CN109400713B - Use of novel chimeric antigen receptor modified T cells for the treatment of cancer - Google Patents

Abstract

The invention discloses a novel chimeric antigen receptor and application of a T cell modified by the same in treating cancer. The novel chimeric antigen receptor consists of an extracellular signal peptide, an antigen binding domain, an intracellular first conduction domain and an intracellular second conduction domain, and comprises an intracellular first conduction domain NKp44 or an intracellular first conduction domain TREM 1. The invention separates and purifies a plurality of chimeric antigen receptor nucleotide sequences and provides a chimeric antigen receptor and CAR-T cells which are specifically aimed at CD19 malignant hematological malignancy and mesothelin malignant solid tumor antigens. In a killing test of blood tumor and solid tumor, the killing capability of CAR-T cells to tumor cells is obviously enhanced, and good safety and anti-tumor activity are shown in clinical application.

Description

Use of novel chimeric antigen receptor modified T cells for the treatment of cancer

Technical Field

The invention relates to the technical field of tumor immunotherapy and provides a composition and a method for treating human cancers. The invention includes T cells that involve genetic engineering modifications to express a CAR, wherein the CAR includes an antigen binding domain, a first conduction domain, and a second conduction domain. The invention also relates to nucleic acids encoding such transmembrane polypeptides, vectors, and immune cells expressing the CAR on their surface for use in immunotherapy. The present invention opens the way to effective adoptive immunotherapy strategies for the treatment of cancer.

Background

With the development of tumor immunology theory and clinical technology, chimeric antigen receptor T-cell therapy (CAR-T) is one of the most promising tumor immunotherapy at present [ Schmitz M, et al. 10.1155/2010/956304.]. Chimeric Antigen Receptors (CARs) are a core component of CAR-T, CARs can redirect their specificity and reactivity towards selected immune cells, thus conferring on T cells the ability to recognize tumor antigens in an HLA-independent manner, which makes CAR-engineered T cells capable of activating and proliferating, and thus capable of efficiently killing tumor cells, unlike normal T Cell Receptor (TCR) responses, independent of MHC restriction. Chimeric Antigen Receptors (CARs) express synthetic proteins on T cells that fuse antigen recognition fragments of antibodies (e.g., antibody single chain variable fragments) to intracellular signaling domains. Upon interaction with a target cell expressing a cognate antigen of the scFv, expression of the CAR expressed on the T cell causes the T cell to activate killing of the target cell. Current CAR these studies employ second generation CAR-T cells based on fusion of scFv and the intracellular domain of CD3 zeta chain to form a chimeric receptor, with the addition of a 4-1BB or CD28 costimulatory molecule, and third generation CAR-T cells based on second generation CAR-T cells, with two costimulatory molecules 4-1BB and CD28 in tandem. Adoptive immunotherapy based on T lymphocytes achieves a certain effect in some tumors, and this immunotherapy method can overcome the defects of antibody therapy, but the curative effect in most tumors is still unsatisfactory [ Grupp SA, et al, additive cellular therapy. curr topicrobiol immunol.,. 2011; 344: 149-72.]. These CARs are designed based on a single chimeric molecule capable of eliciting an antigen-specific T cell response, but the innate immune receptor is typically a multi-chain complex composed of separate ligand binding regions and an ITAM-containing signal chain, such as the T cell receptor TCR-CD3 complex. The potential benefits of multi-chain immunoreceptor complexes are manifold, including greater signal diversity achieved through multiple interactions between ligand-binding and signaling molecules, as well as sustained ITAM signaling. Related studies report that a new Generation of CARs uses the transmembrane receptor DAP12 in combination with a killer immunoglobulin-like receptor (KIR) to form the signaling structure of CARs [ Generation of patent T-cell stimulating for cancer using DAP12-Based multiple immunoglobulin transduction receptors, cancer Immunology Research,2015,3(7):815-826 ], among many ligands, DAP12 is of great interest because of its ability to bind to multiple activating receptors to form an immunoactive complex and thereby mediate the activated signal by phosphorylation Syk or Zap 70.

The invention provides a novel CAR structure comprising an intracellular second transduction domain, T2A, an extracellular signal peptide, an antigen binding domain, an intracellular first transduction domain, e.g. intracellular second transduction domains DAP12, T2A, CD8 α signal peptide, a tumor antigen binding domain, an intracellular first transduction domain TREM1(DAP12-T2A-CD8 α signal peptide-VL-Linker-VH-TREM 1). The intracellular first signal conduction structure and the intracellular second signal conduction structure are used, so that CAR-T cells are facilitated to secrete lower-level cytokines when being stimulated by extracellular tumor antigens, the safety of clinical application can be well guaranteed, the CAR-T cells have good tumor killing effect in vitro and in vivo, the CAR-T cells also show remarkable curative effect in clinical treatment of solid tumors and blood tumor patients, and the cytokine release response is milder.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a novel chimeric antigen receptor with better safety and more obvious curative effect and application thereof.

It is also an object of the present invention to provide a nucleic acid encoding the chimeric antigen receptor.

The invention also aims to provide a cell containing the chimeric antigen receptor and application thereof.

A novel chimeric antigen receptor comprising an extracellular signal peptide, an antigen binding domain, an intracellular first conducting domain selected from one of NKp44, NKp46, NKp30, NKG2D, TREM1 or TREM2, and an intracellular second conducting domain selected from one of DAP12 or FcR γ.

The amino acid sequence of the first conduction domain NKp44 is preferably shown in SEQ ID NO.9, or a polypeptide having 85-99% identity with the amino acid sequence thereof.

The amino acid sequence of the first conduction domain NKp46 is preferably shown in SEQ ID NO.23, or a polypeptide having 85-99% identity with the amino acid sequence thereof.

The amino acid sequence of the first conduction domain NKp30 is preferably shown in SEQ ID NO.24, or a polypeptide having 85-99% identity with the amino acid sequence thereof.

The amino acid sequence of the first conduction domain NKG2D is preferably shown in SEQ ID NO.25, or a polypeptide having 85-99% identity with the amino acid sequence thereof.

The amino acid sequence of the first conduction domain TREM1 is preferably shown in SEQ ID NO.10, or a polypeptide with 85-99% identity with the amino acid sequence thereof.

The amino acid sequence of the first conduction domain TREM2 is preferably shown in SEQ ID NO.26, or a polypeptide with 85-99% identity with the amino acid sequence thereof.

