HK40061062B - Immune cell containing tumor antigen recognition receptor and application thereof - Google Patents

Description

An immune cell containing a tumor antigen recognition receptor and its application

Technical Field

This application relates to the field of biomedicine, specifically to a modified immune effector cell and its application, particularly an immune effector cell in which the expression and/or activity of the S1PR1 protein are upregulated.

Background Technology

In CAR-T (or TCR-T) therapy, memory CAR-T cells, like memory T cells, tend to home to lymph nodes (https://doi.org/10.1124/jpet.118.252858). However, because CAR-T cell activation depends on the direct recognition of target cell membrane antigens by the antigen recognition region, memory CAR-T cells home to lymph nodes often fail to be effectively activated. Prolonged residence within lymph nodes without contact with target cells negatively impacts the therapeutic effect.

Central memory T lymphocytes, due to the expression of homing receptors such as CCR7 and adhesion molecule receptor CD62L, tend to hom to lymph nodes in the peripheral circulation. Once in the lymph node, if activated by antigen-presenting cells (such as dendritic cells), they proliferate and differentiate into effector cells, reducing homing receptor expression while upregulating the expression of receptors such as S1PR1 (sphingosine 1 phosphate receptor 1), thus migrating out of the lymph node to reach the treatment site. In contrast, CAR-T cells, because their activation depends on binding to target cells rather than on antigen-presenting cells, are difficult to activate and migrate out of the lymph node in the absence of target cells. Therefore, they remain within the lymph node and cannot perform their function.

Nevertheless, there is no existing technology on how to promote the migration of CAR-T (or TCR-T) cells outside the lymph nodes.

Summary of the Invention

This application provides a modified immune effector cell and its use therein. The expression and/or activity of the S1PR1 protein in the modified immune effector cell are upregulated. The immune effector cells of this application have a stronger ability to migrate from lymph nodes, are more likely to reach the treatment site to exert their function, and have enhanced immune effector function, thereby possessing enhanced anti-tumor ability. This application also provides a cell population comprising the said immune effector cells and a pharmaceutical composition.

On the one hand, this application provides a modified immune effector cell in which the expression and/or activity of the S1PR1 protein is upregulated compared with that of an unmodified immune effector cell.

In some embodiments, the immune effector cells include T cells, lymphocytes, granulocytes, and/or peripheral blood mononuclear cells.

In some embodiments, the immune effector cells include memory T cells.

In some embodiments, the expression and/or activity of nucleic acid molecules encoding the S1PR1 protein are upregulated in the modified immune cells.

In some embodiments, the modified immune cells additionally contain the S1PR1 protein.

In some embodiments, the modified immune effector cells additionally contain the nucleic acid molecule encoding the S1PR1 protein.

In some embodiments, the modified immune effector cells comprise a vector comprising the nucleic acid molecule encoding the S1PR1 protein.

In some embodiments, the immune effector cells express a chimeric antigen receptor (CAR).

In some embodiments, the modified immune effector cells comprise a vector that includes a nucleic acid molecule encoding the chimeric antigen receptor.

In some embodiments, the nucleic acid molecule encoding the chimeric antigen receptor and the nucleic acid molecule encoding the S1PR1 protein are located in the same vector.

In some embodiments, the nucleic acid molecule encoding the chimeric antigen receptor is located at the 5' end of the nucleic acid molecule encoding the S1PR1 protein; or, the nucleic acid molecule encoding the chimeric antigen receptor is located at the 3' end of the nucleic acid molecule encoding the S1PR1 protein.

In some embodiments, the chimeric antigen receptor includes a chimeric antigen receptor that targets a tumor-specific antigen selected from the group consisting of: EpCAM, Mesothelin (MSLN), CEA, IL13, PDPN, VEGF, EGFR, EGFRvIII, PSMA, FAP, CD171, GD2, Glypican-2, Glypican-3, HER2, HPV antigen, cyclin D1, p53, MMP-7, IL13Ralpha2, MMP-2, MUC-1, G250, L1CAM, ROR1, and GPC3.

In some embodiments, the chimeric antigen receptor includes an antigen-binding domain that specifically binds to the tumor-specific antigen.

In some embodiments, the antigen-binding domain includes a single-chain antibody.

In some embodiments, the antigen-binding domain includes a single-chain antibody targeting GPC3 or MSLN.

In some embodiments, the single-chain antibody comprises the amino acid sequence shown in any one of SEQ ID NO: 1-2.

In some embodiments, the chimeric antigen receptor includes a transmembrane domain.

In some embodiments, the transmembrane domain comprises a transmembrane domain derived from a selection of proteins including CD3ζ, CD28, 4-1BB, OX40, SLAMF4, CD127, NKG2D, ICOS, and FcγRIIIa.

In some embodiments, the chimeric antigen receptor includes a co-stimulatory domain.

In some embodiments, the co-stimulatory domain comprises a co-stimulatory domain selected from the following proteins or combinations thereof: CD137, CD28, OX40, ICOS, DAP10, 2B4, CD27, CD30, CD40, CD40L, TIM1, CD226, DR3, SLAM, NKG2D, CD244, FceRIγ, BTLA, GITR, HVEM, CD2, NKG2C, LIGHT, and DAP12.

In some embodiments, the chimeric antigen receptor includes a hinge region connecting the antigen-binding domain and the transmembrane domain.

In some embodiments, the hinge region comprises a hinge region derived from proteins selected from: CD8, CD28, IgG, 4-1BB, CD4, CD27, CD7, PD-1, and CH2CH3.

In some embodiments, the chimeric antigen receptor comprises the amino acid sequence shown in any one of SEQ ID NO: 5-6.

In some embodiments, the immune effector cells include nucleic acid molecules encoding the chimeric antigen receptor.

In some embodiments, the vector comprising the nucleic acid molecule encoding the chimeric antigen receptor and the nucleic acid molecule encoding the S1PR1 protein comprises the nucleotide sequence shown in any one of SEQ ID NO: 7-9.

On the other hand, this application provides a cell population comprising the aforementioned immune effector cells.

On the other hand, this application provides pharmaceutical compositions comprising the aforementioned immune effector cells and/or the aforementioned cell populations, and optionally pharmaceutically acceptable carriers.

On the other hand, this application provides the use of the said immune effector cells, the said cell population and/or the said pharmaceutical composition in the preparation of a medicament for the treatment of tumors.

In some implementations, the tumor includes a solid tumor.

In some embodiments, the tumor includes pancreatic cancer, glioma, liver cancer, and/or colon cancer.

Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive.

Attached Figure Description

The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

Figure 1 shows the CAR T cell positivity rate described in this application;

Figure 2 shows the flow cytometry results of GFP-positive lower chamber cells of the CAR T cells described in this application in the migration assay at gradient concentrations of S1P;

Figure 3 shows the GFP positivity rate of the lower chamber cells of the CAR T cells described in this application under gradient concentrations of S1P in the migration assay;

Figure 4 shows the migration efficiency of the CAR T cells described in this application in the migration experiment of the lower chamber cells;

Figure 5 shows the killing efficiency of the CAR T cells against target cells described in this application;

Figure 6 shows the killing efficiency of the CAR T cells against target cells described in this application;

Figure 7 shows the killing efficiency of the CAR T cells against target cells described in this application;

Figure 8 shows the cytokines secreted by the CAR T cells described in this application under the stimulation of target cells, A: IL-2, B: IFN-γ;

Figure 9 shows the in vivo inhibition of tumor growth by CAR T cells as described in this application. A: tail vein administration, B: intratumoral injection administration;

Figure 10 shows the T cell content in peripheral blood on day 28 of the in vivo migration experiment;

Figure 11 shows the in vivo inhibition of tumor growth by CAR T cells as described in this application;

Figure 12 shows the relationship between peripheral blood CD3 ⁺ T cell content and tumor volume. A: GC33CAR, B: GC33CAR-IRES-S1PR1.

Detailed Implementation

The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

Terminology Definition

In this application, the term "S1PR1" generally refers to sphingosine-1-phosphate receptor 1 (EDG1), also known as endothelial differentiation gene 1. S1PR1 is a G protein-coupled receptor that binds to the biologically active signaling molecule sphingosine-1-phosphate (S1P). This term may include natural S1PR1 or its variants, and synthetically produced S1PR1. The S1PR1 may also include full-length S1PR1, truncated versions of S1PR1, spliced forms, or functional fragments thereof. In some cases, the S1PR1 in this application may be full-length human S1PR1. The nucleotide sequence of the full-length human S1PR1 gene is shown under NCBI accession number NM_001400, and the amino acid sequence of the S1PR1 protein is shown under NCBI accession number NP_001391.

In this application, the terms “MSLN,” “Mesothelin,” and “mesothelin” are used interchangeably and generally refer to a glycophosphatidylinositol-linked cell (e.g., tumor cell) surface glycoprotein. The term “MSLN” may include mature MSLN protein, various isoforms of MSLN protein and their precursor proteins, as well as native MSLN protein or its variants, cleaved forms, truncated forms, and synthetic MSLN protein. MSLN precursor proteins may include mesothelin isoform 1 precursor protein and mesothelin isoform 2 precursor protein, which may be processed (e.g., enzymatically cleaved) to generate mature MSLN. MSLN may be linked to polymeric cell-enhancing factor (MPF). The amino acid sequence of mesothelin isoform 1 precursor protein is shown under NCBI accession numbers NP_001170826 or NP_005814, and the amino acid sequence of mesothelin isoform 2 precursor protein is shown under NCBI accession number NP_037536.

In this application, the term "GPC3" is also referred to as Glypican-3, which generally refers to a complex formed by the covalent linkage of a protein, sugar, and lipid, which can be expressed on the cell surface via glycosylphosphatidyl inositol (GPI). This term may include various isoforms (e.g., isoform 1, isoform 2, isoform 3, and isoform 4), variants, precursors, spliced forms of natural GPC3, as well as synthetically produced GPC3 or functional fragments thereof. For example, the amino acid sequence of the precursor form of GPC3 subtype 1 can be found under NCBI accession number NP_001158089, the amino acid sequence of the precursor form of GPC3 subtype 3 can be found under NCBI accession number NP_001158090, the amino acid sequence of the precursor form of GPC3 subtype 4 can be found under NCBI accession number NP_001158091, and the amino acid sequence of the precursor form of GPC3 subtype 2 can be found under NCBI accession number NP_004475.

In this application, the term "Chimeric Antigen Receptor (CAR)" generally refers to a fusion protein comprising an extracellular domain capable of binding an antigen and at least one intracellular domain. The CAR is a core component of chimeric antigen receptor T cells (CAR-T cells) and may include an antigen-binding domain (e.g., tumor-specific antigens and/or tumor-associated antigens), a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain. In this application, the CAR may be combined with the T cell receptor-activating intracellular domain based on the antigen specificity of an antibody (e.g., CD70). Genetically modified T cells expressing the CAR can specifically recognize and eliminate malignant cells expressing the target antigen. For descriptions of CAR and CAR-T cells, see, for example, Sadelain M, Brentjens R, Rivière I. The basic principles of chimeric antigen receptor design. Cancer Discov. 2013; 3(4): 388-398; Turtle CJ, Hudecek M, Jensen MC, Riddell SR. Engineered T cells for anti-cancer therapy. y.Curr Opin Immunol2012;24(5):633-639;Dotti G, Gottschalk S, Savoldo B, Brenner MK.Design and development of therapy es using chimeric antigen receptor-expressing Tcells. Immunol Rev. 2014; 257(1): 107-126; and WO2013154760, WO2016014789.

