEQ NO: 7).

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.

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, it is the CD8 transmembrane domain;

The CTR molecules specifically provided by the present invention include:

αCD19-CD8-CD8-CD3ε

αCD19-CD28-CD28-CD3ε

PD1-PD1-PD1-CD3ε.

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

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

The present invention also provides a nucleic acid construct, wherein the nucleic acid construct comprises the above-mentioned nucleic acid sequence;

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 above-mentioned nucleic acid sequence.

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

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.

The present invention also provides a pharmaceutical composition or a kit, which comprises 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 or the above-mentioned gene-modified cell.

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 the preparation of 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.

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 or the above-mentioned genetically modified cell in the preparation of any one or more of the following use products: (1) treating cancer; (2) inhibiting the 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.

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.

Explanation of relevant technical terms in the present invention:

Lck or its variants

Lck belongs to the Src family kinase (SFK), 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.

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

In the present invention, the Lck phosphorylation variant refers to a variant in which the corresponding tyrosine is phosphorylated, such as Y394 and Y505, or is expressed as pY394 or pY505, wherein pY394 is an activated form of Lck.

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.

In the present invention, Lck regulatory domain construct refers 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.

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

CD3ε and its variants

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

The CD3ε cytoplasmic domain variants of the present invention include:

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 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 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. A tag or amino acid such as cysteine added to the C or N terminus for labeling or separation also belongs to this type of variant. Those skilled in the art will appreciate that the above-mentioned tag or amino acid is removed when designing or preparing the CTR molecule;

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.

The specific embodiments of the present invention include the following variations:

CD3ε1 (SEQ NO:35)

CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLY SGLNQRRI;

CD3ε-pY1 (SEQ NO:36)

CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPD-pY-EPIRKGQRD LYSGLNQRRI;

CD3ε-pY2 (SEQ NO:37)

CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDL-pY-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.

A person 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 for the construction of CTR chimeric molecules or for the verification and study of the phase separation mechanism.

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 solutions, 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.

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

The "CTR (Chimeric trigger receptor) molecule" in the present invention is an artificially modified molecule that can anchor specific molecules (such as antibodies) that recognize tumor cell surface antigens on immune cells (such as T cells), allowing immune cells to recognize tumor antigens or viral antigens, and activate immune cells through the CD3ε cytoplasmic domain as a switch for TCR activation. The CTR (Chimeric trigger receptor) molecule includes:

The extracellular domain, hinge region, transmembrane domain and intracellular domain are connected in sequence;

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

The intracellular domain contains only the CD3ε cytoplasmic domain as a switch for TCR activation.

Among them, the signal peptide is located at the extracellular end of the CTR molecule, and its function is to guide the newly generated CAR protein into the endoplasmic reticulum of the cell, where the CTR protein is glycosylated, and the glycosylated CTR can appear on the T cell membrane. The signal peptide sequence in any animal cell can be used on the CTR molecule. In the present invention, the signal peptide is optional, that is, it can be used or not.

Among them, the antigen recognition region recognizes tumor surface antigens, and the antigen recognition region is preferably a single-chain antibody against tumor surface antigens, 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.

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.

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

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.

Wherein, "hinge region" refers to the hydrophilic region between the antigen recognition domain and the transmembrane domain. In the present invention, the hinge region is optional, that is, it can be used or not. The hinge region can use the hinge region of various antibodies or antigen receptors, especially the hinge region of CD molecules. In a specific embodiment, the hinge region can be selected from, for example, CD4, CD8α, CD28, IgG1, IgG4, CD279 (PD-1).

Among them, 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 the 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, epidermal growth factor receptor) or GITR. The transmembrane region of the present invention is preferably the transmembrane region of a type I single transmembrane molecule or a homologous multimer thereof; in a preferred embodiment of the present invention, the CD8 transmembrane domain is used.

In the present invention, the term "identity" or "homology" generally refers to the proportion of nucleotide bases or amino acid residues in a candidate sequence that are identical to the corresponding sequence after alignment and, if necessary, introduction of intervals to achieve the maximum percentage of identity of the entire sequence and not considering any conservative substitutions as part of the sequence identity. Neither N-terminal nor C-terminal extensions or insertions should be construed as reducing the identity or homology. Methods and computer programs for alignment are available and well known in the art, for example, sequence identity can be determined by sequence analysis software.

Nucleotide sequences and vectors

The polynucleotide sequence of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. The present invention also includes degenerate variants of the polynucleotide sequence encoding the fusion protein, i.e., nucleotide sequences encoding the same amino acid sequence but with different nucleotide sequences.

The polynucleotide sequences of the present invention can usually be obtained by PCR amplification. Specifically, primers can be designed based on the nucleotide sequences disclosed herein, especially the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art are used as templates to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified in each amplification in the correct order.

The nucleic acid construct provided by the present invention also includes one or more regulatory sequences operably linked to the aforementioned polynucleotide sequence. The coding sequence of the CTR (Chimeric trigger receptor) molecule described in the present invention can be manipulated in a variety of ways to ensure the expression of the protein. Before inserting the nucleic acid construct into a vector, the nucleic acid construct can be manipulated according to the different or required expression vectors. The technology of using recombinant DNA methods to change polynucleotide sequences is known in the art.

The regulatory sequence may be a suitable promoter sequence. The promoter sequence is usually 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 genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

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 can be used in the present invention.

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.

Preferably, the nucleic acid construct is a vector.

Usually, the polynucleotide sequence encoding CAR is operably connected to the promoter, and the construct is incorporated into the expression vector to achieve the expression of the polynucleotide sequence encoding CTR (Chimeric trigger receptor) molecule. 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.

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. Further, 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 works in at least one organism, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers.

More preferably, the nucleic acid construct is a lentiviral vector containing a replication initiation site, 3'LTR, 5'LTR and the aforementioned polynucleotide sequence.

The promoter may use a constitutive promoter sequence, including but not limited to the 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 may also be considered. The use of inducible promoters provides a molecular switch that is capable of turning on the expression of a polynucleotide sequence operably connected to an inducible promoter when the expression is limited, and turning off the expression when the expression is not desired. Examples of inducible promoters include but are not limited to metallothionein promoters, glucocorticoid promoters, progesterone promoters and tetracycline promoters.

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

Reporter genes are used to identify cells of potential transfection and to evaluate the functionality of regulatory sequences. After DNA has been introduced into recipient cells, the expression of reporter genes is measured at the appropriate time. Suitable reporter genes may 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 techniques to prepare or commercially obtain.