The antigen binding domain binds an antigen associated with a malignant tumor selected from the group consisting of CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CEA, CTLA4, BCMA, CS1, c-Met, EPCAM, EGFR/EGFRvIII, gp100, GPC3, GD-2, IGF1R, IGF-I receptor, MAGEA3, mesothelin, MUC1, NY-ESO-1, HER2, PD1, PSMA, ROR1, WT1, glycolipid F77 or any other tumor antigen or other modification type and any combination thereof.

The antigen binding domain binding antigen is further preferably one of CD19 or mesothelin; the amino acid sequence of the CD19 antigen binding domain is shown as SEQ ID NO.12, and the amino acid sequence of the mesothelin antigen binding domain is shown as SEQ ID NO. 14.

The antigen binding domain consists of an antibody fragment, preferably a monoclonal antibody, Fab, scFv, VHH or other antibody fragment. The antigen binding domain antibody may be from murine, camelid or humanized antibodies. In particular embodiments, the antigen binding domain of the chimeric antigen receptor consists of a specific single chain antibody fragment of CD19 or mesothelin, including the light chain variable region (VL) and the heavy chain variable region (VH) of a single chain antibody.

In one embodiment, the variable light chain region (VL) and the variable heavy chain region (VH) of the single chain antibody of the antigen binding domain are linked by a Linker peptide (Linker), comprising a GS Linker such as (G Linker)₃S)₄Or (G)₄S)₃Preferably (G)₄S)₃A linker peptide.

The intracellular signaling domain preferably the first signaling domain comprises a sequence of NKp44, NKp46, NKp30, NKG2D, TREM1 or TREM 2. Further preferably, the second signaling domain comprises the sequence of DAP12 or FcR γ.

The intracellular second transduction domain DAP12 is in tandem with the extracellular signal peptide and the antigen binding domain via T2A. Preferably, the amino acid sequence of DAP12 is shown in SEQ ID NO. 2; the T2A amino acid sequence is shown in SEQ ID NO. 4.

In a particular embodiment, the chimeric antigen receptor preferably comprises an intracellular second signaling domain DAP12, T2A, CD8 a signal peptide, an antigen binding domain, an intracellular first signaling domain NKp44, connected in series.

In a particular embodiment, the chimeric antigen receptor preferably comprises the intracellular second signaling domain DAP12, T2A, CD8 a signal peptide, antigen binding domain, intracellular first signaling domain TREM1, connected in series.

The chimeric antigen receptor of the invention has the following structure:

DAP12-T2A-CD8α signal peptide-VL-Linker-VH-NKp44

DAP12-T2A-CD8α signal peptide-VL-Linker-VH-NKp46

DAP12-T2A-CD8α signal peptide-VL-Linker-VH-NKp30

DAP12-T2A-CD8α signal peptide-VL-Linker-VH-NKG2D

DAP12-T2A-CD8α signal peptide-VL-Linker-VH-TREM1

DAP12-T2A-CD8α signal peptide-VL-Linker-VH-TREM2

where the antigen binding domain may consist of VH-Linker-VL or VL-Linker-VH or VHH1-Linker-VHH2, VHH1 and VHH2 may recognize the same antigen, or different antigens.

Preferably, the chimeric antigen receptor comprises an amino acid sequence shown in SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21 or SEQ ID NO.22, or an amino acid sequence having 85% to 99% identity thereto.

Nucleic acid molecules encoding the aforementioned chimeric antigen receptors.

The nucleic acid molecule preferably comprises the nucleotide sequence shown in SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, or SEQ ID NO. 18.

A recombinant expression vector, preferably a lentiviral expression vector, comprising a nucleotide sequence encoding SEQ ID No.15, SEQ ID No.16, SEQ ID No.17, or SEQ ID No. 18.

A cell expressing a chimeric antigen receptor, preferably an immune cell; further preferred are immune cells differentiated by culture of T lymphocytes, NK cells, NKT cells, macrophages, mesenchymal stem cells, hematopoietic stem cells, pluripotent stem cells or embryonic stem cells.

A method for preparing a novel chimeric antigen receptor-modified T cell, which comprises isolating and activating a T cell to be modified, and then transducing the T cell with the aforementioned expression vector.

Contains the novel chimeric antigen receptor, an expression vector and the application of the cell in preparing a medicament for treating tumors.

Preferably, the tumor comprises brain cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, liver cancer, kidney cancer, lymphoma, leukemia, lung cancer, melanoma, metastatic melanoma, mesothelioma, neuroblastoma, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, skin cancer, thymoma, sarcoma, non-hodgkin's lymphoma, uterine cancer and any combination thereof.

In a specific embodiment, the invention relates to the use of the compounds for the preparation of a medicament for the treatment of solid tumors and hematological tumors.

The invention discloses a novel chimeric antigen receptor, and relates to application in preparation of antitumor drugs and cell immunotherapy.

The immune effector cell of the invention relates to the application in preparing antitumor drugs and cell immunotherapy.

The invention has the beneficial effects that:

the invention provides a novel CAR structure comprising an intracellular second transduction domain, T2A, an extracellular signal peptide, an antigen binding domain, an intracellular first transduction domain, e.g. intracellular second transduction domains DAP12, T2A, CD8 α signal peptide, a tumor antigen binding domain, an intracellular first transduction domain TREM1(DAP12-T2A-CD8 α signal peptide-VL-Linker-VH-TREM 1). The intracellular first signal conduction structure and the intracellular second signal conduction structure are used, so that CAR-T cells are facilitated to secrete lower-level cytokines when being stimulated by extracellular tumor antigens, the safety of clinical application can be well guaranteed, the CAR-T cells have good tumor killing effect in vitro and in vivo, the CAR-T cells also show remarkable curative effect in clinical treatment of solid tumors and blood tumor patients, and the cytokine release response is milder. By taking CD19 and mesothelin antigen targets as examples, compared with the prior art, the novel chimeric antigen receptor shows higher anti-tumor capability, immune cells modified by the novel chimeric antigen receptor have higher capability of identifying tumor antigens in a targeted manner, and the killing activity to tumor cells is enhanced.

Drawings

Figure 1 is a CAR structure containing different antigen binding domains or intracellular second transduction domains, demonstrating the structural diagram of various chimeric antigens involved in the present invention.

