Cell engineering method for reversing PD1 signals of T and CAR-T cells by using CD27

Abstract

The invention discloses a cell engineering method for reversing PD1 signals of T and CAR-T cells by using CD27. Specifically, the invention discloses a fusion protein PD1-CD27 (SEQ ID No. 4) and a nucleic acid molecule (SEQ ID No. 1), and further constructs a modified T cell for different antigens. The modified T cell has small influence on the phenotypes of CD4 and CD8-T cells of a patient, the proportion of memory T cells is increased, and the modified T cell can survive for a long time, specifically recognizes and kills tumor cells expressing tumor-associated antigens (mesothelin, HER2 and the like), has no killing effect on negative cells, and has the effects of resisting a tumor cell microenvironment and resisting T cell depletion. The CD27 is used for reversing an inhibition signal of PD1, and the CD27 has practical significance and potential clinical application value for overcoming a tumor microenvironment, playing a cytotoxic role in the microenvironment and prolonging the survival time of CAR-T.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to an engineered cell method for reversing PD1 signals of T and CAR-T cells using CD27. Background Art

[0002] Chimeric antigen receptor (CAR) technology uses genetic engineering methods to modify the patient's autologous T cells to express proteins that can recognize tumor cell surface antigens, and specifically activate T cells and kill tumor cells through costimulatory factors and activation signal transduction domain CD3ζ, without the need for antigen presentation and without the restriction of MHC-I class molecules (human leukocyte antigen, HLA). The structure of CAR consists of an extracellular recognition domain and an intracellular signal transduction domain: the extracellular recognition domain is ScFV (Single chain fragment variable), which is composed of the heavy chain (VH), light chain (VL) and linker of a monoclonal antibody targeting tumor-associated antigens, and can specifically recognize tumor surface specific proteins (antigens). The intracellular signal activation transduction domain or costimulatory domain is used to initiate the immune response of the patient's autologous T lymphocytes after recognizing tumor cell (target cell) surface proteins, exerting cytotoxic effects and thus killing target cells.

[0003] The DNA encoding CAR consists of a signal peptide (SP), a single-chain antibody domain (scFv) that recognizes the antigen, a linker, a hinge, and a transmembrane region (TM) to form an extracellular structure; the intracellular domain is composed of a costimulatory molecule and an intracellular segment of the activation domain CD3ζ (CD3z). The combined protein is a type of transmembrane protein. From the extracellular end of the amino terminus to the carboxyl end of the cell, the precursor protein will remove the signal peptide in the endoplasmic reticulum and become a mature transmembrane protein. According to the presence or absence of costimulatory factors and the number of costimulatory factors, it is divided into the first generation of CAR (no costimulatory factors), the second generation of CAR containing one costimulatory molecule (CD28, 4-1BB, OX40, ICOS, etc.), and the third generation of CAR containing two costimulatory molecules.

[0004] PD1 is an inhibitory receptor that activates T cells. It can directly inhibit T cell activation by binding to its ligand PD-L1 or PD-L2 through the extracellular domain, and plays a key role in inducing and maintaining immune tolerance to itself. The specific mechanism is that after PD1 binds to its ligand, the ITSM motif in the intracellular region is phosphorylated, leading to the recruitment of protein tyrosine phosphatase PTPN11 / SHP-2, which mediates the dephosphorylation of key TCR proximal signaling molecules, such as ZAP70, PKCθ and CD247 / CD3ζ, thereby inhibiting T cell activation. Structurally, PD1, PD-L1, CD28, CD80, CTLA4 and CD86 are all members of the B7 family, which is different from the structure of the tumor necrosis factor family. For details, see Chen L, Flies DB. Molecular mechanisms of T cell co-stimulation and co-inhibition. Nat Rev Immunol. 2013; 13(4): 227-42.

[0005] CD27 (also known as TNFRSF7) belongs to the tumor necrosis factor family, which also includes CD30, OX-40 (also known as CD134) and 4-1BB (also known as CD 137, TNFRSF9), which have the function of regulating lymphocyte action and belong to a different family from CD28. Studies have found that CD27 is a factor necessary for T cell survival and long-term maintenance of T cell function; other prominent members of this family are RANK and CD40, which also regulate the response of antigen-presenting cells such as dendritic cells. CD27, CD30, 4-1BB and OX-40 are expressed only on lymphoid cells, mostly in an activation-specific manner, and all enhance T cell receptor (TCR)-induced T cell expansion (De-Gang Song, Qunrui Ye, Mathilde Poussin, Gretchen M. Harms, Mariangela Figini, Daniel J. Powell; CD27 costimulation augments the survival and antitumor activity of redirected human T cells in vivo. Blood 2012; 119 (3): 696–706.). CD27 costimulation can enhance the survival and antitumor activity of redirected human T cells in vivo.

[0006] Both PD1 and CD27 are type I single-transmembrane proteins, and their intracellular or extracellular regions can be replaced through genetic engineering. Summary of the invention

[0007] The technical problem to be solved by the present invention is how to use CD27 to reverse the PD1 signal of T and CAR-T cells and improve the anti-tumor ability of T and CAR-T cells. The technical problem to be solved is not limited to the described technical subject matter, and those skilled in the art can clearly understand other technical subjects not mentioned in this article through the following description.

[0008] Furthermore, the technical problem to be solved by the present invention is that tumor cells, when under pressure or in a tumor microenvironment, will upregulate the expression of their cell membrane proteins PD-L1 or PD-L2. The upregulated PD-L1 or PD-L2 is the ligand of T cell PD1 (PD1 is the receptor of PD-L1 or PD-L2). After the ligand of the tumor cell (PD-L1 or PD-L2) binds to the receptor of the T cell (PD1), it will inhibit the cytotoxic effect of T cells killing tumors, showing immune tolerance. The present invention separates the extracellular domain and the intracellular domain of T cell PD1, the extracellular cell domain remains unchanged, and still binds to the PD-L1 or PD-L2 ligand; the intracellular domain contains the TCR inhibitory ITSM motif, which is replaced by the intracellular domain of CD27. That is, after PD1 and PD-L1 are combined, the T cell inhibition signal is converted into the T / CAR-T cell survival signal of PD1-CD27, so that T / CAR-T cells can survive and exert cytotoxic effects under pressure or in the tumor microenvironment. In order to solve the above technical problems, the present invention forces the exogenous combined gene PD1-CD27 to be expressed in T / CAR-T cells through viral infection.

[0009] To solve the above technical problems, the present invention first provides a fusion protein named PD1-CD27 (also known as combined protein PD1-CD27), wherein the fusion protein comprises the extracellular region and transmembrane region of PD1 and the intracellular region of CD27, and the amino acid sequence of the fusion protein can be shown as SEQ ID No.4.

[0010] The present invention also provides a fusion gene, which encodes the fusion protein PD1-CD27. The nucleotide sequence of the fusion gene can be shown as SEQ ID No.1.

[0011] The fusion gene PD1-CD27 (also called the combined gene PD1-CD27) of the present invention is characterized in that the encoded extracellular region and transmembrane region are the original sequences of PD1, which can bind to its ligand PD-L1 or PD-L2. The encoded intracellular region is the intracellular region sequence of CD27, which exerts the function of CD27; after PD1 and PD-L1 are combined, the T / CAR-T cell inhibition signal can be theoretically converted into the T / CAR-T cell survival signal of CD27.

[0012] The combination gene PD1-CD27 can be expressed in T cells alone, or CAR can be expressed first and then PD1-CD27 to form the second-generation CAR-PD1-CD27. Taking the CAR targeting mesothelin (Meso) as an example, the Meso-CAR structure is as follows Figure 1 As shown in B, the DNA sequence is shown in SEQ ID No.2, and the corresponding amino acid sequence is shown in SEQ ID No.5.

[0013] The present invention also provides a nucleic acid molecule, which may include the fusion gene and a gene encoding a chimeric antigen receptor, wherein the gene encoding the chimeric antigen receptor and the fusion gene are connected via a gene encoding a self-cleaving peptide, and the chimeric antigen receptor may target mesothelin, EpCAM, HER2 or EGFR but is not limited thereto.

[0014] Further, the self-cleaving peptide may include P2A, F2A, T2A or E2A.

[0015] Furthermore, the self-cleaving peptide may be P2A, and the nucleotide sequence and amino acid sequence of P2A are shown as SEQ ID No.7 and SEQ ID No.8, respectively.