Methods for introducing genes into cells and expressing genes into 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.

Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. 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.

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, lentiviral vectors are used.

The lentiviral vector system provided by the present invention contains the aforementioned nucleic acid construct and lentiviral vector auxiliary components. The lentiviral auxiliary components include lentiviral packaging plasmids and cell lines. The lentiviral vector system is formed by viral packaging of the aforementioned nucleic acid construct with the assistance of lentiviral packaging plasmids and cell lines. The method for constructing the lentiviral vector system is a commonly used method in the art.

Cell therapy

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

The anti-tumor immune response caused 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 the CTR-T cells induce an immune response specific for the antigen-binding portion of the CTR molecule.

Treatable cancers may be non-solid tumors, such as blood tumors, for example leukemias and lymphomas.

The present invention provides a genetically modified T cell or a pharmaceutical composition containing the genetically modified T cell, wherein the cell contains the aforementioned polynucleotide sequence, or contains the aforementioned nucleic acid construct, or is infected with the aforementioned lentiviral vector system.

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 with other components such as related cytokines or cell populations. Briefly, the pharmaceutical composition of the present invention may include CTR cells as described herein, combined 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, etc.; 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.

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 such factors as the patient's condition, and the type and severity of the patient's disease.

When an "immunologically effective amount", "anti-tumor effective amount", "tumor-inhibitory effective amount" or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, which 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 the pharmaceutical composition comprising the T cells described herein can be administered at a dose of 10 ⁴ to 10 ⁹ cells/kg body weight, preferably 10 ⁵ to 10 ⁶ cells/kg body weight. The T cell composition can also be administered multiple times at these doses. The cells can be administered by using infusion techniques well known in immunotherapy. The optimal dosage and treatment regimen for a particular patient can be easily determined by a person skilled in the medical field by monitoring the patient for signs of disease and adjusting the treatment accordingly.

Administration of the subject composition 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 composition of the present invention is administered to the patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the present invention is preferably administered by intravenous injection. The composition of the T cell can be directly injected into a tumor, lymph node or infection site.

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. The 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, FK506, fludarabine, rapamycin, and mycophenolic acid, etc. In further embodiments, the cell compositions of the present invention are administered to patients in combination (e.g., before, simultaneously, or after) with bone marrow transplantation, T cell ablation therapy using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH.

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.

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

Optionally, the tumor is selected from one or more of leukemia or solid tumors.

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.

Technical Effects of the Invention

There are currently three generations of CAR. "First generation" CAR is usually a single-chain polypeptide consisting of a transmembrane domain fused to a cytoplasm/intracellular domain as an antigen binding domain by scFv, and the cytoplasm/intracellular domain 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" CAR 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 independently of HLA-mediated antigen presentation. "Second generation" CAR adds the intracellular domain 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" CAR includes a fragment providing costimulation (e.g., CD28 or 4-IBB) and activation (e.g., CD3 ζ). Preclinical studies have shown that "second generation" CAR 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., CD3ζ). It can be seen that the "CTR (Chimeric trigger receptor) molecule" of the present invention is different from the structures of the above three generations of CAR (the chimeric antigen receptor disclosed in CN 112500492A contains a 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 self-inhibition property of CD3e, the CTR molecule has a higher specific ligand dependence (the excessively long intracellular domain of the traditional CAR molecule may cause the instability of its membrane-attaching self-inhibition state, thereby leading to a stronger endogenous signal); second, since 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 in the form of phase separation. Therefore, compared with traditional CAR-T, it has richer intracellular signals and is closer to primitive 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

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

Left: The ability of Lck _UD-SH3-SH2 to form aggregates in solution with the indicated intracellular portions of CD3 molecules. CD3-Alexa647 was mixed with Lck _UD-SH3-SH2 -Alexa488 in PBS containing 0.1% BSA for 1 h. Scale bar, 20 μm.

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

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

A: Schematic diagram of the experimental design. CD3ε-wt and CD3ζ were mixed with Lck _UD-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 remained in solution in the case of CD3ε-mBRS. ATP-Mg ²⁺ was then provided to initiate the phosphorylation reaction.

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

Figure 3. Phosphorylation of CD3ε promotes phase separation of CD3ε/Lck

Figure 4. Phase separation of CD3ε and Lck on the two-dimensional phospholipid membrane reconstituted in vitro

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

Figure 6. Csk dissolves CD3ε/Lck aggregates

Figure 7. Domain mutations of CD3ε affect the phase separation of CD3ε/Lck (CD3ε-null, referring to simultaneous mutations in the three domains of BRS/PRS/RK)

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

Figure 9. CD3ε intracellular domain-mediated T cell activation depends on the engagement of endogenous TCR

Wild-type Jurkat cells (A, B) and TCR-deficient Jurkat cells (C, D) containing chimeric molecules of 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.

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

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.

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

A: Antibody activates Jurkat cells engineered with CD3ε cytoplasmic domain. Jurkat cells overexpressing chimeric molecules composed of PD-1 extracellular domain, transmembrane domain and CD3ε cytoplasmic domain were co-cultured with beads connected to the response antibody. The beads connected to the PD-1 antibody can effectively activate Jurkat cells expressing the chimeric molecule.

B: Raji cells stimulated Jurkat cells engineered with CD3ε cytoplasmic domain to secrete IL-2 based on ligand-receptor interaction. The corresponding number of Raji cells overexpressing PD-L1 were co-cultured with Jurkat cells expressing chimeric molecules (PD-1 extracellular domain, CD8 transmembrane domain, and CD3ε cytoplasmic domain).

DETAILED DESCRIPTION

The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

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 the 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.

When the embodiments give 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 can 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 grasp of the prior art by those skilled in the art and the record 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 can also be used to realize the present invention.

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

1. Materials and Methods

Antibody

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

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).

Chimeric molecule construction

All chimeric molecules are carried by lentiviral vectors, and 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 cleavage, and the extracellular domain is a signal peptide of CD8α using the sequence of a CD19 single-chain antibody (clone number FMC63).

Jurkat cells

The relevant chimeric molecules were transferred into Jurkat cells by lentiviral infection for overexpression, and the successfully infected cells were sorted by flow cytometry and used for subsequent functional experiments. All Jurkat cells were cultured in RPMI1640 medium containing 100U/mL streptomycin, 100ug/mL penicillin and 10% fetal bovine serum at 37°C and 5% CO2.