In this application, the term "antigen-binding domain" generally refers to a domain capable of binding to a target antigen. An antigen-binding domain may comprise a chimeric antigen receptor or fragment thereof capable of specifically binding to an antigen, an antibody or its antigen-binding fragment. The antigen-binding domain may be of natural, synthetic, semi-synthetic, or recombinant origin. In some cases, the antigen-binding domain may comprise a single-chain antibody.

In this application, the term "antibody" generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a particular antigen. For example, an antibody may comprise an immunoglobulin consisting of at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and includes any molecule containing its antigen-binding portion. The term "antibody" includes monoclonal antibodies, antibody fragments, or antibody derivatives, including but not limited to human antibodies, humanized antibodies, chimeric antibodies, single-domain antibodies (e.g., dAb), single-chain antibodies (e.g., scFv), and antigen-binding antibody fragments (e.g., Fab, Fab', and (Fab)2 fragments). The term "antibody" also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and any antigen-binding antibody fragments and their derivatives described in this application. Each heavy chain may consist of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain may consist of a light chain variable region (VL) and a light chain constant region. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are scattered within more conserved regions called framework regions (FRs). Each VH and VL region may consist of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen.

In this application, the term "single-chain antibody (scFv)" can refer to an antibody consisting of the heavy chain variable region and the light chain variable region, or comprising an antibody linked by a linker.

In this application, the term "transmembrane domain" generally refers to a domain in a CAR that crosses the cell membrane and connects with intracellular signal transduction domains, playing a role in transmitting signals.

In this application, the term "co-stimulatory domain" generally refers to an intracellular domain that can provide immune co-stimulatory molecules, which are cell surface molecules required for an effective lymphocyte response to an antigen.

In this application, the term "hinge region" generally refers to the connection region between the antigen-binding domain and the transmembrane region.

In this application, the term "signal transduction domain" generally refers to a domain located inside the cell that can transduce signals. In this application, the intracellular signal transduction domain can transduce signals into the cell. Typically, a signal transduction domain is any continuous amino acid sequence used to guide protein target acquisition. For example, the intracellular signal transduction domain is the intracellular signal transduction domain of the chimeric antigen receptor.

In this application, the term "immune effector cell" generally refers to immune cells that participate in the immune response and perform effector functions, such as clearing foreign antigens and promoting the immune response. In some cases, immune effector cells can be stimulated by antigens on the surface of target cells to produce an immune response, for example, by secreting cytokines. Examples of immune cells include, but are not limited to, plasma cells, T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, granulocytes, monocytes, lymphocytes, dendritic cells, and macrophages. The term also includes engineered immune cells, such as immune cells that have been genetically modified by adding exogenous genetic material in the form of DNA or RNA to the total genetic material of the cell.

In this application, the term "T-cell receptor (TCR)" generally refers to a specific receptor on the surface of T cells. The T-cell receptor is a heterodimer and can be composed of two distinct subunits. Most T-cell receptors (e.g., 95% or more, 96% or more, 97% or more, etc.) are composed of α and β subunits, and each polypeptide chain can be further divided into variable (V) region, constant (C) region, transmembrane region, and cytoplasmic region. In this application, the terms "polypeptide," "peptide," "protein," and "protein" are used interchangeably and generally refer to a polymer of any length of amino acids. The polymer can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acid components. These terms also cover polymers containing modified amino acids. These modifications can include: disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation (such as binding to a labeled component). The term "amino acid" includes natural and/or non-natural or synthetic amino acids, including glycine and its D and L optical isomers, as well as amino acid analogs and peptide mimics.

In this application, the terms "polynucleotide," "nucleotide," "nucleotide sequence," "nucleic acid," and "oligonucleotide" are used interchangeably and generally refer to a polymeric form of nucleotides of any length, such as deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, multiple loci (one locus) as defined by ligation analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, the nucleotide structure may be modified before or after polymer assembly. The sequence of a nucleotide may be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with labeled components.

In this application, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host for transferring inserted nucleic acid molecules into host cells and/or between host cells. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and/or translation of DNA or RNA. The vector also includes vectors having multiple of the aforementioned functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the vector, the vector can produce the desired expression product.

In this application, the term "lentiviral vector" generally refers to an RNA viral vector. A lentiviral vector is based on the genome of a lentivirus, with multiple sequences related to viral activity removed to ensure its biological safety. The sequence and expression structure of the target gene required for the experiment are then introduced into this genomic backbone, and the vector is prepared. Through lentiviral vector transfection, the host genome and its carried foreign genes can be randomly and stably integrated into the host cell genome; for example, CAR molecules can be integrated into host cells.

In this application, the term "treatment" generally refers to slowing or improving the progression, severity, and/or duration of a proliferative condition, or improving one or more symptoms of a proliferative condition (e.g., one or more identifiable symptoms), due to the application of one or more therapies (e.g., one or more therapeutic agents such as the nucleic acid molecules and/or immune effector cells of this application). In this application, the term "treatment" may also refer to improving at least one measurable physical parameter of a proliferative condition, such as tumor growth, which need not be identifiable by the patient. The term "treatment" in this application may also refer to inhibiting the progression of a proliferative condition physically, for example, by stabilizing identifiable symptoms, physiologically, for example, by stabilizing physical parameters, or both. In some cases, the term "treatment" may refer to reducing or stabilizing tumor size or cancer cell count.

In this application, the term "and/or" should be understood to mean any one of the options or both of the options.

In this application, the term "comprising" generally means including the explicitly specified features, but does not exclude other elements.

In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

Invention Details

The technical problem this application aims to solve is to address the deficiency in existing technologies where CAR-T (or TCR-T) cells reside within lymph nodes for extended periods, preventing them from contacting target cells and thus affecting therapeutic efficacy. This application provides an immune cell containing a tumor antigen recognition receptor and its application. The immune cells overexpressing S1PR1 and containing the tumor antigen recognition receptor (e.g., CAR/TCR-T cells) of this application exhibit stronger migration ability from lymph nodes, making it easier to reach the treatment site and perform their function; furthermore, the immune cells have strong survival capabilities.

The inventors of this application have creatively overexpressed S1PR1 in immune cells containing tumor antigen recognition receptors (such as memory CAR-T cells). This preserves the memory function of CAR-T cells while preventing them from remaining in lymph nodes for extended periods and thus avoiding contact with target cells, making it easier for them to migrate from lymph nodes to the treatment site. Furthermore, it was unexpectedly discovered that when the CAR and S1PR1 proteins are linked, overexpression of S1PR1 increases the survival rate of CAR-T cells. Compared to existing technologies, the technical solution of this application effectively enhances the formation and subsequent function of CAR-T cell memory, thereby enhancing its anti-tumor effect.

This application mainly solves the above-mentioned technical problems through the following technical solutions.

This application provides an immune cell containing a tumor antigen recognition receptor, wherein the immune cell overexpresses S1PR1.

The tumor antigen recognition receptor may be a conventional tumor antigen recognition receptor in the art, such as a chimeric antigen receptor (CAR) or a T-cell receptor (TCR).

The CAR described herein may be a conventional CAR in the art, for example, it may include an intracellular region, a hinge region, and a transmembrane region, all of which are conventional in the art. Examples of intracellular domains used in this application include, but are not limited to, the cytoplasmic portion of a surface receptor, co-stimulatory molecules, and any molecules that act in concert to initiate signal transduction in T cells, as well as any derivatives or variants of these elements and any synthetic sequences having the same functional capabilities.

The intracellular domains described in this application include, for example, those derived from TCR, CD3ζ, CD3γ, CD3δ, CD3ε, CD86, common FcRγ, FcRβ (FcεR1b), CD79a, CD79b, FcγRIIa, DAP10, DAP12, T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), OX9, OX40, CD30, CD40, PD-1, ICOS, KIR family proteins, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligands that specifically bind to CD83, and C. DS, ICAM-1, GITR, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80(KLRF1), CD127, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49 a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(B Y55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME(SLAMF8), SELPLG(CD162), LTBR, LAT, GADS, SL Fragments or domains of P-76, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, or other co-stimulatory molecules may be combined with any antigen-binding domain described herein, any transmembrane domain described herein, or any other domain described herein that may be included in the CAR; for example, they may be derived from one or more of CD3ζ, CD28, 4-1BB, OX40, SLAMF4, CD127, NKG2D, ICOS, and FcγRIIIa.

For example, the intracellular region of the CAR may include the human 4-1BB intracellular region and/or the human CD28 intracellular region, as well as the human CD3ζ intracellular region; for example, the intracellular region of the CAR may include the human 4-1BB intracellular region, the human CD28 intracellular region, and the human CD3ζ intracellular region.

The antigen recognized by the CAR may be a conventional antigen in the art, such as EpCAM, Mesothelin, CEA, IL13, PDPN, VEGF, EGFR, EGFRvIII, PSMA, FAP, CD171, GD2, Glypican-2, Glypican-3, HER2, HPV antigen, cyclin D1, p53, MMP-7, IL13Ralpha2, MMP-2, MUC-1, G250, L1CAM, ROR1, GPC3, or MSLN, etc. In some cases, the antigen recognized by the CAR in this application may be selected from GPC3, mesothelin, EGFRvIII, IL13Ra2, GPC, or CEA. For example, the CAR gene against GPC3 may contain the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing.

The S1PR1 protein may contain an amino acid sequence as shown in NCBI accession number NP_001391, and the S1PR1 gene may contain a nucleotide sequence as shown in NCBI accession number NM_001400.

In the immune cells, the S1PR1 gene and the CAR or TCR may be located on the same expression vector or on different expression vectors.

When the S1PR1 gene is located on the same vector as the CAR or the TCR, the expression vector may sequentially contain the nucleotide sequence encoding S1PR1, IRES, and the nucleotide sequence encoding CAR; or sequentially contain the nucleotide sequence encoding S1PR1, IRES, and the nucleotide sequence encoding TCR; or sequentially contain the nucleotide sequence encoding S1PR1, 2A, and the nucleotide sequence encoding CAR; or sequentially contain the nucleotide sequence encoding S1PR1, 2A, and the nucleotide sequence encoding TCR; or sequentially contain the nucleotide sequence encoding CAR, IRES, and the nucleotide sequence encoding S1PR1; or sequentially contain the nucleotide sequence encoding TCR, IRES, and the nucleotide sequence encoding S1PR1; or sequentially contain the nucleotide sequence encoding CAR, 2A, and the nucleotide sequence encoding S1PR1; or sequentially contain the nucleotide sequence encoding TCR, 2A, and the nucleotide sequence encoding S1PR1. The nucleotide sequence of IRES may be as shown in SEQ ID NO: 13 in the sequence listing, and the nucleotide sequence of 2A may be as shown in SEQ ID NO: 14 in the sequence listing.

The immune cells described in this application can be T cells, for example, memory T cells.

This application also provides the use of the immune cells described above in the preparation of a drug for treating tumors; the tumors preferably include pancreatic cancer, glioma, liver cancer, or colon cancer.

This application also provides a pharmaceutical composition comprising the immune cells described above.

This application also provides the use of S1PR1 in the preparation of immune cells containing tumor antigen recognition receptors. For example, the tumor antigen recognition receptor can be a chimeric antigen receptor or a T-cell receptor.

Based on common knowledge in this field, the above conditions can be combined arbitrarily to obtain various examples of this application.

All reagents and raw materials used in this application are commercially available.