Figure 2 is the positive rate of expression of CAR structures recognized on the T cell surface by flow cytometry 7 days after infection of CAR1, CAR2, CAR3, CAR4CAR-T cells with lentivirus.

FIG. 3 is cell proliferation after infection of CAR-T cells with different CAR lentiviruses, CAR-T and NTD cells expanded in vitro for 10 days, where NTD is a T cell that is not transfected with CAR lentivirus.

Figure 4 is the secretion of IFN- γ by CAR1 and CAR2CAR-T cells under stimulation with CD19 antigen, CAR1 and CAR2 were co-cultured with target cells for 24h, respectively.

FIG. 5 is the secretion of IFN- γ by CAR3 and CAR4CAR-T cells under mesothelin antigen stimulation, CAR3 and CAR3 were co-cultured with target cells for 24h, respectively.

Figure 6 is the killing effect of CAR1 and CAR2CAR-T cells on CD19 antigen positive cell lines, CAR1 and CAR2, respectively, co-cultured with target cells for 8 h.

FIG. 7 is a graph of the killing effect of CAR3 and CAR4CAR-T cells on mesothelin antigen positive cell lines, and CAR3 and CAR4 were co-cultured with target cells for 8 h.

FIG. 8 is a graph of the release level of human interleukin 6, and after the CAR1-T1 patient returned CAR1 cells, the body temperature began to rise 3 days after the last return, and the cytokine detection result shows that the secretion level of interleukin 6(IL-6) is obviously increased by 100 times.

FIG. 9 is a photograph of before and after imaging evaluation of a patient with advanced pancreatic cancer.

Detailed Description

The present invention provides a novel chimeric antigen receptor, an immune effector cell and the application thereof in the clinical treatment of hematological tumors and solid tumors, and the present invention is further illustrated below with reference to specific examples.

The novel chimeric antigen receptors of the invention comprise an extracellular signal peptide structure, such as the CD8 alpha signal peptide, the 4-1BB signal peptide, the GM-CSFR alpha signal peptide or the CD4 signal peptide, preferably the CD8 alpha signal peptide.

The term "intracellular signaling domain" as used herein refers to a region of a protein structure capable of signaling cellular effector functions and directing the cell to perform a specific function. The intracellular signaling domain may include a first signaling domain, a second signaling domain, and/or a transmembrane domain.

As used herein, the term "identity" of amino acid sequences is used interchangeably with "similarity" and refers to the degree of similarity between amino acid sequences as determined by sequence alignment software, such as BLAST. Methods and software for amino acid sequence alignment are well known to those skilled in the art. An engineered amino acid sequence may be obtained by substitution, deletion and/or addition of one or several (e.g., 1-15, e.g., 2, 3, 5, 8, 10 or 12) amino acid residues to a known amino acid sequence. For example, by modifying the intracellular first signaling domain of NKp44 shown in SEQ ID No.7 of the present invention by conventional protein engineering means (e.g., amino acid conservative substitution, etc.), a variant sequence having at least 85% (e.g., 85% to 99% or 90% to 99% or 95% to 99%) sequence identity with SEQ ID No.7 and having substantially the same signaling domain can be obtained.

The term "antigen binding domain" as used herein is intended to include the variable region of a functional antibody portion, preferably an antigen binding and/or intact antibody. Antibody fragments include Fab, Fab ', F (ab') 2, Fv fragments, single chain antibody scFv, single domain antibody VHH, and multispecific antibodies.

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The experimental procedures, for which specific conditions are not indicated in the examples, are generally carried out according to conventional conditions, for example those described in the molecular cloning protocols (third edition, sambrook et al) or according to the manufacturer's recommendations. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

Example 1 chimeric antigen receptor preparation

The present invention provides a novel chimeric antigen receptor. The chimeric antigen receptor of the invention is composed of an intracellular second conduction domain-T2A-extracellular signal peptide-antigen binding domain-intracellular first conduction domain which are connected in series in sequence. Therefore, it is necessary to construct viral vectors containing different combinations of stimulatory signals separately. In this example, the CD 19-targeting single-chain antibody and the human mesothelin-targeting single-chain antibody are used as the structures of the unified extracellular recognition antigen, and the following 4 chimeric antigen receptors (fig. 1) need to be constructed:

DAP12-T2A-CD8 alpha signal peptide-CD 19 antigen binding domain-NKp 44(CAR1)

DAP12-T2A-CD8 alpha signal peptide-CD 19 antigen binding domain-TREM 1(CAR2)

DAP12-T2A-CD8 alpha signal peptide-mesothelin antigen binding domain-NKp 44(CAR3)

DAP12-T2A-CD8 alpha signal peptide-mesothelin antigen binding domain-TREM 1(CAR4)

1. Gene sequence containing novel chimeric antigen receptor targeting CD19 and mesothelin

The design sequentially contains a natural killer activated receptor (DAP12 for short), T2A, CD8 alpha signal peptide, a CD19 antigen binding domain, a mesothelin antigen binding domain, a natural cytotoxic receptor (NKp 44 for short) and a myeloid cell triggering receptor (TREM 1 for short), and the structure of the receptor is shown in figure 1. Wherein the nucleotide sequence of DAP12 is shown as SEQ ID NO.1, the amino acid sequence is shown as SEQ ID NO.2, the nucleotide sequence of T2A is shown as SEQ ID NO.3, the amino acid sequence is shown as SEQ ID NO.4, the nucleotide sequence of CD8 alpha signal peptide is shown as SEQ ID NO.5, the amino acid sequence is shown as SEQ ID NO.6, the nucleotide sequence of CD19 antigen binding domain is shown as SEQ ID NO.11, the amino acid sequence is shown as SEQ ID NO.12, the nucleotide sequence of CD19 antigen binding domain is shown as SEQ ID NO.13, the amino acid sequence is shown as SEQ ID NO.14, the nucleotide sequence of NKp44 is shown as SEQ ID NO.7, the amino acid sequence is shown as SEQ ID NO.9, the nucleotide sequence of TREM1 is shown as SEQ ID NO.8, and the amino acid sequence is shown as SEQ ID NO. 10.