Cell function experiments

1×10 ⁴ corresponding Jurkat cells were inoculated in a U-shaped 96-well culture plate 30 minutes in advance, and the corresponding number of antibody-linked beads or Raji cells were added, with a total system of 200 μl, and placed in a 37°C incubator for 16-24 hours. The culture supernatant was collected and the secretion of IL-2 was detected by ELISA. Each experiment was repeated three times in parallel and three independent repeated experiments were performed.

Peptides

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

Recombinant protein expression and labeling

LqCy

Full-length Lck, Lck regulatory domain constructs (various combinations of UD, SH3 and SH2 domains) or Lck kinase domain were expressed in Expi293F cells, bacteria and insect cells, respectively. To express full-length Lck, cDNA encoding human wild-type and mutant Lck with N-terminal 10×His tags (3-509a.a., Lck-wt; K273R & Y505F, Lck-open) were subcloned into pHAGE vectors. pHAGE-Lck-wt or pHAGE-Lck-open plasmids were transiently transfected into Expi293F cells by polyethyleneimine (PEI) (Polysciences, 23966). After 48 hours of expression, cells were collected and lysed in 50mM Na2HPO4, 300mMNaCl, 1mM EDTA, 1mM DTT, 1X protease inhibitor cocktail, 1mM 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 clear lysate containing the recombinant protein.

To express Lck regulatory domain constructs, cDNA encoding UD (3-60a.a.), UD-SH3 (3-121a.a.), UD-SH3-SH2 (3-226a.a.) or SH3-SH2 (59-226a.a.) human Lck domain with an N-terminal 10×His tag was subcloned into 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 expression of recombinant proteins. Bacteria were harvested and lysed by ultrasonic cell disruptor in 50mM Na ₂ HPO ₄ , 300mM NaCl, 1mM EDTA, 1mM DTT, 1X protease inhibitor cocktail, 1mM PMSF, pH=8.0. For UD recombinant proteins, the lysate was first treated with an additional 95°C boiling for 30 minutes. The lysate was centrifuged and filtered through a 0.22 μm membrane filter to obtain a clear lysate containing the recombinant protein.

To express the Lck kinase domain (245-501a.a.), the coding cDNA was inserted into the pFast Bac1-MBP vector with N-terminal MBP and HRV 3C sites and 10×His sequences. Recombinant baculovirus containing the expression plasmid was obtained by 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 collected and lysed by ultrasonic cell disruptor in 50mM Na2HPO4, 300mM NaCl, 1mM EDTA, 1mM DTT, 1X protease inhibitor cocktail, 1mMPMSF, pH=8.0. The lysate cleared by centrifugation was incubated with dextrin beads (Smart-lifesciences, SA026025). MBP was removed with 3C protease at 4°C for 12 hours. The cleaved recombinant Lck kinase was obtained after centrifugation.

Csk

Full-length and truncated Csk were constructed into pCold plasmids with N-terminal GST and 8×His tags. All constructs were transduced into E. coli BL21 (DE3) and induced with 1mM IPTG at 15°C for 18 hours. 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 (Hiload16/600, superdex200, GE Healthcare).

CD3ε

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

CD3ζ

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 by GST affinity column and then the GST tag was removed by TEV cleavage. The protein was then subjected to HPLC purification.

Fluorescent dye labeling

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

Phase separation on loaded lipid bilayers

Small unilamellar vesicles (SUV)

Phospholipids (95% DOPC, 5% DGS-NTA-Ni ²⁺ and 0.1% DSPE-PEG5000) were dried in a 37°C water bath under a stream of nitrogen. The dried lipid film was further dehydrated in a desiccator for more than 2 hours and resuspended with 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 21,380g for 60 minutes at 4°C. The supernatant containing SUV was then collected.

Loaded lipid bilayer (SLB)

The glass-bottomed 96-well plate was washed with 5% Hellmanex III and rinsed thoroughly with MilliQ H2O three times. Then it was washed with 6M NaOH at 50°C for 2 hours, then rinsed thoroughly with MilliQ H2O, and then equilibrated with PBS for 30 minutes. 15 μl of freshly made SUV was added and incubated at 37°C for 1 hour to induce SLB formation. SLBs were then washed three times with aggregation buffer (PBS containing 1% BSA) and incubated at room temperature for 30 minutes.

Phase separation assay on membrane

Typically, 2 μM His-tagged wild-type or truncated Lck was incubated with preformed SLBs for 1 hour at room temperature. Unbound proteins were removed by washing three times with aggregation buffer. After another 30-min incubation, 2 μM His-tagged CD3ε or 10 μM CD3 molecules (CD3ε, CD3δ, CD3γ, and CD3ζ without 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.

For Lck recruitment and Csk dissolution assays, 2 μM Alexa-488-labeled Lck _UD-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 the dissolution of clusters.

In the signal reconstitution analysis, 2.5 μM wild-type His-tagged CD3ε was used to trigger the phase separation of SLB-bound Lck _UD-SH3-SH2 in the signal buffer. Then 1 μM Alexa-647-labeled Lck-wt was added to the solution and incubated for 30 minutes, and the unbound Lck-wt was removed by washing with the signal buffer. 1 mM ATP-Mg ²⁺ or carrier mimics were injected to trigger Lck kinase activity, and the whole system was incubated at room temperature for 1 hour. Then 5 μM Csk protein was added and mixed thoroughly.

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

Phase separation determination in solution

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

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

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 Lck _UD-SH3-SH2 in signal buffer (35 mM HEPES, 60 mM NaCl, 30 mM KCl, 1 mM MgCl ₂ , pH 7.2). Subsequently, 10 μM Lck-wt was added and incubated for 30 minutes. 1 mM ATP-Mg ²⁺ was then added to trigger the phosphorylation reaction 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.

2. Specific embodiments

Example 1

Our results show that the CD3ε subunit in the TCR complex can undergo liquid-liquid phase separation (LLPS) with the kinase Lck molecule, while other subunits (CD3ζ, CD3γ and CD3δ) cannot undergo phase separation with Lck, but can be recruited by the phase separation droplets of CD3ε/Lck (Figure 1). At the same time, we found that the phase separation of CD3ε/Lck can promote the phosphorylation of tyrosine 394 of Lck, and the phosphorylation of Lck-Y394 is a sufficient condition for Lck to enter the activated conformation. Therefore, the phase separation of CD3ε/Lck will enhance the phosphorylation kinase activity of Lck and promote the phosphorylation of the cytoplasmic domains of each subunit of the TCR complex (Figure 2). Next, by testing different Lck truncations and CD3ε in different phosphorylation states, we found that Lck in the activated state and CD3ε with the first tyrosine phosphorylated on the ITAM motif can 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 and thus lead to the activation of the entire T cell.