The positive and progressive effects of this application are as follows:

The immune cells overexpressing S1PR1 and containing tumor antigen recognition receptors (such as CAR/TCR-T cells) have a stronger ability to migrate from lymph nodes, making it easier to reach the treatment site and perform their functions. They can also increase the in vivo and in vitro survival of immune cells containing tumor antigen recognition receptors (such as CAR-T cells); and are beneficial to enhancing the formation of memory and subsequent function of CAR-T cells, thereby enhancing their anti-tumor effect.

S1PR1

On one hand, this application provides a modified immune effector cell, in which the expression and/or activity of S1PR1 protein is upregulated compared to unmodified immune effector cells. In some cases, the unmodified immune effector cells may not express S1PR1 protein. In other cases, the unmodified immune effector cells may express a low level of S1PR1 protein, rendering them unable to perform their S1PR1 function. For example, compared to unmodified immune effector cells, the modified immune effector cells show upregulated expression of S1PR1 protein and upregulated expression of S1PR1 signaling pathway-related proteins. The protein expression level of the cells can be detected using protein quantification methods, such as BCA quantification, Bradford quantification, Lowry assay, spectrophotometry, etc. For example, compared to unmodified immune effector cells, the modified immune effector cells show increased S1PR1 protein activity, which can be reflected in increased affinity of the modified immune effector cells for S1PR, upregulated S1PR1 signaling pathway activity, and enhanced immune effector function.

For example, an agent can be administered to the immune effector cells that upregulates the expression and/or activity of the S1PR1 protein. The agent can be a macromolecule (e.g., DNA, RNA, polypeptide, or protein), a virus, a plasmid, an organic or inorganic small molecule, or a combination thereof.

In some cases, the modified immune effector cells may additionally contain the S1PR1 protein. The immune effector cells may not contain the S1PR1 protein before modification; after modification, S1PR1 protein expression can be detected in the modified immune effector cells. In some cases, S1PR1 can be directly overexpressed at the protein level in the modified immune effector cells. For example, the S1PR1 protein can be introduced into immune effector cells that do not express S1PR1, causing them to express the S1PR1 protein. For example, the S1PR1 protein can be introduced into immune effector cells that express low levels of S1PR1, increasing the level of S1PR1 protein expression. For example, the S1PR1 gene can be introduced into immune effector cells that do not express S1PR1, causing them to express the S1PR1 protein. For example, the S1PR1 gene can be introduced into immune effector cells that express low levels of S1PR1, increasing the level of S1PR1 protein expression.

In this application, compared with unmodified immune effector cells, the expression and/or activity of nucleic acid molecules encoding the S1PR1 protein are upregulated in the modified immune effector cells. In some cases, the immune effector cells may not contain nucleic acid molecules encoding the S1PR1 protein before modification; in other cases, the immune effector proteins may contain nucleic acid molecules encoding the S1PR1 protein before modification but are not expressed; and in still other cases, the immune effector proteins may contain nucleic acid molecules encoding the S1PR1 protein before modification but are expressed at a low level.

In some cases, the modified immune effector cells additionally contain the nucleic acid molecule encoding the S1PR1 protein. For example, the modified immune cells can additionally contain the nucleic acid molecule encoding the S1PR1 protein by introducing the nucleic acid molecule encoding S1PR1 into the immune effector cells (e.g., via vectors, such as those described below).

For example, an agent can be administered to the immune effector cells that enhances the translation or transcription of nucleic acid molecules encoding the S1PR1 protein. For example, S1PR1 in the modified immune effector cells can be overexpressed on DNA and/or RNA. For example, the agent can be a macromolecule (e.g., DNA, RNA, polypeptide, or protein), a virus, a plasmid, an organic or inorganic small molecule, or a combination thereof.

For example, the S1PR1 gene can be introduced into immune effector cells, causing the immune cells to additionally contain the nucleic acid molecule encoding the S1PR1 protein. For example, the S1PR1 gene can be a nucleic acid molecule containing the full-length S1PR1 protein or a nucleic acid molecule containing the S1PR1 coding region. For example, the S1PR1 gene can be constructed into a plasmid or viral vector and then introduced into immune cells.

The S1PR1 protein can be a full-length S1PR1 protein, or a truncated, spliced, or functional fragment thereof. For example, the S1PR1 protein is a full-length human S1PR1 protein, and the nucleic acid molecule encoding the full-length human S1PR1 protein may contain the nucleotide sequence shown in NCBI accession number NM_001400.

CAR

In this application, the immune cells described herein may include chimeric antigen receptors (CARs) and/or T-cell receptors (TCRs).

In this application, the CAR may comprise a chimeric antigen receptor that targets a tumor-specific antigen, wherein the tumor-specific antigen may be selected from the group consisting of: EpCAM, Mesothelin (MSLN), CEA, IL13, PDPN, VEGF, EGFR, EGFRvIII, PSMA, FAP, CD171, GD2, Glypican-2, Glypican-3, HER2, HPV antigen, cyclin D1, p53, MMP-7, IL13Ralpha2, MMP-2, MUC-1, G250, L1CAM, ROR1, and GPC3.

In some cases, the CAR may include an antigen-binding domain. This antigen-binding domain can specifically bind to a tumor-specific antigen selected from the group consisting of: EpCAM, Mesothelin (MSLN), CEA, IL13, PDPN, VEGF, EGFR, EGFRvIII, PSMA, FAP, CD171, GD2, Glypican-2, Glypican-3, HER2, HPV antigen, cyclin D1, p53, MMP-7, IL13Ralpha2, MMP-2, MUC-1, G250, L1CAM, ROR1, and GPC3. In some cases, the antigen-binding domain may include a single-chain antibody scFv. For example, the single-chain antibody may include a single-chain antibody GC33 targeting GPC3, and the single-chain antibody may contain the amino acid sequence shown in SEQ ID NO: 1. For example, the single-chain antibody may include a single-chain antibody P4G2 targeting MSLN, and for example, the single-chain antibody may contain the amino acid sequence shown in SEQ ID NO: 2.

In this application, the CAR may include a transmembrane domain. In some cases, the transmembrane domain may include a transmembrane domain derived from a protein selected from the following proteins: CD3ζ, CD28, 4-1BB, OX40, SLAMF4, CD127, NKG2D, ICOS, and FcγRIIIa.

In this application, the CAR may include a co-stimulatory domain. In some cases, the co-stimulatory domain may include a co-stimulatory domain selected from the following proteins or a combination thereof: CD137, CD28, OX40, ICOS, DAP10, 2B4, CD27, CD30, CD40, CD40L, TIM1, CD226, DR3, SLAM, NKG2D, CD244, FceRIγ, BTLA, GITR, HVEM, CD2, NKG2C, LIGHT, and DAP12.

In this application, the CAR may include a hinge region that connects the antigen-binding domain and the transmembrane domain. In some cases, the hinge region may include a hinge region derived from proteins selected from: CD8, CD28, IgG, 4-1BB, CD4, CD27, CD7, PD-1, and CH2CH3.

In this application, the CAR may include an antigen-binding domain, a hinge region, a transmembrane domain, a co-stimulatory domain, and/or a signal transduction domain. For example, the CAR may contain the amino acid sequence shown in any one of SEQ ID NO: 5-6.

The modified immune cells described in this application may include the CAR. For example, the CAR may contain the amino acid sequence shown in any one of SEQ ID NO: 5-6.

The modified immune cells described in this application may include nucleic acid molecules encoding the CAR. For example, the nucleic acid molecule encoding the CAR may contain the nucleotide sequence shown in any one of SEQ ID NO: 3-4.

Immune effector cells and cell populations

In this application, the modified immune effector cells may comprise a vector, and the vector may include the nucleic acid molecule encoding the S1PR1 protein. Alternatively, the modified immune effector cells may comprise a vector, and the vector may include a nucleic acid molecule encoding the chimeric antigen receptor. In some cases, the nucleic acid molecule encoding the chimeric antigen receptor and the nucleic acid molecule encoding the S1PR1 protein may reside in the same vector. In other cases, the nucleic acid molecule encoding the chimeric antigen receptor and the nucleic acid molecule encoding the S1PR1 protein may reside in different vectors.

When the nucleic acid molecule encoding the chimeric antigen receptor and the nucleic acid molecule encoding the S1PR1 protein are located in the same vector, in some cases, the nucleic acid molecule encoding the chimeric antigen receptor may be located at the 5' end of the nucleic acid molecule encoding the S1PR1 protein; in other cases, the nucleic acid molecule encoding the chimeric antigen receptor may be located at the 3' end of the nucleic acid molecule encoding the S1PR1 protein. The modified immune effector cells...

The nucleic acid molecule encoding the chimeric antigen receptor and the nucleic acid molecule encoding the S1PR1 protein can be linked by any feasible linker (e.g., a cleavage peptide, such as IRES or 2A). For example, the nucleotide sequence of IRES can be as shown in SEQ ID NO: 13 in the sequence listing, and the nucleotide sequence of 2A can be as shown in SEQ ID NO: 14 in the sequence listing.

For example, the vector may sequentially comprise, from its 5' end to its 3' end, a nucleic acid molecule encoding the S1PR1 protein, a linker, and a nucleic acid molecule encoding the chimeric antigen receptor. For example, the vector may sequentially comprise, from its 5' end to its 3' end, a nucleic acid molecule encoding the chimeric antigen receptor, a linker, and a nucleic acid molecule encoding the S1PR1 protein. For example, the vector may comprise the nucleotide sequence shown in any one of SEQ ID NO: 7-9.

The modified immune effector cells described in this application include cases with different types of linkers and different linking sequences, all of which possess the beneficial effects described in this application. In the carrier of the modified immune effector cells, when the nucleic acid molecule encoding the S1PR1 protein is linked to the N-terminus and C-terminus of the antigen-chimeric receptor, or when various types of linkers are applied, the modified immune effector cells exhibit anti-tumor effects both in vivo and in vitro. For example, in in vitro killing experiments, the modified immune effector cells demonstrate enhanced ability to kill target cells, secrete cytokines, and migrate; for example, in in vitro experiments, the modified immune effector cells enhance their ability to inhibit tumor growth and migrate out of lymph nodes.

The immune effector cells may include T cells, B cells, natural killer (NK) cells, macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, and/or peripheral blood mononuclear cells. In some cases, the immune cells may include T lymphocytes. The T lymphocytes may include thymocytes, native T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. The T cells may be helper T cells (Th), such as helper T cell 1 (Th1) or helper T cell 2 (Th2). The T lymphocytes may be CD4+ helper T cells (HTL; CD4+ T cells), cytotoxic T cells (CTL; CD8+ T cells), tumor-infiltrating cytotoxic T cells (TIL; CD8+ T cells), CD4+/CD8+ T cells, CD4-/CD8- T cells, or any other T lymphocyte subtype. The T lymphocytes may be memory T cells, such as central memory T cells (TCM cells), effector memory T cells ( _TEM cells), tissue-resident memory T cells ( _{TRM cells} ), virtual memory T cells (TVM cells), stem memory T cells ( _TSCM cells), or other CD4 ⁺ or CD8 ⁺ memory T cells that typically express CD45RO but may also lack CD45RA.