[0016] Furthermore, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID No.3, SEQ ID No.9, SEQ ID No.10 or SEQ ID No.11.

[0017] Wherein: when the chimeric antigen receptor targets mesothelin, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID No.3; when the chimeric antigen receptor targets EpCAM, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID No.11; when the chimeric antigen receptor targets HER2, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID No.9; when the chimeric antigen receptor targets EGFR, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID No.10.

[0018] The present invention also provides modified T cells, wherein the modified T cells are T cells modified by the fusion protein PD1-CD27, or T cells modified by a fusion gene formed by a gene encoding the fusion protein PD1-CD27 and a gene encoding a chimeric antigen receptor.

[0019] The PD1 extracellular region expressed by the modified T cells can bind to PD-L1 or PD-L2 expressed by tumor cells, and the effect played is not the PD1 signal, but the CD27 signal.

[0020] Furthermore, the modified T cell contains the fusion gene (SEQ ID No. 1), or contains any nucleic acid molecule described herein.

[0021] In one or more embodiments of the present invention, the modified T cells are PD1-CD27 T cells or Meso-CAR PD1-CD27 T cells.

[0022] The PD1-CD27 T cells are recombinant cells obtained by packaging the recombinant lentiviral vector pLV-PD1-CD27 through packaging cells to obtain recombinant lentivirus, and then introducing the recombinant lentivirus into T cells. The packaging cells can be HEK293T cells. The PD1-CD27 T cells contain or express the PD1-CD27 gene shown in SEQ ID No.1. The recombinant lentiviral vector pLV-PD1-CD27 is a recombinant lentiviral vector obtained by cloning the PD1-CD27 gene (SEQ ID No.1) into the pLV vector.

[0023] The Meso-CAR PD1-CD27 T cell is a recombinant cell obtained by packaging the recombinant lentiviral vector pLV-Meso-CAR PD1-CD27 through packaging cells to obtain a recombinant lentivirus, and then introducing the recombinant lentivirus into T cells, and the packaging cells may be HEK293T cells. The Meso-CAR PD1-CD27 T cell contains or expresses the nucleic acid molecule shown in SEQ ID No.3. The recombinant lentiviral vector pLV-Meso-CAR PD1-CD27 is a recombinant lentiviral vector obtained by cloning the Meso-CAR PD1-CD27 gene (SEQID No.3) into the pLV vector.

[0024] The present invention also provides any of the following uses of the fusion protein, the fusion gene and / or any of the nucleic acid molecules described herein:

[0025] A1) Application in converting the PD1 inhibitory signal of T cells or CAR-T cells into a survival signal of T cells or CAR-T cells of CD27;

[0026] A2) Use in the preparation of products for immunosuppression and / or T cell depletion against tumor microenvironment;

[0027] A3) Use in the preparation of T cells or CAR-T cells for preventing or treating tumors;

[0028] A4) Use in the preparation of products that enhance the tumor killing activity of T cells or CAR-T cells;

[0029] A5) Application in the preparation of products that promote the release of cytokine IFN-γ, thereby improving the anti-tumor effect of T cells or CAR-T cells;

[0030] A6) Use in the preparation of drugs for preventing or treating tumors;

[0031] A7) Use in the preparation of drugs for inhibiting and / or killing tumor cells.

[0032] In the above application, the tumor may be a tumor expressing mesothelin antigen, EpCAM antigen, HER2 antigen or EGFR antigen.

[0033] The present invention also provides an engineered cell method for reversing T cell and / or CAR-T cell PD1 signals using CD27, the method comprising replacing the intracellular domain of T cell and / or CAR-T cell PD1 with the intracellular domain of CD27, the replacement method comprising modifying T cells using the fusion protein, the fusion gene or any nucleic acid molecule described herein.

[0034] The T cells may be autologous cells from a tumor patient or volunteer, or T lymphocytes separated from autologous cells.

[0035] Furthermore, the method may include the following steps:

[0036] C1) constructing a recombinant lentiviral vector expressing the Meso-CAR PD1-CD27 gene, wherein the nucleotide sequence of the Meso-CAR PD1-CD27 gene is SEQ ID No. 3;

[0037] C2) lentiviral packaging to obtain a recombinant lentivirus expressing the Meso-CAR PD1-CD27 gene;

[0038] C3) The recombinant lentivirus infects T cells to obtain recombinant T cells expressing the Meso-CAR PD1-CD27 gene, i.e., CAR-T cells targeting the mesothelin antigen.

[0039] Further, the tumor expressing mesothelin antigen described herein may include mesothelioma, ovarian cancer, pancreatic cancer, gastric cancer, lung cancer, breast cancer or endometrial cancer, but not limited thereto; the tumor expressing EpCAM antigen may include esophageal cancer, gastric cancer, colon cancer, prostate cancer, lung cancer, ovarian cancer, breast cancer, etc., but not limited thereto; the tumor expressing HER2 antigen may include breast cancer, ovarian cancer, cervical cancer, lung cancer, colon cancer, prostate cancer, etc., but not limited thereto; the tumor expressing EGFR antigen may include breast cancer, head and neck cancer, non-small cell lung cancer, kidney cancer, ovarian cancer, colon cancer, etc., but not limited thereto. To select which antigen to target, it is necessary to perform immunohistochemical detection on the patient sample, and select the membrane protein (antigen) that is relatively highly expressed in tumor cells and relatively low in normal tissues.

[0040] The present invention replaces the structure of the intracellular region of PD1 by the intracellular region of CD27, reverses the inhibitory signal of T cells, and achieves the survival of T cells in the tumor microenvironment; it can also be combined with chimeric antigen receptor technology to achieve an immunotherapy method targeting surface antigens of solid tumors.

[0041] After extensive and in-depth research, the inventors of the present application first designed a combination gene PD1-CD27, the structural diagram of which is shown in FIG. Figure 1 As shown in A, it contains the signal peptide, extracellular region, transmembrane region of PD1, and the intracellular region and tag HA of CD27. The combined gene and a CAR targeting tumor-associated antigens are further connected through self-cleavage protein and named CAR PD1-CD27 gene. The CAR PD1-CD27 gene consists of a signal peptide, scFv (tumor antigen binding region, i.e. single-chain antibody region), CD8α hinge region and transmembrane region, 4-1BB (or CD28, intracellular signal region), CD3ζ (intracellular signal region), P2A, PD1 extracellular region and transmembrane region, CD27 intracellular region, and HA from N-terminus to C-terminus. Figure 1 C. Wherein: the tumor antigen binding region (antigen recognition region) can be a single-chain variable region (scFv) of an antibody that specifically recognizes tumor cell surface antigens EpCAM, HER2, EGFR or mesothelin, etc., which is composed of a heavy chain (VH), a connecting region (linker) and a light chain (VH).

[0042] Furthermore, the present invention designs and constructs a Meso-CAR PD1-CD27 gene targeting mesothelin, constructs a lentiviral vector containing the gene, and obtains Meso-CAR PD1-CD27 T cells by lentiviral packaging and infection of human T cells, so that the second-generation CAR (SEQ ID No. 2) and PD1-CD27 (SEQ ID No. 1) are exogenously expressed, and after in vitro amplification, can be used to prepare a cell therapy preparation that can be re-infused into tumor patients. The second-generation CAR part expressed by Meso-CAR PD1-CD27T cells is used to recognize tumor-associated antigens (such as mesothelin), specifically kill tumor cells expressing mesothelin, and initiate tumor killing; the PD1-CD27 part expressed by Meso-CAR PD1-CD27 T cells is used to reverse the inhibitory signal of T cells and enhance the tumor killing effect.

[0043] Furthermore, in order to verify that the fusion protein PD1-CD27 of the present invention can be applied to different antigens (EpCAM, HER2 or EGFR) in addition to mesothelin, the present invention further designed and constructed a HER2 second-generation CAR with CD28 as a co-stimulatory factor, and an EGFR and EpCAM second-generation CAR with 4-1BB as a co-stimulatory factor. The results showed that CAR-T cells modified with PD1-CD27 can be cultured in vitro for a long time, can specifically recognize and kill tumor cells expressing these tumor-associated antigens (HER2, EGFR or EpCAM), and have the effect of resisting the tumor cell microenvironment and T cell dysfunction or exhaustion, thereby enhancing the tumor killing effect.