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

Example 2

In addition, we also verified the full-length Lck on a two-dimensional phospholipid membrane reconstituted in vitro. We first connected Lck to the phospholipid membrane and then added CD3ε. We could observe obvious phase separation by confocal microscopy (Figure 4). However, when CD3ε was first connected to the phospholipid membrane and then Lck molecules were added, no phase separation could be observed; at this time, we added CD3ε to the system and it could still effectively initiate the phase separation. Moreover, we also found that the CD3ε molecules originally added and connected to the membrane would also be recruited into the microclusters generated by phase separation (Figure 5). Our results further verified that CD3ε of T cells in the resting state is in a self-inhibitory state attached to the membrane; at the same time, we also found that CD3ε/Lck phase separation microclusters can recruit CD3ε in a state of attachment to the membrane (self-inhibitory state). Based on this, we speculate that CD3ε plays a "switch" role in the entire TCR complex. CD3ε can recruit endogenous TCR in T cells through phase separation with Lck, which in turn leads to the phosphorylation of the cytoplasmic domains of each subunit of the endogenous TCR by Lck, causing the activation of the entire T cell.

Example 3

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 resulted in rapid solubilization of pCD3ε-Lck aggregates, whereas this effect was not observed with CD3ε-Lck aggregates. (Figure 6)

Example 4

To further study the molecular mechanism of phase separation between CD3ε and Lck, we designed a series of mutants based on the amino acid sequence characteristics of CD3ε. The results showed that the BRS domain of CD3ε is very important for CD3ε/Lck phase separation (Figure 7). However, mutations in the PRS domain and the RK domain did not cause significant changes in the CD3ε/Lck phase separation phenomenon (Figure 7). In addition, tyrosine mutations in the ITAM domain did not change the phase separation of CD3ε/Lck (Figure 7). Moreover, since tyrosine mutations would prevent CD3ε from being phosphorylated, Csk would not be recruited, leading to the dissolution of phase-separated microclusters.

Example 5

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 from T cells mediated by the intracellular domains of CD3ε and CD3ζ were comparable (Figure 8), while the secretion levels of IL-2 from T cells mediated by CD3δ and CD3γ were very low. Among them, the phosphorylation of CD3ζ to recruit Zap70 and transmit activation signals to the downstream is a known TCR activation mechanism, but there is currently no research to prove that CD3ε can also recruit Zap70 after phosphorylation, so the mechanism by which the CD3ε intracellular domain mediates the activation of TCR downstream signals is not clear.

Example 6

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 T cell activation. 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 identified the functions of the four cells (Figure 9). The results show that in wild-type Jurkat cells, the IL-2 secretion levels caused by CD3ε and CD3ζ intracellular domains are comparable (Figure 9A-B); however, in Jurkat cells with TCR deficiency, only CD3ζ can effectively mediate the secretion of IL-2 (Figure 9C-D). These results show that CD3ε intracellular domain-mediated T cell activation depends on the participation of endogenous TCR, and it itself cannot directly lead to the activation of T cells.

Example 7

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 effectively mediate T cell activation (Figure 10).

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

Sequence information

1.CD8A signal peptide sequence

Amino acid sequence (SEQ NO: 1)

MALPVTALLLPLALLLHAAR DNA sequence (SEQNO:2)

atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccagg

2. Single-chain antibody sequence

CD19 single chain antibody (FMC63)

Amino acid sequence (SEQ NO:3)

PDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSR

FSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSE

VKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSA

LKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDNA sequence (SEQNO:4)

ccggacatccagatgacacagactacatcctccctgtctgcctctctgggagacagtcaccatcagttgcagggcaagtcaggacattagta

aatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatcaagattacactcaggagtcccatcaaggttcagt

ggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgt

acacgttcggaggggggaccaagctggagatcacaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctgaggtgaaac

tgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaag

ctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagac

tgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacatta

ttactacggtggtagctatgctatggactactggggccaaggaacctcagtcaccgtctcctcaBCMA single-chain antibody

Amino acid sequence (SEQ NO: 5)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNHIIHWVRQAPGQCLEWMGYINPYPGYHA

YNEKFQGRATMTSDTSTSTVYMELSSLRSEDTAVYYCARDGYYRDTDVLDYWGQGTLVT

VSSGGGGSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCQASQDISNYLNWYQQKPGK

APKLLIYYTSRLHTGVPSRFSGSGSGTDFTFTISSLEPEDIATYYCQQGNTLPWTFGCGTKVE

IK

DNA sequence (SEQ NO:6)

caagtgcagctggtgcagtccggcgccgaggtgaagaagcccggcgcaagcgtgaaggtgagctgcaaggcaagcggctacaccttcacc

aaccacatcatccactgggtgagacaagcccccgggcagtgtctggagtggatgggctacatcaacccctaccccggctaccacgcctacaa

cgagaagttccaaggcagagccaccatgacaagcgacacaagcacaagcaccgtgtacatggagctgagcagcctgagaagcgaagacac

tgccgtgtactattgcgctagagacggctactacagagacaccgacgtgctggactattggggccaaggcacattagtcaccgtcagcagcggt

gggggaggtagtggaggcggaggctctggtggtggtgggtccgacattcagatgacacagagccctagcagcctgagcgcaagcgtgggc

gacagagtgaccatcacctgccaagcaagccaagacatcagcaactacctgaactggtatcagcagaagccgggcaaagcccccaagctgctgatctactacacaagcagactgcacaccggcgtgcctagcagattcagcggcagcggcagcggcaccgacttcacctttaccatcagcagcagtcttgagcccgaggacatcgccacctattattgtca gcaaggcaacaccctgccctggaccttcggctgcggcaccaaagtggagataaagEGFR single chain antibody

Amino acid sequence (SEQ NO:7)

EIQLVQSGAEVKKPGESLRISCKGSGFNIEDYYIHWVRQMPGKGLEWMGRIDPENDETKYGPIFQGHVTISADTSINTVYLQWSSLKASDTAMYYCAFRGGVYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPDSLAVSLGERATINCKSSQSLLDSDGKTYLNWLQQKPGQPPKRLISLVSKLDSGVPDRFSGSGS GTDFTLTISSLQAEDVAVYYCWQGTHFPGTFGGGTKVEIK