On the other hand, this application provides a cell population that may contain the immune effector cells described in this application. In certain cases, when the immune effector cells in the cell population reach 20% or more (e.g., 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more or more), the cell population may have the ability to migrate to the tumor site (e.g., inside the tumor tissue). For example, in in vivo experiments, when mice are injected with the cell population described in this application, an increase in peripheral blood T cell content is detected in the group containing the cell population described in this application compared to a CAR T cell population that does not express S1PR1. For example, in in vitro experiments, CAR T cells expressing S1PR1 are able to migrate to the lower chamber. For example, in in vivo experiments, mice were injected with the cell population and the T cell content in peripheral blood was measured. T cells in the CAR T cell population expressing S1PR1 were more distributed in the peripheral blood and inside the tumor tissue, while T cells in the CAR T cell population not expressing S1PR1 were more distributed in the peripheral blood, lymph nodes and inside the tumor tissue.

Pharmaceutical compositions, methods, uses and carriers

On the other hand, this application provides a pharmaceutical composition. The pharmaceutical composition may comprise the immune effector cells and/or cell populations of this application, and optionally a pharmaceutically acceptable carrier. In this application, the term "pharmaceutically acceptable carrier" generally refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents that are compatible with the immune effector cells and/or cell populations of this application. Unless incompatible with the immune effector cells and/or cell populations described in this application, any conventional media or reagents may be considered for use in the pharmaceutical composition of this application.

In some aspects, the methods of this application may include administering one or more of the CARs and/or a vector containing the CARs to host cells (e.g., immune effector cells). In some aspects, the methods of this application may include administering one or more of the TCRs and/or a vector containing the TCRs to host cells (e.g., immune effector cells). In some cases, the methods of this application may include administering S1PR1 and/or a vector containing the S1PR1 to host cells (e.g., immune effector cells). The methods of this application may also include delivering one or more of the CARs and/or TCRs, and a vector containing S1PR1, to host cells (e.g., immune cells). In some aspects, this application further provides cells generated by the methods and organisms comprising or generated from the cells (e.g., animals, plants, or fungi). In some cases, a CAR linked to S1PR1 can be delivered to the cells. Nucleic acid sequences can be introduced into mammalian cells or target tissues using conventional viral and non-viral gene transfer methods.

On the other hand, this application provides a carrier that can contain the isolated nucleic acid molecules.

In this application, the vector may be selected from one or more of plasmids, retroviral vectors, and lentiviral vectors. For example, the vector of this application may contain the following nucleotide sequences from its 5' end: a gene encoding S1PR1, a gene encoding IRES, and a gene encoding CAR. As another example, the vector of this application may contain the following nucleotide sequences from its 5' end: a gene encoding S1PR1, a gene encoding 2A, and a gene encoding CAR. As another example, the vector of this application may contain the following nucleotide sequences from its 5' end: a gene encoding CAR, a gene encoding IRES, and a gene encoding S1PR1. As another example, the vector of this application may contain the following nucleotide sequences from its 5' end: a gene encoding CAR, a gene encoding 2A, and a gene encoding S1PR1. Furthermore, the vector may also contain other genes, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. Additionally, the vector may contain expression control elements that allow the coding region to be correctly expressed in an appropriate host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements regulating gene transcription or mRNA translation. In some embodiments, the expression control sequence is a tunable element. The specific structure of the expression control sequence may vary depending on the function of the species or cell type, but typically includes 5' non-transcriptional sequences and 5' and 3' non-translational sequences, respectively, involved in transcription and translation initiation, such as TATA boxes, capping sequences, CAAT sequences, etc. For example, the 5' non-transcriptional expression control sequence may include a promoter region, which may contain a promoter sequence for transcriptionally controlling the functional linking of nucleic acids. One or more nucleic acid molecules described in this application can be operatively linked to the expression control element.

Non-viral methods for nucleic acid delivery include lipid transfection, nuclear transfection, microinjection, gene guns, viral particles, liposomes, immunoliposomes, polycationic or lipid-nucleic acid conjugates, naked DNA, artificial virions, and reagents that enhance DNA uptake. Nucleic acids can be delivered using RNA or DNA virus-based systems, for example, by utilizing the ability of viruses to target specific cells in vivo to efficiently deliver viral payloads into the cell nucleus. Viral vectors can be administered directly to patients (in vivo) or indirectly, for example, by treating cells in vitro with viruses and then administering the treated cells to patients (ex vivo). Conventional virus-based systems can include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus vectors for gene transfer. In some cases, retroviral, lentiviral, and adeno-associated virus methods can be used to integrate genes into the host genome, enabling long-term expression of the inserted genes. Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and typically producing high viral titers. Lentiviral vectors may contain long terminal repeat (5'LTR) sequences and truncated 3'LTRs, RREs, rev response elements (cPPTs), central termination sequences (CTSs), and/or post-translational regulatory elements (WPREs). These molecules can be constructed onto lentiviral vectors via BamHI and XbaI digestion. The selection of a retroviral gene transfer system will depend on the target tissue.

The method of this application may include introducing the vector described in this application into immune effector cells. For example, the vector described in this application may be introduced into the immune effector cells, such as T cells, lymphocytes, granulocytes, and/or peripheral blood mononuclear cells. In some embodiments, each type or each cell may contain one or more vectors described in this application. In some embodiments, each type or each cell may contain multiple (e.g., two or more) or more types (e.g., two or more) vectors described in this application. For example, the vector described in this application may be introduced into the cells. For example, immune effector cells can be transfected using a retroviral vector, allowing the viral genome carrying the nucleic acid encoding the fusion protein to integrate into the host genome, ensuring long-term and stable expression of the target gene. In this application, the vector carrying the nucleic acid encoding the fusion protein described in this application may be introduced into the cells using methods known in the art, such as electroporation (Neon electroporator), liposome transfection, etc.

On the other hand, this application provides the use of the described immune effector cells, the described cell population, and/or the described pharmaceutical composition in the preparation of a medicament for treating tumors. In some cases, the tumor may include a solid tumor. For example, the tumor may include pancreatic cancer, glioma, liver cancer, and/or colon cancer.

The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the fusion protein, preparation method and use of this application, and are not intended to limit the scope of the invention.

Example

Example 1: Construction and Packaging of Lentiviral Vectors

1.1 Construction of Lentiviral Vector

The S1PR1 gene (accession number NM_001400 in NCBI GenBank), IRES gene (nucleotide sequence of IRES as shown in SEQ ID NO: 13 in the sequence listing), and GFP gene (nucleotide sequence of GFP as shown in SEQ ID NO: 15 in the sequence listing) were synthesized in sequence using a whole-gene synthesis method. A BamHI restriction site sequence was added downstream of S1PR1, and an XbaI restriction site sequence was added upstream of the GFP gene. The resulting plasmid was cloned into the vector pLVX-EF1α-IRES-Puro EcoRI/MluI between the two restriction sites to construct the S1PR1-IRES-GFP plasmid (nucleotide sequence as shown in SEQ ID NO: 10).

The anti-GPC3 CAR gene GC33CAR was synthesized from the whole genome. It consists of the anti-GPC3 single-chain antibody GC33 (amino acid sequence shown in SEQ ID NO: 1), the CD8a hinge region, the CD8 transmembrane region, the CD137(4-1BB) intracellular region, and the CD3z intracellular region, sequentially linked together. The nucleotide sequence of GC33CAR is shown in SEQ ID NO: 3. The S1PR1 and GFP in S1PR1-IRES-GFP were replaced with GC33CAR, respectively, to construct plasmids GC33CAR-IRES-GFP (nucleotide sequence shown in SEQ ID NO: 11) and S1PR1-IRES-GC33CAR (nucleotide sequence shown in SEQ ID NO: 12).

Replace IRES in S1PR1-IRES-GC33CAR with 2A (nucleotide sequence as shown in SEQ ID NO: 14), insert into the vector, and obtain the plasmid S1PR1-2A-GC33CAR (nucleotide sequence as shown in SEQ ID NO: 8).

Replace the GFP in GC33CAR-IRES-GFP with S1PR1 and insert it into the vector to obtain the plasmid GC33CAR-IRES-S1PR1 (nucleotide sequence as shown in SEQ ID NO: 7).

The GC33CAR gene was inserted into the vector to obtain the GC33CAR plasmid.

The anti-MSLN CAR gene P4G2CAR was synthesized from the whole genome. It consists of the anti-MSLN single-chain antibody P4G2 (amino acid sequence shown in SEQ ID NO: 2), the CD8a hinge region, the CD8 transmembrane region, the CD137(4-1BB) intracellular region, and the CD3z intracellular region, sequentially linked together. The nucleotide sequence of P4G2CAR is shown in SEQ ID NO: 4. Replacing GC3CAR in S1PR1-2A-GC33CAR with P4G2CAR yielded the plasmid S1PR1-2A-P4G2CAR (nucleotide sequence shown in SEQ ID NO: 9).

The P4G2 gene was inserted into the vector to obtain the P4G2 plasmid.

1.2 Lentiviral Packaging

Lentiviral packaging is performed using a three-plasmid system.

Example 2 CAR Expression Validation

CAR-T cells were constructed by infecting activated PBMC cells with lentiviruses of GC33CAR-IRES-GFP, S1PR1-IRES-GC33CAR, and S1PR1-IRES-GFP (CD3 and CD28 activation). The CAR positivity rate of virus-infected CAR-T cells was detected by flow cytometry. For immune T cells transfected with S1PR1-IRES-GFP, GFP was used as a substitute for CAR positivity rate. The results are shown in Table 1 and Figure 1. The results showed that the CAR positivity rate of CAR-T cells containing GC33CAR-IRES-GFP was 56.68% (Figure 1A), the positivity rate of immune T cells transfected with S1PR1-IRES-GFP (control) was 50.93% (Figure 1B), and the CAR positivity rate of CAR-T cells containing S1PR1-IRES-GC33CAR was the lowest, at 30.24% (Figure 1C).

Table 1. CAR-T (or T) cell positivity rate

[GFP+]%Gated [CAR+]%Gated GC33CAR-IRES-GFP 59.03 56.68 S1PR1-IRES-GFP 50.93 0.2 S1PR1-IRES-GC33CAR 0.69 30.24

Example 3: In vitro S1PR1 migration function verification

Transwell assays were used to verify the migration ability of CAR-T cells treated with gradient concentrations of S1P (0, 1, 10, 100, and 1000 nM). 200 μl of immune T cells (2.5 × ^10⁶ cells) transfected with S1PR1-IRES-GFP and exhibiting a GFP positivity rate of 70% were added to the upper chamber of a 3415 Transwell chamber, with culture medium at 1640. 500 μl of a concentration gradient of S1P was added to the lower chamber, with culture medium at 1.5% FBS and 1640. After 3 h of incubation, the GFP positivity rate of cells migrating to the lower chamber was detected by flow cytometry. The results showed that treatment with 100 nM and 1000 nM S1P significantly increased the proportion of positive CAR-T cells migrating to the lower chamber, indicating that S1PR1-expressing immune T cells can enhance the migration ability of cells affected by S1P (Figures 2 and 3).

One × ^10⁶ immune T cells transfected with GC33CAR or GC33CAR-IRES-S1PR1 were placed in the upper chamber of a 3415 Transwell chamber at 1640 °C. 500 μl of S1P containing 100 nM was added to the lower chamber, and the culture medium was maintained at 1640 °C. The cells were incubated for 3 hours. A control without S1P was used. The cell count in the lower chamber was calculated using a cell counter, and the migration capacity % was calculated as: (cell count in lower chamber / (1 × ^10⁶ ) × 100%). The results showed that CAR T cells expressing S1PR1 enhanced cell migration, and the migration efficiency was high under S1P-positive conditions (Figure 4).