[0044] In summary, the experimental results show that the Meso-CAR PD1-CD27 T cells constructed by the present invention have little effect on the patient's CD4 and CD8-T cell phenotypes, and the expression percentage and intensity of the exhaustion indicators LAG3 and TIM3 of the second-generation CAR T cells are significantly higher than those of PD1-CD27 and CAR PD1-CD27 T cells. If the exogenously infected PD1 is deducted, the expression of the exhaustion indicator PD1 is also significantly higher in the second-generation CAR T cells than in PD1-CD27 and CAR PD1-CD27 T cells. It shows that the three major exhaustion indicators of T and CAR-T cells transformed with PD1-CD27 are significantly lower than those of traditional second-generation CAR-T cells.

[0045] Meso CAR-T cells without PD1-CD27 fusion have a strong killing rate and a high IFN-γ secretion level against target cells under normal culture conditions, and are slightly higher than Meso CAR PD1-CD27 T cells. However, in tumor culture fluid (tumor microenvironment), the killing rate of target cells and the IFN-γ secretion level of Meso-CAR T cells dropped significantly, far lower than the similar indicators of Meso-CAR PD1-CD27 T. This suggests that in a simulated tumor microenvironment, T and CAR-T cells modified with PD1-CD27 have a lower degree of exhaustion and increased activity. From the perspective of long-term in vitro culture, CAR-T cells modified with PD1-CD27 have a higher proportion of memory T cells, suggesting that these cells can survive for a longer period of time. In summary, CAR-T cells modified with PD1-CD27 can specifically kill tumor cells in vitro. These tumor cells highly express tumor-related antigens, such as mesothelin, epidermal growth factor (EGFR), HER2 and EpCAM, but have no killing effect on negative cells. The nucleic acid molecules designed by the present invention improve the anti-exhaustion effect of T cells and CAR-T cells, achieve anti-tumor effects in the tumor microenvironment, and have important significance and potential clinical application value for tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The design and structure diagram of the PD1-CD27 gene. Figure 1 A in the middle is the combined PD1-CD27 structure; Figure 1 Middle B is the structure of chimeric antigen receptor (CAR); Figure 1 C in the middle is the structure of the second-generation CAR PD1-CD27 gene.

[0047] Figure 2 The plasmid maps of the three recombinant lentiviral vectors constructed in Example 3 are shown below. Figure 2 Middle A is pLV-PD1-CD27; Figure 2 Middle B is pLV-Meso-CAR; Figure 2 Center C is pLV-Meso-CAR PD1-CD27.

[0048] Figure 3 It is the positive rate of CD3-positive T cells after PBMC stimulation.

[0049] Figure 4 CD3 + The positive rate of T cell PD1-CD27 CAR and CAR-T cell lentivirus infection. Figure 4A in the middle is the positive rate of Meso-CAR PD1-CD27 T cells detected by Anti-human F(ab')2 antibody; Figure 4 Middle B is the positive rate of Meso-CAR PD1-CD27 T cells detected by the tag protein HA.

[0050] Figure 5 For tumor cells and CAR-T cells ( Figure 5 A) or fused Meso-CAR PD1-CD27 T cells ( Figure 5 Middle B) Schematic diagram of the action.

[0051] Figure 6 The CD4 and CD8 phenotypes of uninfected T cells, Meso-CAR T cells, PD1-CD27 T cells, and Meso-CARPD1-CD27 T cells were analyzed by flow cytometry.

[0052] Figure 7 CD3 for flow cytometry analysis + Expression of PD1 on T cells.

[0053] Figure 8 Flow cytometry was used to analyze the cell phenotypes of Meso-CAR T cells and Meso-CAR PD1-CD27 T cells cultured in vitro for different time periods. CD62L and CD45RO were used as phenotypic analysis indicators.

[0054] Fig. 9 Indicator analysis for T cells, Meso-CAR T cells, PD1-CD27 T cells, and Meso-CAR PD1-CD27 T cell exhaustion.

[0055] Fig.10 Analysis of PD1 expression in CD3 negative and positive cells. Fig.10 A in the middle shows the expression of PD1 in CD3-positive cells; Fig.10 Middle B is the PD1 expression analysis of CD3-negative and HLA-DR-positive cells.

[0056] Fig.11 It is the positive expression rate of PD-L1 after tumor cells were treated with IFN-γ.

[0057] Fig.12 is the positive expression rate of tumor cell target antigen ( Fig.12 A, B, C) and the positive rate of mesothelin expression in cells overexpressing mesothelin antigen ( Fig.12 (middle D).

[0058] Fig.13The results of in vitro killing experiments of normal cultured T, Meso-CAR and Meso-CAR PD1-CD27 T cells. The horizontal axis is the effector-target ratio (E:T), the vertical axis is the tumor cell killing rate (%), and the T cells not infected with CAR (NormalT) are used as the control to compare the killing rates of Meso-CAR T cells and Meso-CAR PD1-CD27 T cells.

[0059] Fig.14 The results of in vitro killing experiments of T, Meso-CAR and Meso-CAR PD1-CD27 T cells cultured in tumor culture medium (tumor microenvironment).

[0060] Fig.15 This is the cytokine IFN-γ release level of each group in Example 6.

[0061] Fig.16 These are the results of in vitro killing experiments of T cells, HER2-CAR, and HER2-CAR PD1-CD27 T cells cultured in normal culture and tumor culture medium.

[0062] Fig.17 Flow cytometric analysis of T cells infected with PD1-CD27 and EGFR-CAR (or EpCAM-CAR).

[0063] Fig.18 The cytokine IFN-γ release level of T cells infected with PD1-CD27 and EGFR-CAR.

[0064] Fig.19 The levels of cytokine IFN-γ released by T cells infected with PD1-CD27 and EpCAM-CAR. DETAILED DESCRIPTION

[0065] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0066] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0067] The packaging plasmids psPAX2 and pMD2.G in the following examples are vectors preserved in the applicant's laboratory. For details, see the non-patent literature, Deng C, Zhao J, Zhou S, Dong J, Cao J, Gao J, Bai Y, Deng H. The Vascular Disrupting Agent CA4P Improves the Antitumor Efficacy of CAR-T Cells in Preclinical Models of Solid Human Tumors. Mol Ther. 2020 Jan 8; 28(1): 75-88.

[0068] The human peripheral blood mononuclear cells (PBMC) in the following examples were derived from the peripheral blood of healthy volunteers.

[0069] The lentiviral vector pRRL-SIN-GFP.WPRE is recorded in the following document: Zhao Y, Stepto H, Schneider CK. Development of the First World Health Organization Lentiviral Vector Standard: Toward the Production Control and Standardization of Lentivirus-Based Gene Therapy Products. Human Gene Therapy Methods. 2017; 28 (4): 205-214. The pRRL-SIN-GFP.WPRE plasmid used in this application is a self-inactivating (SIN) lentiviral vector, which is safer than traditional lentiviral vectors. The original promoter PGK was replaced with elongation factor-1α (EF1A) using restriction endonucleases BamHI and BstXI, called pRRL-SIN-EF1A-GFP.WPRE vector, named pLV-EF1A lentiviral vector (referred to as pLV lentiviral vector).

[0070] Example 1. Design and structure of combined PD1-CD27 gene

[0071] Combined PD1-CD27 structure and coding sequence: The combination consists of PD1 signal peptide, extracellular region (PD1-ect) and transmembrane region (TM), as well as CD27 intracellular region and tag protein hemagglutinin (HA) (structure as shown in Figure 1 The nucleotide sequence of the PD1-CD27 gene is shown in SEQ ID No. 1.

[0072] Positions 1-60 of SEQ ID No. 1 are the signal peptide coding sequence of PD1;

[0073] Positions 61-510 of SEQ ID No. 1 are the coding sequence of the extracellular region of PD1;

[0074] Positions 511-579 of SEQ ID No. 1 are the coding sequence of the transmembrane region of PD1;

[0075] Positions 580-723 of SEQ ID No. 1 are the coding sequence of the intracellular region of CD27;

[0076] Positions 724-750 of SEQ ID No. 1 are the coding gene of HA.

[0077] Example 2. Design and structure of Meso-CAR PD1-CD27 gene

[0078] 1. CAR design: Chimeric antigen receptor (CAR) is composed of CD8α or immunoglobulin light chain signal peptide (SP), the extracellular region of the single-chain antibody domain (scFv) that recognizes the antigen, the hinge region (Hinge) and transmembrane region (TM) of CD8α, the intracellular co-stimulatory molecule 4-1BB (or CD28) and the signal domain CD3ζ (CD3z) (structure as shown in Figure 2). Figure 1 (shown in B).