DNA sequence (SEQ NO:8)

gagattcagctcgtgcaatcgggagcggaagtcaagaagccaggagagtccttgcggatctcatgcaagggtagcggctttaacatcgaggattactacatccactgggtgaggcagatgccggggaagggactcgaatggatgggacggatcgacccagaaaacgacgaaactaagtacggtccgatcttccaaggccatgtgactattagcg ccgatacttcaatcaataccgtgtatctgcaatggtcctcattgaaagcctcagataccgcgatgtactactgtgctttcagaggaggggtctactggggacagggaactaccgtgactgtctcgtccggcggaggcgggtcaggaggtggcggc agcggaggaggagggtccggcggaggtgggtccgacgtcgtgatgacccagcctgacagcctggcagtgagcctgggcgaaagagctaccattaactgcaaatcgtcgcagagcctgctggactcggacggaaaaacgtacctcaattggctgcagcaaaaagcctggccagccaccgaagcgccttatctcactgg tgtcgaagctggattcgggagtgcccgatcgcttctccggctcgggatcgggtactgacttcaccctcactatctcctcgcttcaagcagaggacgtggccgtctactactgctggcagggaacccactttccgggaaccttcggcggagggacgaaagtggagatcaag

Mesothelin scFv

Amino acid sequence (SEQ NO:9)

PQLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQTPEKGLEWIAYIYNSGTTKFNPSLKGRVTISMDASKNQLSMKLSSVTSADTAVYFCARDQGNSPYPDAFDVWGQGTLVTVSSGGGGSGGGGSGGGGSQSALTQPPSASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSKRPSGVPDR FSGSKSGNTASLTVSGLQAEDEADYYCSSYAGSNNYVFGTGTKVTVL

DNA sequence (SEQ NO:10)

ccccagctgcagctgcaggagagcggccccggcctggtgaagcccagcgagaccctgagcctgacctgcaccgtgagcggcggcagcatcagcagcagcagctactactggggctggatccgccagaccccgagaagggcctggagtggatcgcctacatctacaacagcggcaccaccaagttcaaccccagcctgaagggccgcgt gaccatcagcatggacgccagcaagaaccagctgagcatgaagctgagcagcgtgaccagcgccgacaccgccgtgtacttctgcgcccgcgaccagggcaacagcccctaccccgacgccttcgacgtgtggggccagggcaccc tggtgaccgtgagcagcggcggcggcggcagcggcggcggcggcagcggcggcggcggcagccagagcgccctgacccagccccccagcgccagcggcagccccggccagagcgtgaccatcagctgcaccggcaccagcagcgacgtgggcggctacaactacgtgagctggtaccagcagcaccccggcaaggcc cccaagctgatgatctacgaggtgagcaagcgccccagcggcgtgcccgaccgcttcagcggcagcaagagcggcaacaccgccagcctgaccgtgagcggcctgcaggccgaggacgaggccgactactactgcagcagctacgccggcagcaacaac

3.CD8A hinge region and transmembrane region sequence

Amino acid sequence (SEQ NO:11)

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCK

DNA sequence (SEQ NO:12)

accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactgg ttatcaccctttatactgcaaa

CD

4.CD28 hinge region and transmembrane region sequence

Amino acid sequence (SEQ NO:13)

IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWV

DNA sequence (SEQ NO:14)

attgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttggtggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtg

5.CD3 intracellular domain sequence

CD3ε

Amino acid sequence (SEQ NO:15)

KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI

DNA sequence (SEQ NO:16)

aagaatagaaaggccaaggccaagcctgtgacacgaggagcgggtgctggcggcaggcaaaggggacaaaacaaggagaggccaccacctgttcccaacccagactatgagcccatccggaaaggccagcgggacctgtattctggcctgaatcagagacgcatc

CD3ζ

Amino acid sequence (SEQ NO:17)

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

DNA sequence (SEQ NO:18)

agagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgcagagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggagg cctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccaccaaga

CD3δ

Amino acid sequence (SEQ NO:19)

GHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK

DNA sequence (SEQ NO:20)

ggacatgagactggaaggctgtctggggctgccgacacacaagctctgttgaggaatgaccaggtctatcagcccctccgagatcgagatgatgctcagtacagccaccttggaggaaactgggctcggaacaag

CD3γ

Amino acid sequence (SEQ NO:21)

GQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN

DNA sequence (SEQ NO:22)

ggacaggatggagttcgccagtcgagagcttcagacaagcagactctgttgcccaatgaccagctctaccagcccctcaaggatcgagaagatgaccagtacagccaccttcaaggaaaccagttgaggaggaat

6. CTR-T chimeric molecule sequence

αCD19-CD8-CD8-CD3ε

Amino acid sequence (SEQ NO:23)

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIK DNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI

DNA sequence (SEQ NO:24)

atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggacatccagatgacacagactacatcctccctgtctgcctctctgggagacagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacat caagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagc aacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggaccaagctggagatcacaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctgaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgc actgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaata tggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggccaaggaacctcagtcaccgtctcctcaaccacgacgccagcgcc gcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccgg ccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttatactgcaaaaagaatagaaaggccaaggccaagcctgtgacacgaggagcgggtgctggcggcaggcaaaggggacaaa acaaggagaggccaccacctgttcccaacccagactatgagcccatccggaaaggccagcgggacctgtattctggcctgaatcagagacgcatc

αCD19-CD8-CD8-CD3ζ

Amino acid sequence (SEQ NO:25)

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTII KDNSKSQVFL KMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR

DNA sequence (SEQ NO:26)

atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggacatccagatgacacagactacatcctccctgtctgcctctctgggagacagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaa

ctgttaaactcctgatctaccatacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccatt

agcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggaccaagctggagatca

caggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctgaggtgaaactgcaggagtcaggacctggcctggtggcgccct

cacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggag

tggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagt

tttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggg

gccaaggaacctcagtcaccgtctcctcaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtc

cctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcc

cttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaaagagtgaagttcagcaggagcgcagacgcccccgcgt

accagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccct

gagatggggggaaagccgcagagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagt

gagattggggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgac

gcccttcacatgcaggcctgccaccaagaαCD19-CD8-CD8-CD3δ

Amino acid sequence (SEQ NO:27)