Example 4: Verification of Target Cell Killing Ability

4.1 Killing ability of T cells infected with S1PR1-IRES-GFP, GC33CAR-IRES-GFP and S1PR1-IRES-GC33CAR

The killing ability of T (or CAR-T) cells infected with S1PR1-IRES-GFP, GC33CAR-IRES-GFP, and S1PR1-IRES-GC33CAR lentiviruses against Huh7 target cells (purchased from the Chinese Academy of Sciences Cell Bank) expressing luciferase protein (Luc) was verified by T cell killing assays. The effector cell to target cell ratios were 20:1, 10:1, 5:1, 2.5:1, 1.25:1, and 0.625:1. A blank control group was established using DMEM (2% FBS) culture medium; a CAR-T effector cell group; an effector cell and Huh7 target cell group (1 × ^10⁴ cells per well, effector cells added according to the effector-to-target ratio); a target cell group (Huh7) and a maximum lysis rate group (Huh7-max) (1 × ^10⁴ cells per well). Cells were cultured at 37°C for 4 hours. After a 3.5-hour treatment, 20 μl of Lysis Buffer was added to each well of the Huh7-max group. Continue culturing for another half hour, then centrifuge at 300g for 5 minutes. Following the original plate order, transfer 50 μl of supernatant from each well to a COSTAR 3300 plate. Add 50 μl of substrate, incubate at room temperature for 30 min, then add 50 μl of STOP Buffer (cytoTox 96 Non-radioactive Cytotoxicity kit, Promega). Detect OD490 readings and calculate the CAR-T cell killing efficiency against target cells [Calculation formula: Specific killing % = (Experimental group - Effector cell self-release - Target cell self-release) / (Target cell maximum release - Target cell self-release) × 100%]. Results showed that CAR-T cells containing (or transfected with) S1PR1-IRES-GC33CAR exhibited the strongest killing ability against target cells, followed by CAR-T cells containing GC33CAR-IRES-GFP, while immune T cells transfected with S1PR1-IRES-GFP showed no significant killing ability against target cells (Figure 5).

4.2 The killing ability of GC33CAR-IRES-S1PR1 and S1PR1-2A-GC33CAR and GC33CAR-infected T cells

Following the method described in section 4.1, the killing ability of GC33CAR-IRES-S1PR1, S1PR1-2A-GC33CAR, and GC33CAR lentivir-infected T (or CAR-T) cells against target cells Huh7-Luc was verified. GC33CAR-infected T cells and wild-type T cells were used as controls. The results are shown in Figure 6. T cells with CARs expressing S1PR1 at the 5' end and CARs expressing S1PR1 at the 3' end both exhibited killing ability against target cells. T cells using different linker peptides between S1PR1 and CAR all exhibited killing ability against target cells.

4.3 Killing ability of S1PR1-2A-P4G2CAR-infected T cells

Following the method described in section 4.1, the killing ability of T cells infected with S1PR1-2A-P4G2CAR lentivirus against target cells Huh7-Luc was verified. P4G2CAR-infected T cells were used as a control. The results, shown in Figure 7, indicate that CAR T cells expressing S1PR1 possessed the ability to kill target cells, and this ability was higher than that of control CAR T cells.

Example 5: Validation of in vitro cytokine secretion

CAR-T cells were constructed by infecting activated PBMC cells with P4G2 and S1PR1-2A-P4G2CAR lentiviruses (CD3 and CD28 activation) to verify the ability of T cells to secrete cytokines in vitro.

In summary, we first constructed CF-APC-1 cells expressing MSLN (CF-APC-1-MSLN), obtained the MSLN nucleotide sequence from the NCBI database accession number NM_005823, synthesized MSLN-IRES-puro, packaged lentivirus overexpressing MSLN-IRES-puro, and infected CF-APC-1 cells (Chinese Academy of Sciences Cell Bank, TCHU112) with the obtained lentivirus. After 48 hours, stable transfected cells were obtained by screening with 5 μg/ml puromycin.

CF-APC-1 cells (Chinese Academy of Sciences Cell Bank, THu112), CF-APC-1-MSLN cells, human pancreatic cancer cells (PANC, sourced from Chinese Academy of Sciences Cell Bank), and MSLN-expressing PANC cells (Chinese Academy of Sciences Cell Bank, THu 98) were used as target cells. After washing, the cells were suspended at 1× ^{10⁵ cells} /mL in DMEM (containing 2% FBS) medium, and each target cell was added to a 96-well plate at a concentration of 1×10⁵ cells/well. CAR-T cells were washed and suspended at 1× ^10⁵ cells/well in DMEM (containing 2% FBS) medium. Wild-type T cells that do not express CAR were used as a control. The 96-well plates were incubated at 37°C for 24 hours. The supernatant was then harvested, and flow cytometry was used to measure IL-2 and IFN-γ using a human TH1/TH2 cytokine assay kit (BD CBA, Cat#551809).

The results showed that, when stimulated by target cells expressing MSLN, T cells expressing S1PR1-2A-P4G2CAR had a greater ability to secrete cytokines IL-2 (Fig. 8A) and IFN-γ (Fig. 8B) compared to T cells expressing only P4G2CAR.

Example 6: In vivo tumor inhibition experiment verification

CAR-T cells were constructed by infecting activated PBMC cells with GC33CAR and GC33CAR-IRES-S1PR1 lentiviruses (CD3 and CD28 activation).

In summary, self-constructed target tumor cells expressing luciferase were subcutaneously injected into NSG mice to induce tumor formation. Two × ^10⁶ of these target cells were resuspended in 100 μl of phosphate-buffered saline solution and injected subcutaneously. Alternatively, self-constructed target tumor cells expressing luciferase were intravenously injected into NSG mice to induce tumor formation. Two × ^10⁶ of these target cells were resuspended in 100 μl of phosphate-buffered saline solution and injected subcutaneously into the mice. Tumor volume was measured every 3-4 days using calipers. Tumor volume = length * width * width / 2

Fourteen days after tumor formation, CAR T cells washed with PBS were resuspended in serum-free culture medium and the cell density was adjusted to 1 × ^10⁷ /ml. The cells were then administered intravenously (iv) to mice at a rate of 200 μl/mouse, or intratumorally (it) to mice at a rate of 50 ^μl /mouse. Wild-type T cells were used as a control. Tumor volume was measured every 3 days after injection. Tumor volume was calculated as: tumor volume ( ^mm³ ) = 0.5 × tumor long axis × tumor short ^axis² . Peripheral blood was collected from mice on day 28, and the CD3 ⁺ cell count was detected by flow cytometry.

The results showed that both GC33CAR T cells and GC33CAR-IRES-S1PR1 T cells effectively inhibited tumor growth (Figure 8). Figures 9A-9B show the reduction in tumor volume in mice after injection of CAR T cells via different injection routes.

Example 7: In vivo S1PR1 migration function verification

The mouse model was established according to the method in Example 6. The mice were divided into groups, and GC33CAR T cells and GC33CAR-IRES-S1PR1 T cells were injected 14 days after tumor formation. Peripheral blood was collected from the mice on day 28, and the CD3 ⁺ cell count was detected by flow cytometry.

The results showed that mice treated with GC33CAR-IRES-S1PR1 T cells had a higher number of T cells in their peripheral blood than the group treated with GC33CAR T cells (Figure 10). CAR T cells expressing S1PR1 can enhance cell migration ability.

Example 8: Validation of the synergistic effect of S1PR1 and CAR

A mouse tumor model was established according to the methods in Examples 7 and 8. CAR T cells were injected 14 days after tumor formation, and the tumor volume was measured every 3 days. Peripheral blood was collected to detect the CD3 ⁺ T cell content.

Figure 11 shows the changes in tumor volume from the onset of tumor formation in mice. After injection of the CAR T cells described in this application, the tumor volume decreased. In the GC33CAR-IRES-S1PR1 T cell group, the amount of CD3 ⁺ T cells in peripheral blood was negatively correlated with tumor volume (Figure 12B), possibly indicating that GC33CAR-IRES-S1PR1 T cells were distributed more predominantly in the peripheral blood and tumor tissue, while no correlation was found in GC33CAR T cells (Figure 12A), possibly because GC33CAR T cells tend to be distributed in the peripheral blood, lymph nodes, and tumor tissue. These data suggest that S1PR1 and CAR have a synergistic anti-tumor effect in vivo.

sequence list

<110> Shanghai Pharmaceuticals Holding Co., Ltd.

<120> An immune cell containing a tumor antigen recognition receptor and its application

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<400> 3

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggacgtgg tgatgaccca gagcccttta tctttacccg ttacacccgg tgagcccgct 120