[0079] 2. The CAR designed in step 1 is connected to the combined PD1-CD27 structure designed in Example 1 through the self-cleaving peptide P2A to obtain the CAR PD1-CD27 gene of the present invention.

[0080] The CAR PD1-CD27 gene consists of CD8α signal peptide, scFv (tumor antigen binding region, i.e. single-chain antibody region), CD8α hinge region and transmembrane region, 4-1BB (or CD28, intracellular signal region), CD3ζ (intracellular signal region), P2A, PD1 extracellular region and transmembrane region, CD27 intracellular region, and HA from N-terminus to C-terminus. The structural diagram is shown in the figure below. Figure 1 As shown in C.

[0081] Among them: the tumor antigen binding region (antigen recognition region) can be a single-chain variable region (scFv) of an antibody that specifically recognizes tumor cell surface antigens EpCAM, HER2, EGFR or mesothelin, etc., which is composed of a heavy chain (VH), a linker, and a light chain (VH). The extracellular region of PD1 recognizes its ligand PD-L1 or PD-L2.

[0082] The co-stimulatory signal molecules selected are the intracellular segments of CD137 (4-1BB, a member of the TNF receptor superfamily) or CD28, and the intracellular signal transduction region of CD3ζ (CD3z). P2A is connected to the extracellular region and transmembrane region of PD1, and the intracellular region of CD27. HA is added to the carboxyl end of the combined encoded protein to distinguish endogenous and exogenous PD1 and CD27.

[0083] P2A (GSGATNFSLLKQAGDVEENPGP, SEQ ID No. 8) is a "self-cleaving" peptide. The "self-cleaving" function of the P2A peptide forms two proteins, an upstream product and a downstream product, by self-cleavage. Those skilled in the art may replace the P2A gene in the CAR PD1-CD27 gene, for example, using F2A (VKQTLNFDLLKLAGCVESNPG), T2A (EGRGSLLTCGDVEENPG), E2A (QCTNYALLKLAGDVESNPG) and the like having the same "self-cleaving" function as the P2A gene.

[0084] 3. Sequence of the combined Meso-CAR PD1-CD27 gene

[0085] Based on the design of steps 1-2 above, this embodiment constructs a specific CAR PD1-CD27 gene, named: Meso-CAR PD1-CD27 gene. The antigen recognition region of the Meso-CAR PD1-CD27 gene is a single-chain variable region (scFv) of an anti-Mesothelin antibody, which specifically recognizes the antigen Mesothelin.

[0086] The Meso-CAR PD1-CD27 gene is composed of CD8α signal peptide, Mesothelin scFv, CD8α hinge region and transmembrane region, 4-1BB, CD3ζ, P2A, PD1 extracellular region and transmembrane region, CD27 intracellular region and HA tag from N-terminus to C-terminus. The nucleotide sequence of the Meso-CARPD1-CD27 gene is shown in SEQ ID No.3 (the corresponding encoded amino acid sequence is shown in SEQ ID No.6). Under the action of the "self-cleavage" function of the P2A peptide, it expresses the Meso-CAR protein with an amino acid sequence of SEQ ID No.5 and the combined protein PD1-CD27 with an amino acid sequence of SEQ ID No.4. The Meso-CAR protein is encoded by the Meso-CAR gene (SEQID No.2); the combined protein PD1-CD27 is encoded by the PD1-CD27 gene (SEQ ID No.1). Among them:

[0087] Positions 1-63 of SEQ ID No. 3 are the coding gene for CD8α signal peptide;

[0088] Positions 64-789 of SEQ ID No. 3 are the coding gene of Mesothelin scFv;

[0089] Positions 790-996 of SEQ ID No. 3 are the genes encoding the hinge region and transmembrane region of CD8α;

[0090] Positions 997-1122 of SEQ ID No. 3 are the coding gene of 4-1BB;

[0091] Positions 1123-1458 of SEQ ID No. 3 are the coding gene of CD3ζ;

[0092] Positions 1459-1524 of SEQ ID No. 3 are the coding gene of P2A;

[0093] Positions 1525-1539 of SEQ ID No. 3 are restriction enzyme cleavage sites and connection sequences;

[0094] Positions 1540-1599 of SEQ ID No.3 are the coding genes for the PD1 signal peptide

[0095] Positions 1600-2118 of SEQ ID No. 3 are the genes encoding the extracellular and transmembrane regions of PD1;

[0096] Positions 2119-2262 of SEQ ID No. 3 are the genes encoding the intracellular region of CD27;

[0097] Positions 2263-2289 of SEQ ID No. 3 are the coding gene of HA.

[0098] Positions 1 to 1458 of SEQ ID No. 3 are the coding gene (Meso-CAR gene) of Meso-CAR (amino acid sequence is SEQ ID No. 5);

[0099] Positions 1540-2289 of SEQ ID No. 3 are the coding gene for the combined protein PD1-CD27 (amino acid sequence is SEQ ID No. 4).

[0100] Example 3. Construction of CAR-T cells

[0101] This example takes the mesothelin target as an example to describe the preparation of common second-generation CAR-T cells with 4-1BB as a co-stimulatory factor and fused PD1-CD27 CAR-T cells of the present invention. The Meso-CAR PD1-CD27 gene (SEQ ID No. 3) and the Meso-CAR gene (SEQ ID No. 2) were transferred into T cells for stable expression, and the obtained CAR-T cells were named Meso-CAR PD1-CD27 T cells and Meso-CAR T cells. The specific steps are as follows:

[0102] 1. Construction of recombinant lentiviral vector

[0103] The lentivirus pLV was selected as the basic vector, and the PD1-CD27 gene (SEQ ID No.1, synthesized by Beijing Liuhe BGI Gene Technology Co., Ltd.) and Meso-CAR gene (SEQ ID No.2, synthesized by Beijing Hesheng Biotechnology Co., Ltd.) were cloned into the pLV lentivirus basic vector using restriction endonucleases XbaⅠ and SalⅠ, respectively. Finally, two recombinant lentivirus vectors were obtained, which were named as: pLV-PD1-CD27 ( Figure 2 Middle A) and pLV-Meso-CAR ( Figure 2 Then, using the two endonuclease sites of BamHⅠ and SalⅠ of pLV-Meso-CAR, P2A-PD1-CD27 was linked to pLV-Meso-CAR and named pLV-Meso-CARPD1-CD27 ( Figure 2 Middle C).

[0104] The lentiviral plasmid vector pLV uses the Human elongation factor-1α (EF1A) promoter to start the expression of the combined PD1-CD27, the second-generation CAR gene (i.e., the Meso-CAR gene), or the Meso-CAR PD1-CD27 gene. The combined PD1-CD27 consists of the PD1 signal peptide, the extracellular region and the transmembrane region, the CD27 intracellular region, and HA; the second-generation CAR consists of the CD8α signal peptide that recognizes tumor-associated antigens, scFv, the CD8α hinge region (Hinge) and the transmembrane region (TM), 41-BB, and CD3ζ; the Meso-CARPD1-CD27 consists of the CD8α signal peptide, scFv, the CD8α hinge region and the transmembrane region, 41-BB, CD3ζ, P2A, PD1-CD27, and HA.

[0105] 2. Lentivirus Packaging

[0106] The combined PD1-CD27, second-generation CAR gene (Meso-CAR) or Meso-CAR PD1-CD27 gene are introduced into packaging cells for packaging, completing virus assembly to obtain recombinant lentivirus. The specific steps of lentivirus packaging are as follows:

[0107] 1. Cell culture: HEK293T cells were cultured in DMEM medium containing 10% serum at a rate of about 5×10 6 Cells were plated on a 100 mm culture dish at a density of 100 cells / ml. After the cells adhered to the wall, the confluence was preferably 50%-70%. The cells were placed in a 37°C incubator containing 5% CO2 and incubated for 8-24 hours. When the cells were completely attached to the wall, transfection could be started. The medium was changed 2 hours before transfection (8 ml of fresh complete medium was used to replace the old medium). The preparation method of complete medium is: commercial DMEM medium (Hyclone) was added with fetal bovine serum (Gemini Foundation), and the final concentration was 10%.