2. Construction of Lentiviral vectors expressing chimeric antigen receptors

pELNS-DAP12-T2A-CD 8. alpha. plasmid was stored by Nanjing Katsui medicine science Co., Ltd, or was constructed according to the method disclosed in the literature (Enxiu Wang et al. Generation of patent T-cell immunology for Cancer Using DAP12-Based, Multichain, Chimeric Immunoresists. 2015, Cancer ImmunogyResearch, 3(7):815), CD19 antigen binding domain-NKp 44, CD19 antigen binding domain-TREM 1, mesothelin antigen binding domain-NKp 44, mesothelin antigen binding domain-TREM 1 gene synthesis by Nanjing Kinry Biotech company and provides pUC19-CD19-1, pUC 6326-CD 19-2, pUC 19-mesol-5961, pUC 19-TAI-cDNA 19, and BamHI fragment synthesis by the enzyme digestion reaction of Sal DNA plasmid separately (Takara 23 ), after enzyme digestion, the lentivirus vectors pELNS-DAP12-T2A-CD8 alpha signal peptide-CD 19 antigen binding domain-NKp 44(CAR1 for short), pELNS-DAP12-T2A-CD8 alpha signal peptide-CD 19 antigen binding domain-TREM 1(CAR2 for short), pELNS-DAP12-T2A-CD8 alpha signal peptide-mesothelin antigen binding domain-NKp 44(CAR3 for short), pELNS-DAP12-T2A-CD8 alpha signal peptide-mesothelin antigen binding domain-TREM 1(CAR4 for short) for expressing the chimeric antigen receptor are obtained by connection. 5 mu.L of lentiviral vector was transformed into E.coli TOP10 competent cells (purchased from Nanjing Ande high Biotech Co., Ltd.), and after culturing at 37 ℃ for 16h, monoclonals were picked up, and after culturing at 37 ℃ for 12h, plasmids were extracted with a plasmid extraction kit (purchased from Takara Co., Ltd.), and the specific method is described in the specification.

3. Lentiviral packaging

The slow virus is packaged by a calcium phosphate method, and the method comprises the following specific steps:

(1) alternate day passage of 293T cells

5 × 10 cells were seeded per T150 cell flask⁶And (4) cells. After 48 hours, the cell number should reach 20-25 million/vial.

(2)293T cell spreading bottle

a) Using 1T 150 cell culture flask as an example, the cells were gently washed twice with about 15ml of 1 XPBS

b) 3ml of 0.25% pancreatin-2.21 mM EDTA were added

c) When cells had fallen, 12ml of 10% (wt) FBS (from Gibico) DMEM medium (from corning) was added to the cells that had fallen

d) The cells were collected and transferred to a sterile centrifuge tube, 1000rpm, and centrifuged for 10 minutes

e) The supernatant was aspirated off and the pellet was resuspended in 10ml of 10% (wt) FBS in DMEM.

f) Cell count, calculation from cell concentration 12 × 10⁶Volume required for individual cell

g) The cells were combined with 25ml of 10% (wt) FBS DMEM medium, placed in a T150 cell flask and gently shaken to distribute the cells evenly to the bottom of the cell flask at 37 ℃ and cultured overnight in a 5% CO2 incubator.

(3) Cell transfection

The cells are observed to reach a cell density of about 80% -90%, at which point transfection can begin

a) The culture medium was gently aspirated 30-60 min before transfection.

b) Plasmid DNA and calcium chloride solution were mixed, taking a T150 vial as an example, requiring 28ug pRSV.rev (available from Invitrogen), 28ug pGAG-Pol (available from Invitrogen), 11ug pVSVG (available from Invitrogen), 23ug recombinant lentivirus expression plasmid CAR1/CAR2/CAR3/CAR4, added to 1.5ml of calcium chloride solution and mixed well.

c) Adding 1.5ml of BBS solution into a 15ml sterile centrifuge tube, uniformly mixing the DNA-calcium chloride solution with a 1ml gun head, dropwise adding the mixture into the BBS solution, rapidly mixing the mixture uniformly for 15-20 ℃, and incubating the mixture for 25-30 minutes at room temperature.

d) The DNA-calcium chloride-BBS mixture (available from Shanghai Bintian Biotechnology Co., Ltd.) was added dropwise to the T150 bottle uniformly using a 5ml pipette. Culturing in a cell culture box containing 5% carbon dioxide at 37 deg.C for 6 hr.

e) And changing the liquid after 6 h. The plate was gently shaken several times to suspend some calcium phosphate precipitate sufficiently, the culture solution containing calcium phosphate precipitate was aspirated, 20ml of fresh 5% (wt) FBS DMEM culture solution was added, and the culture was continued.

(4) Initial Collection of viral supernatants

a) The 293T cell culture supernatant transfected the previous day was collected into a centrifuge tube, centrifuged at 1000rpm for 5 minutes, labeled, and stored in a4 ℃ freezer.

b) 20ml of 5% (wt) FBS DMEM medium previously preheated was added to the cell flask and the cell culture was continued overnight in the cell incubator at 37 ℃.

(5) The viral supernatant was collected a second time (48 h/day).

(6) Filtering the supernatant

The supernatants from both collections were pooled together and filtered through a 0.45 μm filter to remove cellular debris.

(7) Concentration of virus

Centrifugation at 12000-24000rpm at 4 ℃ overnight

(8) Virus storage

After centrifugation, the whole supernatant was poured off, resuspended in fresh 5% (wt) FBS DMEM medium, split-packaged for virus and stored rapidly in a-80 ℃ freezer for further use

(9) Lentiviral titer determination

a) Viral infection of 293T cells

The 293T cells were plated in a 24-well plate before infection, 200. mu.L of the purified concentrated virus was added to the 293T cells, and after 24 hours, the medium was replaced with DMEM containing 10% FBS (wt), and after 72 hours of infection, the cells were collected by centrifugation at 1200r/min for 5 minutes, and the genome was extracted.

b) Extraction of genome

The genome extraction kit is purchased from Takara company and operated according to the kit instruction

c) qPCR determination of viral titre

The reaction system is as follows, Probe qPCR Mix 12.5 uL (purchased from Takara), upstream primer 0.5 uL (synthesized by Nanjing Kinsry), downstream primer 0.5 uL (synthesized by Nanjing Kinsry), Probe 1 uL (synthesized by Nanjing Kinsry), template 2 uL, sterilized water 8.5 uL, reaction system 25 uL, reaction conditions are set according to the instruction, after the reaction is finished, analysis software is used for analyzing data, and virus titer is calculated according to a standard curve, and the calculation result shows that the virus titer is 1 × 10⁷TU/ml。