agcatctctt gtagaagcag ccagtcttta gtgcacagca acggcaacac atatttacac 180

tggtatttac agaagcccgg tcagagcccc cagctgctga tctacaaggt gagcaatcgt 240

ttctccggcg tgcccgatag attcagcggc agcggctccg gaaccgactt cactttaaag 300

atcagcagag tggaggccga ggacgtgggt gtgtactact gctcccagaa cacccacgtg 360

ccccctacat tcggtcaagg taccaaactg gagatcaaag gtggcggtgg ctcgggcggt 420

ggtgggtcgg gtggcggcgg atctcaagtt cagctggtgc agagcggcgc cgaggtcaaa 480

aaacccggag ccagcgtgaa ggtgtcttgt aaagccagcg gctacacctt taccgactat 540

gagatgcact gggtgagaca agctcccggt caaggtctgg aatggatggg cgctttagac 600

cccaagactg gtgacacagc ctactcccag aagttcaagg gtcgtgtgac tttaacagcc 660

gacgagtcca ccagcacagc ctacatggaa ctgagctctt taaggagcga ggacaccgcc 720

gtgtattact gcactcgttt ctacagctac acctactggg gccaaggtac tttagtgaca 780

gtgagcagct tcgtgccggt cttcctgcca gcgaagccca ccacgacgcc agcgccgcga 840

ccaccaacac cggcgcccac catcgcgtcg cagcccctgt ccctgcgccc agaggcgtgc 900

cggccagcgg cggggggcgc agtgcacacg aggggctgg acttcgcctg tgatatctac 960

atctgggcgc ccttggccgg gacttgtggg gtccttctcc tgtcactggt tatcaccctt 1020

tactgcaacc acaggaaccg tttctctgtt gttaaacggg gcagaaagaa gctcctgtat 1080

atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1140

tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 1200

gcagacgccc ccgcgtacca gcagggccag aaccagctct ataacgagct caatctagga 1260

cgaagagagg agtacgatgt tttggacaaa agaagaggcc gggaccctga gatgggggga 1320

aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1380

gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1440

ggcctttacc agggtctcag tacagccacc aaggacacct aggacgcct tcacatgcag 1500

gccctgcccc ctcgctaa 1518

<210> 4

<211> 882

<212> DNA

<213> Artificial Sequence

<220>

<223> P4G2CAR

<400> 4

atggctctgc ccgtgaccgc tctgctgctg cctctcgctc tgctgctgca tgccgccaga 60

cccggctccc aagtgcaact ccagcaatcc ggacccggac tggtgacacc ctcccaaaca 120

ctgtccctca catgcgccat cagcggagat tccgtgtcct ccaacagcgc tacatggaat 180

tggattagac agagcccttc cagaggactg gagtggctcg gaagaacata ctacagatcc 240

aagtggtata acgactacgc cgtgtccgtg aagtctagaa tgagcatcaa ccccgacacc 300

agcaagaacc agttttctct gcagctgaac agcgtgacac ccgaggacac cgctgtgtac 360

tactgcgcca gaggcatgat gacctactac tacggcatgg acgtctgggg acaaggcacc 420

accgtgacag tgagcagcgg cattctgggc tccggaggag gcggaagcgg aggcggaggc 480

agcggaggcg gcggaagcca acccgtgctc acacaaagct cctctctgag cgctagcccc 540

ggcgcctccg cttctctgac atgtacactg aggagcggca tcaacgtcgg accctataga 600

atctactggt accagcagaa gcccggcagc cccccccaat atctgctgaa ctacaagtcc 660

gactccgaca agcagcaagg cagcggcgtg ccttctagat tcagcggcag caaggacgct 720

agcgccaacg ccggagtgct gctgatctcc ggactgagaa gcgaggacga ggccgactac 780

tattgcatga tctggcactc cagcgccgct gtctttggag gaggcacaca gctgacagtg 840

ctgagcgcca gcttcgtgcc cgtgttcctc cccgccaaac cc 882

<210> 5

<211> 505

<212> PRT

<213> Artificial Sequence

<220>

<223> GC33CAR

<400> 5

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu

20 25 30

Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln

35 40 45

Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln

50 55 60

Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg

65 70 75 80

Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

85 90 95

Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr

100 105 110

Tyr Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr

115 120 125

Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

145 150 155 160

Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr

165 170 175

Phe Thr Asp Tyr Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly

180 185 190

Leu Glu Trp Met Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr

195 200 205

Ser Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr

210 215 220

Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala

225 230 235 240

Val Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly

245 250 255

Thr Leu Val Thr Val Ser Ser Phe Val Pro Val Phe Leu Pro Ala Lys

260 265 270

Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile

275 280 285

Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala

290 295 300

Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr

305 310 315 320

Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu

325 330 335

Val Ile Thr Leu Tyr Cys Asn His Arg Asn Arg Phe Ser Val Val Lys

340 345 350

Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg

355 360 365

Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro

370 375 380

Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser

385 390 395 400

Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu

405 410 415

Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg

420 425 430

Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln

435 440 445

Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr

450 455 460

Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp

465 470 475 480

Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala

485 490 495

Leu His Met Gln Ala Leu Pro Pro Arg

500 505

<210> 6

<211> 294

<212> PRT

<213> Artificial Sequence

<220>

<223> P4G2CAR

<400> 6

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Pro

20 25 30

Gly Leu Val Thr Pro Ser Gln Thr Leu Ser Leu Thr Cys Ala Ile Ser

35 40 45

Gly Asp Ser Val Ser Ser Asn Ser Ala Thr Trp Asn Trp Ile Arg Gln

50 55 60

Ser Pro Ser Arg Gly Leu Glu Trp Leu Gly Arg Thr Tyr Tyr Arg Ser

65 70 75 80

Lys Trp Tyr Asn Asp Tyr Ala Val Ser Val Lys Ser Arg Met Ser Ile

85 90 95

Asn Pro Asp Thr Ser Lys Asn Gln Phe Ser Leu Gln Leu Asn Ser Val

100 105 110

Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Met Met Thr

115 120 125

Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val

130 135 140

Ser Ser Gly Ile Leu Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Gly Gly Gly Gly Ser Gln Pro Val Leu Thr Gln Ser Ser Ser Leu

165 170 175

Ser Ala Ser Pro Gly Ala Ser Ala Ser Leu Thr Cys Thr Leu Arg Ser

180 185 190

Gly Ile Asn Val Gly Pro Tyr Arg Ile Tyr Trp Tyr Gln Gln Lys Pro

195 200 205

Gly Ser Pro Pro Gln Tyr Leu Leu Asn Tyr Lys Ser Asp Ser Asp Lys

210 215 220

Gln Gln Gly Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Lys Asp Ala

225 230 235 240

Ser Ala Asn Ala Gly Val Leu Leu Ile Ser Gly Leu Arg Ser Glu Asp

245 250 255

Glu Ala Asp Tyr Tyr Cys Met Ile Trp His Ser Ser Ala Ala Val Phe

260 265 270

Gly Gly Gly Thr Gln Leu Thr Val Leu Ser Ala Ser Phe Val Pro Val

275 280 285

Phe Leu Pro Ala Lys Pro

290

<210> 7

<211> 3282

<212> DNA

<213> Artificial Sequence

<220>

<223> GC33CAR-IRES-S1PR1

<400> 7

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggacgtgg tgatgaccca gagcccttta tctttacccg ttacacccgg tgagcccgct 120