[0108] Note: To obtain a higher transfection efficiency, try to use exponentially growing cells and the cell density should not exceed 80% during transfection.

[0109] 2. Transfection reagent VigoFect is a commercial transfection reagent, a product of Vigolas Biotechnology (Beijing) Co., Ltd. Add plasmid DNA to sterile water and package according to the three-plasmid system of lentiviral vector. The target plasmids (pLV-PD1-CD27, pLV-Meso-CAR, pLV-Meso-CAR PD1-CD27), packaging plasmids psPAX2 and pMD2.G are mixed at a ratio of 16μg, 12μg, and 4μg (4:3:1), with a total of 32μg. Add 500μl of serum-free DMEM and mix well. Then add 15μl of VigoFect to 500μl of serum-free DMEM and mix well. Mix the two again and let stand at room temperature for 20 minutes. Then slowly add the DMEM mixed with plasmids and VigoFect to the HEK293T cell culture dish, and place the cells in a cell culture incubator for incubation.

[0110] 3. After 5-7 hours, change the medium and add 8-10 ml of DMEM medium preheated at 37°C. Continue to incubate the cells in the incubator, harvest the recombinant lentiviral supernatant, and observe the transfection efficiency under a fluorescence microscope after 16-40 hours.

[0111] 3. Concentration of recombinant lentivirus

[0112] 1. Preparation of 5×PEG8000 NaCl: weigh 8.766 g of NaCl; dissolve 50 g of PEG8000 in 200 ml of Milli-Q pure water; sterilize with wet heat at 121°C for 30 min; store at 4°C.

[0113] 2. Use a 0.45 μm filter to filter the recombinant lentiviral supernatant.

[0114] 3. Add 7.5 ml of 5×PEG8000 NaCl stock solution to every 30 ml of filtered virus initial solution.

[0115] 4. Mix every 20 to 30 minutes, for a total of 3 to 5 times; leave at 4°C overnight.

[0116] 5. Centrifuge at 4000 g for 20 min at 4°C; discard the supernatant, let the tube stand for 1 to 2 minutes, and then aspirate the remaining liquid.

[0117] 6. Add an appropriate amount of lentivirus dissolution solution to dissolve the lentivirus precipitate; the concentrated virus suspension is divided into 50 μl portions and stored in finished product tubes. The recombinant lentivirus solution is obtained, which is quickly frozen with crushed dry ice and stored at -80°C.

[0118] IV. Determination of recombinant lentivirus titer

[0119] Trypsinize and count HEK293T cells to 2 × 10 5 -3×105 / well was inoculated into a 6-well plate. Place in a 37℃ 5% CO2 incubator and culture until the cells are 50% confluent. Take the recombinant lentiviral solution, add 0.5μl, 1μl, 5μl, and 10μl to the EP tube, add polybrene to a final concentration of 8μg / mL, and add fresh culture medium to a final volume of 1 mL. Discard the HEK293T cell culture medium, add the above-mentioned virus solutions of each concentration (including the stock solution) to the above-mentioned 6-well plate, and incubate in a 37℃ 5% CO2 incubator. After 48h, digest the HEK293T cells and detect the CAR positivity rate of HEK293T cells by flow cytometry.

[0120] Virus titer (TU / mL) = GFP positive cell rate × total number of cells / lentiviral solution volume × 1000. To avoid errors, only wells with a positive rate of 5% to 50% were selected for calculation, and the average value of each group was taken to calculate the titer.

[0121] 5. Recombinant Lentivirus Infection of T Cells

[0122] 1. Acquisition and identification of T cells

[0123] 30 mL of peripheral blood was collected from volunteers, and peripheral blood mononuclear cells (PBMC) were separated by Ficoll. The separated PBMC were stimulated with anti-CD3 and CD28 magnetic beads for 3 days, and the activated T cells were collected and cultured in X-Vivo15 medium (Lonza product, product number BEBP02-054Q) containing 100 IU / mL interleukin 2 (IL2). Flow cytometric analysis showed that CD3-positive T cells accounted for more than 95%.

[0124] Peripheral blood mononuclear cells (PBMCs) isolated from human peripheral blood were stimulated and activated with CD3 and CD28 antibody magnetic beads (anti-CD3 / anti-CD28 Dynabeads, Gibco) for 3 days, and activated T cells were collected and detected by flow cytometry. + ) cells, the positive rate of T cells was 99.2% ( Figure 3 ).

[0125] 2. The steps of recombinant lentivirus infection of T cells are as follows:

[0126] (1) Place the recombinant lentiviral solution stored at -80°C in an ice box and allow it to slowly thaw.

[0127] (2) Take the activated T cells and culture them at 2×10 6 -4×10 6 / well were inoculated into 6-well plates.

[0128] (3) Add polybrene to a final concentration of 8 μg / mL, add the recombinant lentivirus solution dropwise, shake well, and centrifuge at 350 g for 60 minutes at room temperature.

[0129] (4) Place in a 37°C 5% CO2 incubator and culture for 24 hours.

[0130] (5) Repeat step 4 for the second virus infection (optional).

[0131] (6) After culturing in a 37°C 5% CO2 incubator for 24 hours, the cells were transferred to another container and continued to be cultured.

[0132] (7) 72 hours after virus infection, the infection efficiency or positive rate of infected cells was detected by flow cytometry.

[0133] Thus, when the lentivirus pLV is used as the basic vector, the combination of PD1-CD27, Meso-CAR and Meso-CAR PD1-CD27 infects T cells to obtain three types of T cells infected with the recombinant lentivirus, namely, T cells expressing the combination of PD1-CD27 with the nucleotide sequence of SEQ ID No.1, Meso-CAR with SEQ ID No.2 and Meso-CAR PD1-CD27 with SEQ ID No.3.

[0134] The constructed combined PD1-CD27 T cells, Meso-CAR T cells, and Meso-CAR PD1-CD27 T cells were cultured at 37°C using X-Vivo15 medium containing 100 IU / mL interleukin 2 (IL2) for subsequent functional tests.

[0135] 3. Infection efficiency of recombinant lentivirus infecting T cells

[0136] The lentiviral infection rate was detected using Anti-mouse F(ab')2Alexa Flour 647 antibody (Alexa Fluor 647-conjugated goat anti-mouse IgG, F(ab')2fragments specific, Jackson, USA). If the CAR plasmid vector contained green fluorescent protein (GFP), GFP was used for detection. Flow cytometric analysis was performed with cells expressing isotype control IgG as control. The results showed that CD3 + The positive rate was above 90%, the infection rate of Meso-CAR T cells was 34.3%, and the positive rate of Meso-CAR PD1-CD27 T cells was 29.7% ( Figure 4Middle A). At the same time, the antibody of the tag protein HA is used to detect the HA protein in the cell. The cell membrane needs to be punctured and fixed first, which may have a slight effect on the detection of CD3. The Meso-CAR PD1-CD27 T cells detected by HA and the CD3 of PD1-CD27 T cells + The positive rates of T cells were 25.7% and 34.0% ( Figure 4 (middle B).

[0137] 6. The role of tumor cells, CAR-T, and PD1-CD27 fusion CAR-T cells

[0138] Compared with normal cells, tumor cells highly express tumor-associated antigens, such as mesothelin, HER2, EGFR, or EpCAM. Tumor target cells are cell lines that stably express tumor-associated antigens (TAA) (such as SKOV3-Meso). The scFv of the second-generation CAR T can specifically recognize TAA and stimulate a specific killing response. After tumor cells are stimulated by gamma-interferon (IFN-γ) produced by T cells, the expression of PD1-L1 or PD-L2 is upregulated, which can bind to PD1 of T and CAR-T cells, inhibit T cell activation and cytotoxicity, and cause T cell exhaustion ( Figure 5 The fused PD1-CD27 CAR-T cells designed in the present invention recognize target cells similarly to the second-generation CAR T cells. After PD1-L1 or PD-L2 binds to PD1, since a portion of the intracellular signal of PD1 is CD27, the T cell exhaustion signal of PD1 can be reversed. This type of T cell can survive longer and maintain partial cytotoxicity ( Figure 5 (middle B).

[0139] Example 4. Expression of PD1 and T cell phenotype identification in fused PD1-CD27 CAR-T cells and second-generation CAR-T cells

[0140] The test cells were PD1-CD27 T cells, Meso-CAR T cells, Meso-CAR PD1-CD27 T cells and virus-uninfected T cells (Sti-T) constructed in Example 3.