Example 2 viral infection of T cells

1. Isolated activation of T cells and viral infection

(1) Isolation of human peripheral blood mononuclear cells

Collecting peripheral blood by using a blood collection tube containing an anticoagulant, naturally settling for about 30min at room temperature (18-25 ℃), collecting upper plasma, centrifuging the collected upper plasma for 10min at 5000r/min, adding the collected upper plasma to a lymphocyte separation solution (purchased from Tianjin Shangjing Biotechnology Limited liability company) according to a volume ratio of 1:1, carrying out gradient centrifugation for 3000r/min, centrifuging for 30min, and after centrifugation, layering a centrifugal tube from top to bottom: the first layer is a plasma layer; the second layer is a lymphocyte leucocyte layer; the third layer is a transparent separation liquid layer; the fourth layer of red blood cells. Sucking lymphocyte leucocyte membrane layer, washing with PBS for 2 times, centrifuging twice at 1500r/min for 10min, resuspending cells with PBS, adding 5% autologous plasma +300IU/ml recombinant human IL-2+ KBM581 complete culture medium to culture human peripheral blood mononuclear cells.

(2) Lentiviral infection of T lymphocytes

Freshly prepared mononuclear cell PBMC were cultured in complete medium containing 5% autologous plasma +300IU/ml recombinant human IL-2+ KBM581, IL-2 from R & D Systems, KBM581 from Corning, CD3/CD28 Dynabeads immunomagnetic beads (from invitrogen) added on day 0 to activate T cells, lentiviral infection was performed the first 3 days, 0.25MOI corresponding lentiviral vector was added, uninfected T lymphocytes were used as a blank, after 48h the medium was changed to complete medium containing 5% autologous plasma +300IU/ml recombinant human IL-2+ KBM581, and culture was continued for 7-9 days.

2. Detection of CAR positivity in T cells

The virus-infected T cells cultured to day 7 were centrifuged at 1200 rpm for 5min, the supernatant was discarded to collect the cells, the cells were resuspended in a PBS solution containing 1% FBS by volume, and the cells were adjusted to a density of 1 × 10⁵Per ml, adding biotin-labeled goat anti-mouse F (ab)₂(Jackson ImmunoResearch Co.), adding Streptavidin-PE (BDbiosciences Co.), incubating at 4 ℃ for 15min, washing with PBS solution for 2 times, detecting on an up-flow cytometer, and showing that after 7 days of culture, the CAR-T cell CAR positive rate is: the positive rate of the CAR1 virus infection group is 64.5%, the positive rate of the CAR2 virus infection group is 89.6%, the positive rate of the CAR3 virus infection group is 60%, and the positive rate of the CAR4 virus infection group is 20% (fig. 2).

Example 3 Effect of viral infection of CAR-T cells on cell proliferation

After infection of T cells by each group of viruses, the T cells were counted every 1-2 days in complete medium containing 5% volume fraction of autologous plasma +300IU/ml recombinant human IL-2+ KBM 581. T lymphocyte growth was then observed, and the results are shown in FIG. 3. The results show that the cells can still form typical proliferation clonic masses after being infected by the virus expressing the CAR, and the proliferation curves of the CAR1, CAR2, CAR3 and CAR4 are drawn by counting the cells, so that the proliferation of the cells is similar to that of T cells (NTD in figure 3) which are not infected by the virus.

Example 4 detection of cytokine secretion by Virus-infected CD19 and mesothelin CAR-T cells

(1) Cytokine detection was performed by the method of Elisa using a kit of R & D.

(2) And (3) diluting the standard: preparing 7 centrifuge tubes of 1ml, numbering in sequence, firstly adding 500 mu L of standard substance diluent into each centrifuge tube, then adding 500 mu L of original concentration standard substance into 1 numbered centrifuge tube, fully and uniformly mixing, then adding 500 mu L of standard substance into a second centrifuge tube in the centrifuge tube, and fully and uniformly mixing; then taking 500 mu L of the centrifugal tube, adding the centrifugal tube into a third centrifugal tube, and fully and uniformly mixing; then taking 500 mu L of the centrifugal tube, adding the centrifugal tube into a fourth centrifugal tube, and fully and uniformly mixing; then taking 500 mu L of the centrifugal tube, adding the centrifugal tube into a fifth centrifugal tube, and fully and uniformly mixing; then taking 500 mu L of the centrifugal tube, adding the centrifugal tube into a sixth centrifugal tube, and fully and uniformly mixing; then, 500. mu.L of the suspension was added to a seventh centrifuge tube and mixed well.

(3) And standard substance holes are formed in the enzyme-labeled coating plate, and 100 mu L of standard substances with different concentrations are sequentially added, wherein each concentration is 2-3 parallel holes.

(4) Sample adding: respectively arranging blank holes (the blank control hole is replaced by water, the enzyme labeling reagent and the biotin-labeled antibody are operated as before) and sample holes to be detected, adding 100 mu L of sample in the sample holes to be detected on the enzyme labeling coated plate, adding the sample to the bottom of the plate hole of the enzyme labeling plate, keeping the hole wall untouched as far as possible, gently shaking and uniformly mixing

(5) And (3) incubation: standing at room temperature for incubation for 2h

(6) Washing: discarding liquid, spin-drying, adding 200 μ L of washing solution into each well, standing for 30s, discarding, repeating for 3 times, and patting to dry

(7) Adding an antibody: adding 100 mu L of detection antibody on the enzyme-labeled coated plate

(8) And (3) incubation: same operation (5)

(9) Washing: same operation (6)

(10) Marking: 100 μ L of horseradish peroxidase-labeled streptavidin was added to each well

(11) And (3) incubation: incubating at room temperature in dark for 20min

(12) Washing: same operation (6)

(13) Color development: adding 100 μ L of color development liquid into each well, shaking gently, mixing, incubating at room temperature in dark for 20min

(14) And (4) terminating: stop solution (50. mu.L) was added to each well to stop the reaction

(15) And (3) determination: the blank value is used for zero calibration, the absorbance (OD value) of each hole is measured in sequence at the wavelength of 450nm, and the measurement is carried out within 15min after the stop solution is added.