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKP

DGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGT

KLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPP

RKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYY

GGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF

ACDIYIWAPLAGTCGVLLLSLVITLYCKGHETGRLSGAADTQALLRNDQVYQPLRDRDDA

QYSHLGGNWARNK

DNA sequence (SEQ NO:28)

atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggacatccagatgacacagactacatcctccc

tgtctgcctctctggggagacagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaa

ctgttaaactcctgatctaccatacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccatt

agcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggaccaagctggagatca

caggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctgaggtgaaactgcaggagtcaggacctggcctggtggcgccct

cacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggag

tggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagt

tttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggg

gccaaggaacctcagtcaccgtctcctcaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtc

cctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcc

cttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaaggacatgagactggaaggctgtctggggctgccgacac

acaagctctgttgaggaatgaccaggtctatcagcccctccgagatcgagatgatgctcagtacagccaccttggaggaaactgggctcggaa

caag

αCD19-CD8-CD8-CD3γ

Amino acid sequence (SEQ NO:29)

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKP

DGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGT

KLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPP

RKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYY

GGSYAMDYWGQGTSVTVSSTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF

ACDIYIWAPLAGTCGVLLLSLVITLYCKGQDGVRQSRASDKQTLLPNDQLYQPLKDREDD

QYSHLQGNQLRRN

DNA sequence (SEQ NO:30)

atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggacatccagatgacacagactacatcctccc

tgtctgcctctctggggagacagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaa

ctgttaaactcctgatctaccatacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccatt

agcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggaccaagctggagatca

caggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctgaggtgaaactgcaggagtcaggacctggcctggtggcgccct

cacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggag

tggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagt

tttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggg

gccaaggaacctcagtcaccgtctcctcaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtc

cctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcc

cttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaaggacaggatggagttcgccagtcgagagcttcagacaa

gcagactctgttgcccaatgaccagctctaccagcccctcaaggatcgagaagatgaccagtacagccaccttcaaggaaaccagttgaggag

gaat

αCD19-CD28-CD28-CD3ε

Amino acid sequence (SEQ NO:31)

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKP

DGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGT

KLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPP

RKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYY

GGSYAMDYWGQGTSVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV

LVVVGGVLACYSLLVTVAFIIFWVKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPD

YEPIRKGQRDLYSGLNQRRI

DNA sequence (SEQ NO:32)

atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggacatccagatgacacagactacatcctccc

tgtctgcctctctggggagacagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaa

ctgttaaactcctgatctaccatacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccatt

agcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggaccaagctggagatca

caggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctgaggtgaaactgcaggagtcaggacctggcctggtggcgccct

cacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggag

tggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagt

tttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggg

gccaaggaacctcagtcaccgtctcctcaattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaa

agggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttggtggagtcctggcttgctatagcttgc

tagtaacagtggcctttattattttctgggtgaagaatagaaaggccaaggccaagcctgtgacacgaggagcgggtgctggcggcaggcaaa

ggggacaaaacaaggagaggccaccacctgttcccaacccagactatgagcccatccggaaaggccagcgggacctgtattctggcctgaat

cagagacgcatcPD1-PD1-PD1-CD3ε

Amino acid sequence (SEQ NO:33)

MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTS

ESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSG

TYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSL

VLLVWVLAVIKNRKAKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSG

LqCy

DNA sequence (SEQ NO:34)

atgcagatcccacaggcgccctggccagtcgtctgggcggtgctacaactgggctggcggccaggatggttcttagactccccagacaggcc

ctggaacccccccaccttctccccagccctgctcgtggtgaccgaaggggacaacgccaccttcacctgcagcttctccaacacatcggagag

cttcgtgctaaactggtaccgcatgagccccagcaaccagacggacaagctggccgccttccccgaggaccgcagccagcccggccaggac

tgccgcttccgtgtcacacaactgcccaacgggcgtgacttccacatgagcgtggtcagggcccggcgcaatgacagcggcacctacctctgt

ggggccatctccctggcccccaaggcgcagatcaaagagagcctgcgggcagagctcagggtgacagagagaagggcagaagtgcccac

agcccaccccagcccctcacccaggccagccggccagttccaaaccctggtggttggtgtcgtgggcggcctgctgggcagcctggtgctgc

tagtctgggtcctggccgtcatcaagaatagaaaggccaaggccaagcctgtgacacgaggagcgggtgctggcggcaggcaaaggggac

aaaacaaggagaggccaccacctgttcccaacccagactatgagcccatccggaaaggccagcgggacctgtattctggcctgaatcagaga

cgcatc

7. Amino acid sequence of CD3ε mutant (synthesized by Shanghai Jier Company)

CD3ε (SEQ NO:35)

CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI CD3ε-pY1(SEQNO:36)

CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPD-pY-EPIRKGQRDLYSGLNQRRI CD3ε-pY2(SEQ NO:37)

CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDL-pY-SGLNQRRI pCD3ε(SEQNO:38)

CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPD-pY-EPIRKGQRDL-pY-SGLNQRRI CD3ε-YYFF(SEQ NO:39)

CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDFEPIRKGQRDLFSGLNQRRI CD3ε-mPRS(SEQ NO:40)

CKNRKAKAKPVTRGAGAGGRQRGQNKERSSSVSNSDYEPIRKGQRDLYSGLNQRRI CD3ε-mBRS(SEQ NO:41)

CSNSSASASPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI CD3ε-dBRS(SEQ NO:42)

CPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI CD3ε-mRK(SEQ NO:43)

CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPISSGQRDLYSGLNQRRI CD3ε-null(SEQ NO:44)

CSNSSASASPVTRGAGAGGRQRGQNKERSSSVSNSDYEPISSGQRDLYSGLNQRRI

8.Lck-related protein sequences

LckUD-SH3-SH2

Amino acid sequence (SEQ NO:45)