[0141] 1. Phenotypic identification:

[0142] The cells were cultured in vitro for 10 days and the CD3 +T cell phenotype identification: CD8 positivity of uninfected T cells was approximately 28.3%, and CD4 positivity of T cells was 51.6%; CD8 positivity of PD1-CD27 T cells was approximately 27.2%, and CD4 positivity of T cells was 52.9%; CD8 positivity of Meso-CAR T cells was approximately 31.5%, and CD4 positivity of T cells was 50.9%; CD8 positivity of Meso-CAR PD1-CD27 T cells was approximately 30.0%, and CD4 positivity of T cells was 52.6% ( Figure 6 ), Meso-CAR PD1-CD27 T cells CD4 + The signal of some cells showed an obvious left shift.

[0143] 2. Expression of PD1 in CD3-positive T cells cultured in vitro for 10 days:

[0144] 2-1. Grouping: CD3 stimulated by CD3 and CD28 antibody magnetic beads + T cell (Sti-T) group, second-generation Meso-CAR T cell group, PD1-CD27 T cell group, and Meso-CAR PD1-CD27 T cell group. More than 95% of the cells in these groups were CD3 + The PD1 positivity rates of Sti-T, Meso-CAR T, PD1-CD27 T, and Meso-CAR PD1-CD27 T were 37.4%, 69.5%, 59.8%, and 53.5%, respectively. Figure 7 ), PD1 antibodies cannot distinguish endogenous PD1 from exogenously infected PD1. If we refer to the HA test results, PD1-CD27 T, Meso-CAR PD1-CD27 CD3 + The infection positive rates of T cells were 34.0% and 25.7% respectively ( Figure 4 In B), the positivity rates of the two engineered T cells PD1 were estimated to be 25.8% and 27.8%, respectively, which were significantly lower than those of Meso-CAR PD1-CD27 CD3 + 69.5% of T cells.

[0145] 2-2. Phenotypic analysis of T cells cultured in vitro at different times:

[0146] Flow cytometry was used to analyze the phenotypes of Meso-CAR T cells and Meso-CAR PD1-CD27 T cells after 7 days of in vitro culture. The CD62L and CD45RO double-positive cells were memory (Tmem) cells, and CD62L+CD45RO- were naive cells. T cells, CD62L-CD45RO+ are effector (Teff) CD3+ T cells. The percentage of CD62L and CD45RO double positive cells in the Meso-CAR T cell group was 70.7%, while in the Meso-CAR PD1-CD27 T cell group, the percentage of CD62L and CD45RO double positive cells was 71.9%, which was not much different, and the latter was slightly higher than the former ( Figure 8 ), CD62L positive percentages were 93.2% and 97.9%, respectively, and the Meso-CAR PD1-CD27 T cell group was not much different from the Meso-CAR T cell group. After 21 days of in vitro culture, the double positive percentages of CD62L and CD45RO in the Meso-CAR T cell group and the Meso-CAR PD1-CD27 T cell group were 26.9% and 42.6%, respectively; after 28 days of in vitro culture, the double positive percentages of CD62L and CD45RO in these two groups of cells were 3.88% and 34.5%, respectively, which were very significant differences ( Figure 8 ). It showed that the number of CD62L and CD45RO double-positive memory T cells in long-term in vitro cultured fusion Meso-CAR PD1-CD27 T cells was significantly higher than that of Meso-CAR T cells.

[0147] 2-3. Analysis of cell exhaustion degree:

[0148] Since PD1 in T or CAR-T cells can be produced endogenously or from exogenous infection, we first used flow cytometry to analyze the exhaustion indicators of T cells LAG3 and TIM3 cultured in vitro for 14 days. The three groups were CD3 / CD28 magnetic bead-stimulated T cells (Sti-T), Meso-CAR T cells, PD1-CD27 T cells, and Meso-CAR PD1-CD27 T cells. The expression of LAG3 and TIM3 in CD3-positive T cells was analyzed. + The expression of LAG3 and TIM3 in Sti-T cells was 3.5% and 18.7%, respectively, and CD3 + The percentages of these two indicators of Meso-CAR T cells reached 47.6% and 47.1%, and CD3 + The percentages of PD1-CD27 T cells were 28.1% and 22.6%, respectively, and CD3 + The percentages of these two indicators of Meso-CAR PD1-CD27 T cells were 30.2% and 23.6% respectively ( Fig. 9The results showed that the expression percentage and intensity of LAG3 and TIM3 in Meso-CAR T cells were significantly higher than those in PD1-CD27 T cells and Meso-CAR PD1-CD27 T cells, indicating that the degree of exhaustion of fusion PD1-CD27 T cells and Meso-CAR PD1-CD27 T cells was significantly lower than that of Meso-CAR T cells.

[0149] 2-4. Analysis of PD1 expression in CD3-positive and CD3-negative cases:

[0150] In order to analyze cells other than T cells in human peripheral blood mononuclear cells (PBMC), we used a human T cell isolation kit (T cell isolation kit, Stemcell Technologies) to sort CD3-positive and -negative cells in human peripheral blood mononuclear PBMC, cultured and infected with viruses, and detected PD1, an indicator of T cell exhaustion. Since the PD1 antibody cannot distinguish between endogenous PD1 expression and PD1 expressed by exogenous lentivirus infection, we used the expression of the tag protein HA to estimate the exogenous Meso-CAR PD1-CD27 and PD1-CD27 to be 25.7% and 34.0% ( Figure 4 (middle B).

[0151] After 21 days of culture, the expression of PD1 in CD3 positive T cells was analyzed. + The expression of PD1 in the Sti-T cells, Meso-CART cells, PD1-CD27 T cells, and Meso-CAR PD1-CD27 T cells groups was 18.7%, 69.6%, 64.9%, and 64.8%, respectively. After deducting exogenous expression, the PD1 positive rates of PD1 in PD1-CD27 T cells and Meso-CAR PD1-CD27 T cells were 30.9% and 39.1%, respectively, which were significantly lower than the 69.6% PD1 positive rate of Meso-CAR T cells ( Fig.10 After 21 days of culture, the percentages of PD1 and HLA-DR double-positive cells in CD3-negative cells and CD3-negative lymphocytes infected with Meso-CAR, PD1-CD27, and Meso-CAR PD1-CD27 were 36.7%, 80.6%, 49.1%, and 49.5%, respectively ( Fig.10 In B), HLA-DR is one of the markers of antigen presenting cells. This suggests that the expression of PD1 can also be reduced by engineering CD3-negative lymphocytes (such as antigen presenting cells) with PD1-CD27.

[0152] Example 5. In vitro killing experiment of fused PD1-CD27 CAR-T cells and second-generation CAR-T cells

[0153] 1. Target cell preparation and related testing

[0154] 1-1. Preparation of target cells: The target cells are ovarian cancer cell line SKOV3-Meso (also known as Luciferase tumor cell line SKOV3-Meso) expressing mesothelin and luciferase. The preparation method is as follows: The full-length cDNA of mesothelin (MSLN, Mesothelin) (purchased from Sino Biological Beijing Co., Ltd., catalog number: HG13128-G) is cloned into the lentiviral expression vector pLV, and the coding sequence of luciferase is amplified by PCR using pGL4.17 (Promega) as a template, and then connected to the pLV lentiviral vector using restriction endonucleases. First, the luciferase coding gene was packaged into lentivirus using a lentivirus packaging system, and then SKOV3 cells (cell lines preserved in the inventor's laboratory, see Deng C, Zhao J, Zhou S, Dong J, Cao J, Gao J, Bai Y, Deng H. The Vascular Disrupting Agent CA4P Improves the Antitumor Efficacy of CAR-T Cells in Preclinical Models of Solid Human Tumors. Mol Ther. 2020 Jan 8; 28(1): 75-88.) were transfected to establish a SKOV3 cell line expressing luciferase. On this basis, a cell line over-expressing (OE) mesothelin was constructed and named SKOV3-Meso cell line.

[0155] 1-2. Upregulation of PD-L1 (PD1 ligand) expression in tumor cells

[0156] SKOV3-Meso ovarian cancer cells were treated with or without 1000 IU of interferon-γ (IFN-γ) for 48 hours, and the expression of PD-L1 was detected by antibody. The results showed that without IFN-γ, the positive rate of PD-L1 in tumor cells was 34.9%. After IFN-γ was added, the positive rate of PD-L1 in tumor cells increased significantly to 81.3%. Isotype is the same type control of the antibody ( Fig.11 ).