Selecting target cells with different antigen expression levels to co-culture with CAR-T cells of CAR1, CAR2, CAR3 and CAR4, detecting that the CAR-T secretes IFN-gamma level in response to antigen stimulation, selecting 293T-CD19(CD19 positive) and 293T (CD19 negative) from CD19 antigen target cells, and selecting ovarian cancer tumor cell strains OVCAR3(mesothelin positive) and 293T (mesothelin negative) from mesothelin antigen target cells, so as to show that the CAR-T specifically secretes IFN-gamma when stimulated by CD19 or mesothelin antigen, and reflecting that 4 kinds of CAR-T cells generate different responses to target cells with different antigen expression levels. CAR1, CAR2CAR-T cells secreted significantly IFN- γ when co-cultured with CD19 positive target cell 293T-CD19 (figure 4), indicating that CAR1, CAR2CAR-T cells had a response effect on antigen positive tumor cells. CAR3, CAR4CAR-T cells secreted significantly IFN- γ when co-cultured with mesothelin positive target cell OVCAR3 (figure 5), indicating that CAR-T cells of CAR3, CAR4 had a response effect on antigen positive tumor cells.

Example 5 evaluation of the Effect of viral infection targeting CD19 and mesothelin CAR-T cell killing in vitro

(1) Targeted CD19 antigen killing: CAR-T culturing target cells 293T-CD19(CD19 positive), 293T (CD19 negative) and effector cells CAR1, CAR 2;

(2) targeted mesothelin antigen killing: CAR-T of OVCAR3(mesothelin positive), 293T (CD19 negative) and effector cells CAR3, CAR4 were cultured.

(3) Collecting target cells and effector cells, centrifuging at 1500rpm/min for 5min, discarding supernatant

(4) Resuspension of target and effector cells in 10% FBS +1640 complete Medium

(5) Using a real-time cell analysis System (RTCA), 50. mu.L 1640 medium was added to the E-Plate16 in air

(6) Determining normal contact of selected holes using RTCA baseline detection

(7) Setting the effective target ratio to be 0:1, 1:1, 5:1, 10:1

(8) E-Plate16 was removed and 100. mu.L of the well-mixed target cell suspension was added to each well in an effective target ratio so that the number of each cell was 10⁴cells/100μL。

(9) E-Plate16 was placed in an incubator at 37 ℃ under 5% CO2 overnight

(10) The next day, E-Plate16 was removed, 50. mu.L of the corresponding effector cells were added, and the killing rate 8h after the addition of the effector cells was calculated.

(11)

The results of the detection are shown in FIG. 6. The killing effect of CAR1 and CAR2 on CD19 antigen positive cells is obvious and is obviously higher than that of an NTD group. The results of in vitro killing experiments show that the novel chimeric antigen receptor targeted CD19 antigen has strong anti-tumor activity.

The results of the detection are shown in FIG. 7. The CAR-T of CAR3 and CAR4 has obvious killing effect on mesothelin antigen positive cells, and is obviously higher than that of NTD group. The results of in vitro killing experiments show that the novel chimeric antigen receptor targeted mesothelin antigen has strong anti-tumor activity.

Example 6 clinical trial therapeutic efficacy of CAR 1-modified autologous T cells on CD 19-positive acute B-lymphoblastic leukemia patients

Collecting 100ml peripheral blood of acute B lymphocyte leukemia patient, separating and purifying to obtain T cell (example 2), adding 5% autologous plasma, 300IU/ml recombinant human IL-2 and CD3/CD28 magnetic bead to activate in vitro for 1 day, transducing CAR1 slow virus vector with the dose of 0.8-1.5 × 10⁶The culture density of/mL is enlarged for 9-12 days per day.

The CAR1 was transduced into T cells from 5 different acute B-lymphocyte leukemia patients, respectively, and the transduced cells were named CAR1-T1, CAR1-T2, CAR1-T3, CAR1-T4, CAR 1-T5.

The following is an example of the treatment of patients with CAR1-T1 acute B-lymphocytic leukemia.

CAR1-T1 patient:

(1) particle system: the proliferation is active, and the proportion of the neutrophil granulocytes is increased. The proportion of neutral late granulocytes was normal. The proportion of the mature stage is reduced.

(2) Red series:

(3) lymphocyte: the ratio is obviously increased. Primitive lymphocytes were seen, accounting for 26.5%.

(4) Megakaryocytes: the number of megakaryocytes in the whole tablet is 8; classifying 8 megakaryocytes; 0 primitive cells; 0 juvenile megakaryocytes; 7 granular megakaryocytes; 0 platelet-producing megakaryocytes; the number of the naked megakaryocytes is 1. Visualization of blood platelets

(5) Blood slice: the white blood cell count is normal, the granulocytic classification is rare, the lymphocyte proportion is obviously increased, the monocyte proportion is reduced, and the blood platelet is visible.

(6) Impression: acute lymphocytic leukemia relapse

Patients signed informed consent under hospital recommendations, ethical review, and then performed clinical trial studies of CAR1 cell therapy.

The CAR1 modified autologous T cell treatment process comprises collecting peripheral blood of a patient, sorting in vitro to obtain T cells, activating and amplifying the T cells in vitro, preparing CAR1-T1 cell preparation, controlling and releasing the quality of the preparation, infusing twice into the patient through vein at two time points, and infusing about 2.5 × 10 in total⁸CAR-T cells.

After CAR1-T1 patients returned CAR1 cells, mild fever reaction appeared, body temperature began to rise 3 days after the last return, cytokine detection results showed that interleukin 6(IL-6) secretion level increased 100 times compared with baseline period, as shown in fig. 8, patient sustained fever for 3 days did not exceed 39 ℃, and then body temperature gradually decreased and reached normal body temperature. CD19CAR-T treatment reported abroad to treat patients with acute B-lymphoblastic leukemia when the side effects are severe sustained hyperthermia occurs within 3 days. The clinical test result shows that the T cell modified by the novel chimeric antigen receptor has milder reaction in the treatment of B lymphocyte leukemia and has less side effect on patients. Peripheral blood and bone marrow of the patients were examined 14 days after the reinfusion. As shown by the results in table 1, the percentage of CD19 positive (CD19+) cells in peripheral blood was reduced from 16% to 0% before treatment, and the percentage of CD19 positive (CD19+) cells in bone marrow was reduced from 26.5% to < 1% before treatment. The combined hematology detection result shows that: after the patient is treated by the CAR1 cells provided by the invention, the leukemia cells with positive CD19 in peripheral blood and bone marrow are completely eliminated, and clinical complete remission is achieved.