GPENLYFQGMGLNDIFEAQKIEWHEHHHHHHHHHHGGGGSCGCSSHPEDDWMENIDVCE

NCHYPIVPLDGKGTLLIRNGSEVRDPLVTYEGSNPPASPLQDNLVIALHSYEPSHDGDLGFE

KGEQLRILEQSGEWWKAQSLTTGQEGFIPFNFVAKANSLEPEPWFFKNLSRKDAERQLLAP

GNTHGSFLIRESESTAGSFSLSVRDFDQNQGEVVKHYKIRNLDNGGFYISPRITFPGLHELVR

HYTNASDGLCTRLSRPCQTGGGGSDYKDDDDKAHIVMVDAYKPTK

DNA sequence (SEQ NO:46)

gggcccgaaaacttatattttcagggcatgggtctgaacgacatcttcgaggctcagaaaatcgaatggcacgaacaccatcaccaccatcatca

ccaccatcacgggggtggaggctcttgtggctgcagctcacacccggaagatgactggatggaaaacatcgatgtgtgtgagaactgccattat

cccatagtcccactggatggcaagggcacgctgctcatccgaaatggctctgaggtgcgggacccactggttacctacgaaggctccaatccg

ccggcttccccactgcaagacaacctggttatcgctctgcacagctatgagccctctcacgacggagatctgggctttgagaagggggaacag

ctccgcatcctggagcagagcggcgagtggtggaaggcgcagtccctgaccacgggccaggaaggcttcatccccttcaattttgtggccaaa

gcgaacagcctggagcccgaaccctggttcttcaagaacctgagccgcaaggacgcggagcggcagctcctggcgcccgggaacactcac

ggctccttcctcatccgggagagcgagagcaccgcgggatcgttttcactgtcggtccgggacttcgaccagaaccagggagaggtggtgaa

acattacaagatccgtaatctggacaacggtggcttctacatctcccctcgaatcacttttcccggcctgcatgaactggtccgccattacaccaat

gcttcagatgggctgtgcacacggttgagccgcccctgccagaccgggggtggaggctctgattacaaggatgacgatgacaaggcccacat

cgtgatggtggacgcctacaagccgacgaag

Lck-wt

Amino acid sequence (SEQ NO:47)

MGLNDIFEAQKIEWHEHHHHHHHHHHCGCSSHPEDDWMENIDVCENCHYPIVPLDGKGT

LLIRNGSEVRDPLVTYEGSNPPASPLQDNLVIALHSYEPSHDGDLGFEKGEQLRILEQSGEW

WKAQSLTTGQEGFIPFNFVAKANSLEPEPWFFKNLSRKDAERQLLAPGNTHGSFLIRESEST

AGSFSLSVRDFDQNQGEVVKHYKIRNLDNGGFYISPRITFPGLHELVRHYTNASDGLCTRLS

RPCQTQKPQKPWWEDEWEVPRETLKLVERLGAGQFGEVWMGYYNGHTKVAVKSLKQGS

MSPDAFLAEANLMKQLQHQRLVRLYAVVTQEPIYIITEYMENGSLVDFLKTPSGIKLTINKL

LDMAAQIAEGMAFIEERNYIHRDLRAANILVSDTLSCKIADFGLARLIEDNEYTAREGAKFP

IKWTAPEAINYGTFTIKSDVWSFGILLTEIVTHGRIPYPGMTNPEVIQNLERGYRMVRPDNC

PEELYQLMRLCWKERPEDRPTFDYLRSVLEDFFTATEGQYQPQPYPYDVPDYAAHIVMVD

AYKPTK

DNA sequence (SEQ NO:48)

atgggtctgaacgacatcttcgaggctcagaaaatcgaatggcacgaacaccatcaccaccatcatcaccaccatcactgtggctgcagctcac

acccggaagatgactggatggaaaacatcgatgtgtgtgagaactgccattatcccatagtcccactggatggcaagggcacgctgctcatccg

aaatggctctgaggtgcgggacccactggttacctacgaaggctccaatccgccggcttccccactgcaagacaacctggttatcgctctgcac

agctatgagccctctcacgacggagatctgggctttgagaagggggaacagctccgcatcctggagcagagcggcgagtggtggaaggcgc

agtccctgaccacgggccaggaaggcttcatccccttcaattttgtggccaaagcgaacagcctggagcccgaaccctggttcttcaagaacct

gagccgcaaggacgcggagcggcagctcctggcgcccgggaacactcacggctccttcctcatccgggagagcgagagcaccgcgggat

cgttttcactgtcggtccgggacttcgaccagaaccagggagaggtggtgaaacattacaagatccgtaatctggacaacggtggcttctacatc

tcccctcgaatcacttttcccggcctgcatgaactggtccgccattacaccaatgcttcagatgggctgtgcacacggttgagccgcccctgcca

gacccagaagccccgaagccgtggtggggagacgagtgggaggttcccaggggagacgctgaagctggtggagcggctgggggctggac

agttcggggaggtgtggatggggtactacaacgggcacacgaaggtggcggtgaagagcctgaagcagggcagcatgtccccggacgcctt

cctggccgaggccaacctcatgaagcagctgcaacaccagcggctggttcggctctacgctgtggtcacccaggagcccatctacatcatcac

tgaatacatggagaatgggagtctagtggattttctcaagaccccttcaggcatcaagttgaccatcaacaaactcctggacatggcagcccaaat

tgcagaaggcatggcattcattgaagagcggaattatattcatcgtgaccttcgggctgccaacattctggtgtctgacaccctgagctgcaagatt

gcagactttggcctagcacgcctcattgaggacaacgagtacacagccagggagggggccaagtttcccattaagtggacagcgccagaagc

cattaactacgggacattcaccatcaagtcagatgtgtggtcttttgggatcctgctgacggaaattgtcacccacggccgcatcccttacccagg

gatgaccaacccggaggtgattcagaacctggagcgaggctaccgcatggtgcgccctgacaactgtccagaggagctgtaccaactcatga

ggctgtgctggaaggagcgcccagaggaccggcccacctttgactacctgcgcagtgtgctggaggacttcttcacggccacagagggccag

taccagcctcagccttacccatacgatgttccagattacgctgcccacatcgtgatggtggacgcctacaagccgacgaag

10. Csk sequence

Amino acid sequence (SEQ NO:49)