[0157] 1-3. Expression positive rate of tumor cell membrane protein (target antigen) and overexpressed tumor antigen

[0158] Specific antibodies were used to detect the expression positive rates of target antigens HER2, EpCAM and EGFR in SKOV3 ovarian cancer cells and A549 lung adenocarcinoma cells, respectively. Isotype (isotype control) was used as the control for the antibodies. The results of flow cytometry analysis showed that more than 90% of the tumor cells were HER2 positive; the expression positive rate of target antigen EpCAM in A549 lung adenocarcinoma cells was about 77%; the expression positive rate of target antigen EGFR in A549 lung adenocarcinoma cells was about 80% ( Fig.12 (A, B, C).

[0159] The expression rate of mesothelin, the target antigen of tumor cells SKOV3, is very low, and flow cytometry analysis shows that it is only about 3%. However, by using the lentiviral infection method, the gene encoding mesothelin was transferred into SKOV3 cells, and the cells overexpressing mesothelin, namely SKOV3-Meso cells, were obtained. Flow cytometry analysis showed that the expression rate of mesothelin could reach 97.2% ( Fig.12 (middle D).

[0160] 2. In vitro killing experiment

[0161] Effector cells: Meso-CAR PD1-CD27 T cells, Meso-CAR T cells, normal Sti-T cells; Target cells: SKOV3-Meso cells.

[0162] (1) Pipette about 5000 effector cells into a 96-well plate, with a culture volume of 100ul. Add target cells according to different effector-target ratios (1:1, 3:1, 9:1), with 3 replicate wells for each effector-target ratio. Place the added cells in a 37°C incubator for 12-20 hours; and prepare 3 untreated wells as controls (background readings). When the action time comes, gently blow the co-cultured cells, pipette them into EP tubes according to different effector-target ratios, and centrifuge at 1200rpm for 5min.

[0163] (2) Discard the supernatant, aspirate as much as possible, resuspend in 50ul of culture medium and add to a 96-well plate. Pipette 50ul of 150ug / ml D-luciferin and add to a light-proof 96-well plate. Incubate at room temperature for 3-5min and detect chemiluminescence using CentroXS-LB960 (Berthold Technologies, Germany).

[0164] (3) Set the instrument's measurement time, read the data, save the data, output the data to EXCEL table, and perform data processing and calculation:

[0165] Killing rate % = (background reading - experimental group reading) / background reading × 100%.

[0166] The results of in vitro killing experiments showed that Meso-CAR T cells and Meso-CAR PD1-CD27 T cells had a killing effect on tumor cells expressing mesothelin (SKOV3-Meso cells), and the killing rate increased significantly with the increase of the effector-target ratio (T cells: tumor cells), and the former was slightly higher than the latter. However, T cells (Normal T) had no significant killing effect on SKOV3-Meso cells ( Fig.13 The killing rate of Meso-CAR PD1-CD27 T cells against target cells SKOV3-Meso was about 90% ( Fig.13 ).

[0167] 3. In vitro killing experiment under simulated tumor microenvironment culture conditions

[0168] The culture supernatant of tumor cells (SKOV3-Meso cells) was filtered through a 0.45 μm filter to simulate the microenvironment of the tumor. T and CAR-T cells were cultured in this culture medium for 7 days. The remaining conditions were consistent with the method in step 2 of this embodiment, and an in vitro killing experiment was performed.

[0169] Effector cells: Meso-CAR PD1-CD27 T cells, Meso-CAR T cells, normal Sti-T cells; Target cells: SKOV3-Meso cells.

[0170] The results showed that the killing effect of Meso-CAR T cells on SKOV3-Meso cells under the conditions of simulating tumor microenvironment culture was greatly reduced, and the killing rate of 9:1 was about 40%, which was not much different from PD1-CD27 T cells. The killing rate of Meso-CAR PD1-CD27 T cells on the tumor cells decreased by about 20%, and the killing rate of 9:1 was about 70%, which was significantly higher than that of Meso-CAR T cells ( Fig.14 ). It shows that in the tumor microenvironment, CAR PD1-CD27 T cells have a stronger killing effect on tumor cells.

[0171] Example 6: Cytokine IFN-γ Release Test of CAR-T Cells

[0172] The tested effector cells were: Meso-CAR PD1-CD27 T cells, Meso-CAR T cells, virus-uninfected T cells (Sti-T), and GFP-infected T cells (GFP-T); the target cells were: SKOV3-Meso cells.

[0173] The commercial kit (Human precoated ELISA Kit, IFN-γ, produced by Shenzhen Dakoway Biotechnology Co., Ltd., catalog number: DKW12-1000-096) detects the cytokine IFN-γ of T or CAR-T cells. The main detection steps are as follows:

[0174] (1) Effector cells were added to a 96-well plate at a density of 5,000 cells (100 μL per well). After the target cells adhered to the plate the next day, the culture medium was removed. Engineered T cells were added at different effector-target ratios.

[0175] (2) Each effector-target ratio has 3 replicate wells, and the added cells are placed in a 37°C incubator for 20 hours; and 3 untreated wells are prepared as controls (background readings). When the action time comes, gently blow the co-cultured cells, and pipette them into EP tubes according to different effector-target ratios, and centrifuge at 1200 rpm for 5 minutes.

[0176] (3) Discard the supernatant, aspirate as much as possible, resuspend in 50 μl culture medium and add to a 96-well plate. Pipette 50 μl of 150 μg / ml D-luciferin and add to a light-proof 96-well plate. Incubate at room temperature for 3 to 5 min and detect chemiluminescence using CentroXS-LB960 (Berthold Technologies, Germany).

[0177] (4) Set the instrument’s measurement time, reading, saving, outputting EXCEL tables, data processing and calculation.

[0178] Killing rate = (background reading - experimental group reading) / background reading × 100%

[0179] (5) Place the 96-well plate in a 37°C incubator for 20 h, then transfer the culture supernatant into a 1.5 ml EP tube and place it on ice. If cytokines are not being measured temporarily, immediately transfer it to a -80°C refrigerator for storage.

[0180] (6) The supernatant with an effector-target ratio of 9:1 was collected and filtered with a 0.45 μm filter. The cytokine (IFN-γ) determination procedure was strictly performed according to the instructions of the Dakovi ELISA kit; first, the standard curve of IFN-γ was drawn using the standard substance, and then the OD value was used for calculation.

[0181] (7) Detection was performed using an enzyme reader (Spectramax i3, Molecular Devices) and the absorbance OD was measured at 450 nm.

[0182] (8) According to the standard curve, the number of T cells (Sti-T) that were not infected with the virus after CD3 / CD28 magnetic bead stimulation, Meso-CAR T cells and Meso-CAR PD1-CD27 T cells cultured with X-vivo15 + 10% serum, and Con Meso-CAR and Con Meso-CAR PD1-CD27 T cells cultured in tumor cell culture medium for 7 days were calculated, and SKOV3-Meso cell killing was performed, and the supernatant was collected to detect the IFN-γ content.

[0183] The results are as follows Fig.14 As shown in the figure, the IFN-γ content of different groups with an effector-target ratio of 9:1 was detected, and the IFN-γ content of Meso-CAR T cells and Meso-CAR PD1-CD27 T cells in the X-vivo culture medium was significantly higher than that in the Sti-T group, and the Meso-CAR T cells were about 1400pg / mL, slightly higher than the 1100pg / mL of Meso-CAR PD1-CD27 T cells. However, the IFN-γ content of Con Meso-CAR cultured in tumor cell culture medium was 280pg / mL, which was much lower than the 890pg / mL of Con Meso-CAR PD1-CD27 T cells ( Fig.15 ). It showed that in the tumor culture medium (tumor microenvironment), the IFN-γ secreted by Meso-CAR PD1-CD27 T cells was significantly higher than that of Meso-CAR-T cells.