TABLE 1 Change in B lymphocytes before and after treatment of acute B lymphocyte leukemia patients

Detecting items	Peripheral blood (before treatment)	Peripheral blood (after treatment)	Bone marrow (before treatment)	Bone marrow (after treatment)
					CD19+ cells	16％	0％	26.5％	＜1％

Example 7 clinical trial therapeutic Effect of CAR 3-modified autologous T cells on mesothelin-positive patients with advanced pancreatic cancer

Collecting 100ml peripheral blood of a mesothelin-positive patient with advanced pancreatic cancer, separating and purifying to obtain T cells (example 2), adding 5% autologous plasma, 300IU/ml recombinant human IL-2 and CD3/CD28 magnetic beads, activating in vitro for 1 day, and transducing CAR1 lentiviral vector to 0.8-1.5 × 10⁶The culture density of/mL is enlarged for 9-12 days per day.

CAR3 was transduced into T cells from 5 different mesothelin-positive patients with advanced pancreatic cancer, respectively, and the transduced cells were named CAR3-T1, CAR3-T2, CAR3-T3, CAR3-T4, CAR 3-T5.

The following is an example of the treatment of CAR3-T1 for mesothelin-positive patients with advanced pancreatic cancer.

CAR3-T1 patient:

after operation of malignant tumor of pancreatic head, radioactive particle implantation, and cholecystectomy. PET/CT showed the possibility of paraarterial lymph node metastasis in the abdominal cavity. CT reports that suspicious soft tissue foci among abdominal intestinal canals are reinforced and clinically diagnosed as pancreatic cancer. Immunohistochemical detection results show that the mesothelin is positive, and patients sign informed consent through ethical examination after hospital recommendation and then carry out the clinical trial study of CAR3 cell therapy.

The CAR3 modified autologous T cell treatment process comprises collecting peripheral blood of a patient, sorting in vitro to obtain T cells, activating and amplifying the T cells in vitro, preparing CAR3-T1 cell preparation, controlling and releasing the quality of the preparation, infusing the preparation into the patient by vein three times at three time points, and infusing about 1.35 × 10 in total⁸CAR-T cells.

After CAR3-T1 patients had returned CAR3 cells, a mild febrile response occurred, body temperature rose to 37.5 1 days after the last return, followed by a rapid recovery, and cytokine detection showed 4-fold increase in interleukin 6(IL-6) secretion levels over the baseline phase. The side effects of the exogenous reported mesothelin CAR-T treatment on pancreatic cancer are less frequent, but more often, the clinical treatment of mesothelin CAR-T is ineffective. The clinical test result shows that the T cell modified by the novel chimeric antigen receptor has milder response in treating advanced pancreatic cancer and has no side effect on patients. The patient's lesions were evaluated imagewise 1 month after the return infusion. As shown in FIG. 9, the size of the tumor foci before treatment was about 50X 45mm and the size of the tumor foci after treatment was about 43X 39 mm. The results of the combined imaging evaluation show that: after the patient is treated by the CAR3 cells provided by the invention, the tumor is reduced by about 25%, and clinical partial remission is achieved.

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

Sequence listing

<110> Nanjing Kayi medical science and technology Co., Ltd

<120> use of novel chimeric antigen receptor-modified T cells for treating cancer

<130>2018

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

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<212>DNA

<213> Artificial sequence (artificial sequence)

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<213> Artificial sequence (artificial sequence)

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catggaaaga gccttgagtg gattggactt attactcctt acaatggtgc ttctagctac 660