agcatctctt gtagaagcag ccagtcttta gtgcacagca acggcaacac atatttacac 180

tggtatttac agaagcccgg tcagagcccc cagctgctga tctacaaggt gagcaatcgt 240

ttctccggcg tgcccgatag attcagcggc agcggctccg gaaccgactt cactttaaag 300

atcagcagag tggaggccga ggacgtgggt gtgtactact gctcccagaa cacccacgtg 360

ccccctacat tcggtcaagg taccaaactg gagatcaaag gtggcggtgg ctcgggcggt 420

ggtgggtcgg gtggcggcgg atctcaagtt cagctggtgc agagcggcgc cgaggtcaaa 480

aaacccggag ccagcgtgaa ggtgtcttgt aaagccagcg gctacacctt taccgactat 540

gagatgcact gggtgagaca agctcccggt caaggtctgg aatggatggg cgctttagac 600

cccaagactg gtgacacagc ctactcccag aagttcaagg gtcgtgtgac tttaacagcc 660

gacgagtcca ccagcacagc ctacatggaa ctgagctctt taaggagcga ggacaccgcc 720

gtgtattact gcactcgttt ctacagctac acctactggg gccaaggtac tttagtgaca 780

gtgagcagct tcgtgccggt cttcctgcca gcgaagccca ccacgacgcc agcgccgcga 840

ccaccaacac cggcgcccac catcgcgtcg cagcccctgt ccctgcgccc agaggcgtgc 900

cggccagcgg cggggggcgc agtgcacacg aggggctgg acttcgcctg tgatatctac 960

atctgggcgc ccttggccgg gacttgtggg gtccttctcc tgtcactggt tatcaccctt 1020

tactgcaacc acaggaaccg tttctctgtt gttaaacggg gcagaaagaa gctcctgtat 1080

atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1140

tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 1200

gcagacgccc ccgcgtacca gcagggccag aaccagctct ataacgagct caatctagga 1260

cgaagagagg agtacgatgt tttggacaaa agaagaggcc gggaccctga gatgggggga 1320

aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1380

gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1440

ggcctttacc agggtctcag tacagccacc aaggacacct aggacgcct tcacatgcag 1500

gccctgcccc ctcgctaagg atcccgcccc tctccctccc ccccccctaa cgttatactggc 1560

cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg 1620

ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct 1680

aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca 1740

gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg 1800

aacccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct 1860

gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa 1920

tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt 1980

atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc gagttaaaa 2040

aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa 2100

gcttgccaca acccacaatc tagaatgggg cccaccagcg tcccgctggt caaggcccac 2160

cgcagctcgg tctctgacta cgtcaactat gatatcatcg tccggcatta caactacacg 2220

ggaaagctga atatcagcgc ggacaaggag aacagcatta aactgacctc ggtggtgttc 2280

attctcatct gctgctttat catcctggag aacatctttg tcttgctgac catttggaaa 2340

accaagaaat tccaccgacc catgtactat tttatattggca atctggccct ctcagacctg 2400

ttggcaggag tagcctacac agctaacctg ctcttgtctg gggccaccac ctacaagctc 2460

actcccgccc agtggtttct gcgggaaggg agtatgtttg tggccctgtc agcctccgtg 2520

ttcagtctcc tcgccatcgc cattgagcgc tatatcacaa tgctgaaaat gaaactccac 2580

aacgggagca ataacttccg cctcttcctg ctaatcagcg cctgctgggt catctccctc 2640

atcctgggtg gcctgcctat catgggctgg aactgcatca gtgcgctgtc cagctgctcc 2700

accgtgctgc cgctctacca caagcactat atcctcttct gcaccacggt cttcactctg 2760

cttctgctct ccatcgtcat tctgtactgc agaatctact ccttggtcag gactcggagc 2820

cgccgcctga cgttccgcaa gaacatttcc aaggccagcc gcagctctga gaagtcgctg 2880

gcgctgctca agaccgtaat tatcgtcctg agcgtcttca tcgcctgctg ggcaccgctc 2940

ttcatcctgc tcctgctgga tgtgggctgc aaggtgaaga cctgtgacat cctcttcaga 3000

gcggagtact tcctggtgtt agctgtgctc aactccggca ccaaccccat catttacact 3060

ctgaccaaca aggagatgcg tcgggccttc atccggatca tgtcctgctg caagtgcccg 3120

agcggagact ctgctggcaa attcaagcga cccatcatcg ccggcatgga attcagccgc 3180

agcaaatcgg acaattcctc ccacccccag aaagacgaag gggacaaccc agagaccatt 3240

atgtcttctg gaaacgtcaa ctcttcttcc ggaagcggat aa 3282

<210> 8

<211> 2727

<212> DNA

<213> Artificial Sequence

<220>

<223> S1PR1-2A-GC33CAR

<400> 8

atggggccca ccagcgtccc gctggtcaag gcccaccgca gctcggtctc tgactacgtc 60

aactatgata tcatcgtccg gcattacaac tacacgggaa agctgaatat cagcgcggac 120

aaggagaaca gcattaaact gacctcggtg gtgttcattc tcatctgctg ctttatcatc 180

ctggagaaca tctttgtctt gctgaccatt tggaaaacca agaaattcca ccgacccatg 240

tactatttta ttggcaatct ggccctctca gacctgttgg caggagtagc ctacacagct 300

aacctgctct tgtctggggc caccacctac aagctcactc ccgcccagtg gtttctgcgg 360

gaagggagta tgtttgtggc cctgtcagcc tccgtgttca gtctcctcgc catcgccatt 420

gagcgctata tcacaatgct gaaaatgaaa ctccacaacg ggagcaataa cttccgcctc 480

ttcctgctaa tcagcgcctg ctgggtcatc tccctcatcc tgggtggcct gcctatcatg 540

ggctggaact gcatcagtgc gctgtccagc tgctccaccg tgctgccgct ctaccacaag 600

cactatatcc tcttctgcac cacggtcttc actctgcttc tgctctccat cgtcattctg 660

tactgcagaa tctactcctt ggtcaggact cggagccgcc gcctgacgtt ccgcaagaac 720

atttccaagg ccagccgcag ctctgagaag tcgctggcgc tgctcaagac cgtaattatc 780

gtcctgagcg tcttcatcgc ctgctgggca ccgctcttca tcctgctcct gctggatgtg 840

ggctgcaagg tgaagacctg tgacatcctc ttcagagcgg agtacttcct ggtgttagct 900

gtgctcaact ccggcaccaa ccccatcatt tacactctga ccaacaagga gatgcgtcgg 960

gccttcatcc ggatcatgtc ctgctgcaag tgcccgagcg gagactctgc tggcaaattc 1020

aagcgaccca tcatcgccgg catggaattc agccgcagca aatcggacaa ttcctcccac 1080

ccccagaaag acgaagggga caacccagag accattatgt cttctggaaa cgtcaactct 1140

tcttccggaa gcggaggaag cggagagggc agaggaagtc tgctaacatg cggtgacgtc 1200

gaggagaatc ctggacctat ggccttacca gtgaccgcct tgctcctgcc gctggccttg 1260

ctgctccacg ccgccaggcc ggacgtggtg atgacccaga gccctttatc tttacccgtt 1320

acacccggtg agcccgctag catctcttgt agaagcagcc agtctttagt gcacagcaac 1380

ggcaacacat atttacactg gtatttacag aagcccggtc agagccccca gctgctgatc 1440

tacaaggtga gcaatcgttt ctccggcgtg cccgatagat tcagcggcag cggctccgga 1500

accgacttca ctttaaagat cagcagagtg gaggccgagg acgtgggtgt gtactactgc 1560

tcccagaaca cccacgtgcc ccctacattc ggtcaaggta ccaaactgga gatcaaaggt 1620

ggcggtggct cgggcggtgg tgggtcgggt ggcggcggat ctcaagttca gctggtgcag 1680

agcggcgccg aggtcaaaaa acccggagcc agcgtgaagg tgtcttgtaa agccagcggc 1740

tacaccttta ccgactatga gatgcactgg gtgagacaag ctcccggtca aggtctggaa 1800

tggatgggcg ctttagaccc caagactggt gacacagcct actcccagaa gttcaagggt 1860

cgtgtgactt taacagccga cgagtccacc agcacagcct acatggaact gagctcttta 1920

aggagcgagg acaccgccgt gtattactgc actcgtttct acaagctacac ctactggggc 1980

caaggtactt tagtgacagt gagcagcttc gtgccggtct tcctgccagc gaagcccacc 2040

acgacgccag cgccgcgacc accaacaccg gcgcccacca tcgcgtcgca gcccctgtcc 2100

ctgcgcccag aggcgtgccg gccagcggcg gggggcgcag tgcacacgag ggggctggac 2160

ttcgcctgtg attctggtt acccatagga tgtgcagcct ttgttgtagt ctgcattttg 2220

ggatgcatac ttatttgttg gcttacacgt ttctctgttg ttaaacgggg cagaaagaag 2280

ctcctgtata tattcaaaca accattttatg agaccagtac aaactactca agaggaagat 2340

ggctgtagct gccgatttcc agaagaagaa gaaggaggat gtgaactgag agtgaagttc 2400

agcaggagcg cagacgcccc cgcgtaccag cagggccaga accagctcta taacgagctc 2460

aatctaggac gaagagagga gtacgatgtt ttggacaaga gacgtggccg ggaccctgag 2520

atggggggaa agccgagaag gaagaaccct caggaaggcc tgtacaatga actgcagaaa 2580

gataagatgg cggaggccta cagtgagatt gggatgaaag gcgagcgccg gaggggcaag 2640

gggcacgatg gcctttacca gggtctcagt acagccacca aggacacccta cgacgccctt 2700

cacatgcagg ccctgccccc tcgctaa 2727

<210> 9

<211> 2799

<212> DNA

<213> Artificial Sequence

<220>

<223> S1PR1-2A-P4G2CAR

<400> 9

atggggccca ccagcgtccc gctggtcaag gcccaccgca gctcggtctc tgactacgtc 60

aactatgata tcatcgtccg gcattacaac tacacgggaa agctgaatat cagcgcggac 120

aaggagaaca gcattaaact gacctcggtg gtgttcattc tcatctgctg ctttatcatc 180

ctggagaaca tctttgtctt gctgaccatt tggaaaacca agaaattcca ccgacccatg 240

tactatttta ttggcaatct ggccctctca gacctgttgg caggagtagc ctacacagct 300

aacctgctct tgtctggggc caccacctac aagctcactc ccgcccagtg gtttctgcgg 360

gaagggagta tgtttgtggc cctgtcagcc tccgtgttca gtctcctcgc catcgccatt 420

gagcgctata tcacaatgct gaaaatgaaa ctccacaacg ggagcaataa cttccgcctc 480

ttcctgctaa tcagcgcctg ctgggtcatc tccctcatcc tgggtggcct gcctatcatg 540

ggctggaact gcatcagtgc gctgtccagc tgctccaccg tgctgccgct ctaccacaag 600

cactatatcc tcttctgcac cacggtcttc actctgcttc tgctctccat cgtcattctg 660

tactgcagaa tctactcctt ggtcaggact cggagccgcc gcctgacgtt ccgcaagaac 720

atttccaagg ccagccgcag ctctgagaag tcgctggcgc tgctcaagac cgtaattatc 780

gtcctgagcg tcttcatcgc ctgctgggca ccgctcttca tcctgctcct gctggatgtg 840

ggctgcaagg tgaagacctg tgacatcctc ttcagagcgg agtacttcct ggtgttagct 900

gtgctcaact ccggcaccaa ccccatcatt tacactctga ccaacaagga gatgcgtcgg 960

gccttcatcc ggatcatgtc ctgctgcaag tgcccgagcg gagactctgc tggcaaattc 1020

aagcgaccca tcatcgccgg catggaattc agccgcagca aatcggacaa ttcctcccac 1080

ccccagaaag acgaagggga caacccagag accattatgt cttctggaaa cgtcaactct 1140

tcttccggaa gcggaggaag cggagagggc agaggaagtc tgctaacatg cggtgacgtc 1200

gaggagaatc ctggacctat ggctctgccc gtgaccgctc tgctgctgcc tctcgctctg 1260

ctgctgcatg ccgccagacc cggctcccaa gtgcaactcc agcaatccgg acccggactg 1320

gtgacaccct cccaaacact gtccctcaca tgcgccatca gcggagattc cgtgtcctcc 1380

aacagcgcta catggaattg gattagacag agcccttcca gaggactgga gtggctcgga 1440

agaacatact agatccaa gtggtataac gactacgccg tgtccgtgaa gtctagaatg 1500

agcatcaacc ccgacaccag caagaaccag ttttctctgc agctgaacag cgtgacaccc 1560

gaggacaccg ctgtgtacta ctgcgccaga ggcatgatga cctactacta cggcatggac 1620

gtctggggac aaggcaccac cgtgacagtg agcagcggca ttctgggctc cggaggaggc 1680

ggaagcggag gcggaggcag cggaggcggc ggaagccaac ccgtgctcac acaaagctcc 1740

tctctgagcg ctagccccgg cgcctccgct tctctgacat gtacactgag gagcggcatc 1800

aacgtcggac cctatagaat ctactggtac cagcagaagc ccggcagccc cccccaatat 1860

ctgctgaact acaagtccga ctccgacaag cagcaaggca gcggcgtgcc ttctagattc 1920

agcggcagca aggacgctag cgccaacgcc ggagtgctgc tgatctccgg actgagaagc 1980

gaggacgagg ccgactacta ttgcatgatc tggcactcca gcgccgctgt ctttggagga 2040

ggcacacagc tgacagtgct gagcgccagc ttcgtgcccg tgttcctccc cgccaaaccc 2100

accacaacac ccgcccctag acccccctaca cccgccccta ccatcgcttc ccagcctctg 2160

tctctgagac ccgaggcttg tagacccgcc gctggaggag ctgtgcatac aagaggactg 2220

gacttcgctt gcgacatcta tatttgggct cccctcgctg gaacatgcgg agtgctgctg 2280

ctgtctctgg tgatcacact gtactgcaac catagaaaca gattctccgt ggtgaagaga 2340

ggaagaaaga aactgctcta catcttcaag cagcccttca tgaggcccgt gcagaccaca 2400

caagaggagg acggatgtag ctgtagattc cccgaagagg aggagggagg atgtgagctg 2460

agagtgaagt tctctagatc cgccgatgcc cccgcctacc agcaaggcca gaaccagctc 2520

tataacgagc tcaatctggg aagaagagag gagtacgacg tgctggacaa gaggagggga 2580

agagaccccg aaatgggcgg caagcccaga aggaaaaacc cccaagaggg actgtacaac 2640

gaactgcaga aggacaagat ggctgaggcc tacagcgaaa tcggcatgaa aggcgagagg 2700

aggagaggca aaggacacga cggactctat caaggcctca gcaccgccac caaggacacc 2760

tacgatgctc tgcatatgca agctctgccc cctagataa 2799

<210> 10

<211> 2484

<212> DNA

<213> Artificial Sequence

<220>

<223> S1PR1-IRES-GFP

<400> 10

atggggccca ccagcgtccc gctggtcaag gcccaccgca gctcggtctc tgactacgtc 60

aactatgata tcatcgtccg gcattacaac tacacgggaa agctgaatat cagcgcggac 120

aaggagaaca gcattaaact gacctcggtg gtgttcattc tcatctgctg ctttatcatc 180

ctggagaaca tctttgtctt gctgaccatt tggaaaacca agaaattcca ccgacccatg 240

tactatttta ttggcaatct ggccctctca gacctgttgg caggagtagc ctacacagct 300

aacctgctct tgtctggggc caccacctac aagctcactc ccgcccagtg gtttctgcgg 360

gaagggagta tgtttgtggc cctgtcagcc tccgtgttca gtctcctcgc catcgccatt 420

gagcgctata tcacaatgct gaaaatgaaa ctccacaacg ggagcaataa cttccgcctc 480

ttcctgctaa tcagcgcctg ctgggtcatc tccctcatcc tgggtggcct gcctatcatg 540

ggctggaact gcatcagtgc gctgtccagc tgctccaccg tgctgccgct ctaccacaag 600

cactatatcc tcttctgcac cacggtcttc actctgcttc tgctctccat cgtcattctg 660

tactgcagaa tctactcctt ggtcaggact cggagccgcc gcctgacgtt ccgcaagaac 720

atttccaagg ccagccgcag ctctgagaag tcgctggcgc tgctcaagac cgtaattatc 780

gtcctgagcg tcttcatcgc ctgctgggca ccgctcttca tcctgctcct gctggatgtg 840

ggctgcaagg tgaagacctg tgacatcctc ttcagagcgg agtacttcct ggtgttagct 900

gtgctcaact ccggcaccaa ccccatcatt tacactctga ccaacaagga gatgcgtcgg 960

gccttcatcc ggatcatgtc ctgctgcaag tgcccgagcg gagactctgc tggcaaattc 1020

aagcgaccca tcatcgccgg catggaattc agccgcagca aatcggacaa ttcctcccac 1080

ccccagaaag acgaagggga caacccagag accattatgt cttctggaaa cgtcaactct 1140

tcttccggaa gcggataagg atcccgcccc tctccctccc ccccccctaa cgttactggc 1200

cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg 1260

ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct 1320

aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca 1380

gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg 1440

aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct 1500

gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa 1560

tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt 1620

atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc gagttaaaa 1680

aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa 1740

gcttgccaca acccacaatc tagaatggtg agcaagggcg aggagctgtt caccggggtg 1800

gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc 1860

gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc 1920

aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc 1980

agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc 2040

tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag 2100

gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag 2160

gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat 2220

atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc 2280

gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc 2340

cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc 2400

aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc 2460

ggcatggacg agctgtacaa gtaa 2484

<210> 11

<211> 2844

<212> DNA

<213> Artificial Sequence

<220>

<223> GC33CAR-IRES-GFP

<400> 11

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggacgtgg tgatgaccca gagcccttta tctttacccg ttacacccgg tgagcccgct 120