[0184] Example 7: Functional verification of fusion protein PD1-CD27

[0185] In order to further verify that the fusion protein PD1-CD27 of the present invention has the function of converting the PD1 inhibitory signal of T cells or CAR-T cells into the survival signal of T cells or CAR-T cells of CD27, improving the tumor killing activity of T cells or CAR-T cells, and promoting the release of cytokine IFN-γ, thereby improving the anti-tumor effect of T cells or CAR-T cells, and verifying that it can be applied to different antigens (EpCAM, HER2 or EGFR) other than mesothelin, the present invention further designs and constructs HER2-CAR (co-stimulatory factor is CD28) and HER2-CAR PD1-CD27, and also constructs the second-generation CAR of EpCAM and EGFR. When the antigen is EpCAM, the nucleotide sequence of the nucleic acid molecule used to modify T cells is shown in SEQ ID No. 11; when the antigen is HER2, the nucleotide sequence of the nucleic acid molecule used to modify T cells is shown in SEQ ID No. 9; when the antigen is EGFR, the nucleotide sequence of the nucleic acid molecule used to modify T cells is shown in SEQ ID No. 10; the construction method and experimental method are the same as Meso-CARPD1-CD27 T cells.

[0186] The applicant first stimulated T cells (Sti-T) with CD3 / CD28 magnetic beads for 3 days, and infected Sti-T cells with lentivirus containing PD1-CD27 (containing HA tag) to obtain PD1-CD27 T cells. Infect Sti-T cells with EGFR-CAR or EpCAM-CAR to obtain EGFR-CAR or EpCAM-CAR T cells; at the same time, infect PD1-CD27 T cells with lentivirus containing EGFR-CAR or EpCAM-CAR to obtain PD1-CD27+EGFR-CAR or PD1-CD27+EpCAM-CAR T cells, and use Anti-mouse F(ab')2 antibody to detect the positive rate of CAR-T ( Fig.17 ). The results of flow cytometry analysis showed that isotype IgG (Isotype) was used as a control ( Fig.17 A, B left), after infection with PD1-CD27, the percentages of HA (PD1-CD27) positive cells in CD3+ T cells were 65.1% and 60.4% ( Fig.17 A, B middle). After continued infection with EGFR-CAR or EpCAM-CAR, the positive rates of PD1-CD27+EGFR-CAR and PD1-CD27+EpCAM-CAR T cells were 31.2% and 30.4%, respectively ( Fig.17 A, B right).

[0187] Sti-T, PD1-CD27, EGFR-CAR and PD1-CD27+EGFR-CAR T cells with an effector-target ratio of 10:1 were co-cultured with EGFR+ A549 tumor cells. Fig.12 The results showed that most A549 cells were EGFR+ cells. After 24 hours, the level of cytokine IFN-γ was detected by ELISA (the method was the same as that of IFN-γ determination in Example 6). Fig.18 As shown in Figure 2, the IFN-γ secreted by EGFR-CAR and PD1-CD27+EGFR-CAR T cells exceeded 1900 pg / mL, which was significantly higher than 488 pg / mL of PD1-CD27 T cells and 71 pg / mL of Sti-T cells ( Fig.18 ).

[0188] A similar method was also used to detect EpCAM-CAR and PD1-CD27+EpCAM-CAR T cells. The majority of A549 cells were EpCAM-positive cells ( Fig.12). Sti-T, PD1-CD27, EpCAM-CAR and PD1-CD27+EpCAM-CAR T cells with an effector-target ratio of 10:1 were co-cultured with A549 tumor cells. After 24 hours, the level of cytokine IFN-γ was detected by ELISA (the method was the same as that of IFN-γ determination in Example 6). The results are shown in Fig.19 As shown in the figure, the IFN-γ secreted by EpCAM-CAR and PD1-CD27+EpCAM-CAR T cells exceeded 2000pg / mL, which was significantly higher than the 617pg / mL of PD1-CD27 T cells and 109pg / mL of Sti-T cells. The IFN-γ secretion of PD1-CD27 T cells reached 600pg / mL, which was higher than that of the control Sti-T cells ( Fig.19 ).

[0189] It can be seen that the EGFR and EpCAM CAR-T cells engineered with PD1-CD27 interact with target cells to produce cytotoxic cytokine IFN-γ at a level similar to that of CAR-T cells alone, and significantly higher than that of control Sti-T cells and PD1-CD27 cells. This suggests that both EGFR and EpCAM CAR-T cells and the two CAR-T cells engineered with PD1-CD27 have cytotoxic effects.

[0190] The present invention compares the usual culture conditions (X-vivo15 and 10% serum) and the conditions of culturing HER2+ SKOV3 tumor cells in culture medium (simulating tumor microenvironment) for 7 days. Fig.12 Under normal culture conditions, HER2-CAR T cells and HER2-CAR PD1-CD27 T cells have a killing effect on HER2+ tumor cells (SKOV3 cells), and the killing rate increases significantly with the increase of the effector-target ratio (T cells: tumor cells). However, T cells not infected with CAR (Sti-T) have no significant killing effect on SKOV3 cells ( Fig.16 ); Under conditions that simulate the tumor microenvironment, the killing effect of HER2-CAR T cells on HER2+ tumor cells was greatly reduced to less than 30%, while the killing rate of HER2-CAR PD1-CD27 T cells on target cells reached more than 75%, and the difference between the two was significant ( Fig.16 ). This suggests that under conditions that simulate the tumor microenvironment, CAR-T cells engineered with PD1-CD27 have the ability to kill tumor cells (cytotoxic effect), while ordinary CAR-T cells have greatly reduced or almost no toxic effect, indicating that CAR-T is in a state of disability or exhaustion.

[0191] The above results show that CAR-T cells modified with PD1-CD27 can be cultured for a longer period of time in vitro, and the proportion of memory T cells is higher than that of CAR-T cells without PD1-CD27 modification. They can specifically recognize and kill tumor cells expressing these tumor-associated antigens (mesothelin, HER2, etc.), and have the effect of resisting the tumor cell microenvironment and resisting T cell dysfunction or exhaustion.

[0192] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A fusion protein, characterized in that The fusion protein includes the extracellular region and transmembrane region of PD1 and the intracellular region of CD27. The amino acid sequence of the fusion protein is shown in SEQ ID No.

4.

2. A fusion gene, characterized in that: The fusion gene encodes the fusion protein according to claim 1, and the nucleotide sequence of the fusion gene is shown in SEQ ID No.

1.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises the fusion gene of claim 2 and a gene encoding a chimeric antigen receptor, wherein the gene encoding the chimeric antigen receptor and the fusion gene are connected via a gene encoding a self-cleaving peptide, and the chimeric antigen receptor targets mesothelin, EpCAM, HER2 or EGFR.

4. The nucleic acid molecule according to claim 3, characterized in that The self-cleaving peptide includes P2A, F2A, T2A or E2A.

5. The nucleic acid molecule according to claim 3 or 4, characterized in that The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID No.3, SEQ ID No.9, SEQ ID No.10 or SEQ ID No.

11.

6. A modified T cell, characterized in that The modified T cells are T cells modified by the fusion protein of claim 1, or T cells modified by a fusion gene formed by a gene encoding the fusion protein of claim 1 and a gene encoding a chimeric antigen receptor.

7. The modified T cell according to claim 6, characterized in that The modified T cell contains the fusion gene according to claim 2, or contains the nucleic acid molecule according to any one of claims 3-5.

8. Any of the following uses of the fusion protein of claim 1, the fusion gene of claim 2 and / or the nucleic acid molecule of any one of claims 3 to 5: A1) Application in converting the PD1 inhibitory signal of T cells or CAR-T cells into a survival signal of T cells or CAR-T cells of CD27; A2) Use in the preparation of products for immunosuppression and / or T cell depletion against tumor microenvironment; A3) Use in the preparation of T cells or CAR-T cells for preventing or treating tumors; A4) Use in the preparation of products that enhance the tumor killing activity of T cells or CAR-T cells; A5) Application in the preparation of products that promote the release of cytokine IFN-γ, thereby improving the anti-tumor effect of T cells or CAR-T cells; A6) Use in the preparation of drugs for preventing or treating tumors; A7) Use in the preparation of drugs for inhibiting and / or killing tumor cells.

9. The use according to claim 8, characterized in that: The tumor is a tumor expressing mesothelin antigen, EpCAM antigen, HER2 antigen or EGFR antigen.

10. An engineered cell method for reversing PD1 signals of T cells and / or CAR-T cells using CD27, characterized in that: The method comprises replacing the intracellular domain of PD1 of T cells and / or CAR-T cells with the intracellular domain of CD27, and the replacement method comprises modifying T cells using the fusion protein of claim 1, the fusion gene of claim 2, or the nucleic acid molecule of any one of claims 3-5.

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