GPGHHHHHHHHMCEFMSAIQAAWPSGTECIAKYNFHGTAEQDLPFCKGDVLTIVAVTKDP

NWYKAKNKVGREGIIPANYVQKREGVKAGTKLSLMPWFHGKITREQAERLLYPPETGLFL

VRESTNYPGDYTLCVSCDGKVEHYRIMYHASKLSIDEEVYFENLMQLVEHYTSDADGLCT

RLIKPKVMEGTVAAQDEFYRSGWALNMKELKLLQTIGKGEFGDVMLGDYRGNKVAVKCI

KNDATAQAFLAEASVMTQLRHSNLVQLLGVIVEEKGGLYIVTEYMAKGSLVDYLRSRGRS

VLGGDCLLKFSLDVCEAMEYLEGNNFVHRDLAARNVLVSEDNVAKVSDFGLTKEASSTQ

DTGKLPVKWTAPEALREKKFSTKSDVWSFGILLWEIYSFGRVPYPRIPLKDVVPRVEKGYK

MDAPDGCPPAVYEVMKNCWHLDAAMRPSFLQLREQLEHIKTHELHLDNA sequence (SEQ NO:50)

ggcccgggacatcaccatcatcatcatcacatgtgtgaattcatgtcagcaatacaggccgcctggccatccggtacagaatgtattgccaa

gtacaacttccacggcactgccgagcaggacctgcccttctgcaaaggagacgtgctcaccattgtggccgtcaccaaggaccccaactggta

caaagccaaaaacaaggtgggccgtgagggcatcatcccagccaactacgtccagaagcgggagggcgtgaaggcgggtaccaaactcag

cctcatgccttggttccacggcaagatcacacgggagcaggctgagcggcttctgtacccgccggagacaggcctgttcctggtgcgggaga

gcaccaactaccccggagactacacgctgtgcgtgagctgcgacggcaaggtggagcactaccgcatcatgtaccatgccagcaagctcagc

atcgacgaggaggtgtactttgagaacctcatgcagctggtggagcactacacctcagacgcagatggactctgtacgcgcctcattaaaccaa

aggtcatggagggcacagtggcggcccaggatgagttctaccgcagcggctgggccctgaacatgaaggagctgaagctgctgcagaccat

cgggaagggggagttcggagacgtgatgctgggcgattaccgagggaacaaagtcgccgtcaagtgcattaagaacgacgccactgcccag

gccttcctggctgaagcctcagtcatgacgcaactgcggcatagcaacctggtgcagctcctgggcgtgatcgtggaggagaagggcgggct

ctacatcgtcactgagtacatggccaaggggagccttgtggactacctgcggtctaggggtcggtcagtgctgggcggagactgtctcctcaag

ttctcgctagatgtctgcgaggccatggaatacctggagggcaacaatttcgtgcatcgagacctggctgcccgcaatgtgctggtgtctgagga

caacgtggccaaggtcagcgactttggtctcaccaaggaggcgtccagcacccaggacacgggcaagctgccagtcaagtggacagcccct

gaggccctgagagagaagaaattctccactaagtctgacgtgtggagtttcggaatccttctctgggaaatctactcctttgggcgagtgccttatc

caagaattcccctgaaggacgtcgtccctcgggtggagaagggctacaagatggatgcccccgacggctgcccgcccgcagtctatgaagtc

atgaagaactgctggcacctggacgccgccatgcggccctccttcctacagctccgagagcagcttgagcacatcaaaacccacgagctgca

cctg

Claims (27)

1. The use of CD3ε cytoplasmic domain or CD3ε cytoplasmic domain variants in promoting the production of Lck or its variants in liquid-liquid phase separation (LLPS) reagents, 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. The use of the CD3ε cytoplasmic domain or a CD3ε cytoplasmic domain variant for non-therapeutic purposes, wherein the non-therapeutic use includes experimental use or research use, characterized in that the liquid-liquid phase separation (LLPS) of Lck or its variants is promoted, 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. 3. The preparation of CD3ε cytoplasmic domain or CD3ε cytoplasmic domain variants through liquid-liquid phase separation (LLPS) pathway to promote Lck activation and TCR cytoplasmic domain phosphorylation reagents, 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 solution. 4. The use of CD3ε cytoplasmic domain or CD3ε cytoplasmic domain variant for non-therapeutic purposes, including experimental use or research use, which includes CD3ε cytoplasmic domain or CD3ε cytoplasmic domain variant promoting Lck activation and TCR cytoplasmic domain phosphorylation reagent through liquid-liquid phase separation (LLPS) pathway, 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 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, which 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 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: CD3ε1 (SEQ NO:35) CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLY SGLNQRRI; CD3ε-pY1 (SEQ NO:36) CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPD-pY-EPIRKGQRD LYSGLNQRRI; CD3ε-pY2 (SEQ NO:37) CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDL-pY-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) CSNSSASSPVTRGAGAGGRQRGQNKERSSSVSNSDYEPISSGQRDLYS 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: CD3ε1 (SEQ NO:35) CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLY SGLNQRRI; CD3ε-pY1 (SEQ NO:36) CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPD-pY-EPIRKGQRD LYSGLNQRRI; CD3ε-pY2 (SEQ NO:37) CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDL-pY-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) CSNSSASSPVTRGAGAGGRQRGQNKERSSSVSNSDYEPISSGQRDLYS 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, which 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 contains 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 only the CD3ε cytoplasmic domain or its variant 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 CD3ε cytoplasmic domain or a variant thereof as a switch for TCR activation or immune cell activation. 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 the myeloma antigen BCMA recognition domain sequence (SEQ NO: 5) or a 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 comprises 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 to 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 capacity 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 the CD3ε cytoplasmic domain or a variant thereof in the preparation of a T cell or NK cell activation reagent. 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 according to claim 26, comprising: 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: CD3ε1 (SEQ NO:35) CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI; CD3ε-pY1 (SEQ NO:36) CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPD-pY-EPIRKGQRDLYSGLNQRRI; CD3ε-pY2 (SEQ NO:37) CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDL-pY-SGLNQRRI; pCD3ε (SEQ NO:38) CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPD-pY-EPIRKGQRDL-pY-SGLNQRRI; CD3ε-YYFF (SEQ NO:39) CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDFEPIRKGQRDLFSGLNQRRI; CD3ε-mPRS (SEQ NO:40) CKNRKAKAKPVTRGAGAGGRQRGQNKERSSSVSNSDYEPIRKGQRDLYSGLNQRRI; CD3ε-mBRS (SEQ NO:41) CSNSSASASPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI; CD3ε-dBRS (SEQ NO:42) CPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI CD3ε-mRK (SEQ NO:43) CKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPISSGQRDLYSGLNQRRI; CD3ε-null (SEQ NO:44) CSNSSASASPVTRGAGAGGRQRGQNKERSSSVSNSDYEPISSGQRDLYSGLNQRRI; The cysteine in the above variants was used for in vitro labeling and was removed during the design and preparation of the CTR molecule.