agcatctctt gtagaagcag ccagtcttta gtgcacagca acggcaacac atatttacac 180

tggtatttac agaagcccgg tcagagcccc cagctgctga tctacaaggt gagcaatcgt 240

ttctccggcg tgcccgatag attcagcggc agcggctccg gaaccgactt cactttaaag 300

atcagcagag tggaggccga ggacgtgggt gtgtactact gctcccagaa cacccacgtg 360

ccccctacat tcggtcaagg taccaaactg gagatcaaag gtggcggtgg ctcgggcggt 420

ggtgggtcgg gtggcggcgg atctcaagtt cagctggtgc agagcggcgc cgaggtcaaa 480

aaacccggag ccagcgtgaa ggtgtcttgt aaagccagcg gctacacctt taccgactat 540

gagatgcact gggtgagaca agctcccggt caaggtctgg aatggatggg cgctttagac 600

cccaagactg gtgacacagc ctactcccag aagttcaagg gtcgtgtgac tttaacagcc 660

gacgagtcca ccagcacagc ctacatggaa ctgagctctt taaggagcga ggacaccgcc 720

gtgtattact gcactcgttt ctacagctac acctactggg gccaaggtac tttagtgaca 780

gtgagcagct tcgtgccggt cttcctgcca gcgaagccca ccacgacgcc agcgccgcga 840

ccaccaacac cggcgcccac catcgcgtcg cagcccctgt ccctgcgccc agaggcgtgc 900

cggccagcgg cggggggcgc agtgcacacg aggggctgg acttcgcctg tgatatctac 960

atctgggcgc ccttggccgg gacttgtggg gtccttctcc tgtcactggt tatcaccctt 1020

tactgcaacc acaggaaccg tttctctgtt gttaaacggg gcagaaagaa gctcctgtat 1080

atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1140

tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 1200

gcagacgccc ccgcgtacca gcagggccag aaccagctct ataacgagct caatctagga 1260

cgaagagagg agtacgatgt tttggacaaa agaagaggcc gggaccctga gatgggggga 1320

aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1380

gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1440

ggcctttacc agggtctcag tacagccacc aaggacacct aggacgcct tcacatgcag 1500

gccctgcccc ctcgctaagg atcccgcccc tctccctccc ccccccctaa cgttatactggc 1560

cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg 1620

ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct 1680

aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca 1740

gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg 1800

aacccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct 1860

gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa 1920

tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt 1980

atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc gagttaaaa 2040

aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa 2100

gcttgccaca acccacaatc tagaatggtg agcaagggcg aggagctgtt caccggggtg 2160

gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc 2220

gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc 2280

aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc 2340

agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc 2400

tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag 2460

gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag 2520

gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat 2580

atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc 2640

gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc 2700

cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc 2760

aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc 2820

ggcatggacg agctgtacaa gtaa 2844

<210> 12

<211> 3282

<212> DNA

<213> Artificial Sequence

<220>

<223> S1PR1-IRES-GC33CAR

<400> 12

atggggccca ccagcgtccc gctggtcaag gcccaccgca gctcggtctc tgactacgtc 60

aactatgata tcatcgtccg gcattacaac tacacgggaa agctgaatat cagcgcggac 120

aaggagaaca gcattaaact gacctcggtg gtgttcattc tcatctgctg ctttatcatc 180

ctggagaaca tctttgtctt gctgaccatt tggaaaacca agaaattcca ccgacccatg 240

tactatttta ttggcaatct ggccctctca gacctgttgg caggagtagc ctacacagct 300

aacctgctct tgtctggggc caccacctac aagctcactc ccgcccagtg gtttctgcgg 360

gaagggagta tgtttgtggc cctgtcagcc tccgtgttca gtctcctcgc catcgccatt 420

gagcgctata tcacaatgct gaaaatgaaa ctccacaacg ggagcaataa cttccgcctc 480

ttcctgctaa tcagcgcctg ctgggtcatc tccctcatcc tgggtggcct gcctatcatg 540

ggctggaact gcatcagtgc gctgtccagc tgctccaccg tgctgccgct ctaccacaag 600

cactatatcc tcttctgcac cacggtcttc actctgcttc tgctctccat cgtcattctg 660

tactgcagaa tctactcctt ggtcaggact cggagccgcc gcctgacgtt ccgcaagaac 720

atttccaagg ccagccgcag ctctgagaag tcgctggcgc tgctcaagac cgtaattatc 780

gtcctgagcg tcttcatcgc ctgctgggca ccgctcttca tcctgctcct gctggatgtg 840

ggctgcaagg tgaagacctg tgacatcctc ttcagagcgg agtacttcct ggtgttagct 900

gtgctcaact ccggcaccaa ccccatcatt tacactctga ccaacaagga gatgcgtcgg 960

gccttcatcc ggatcatgtc ctgctgcaag tgcccgagcg gagactctgc tggcaaattc 1020

aagcgaccca tcatcgccgg catggaattc agccgcagca aatcggacaa ttcctcccac 1080

ccccagaaag acgaagggga caacccagag accattatgt cttctggaaa cgtcaactct 1140

tcttccggaa gcggataagg atcccgcccc tctccctccc ccccccctaa cgttactggc 1200

cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg 1260

ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct 1320

aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca 1380

gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg 1440

aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct 1500

gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa 1560

tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt 1620

atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc gagttaaaa 1680

aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa 1740

gcttgccaca acccacaatc tagaatggcc ttaccagtga ccgccttgct cctgccgctg 1800

gccttgctgc tccacgccgc caggccggac gtggtgatga cccagagccc tttatcttta 1860

cccgttacac ccggtgagcc cgctagcatc tcttgtagaa gcagccagtc tttagtgcac 1920

agcaacggca acacatattt acactggtat ttacagaagc ccggtcagag cccccagctg 1980

ctgatctaca aggtgagcaa tcgtttctcc ggcgtgcccg atagattcag cggcagcggc 2040

tccggaaccg acttcacttt aaagatcagc agagtggagg ccgaggacgt gggtgtgtac 2100

tactgctccc agaacacca cgtgccccct acattcggtc aaggtaccaa actggagatc 2160

aaaggtggcg gtggctcggg cggtggtggg tcgggtggcg gcggatctca agttcagctg 2220

gtgcagagcg gcgccgaggt caaaaaaccc ggagccagcg tgaaggtgtc ttgtaaagcc 2280

agcggctaca cctttaccga ctatgagatg cactgggtga gacaagctcc cggtcaaggt 2340

ctggaatgga tgggcgcttt agaccccaag actggtgaca cagcctactc ccagaagttc 2400

aagggtcgtg tgactttaac agccgacgag tccaccagca cagcctacat ggaactgagc 2460

tctttaagga gcgaggacac cgccgtgtat tactgcactc gtttctacag ctacacctac 2520

tggggccaag gtactttagt gacagtgagc agcttcgtgc cggtcttcct gccagcgaag 2580

cccaccacga cgccagcgcc gcgaccacca acaccggcgc ccaccatcgc gtcgcagccc 2640

ctgtccctgc gcccagaggc gtgccggcca gcggcggggg gcgcagtgca cacgaggggg 2700

ctggacttcg cctgtgatat ctacatctgg gcgcccttgg ccgggacttg tggggtcctt 2760

ctcctgtcac tggttatcac cctttactgc aaccacagga accgtttctc tgttgttaaa 2820

cggggcagaa agaagctcct gtatatattc aaacaaccat ttatgagacc agtacaaact 2880

actcaagagg aagatggctg tagctgccga tttccagaag aagaagaagg aggatgtgaa 2940

ctgagagtga agttcagcag gagcgcagac gcccccgcgt accagcaggg ccagaaccag 3000

ctctataacg agctcaatct aggacgaaga gaggagtacg atgttttgga caaaagaaga 3060

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 3120

aatgaactgc agaaagataa gatggcggag gcctacagtg agatggggat gaaaggcgag 3180

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 3240

acctacgacg cccttcacat gcaggccctg ccccctcgct aa 3282

<210> 13

<211> 574

<212> DNA

<213> Artificial Sequence

<220>

<223> IRES

<400> 13

cccctctccc tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg 60

tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg 120

gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg 180

aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca 240

aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct 300

ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca 360

cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa 420

ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg 480

cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg 540

ggacgtggtt ttcctttgaa aaacacgatg ataa 574

<210> 14

<211> 54

<212> DNA

<213> Artificial Sequence

<220>

<223> 2A

<400> 14

gagggcagag gaagtctgct aacatgcggt gacgtcgagg agaatcctgg acct 54

<210> 15

<211> 720

<212> DNA

<213> Artificial Sequence

<220>

<223> GFP

<400> 15

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720

Claims (18)

1. Modified immune effector T cells expressing a chimeric antigen receptor (CAR) and an exogenous S1PR1 protein, wherein the expression and/or activity of the S1PR1 protein is upregulated compared with unmodified immune T cells, and the nucleotide sequences of the nucleic acid molecule encoding the chimeric antigen receptor (CAR) and the vector encoding the nucleic acid molecule of the exogenous S1PR1 protein are as shown in any one of SEQ ID NO: 7-9. 2. The immune effector T cell according to claim 1, wherein the immune effector T cell includes memory T cells. 3. The immune effector T cell according to claim 1, wherein the chimeric antigen receptor comprises an antigen-binding domain that specifically binds to tumor-specific antigens. 4. The immune effector T cell according to claim 3, wherein the antigen-binding domain comprises a single-chain antibody. 5. The immune effector T cell of claim 4, wherein the antigen-binding domain comprises a single-chain antibody targeting GPC3 or MSLN. 6. The immune effector T cell according to claim 5, wherein the amino acid sequence of the single-chain antibody is shown in any one of SEQ ID NO: 1-2. 7. The immune effector T cell of claim 1, wherein the chimeric antigen receptor comprises a transmembrane domain. 8. The immune effector T cell of claim 7, wherein the transmembrane domain comprises a transmembrane domain derived from a protein selected from the following proteins: CD3ζ, CD28, 4-1BB, OX40, SLAMF4, CD127, NKG2D, ICOS, and FcγRIIIa. 9. The immune effector T cell of claim 1, wherein the chimeric antigen receptor comprises a co-stimulatory domain. 10. The immune effector T cell of claim 9, wherein the co-stimulatory domain comprises a co-stimulatory domain selected from the following proteins or combinations thereof: CD137, CD28, OX40, ICOS, DAP10, 2B4, CD27, CD30, CD40, CD40L, TIM1, CD226, DR3, SLAM, NKG2D, CD244, FceRIγ, BTLA, GITR, HVEM, CD2, NKG2C, LIGHT, and DAP12. 11. The immune effector T cell of claim 1, wherein the chimeric antigen receptor comprises a hinge region connecting an antigen-binding domain and a transmembrane domain. 12. The immune effector T cell of claim 11, wherein the hinge region comprises a hinge region derived from a protein selected from: CD8, CD28, IgG, 4-1BB, CD4, CD27, CD7, PD-1, and CH2CH3. 13. The immune effector T cell according to claim 1, wherein the amino acid sequence of the chimeric antigen receptor is shown in any one of SEQ ID NO: 5-6. 14. A cell population comprising immune effector T cells according to any one of claims 1-13. 15. A pharmaceutical composition comprising immune effector T cells according to any one of claims 1-13 and/or the cell population according to claim 14, and a pharmaceutically acceptable carrier. 16. Use of any one of the immune effector T cells of claims 1-13, the cell population of claim 14, and/or the pharmaceutical composition of claim 15 in the preparation of a medicament for the treatment of tumors. 17. The use according to claim 16, wherein the tumor comprises a solid tumor. 18. The use according to claim 16, wherein the tumor includes pancreatic cancer, glioma, liver cancer, and/or colon cancer.