aaccagaagt tcaggggcaa ggccacatta actgtagaca agtcatccag cacagcctac 720

atggacctcc tcagtctgac atctgaagac tctgcagtct atttctgtgc aagggggggt 780

tacgacggga ggggttttga ctactggggc caagggacca cggtcaccgt ctcctcaggt 840

ggaggcggtt caggcggcgg tggctctagc ggtggtggat cggacatcga gctcactcag 900

tctccagcaa tcatgtctgc atctccaggg gagaaggtca ccatgacctg cagtgccagc 960

tcaagtgtaa gttacatgca ctggtaccag cagaagtcag gcacctcccc caaaagatgg 1020

atttatgaca catccaaact ggcttctgga gtcccaggtc gcttcagtgg cagtgggtct 1080

ggaaactctt actctctcac aatcagcagc gtggaggctg aagatgatgc aacttattac 1140

tgccagcagt ggagtaagca ccctctcacg tacggtgctg ggacaaagtt ggaaatcaaa 1200

gctagcggtg gcggaggttc tggaggtggg ggttccactg caggagccag acaagcccct 1260

gagtctccat ctaccatccc tgtcccttca cagccacaga actccacgct ccgccctggc 1320

cctgcagccc ccattgccct ggtgcctgtg ttctgtggac tcctcgtagc caagagcctg 1380

gtgctgtcag ccctgctcgt ctggtggggg gacatatggt ggaaaaccat gatggagctc 1440

aggagcctgg atacccaaaa agccacctgc caccttcaac aggtcacgga ccttccctgg 1500

acctcagttt cctcacctgt agagagagaa atattatatc acactgttgc aaggactaag 1560

ataagcgatg atgatgatga acacactttg tga 1593

<210>18

<211>1389

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>18

atggggggac ttgaaccctg cagcaggttc ctgctcctgc ctctcctgct ggctgtaagt 60

ggtctccgtc ctgtccaggt ccaggcccag agcgattgca gttgctctac ggtgagcccg 120

ggcgtgctgg cagggatcgt gatgggagac ctggtgctga cagtgctcat tgccctggcc 180

gtgtacttcc tgggccggct ggtccctcgg gggcgagggg ctgcggaggc agcgacccgg 240

aaacagcgta tcactgagac cgagtcgcct tatcaggagc tccagggtca gaggtcggat 300

gtctacagcg acctcaacac acagaggccg tattacaaag tcgagggcgg cggagagggc 360

agaggaagtc ttctaacatg cggtgacgtg gaggagaatc ccggccctag gatggcctta 420

ccagtgaccg ccttgctcct gccgctggcc ttgctgctcc acgccgccag gccgggatcc 480

caggtacaac tgcagcagtc tgggcctgag ctggagaagc ctggcgcttc agtgaagata 540

tcctgcaagg cttctggtta ctcattcact ggctacacca tgaactgggt gaagcagagc 600

catggaaaga gccttgagtg gattggactt attactcctt acaatggtgc ttctagctac 660

aaccagaagt tcaggggcaa ggccacatta actgtagaca agtcatccag cacagcctac 720

atggacctcc tcagtctgac atctgaagac tctgcagtct atttctgtgc aagggggggt 780

tacgacggga ggggttttga ctactggggc caagggacca cggtcaccgt ctcctcaggt 840

ggaggcggtt caggcggcgg tggctctagc ggtggtggat cggacatcga gctcactcag 900

tctccagcaa tcatgtctgc atctccaggg gagaaggtca ccatgacctg cagtgccagc 960

tcaagtgtaa gttacatgca ctggtaccag cagaagtcag gcacctcccc caaaagatgg 1020

atttatgaca catccaaact ggcttctgga gtcccaggtc gcttcagtgg cagtgggtct 1080

ggaaactctt actctctcac aatcagcagc gtggaggctg aagatgatgc aacttattac 1140

tgccagcagt ggagtaagca ccctctcacg tacggtgctg ggacaaagtt ggaaatcaaa 1200

gctagcggtg gcggaggttc tggaggtggg ggttccactc ctgactctga aatcaacctt 1260

acaaatgtga cagatatcat cagggttccg gtgttcaaca ttgtcattct cctggctggt 1320

ggattcctga gtaagagcct ggtcttctct gtcctgtttg ctgtcacgct gaggtcattt 1380

gtaccctag 1389

<210>19

<211>532

<212>PRT

<213> Artificial sequence (artificial sequence)

<400>19

Met Gly Gly Leu Glu Pro Cys Ser Arg Phe Leu Leu Leu Pro Leu Leu

1 5 10 15

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

20 25 30

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

35 40 45

Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu

50 55 60

Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg

65 70 75 80

Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly

85 90 95

Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr

100 105 110

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

115 120 125

Asp Val Glu Glu Asn Pro Gly Pro Arg Met Ala Leu Pro Val Thr Ala

130 135 140

Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Gly Ser

145 150 155 160

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

165 170 175

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

180 185 190

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

195 200 205

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

210 215 220

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

225 230 235 240

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

245 250 255

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser

260 265 270

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

275 280 285

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

290 295 300

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

305 310 315 320

Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser

325 330 335

Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile

340 345 350

Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln

355 360 365

Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

370 375 380

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

385 390 395 400

Ser Ser Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ala

405 410 415

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

420 425 430

Gln Pro Gln Asn Ser Thr Leu Arg Pro Gly Pro Ala Ala Pro Ile Ala

435 440 445

Leu Val Pro Val Phe Cys Gly Leu Leu Val Ala Lys Ser Leu Val Leu

450 455 460

Ser Ala Leu Leu Val Trp Trp Gly Asp Ile Trp Trp Lys Thr Met Met

465 470 475 480

Glu Leu Arg Ser Leu Asp Thr Gln Lys Ala Thr Cys His Leu Gln Gln

485 490 495

Val Thr Asp Leu Pro Trp Thr Ser Val Ser Ser Pro Val Glu Arg Glu

500 505 510

Ile Leu Tyr His Thr Val Ala Arg Thr Lys Ile Ser Asp Asp Asp Asp

515520 525

Glu His Thr Leu

530

<210>20

<211>464

<212>PRT

<213> Artificial sequence (artificial sequence)

<400>20

Met Gly Gly Leu Glu Pro Cys Ser Arg Phe Leu Leu Leu Pro Leu Leu

1 5 10 15

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

20 25 30

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

35 40 45

Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu

50 55 60

Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg

65 70 75 80

Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly

85 90 95

Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr

100 105 110

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

115120 125

Asp Val Glu Glu Asn Pro Gly Pro Arg Met Ala Leu Pro Val Thr Ala

130 135 140

Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Gly Ser

145 150 155 160

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

165 170 175

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

180 185 190

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

195 200 205

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

210 215 220

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

225 230 235 240

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

245 250 255

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser

260 265 270

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

275280 285

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

290 295 300

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

305 310 315 320

Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser

325 330 335

Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile

340 345 350

Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln

355 360 365

Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

370 375 380

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

385 390 395 400

Ser Ser Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Pro

405 410 415

Asp Ser Glu Ile Asn Leu Thr Asn Val Thr Asp Ile Ile Arg Val Pro

420 425 430

Val Phe Asn Ile Val Ile Leu Leu Ala Gly Gly Phe Leu Ser Lys Ser

435440 445

Leu Val Phe Ser Val Leu Phe Ala Val Thr Leu Arg Ser Phe Val Pro

450 455 460

<210>21

<211>530

<212>PRT

<213> Artificial sequence (artificial sequence)

<400>21

Met Gly Gly Leu Glu Pro Cys Ser Arg Phe Leu Leu Leu Pro Leu Leu

1 5 10 15

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

20 25 30

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

35 40 45

Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu

50 55 60

Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg

65 70 75 80

Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly

85 90 95

Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr

100 105 110

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

115 120 125

Asp Val Glu Glu Asn Pro Gly Pro Arg Met Ala Leu Pro Val Thr Ala

130 135 140

Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Gly Ser

145 150 155 160

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

165 170 175

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

180 185 190

Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

195 200 205

Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe

210 215 220

Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

225 230 235 240

Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

245 250 255

Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Gln Gly

260 265 270

Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

275 280 285

Ser Ser Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro Ala Ile

290 295 300

Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser

305 310 315 320

Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Gly Thr Ser

325 330 335

Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro

340 345 350

Gly Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile

355 360 365

Ser Ser Val Glu Ala Glu Asp Asp Ala Thr Tyr Tyr Cys Gln Gln Trp

370 375 380

Ser Lys His Pro Leu Thr Tyr Gly Ala Gly Thr Lys Leu Glu Ile Lys

385 390 395 400

Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ala Gly Ala

405 410 415

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

420 425 430

Gln Asn Ser Thr Leu Arg Pro Gly Pro Ala Ala Pro Ile Ala Leu Val

435 440 445

Pro Val Phe Cys Gly Leu Leu Val Ala Lys Ser Leu Val Leu Ser Ala

450 455 460

Leu Leu Val Trp Trp Gly Asp Ile Trp Trp Lys Thr Met Met Glu Leu

465 470 475 480

Arg Ser Leu Asp Thr Gln Lys Ala Thr Cys His Leu Gln Gln Val Thr

485 490 495

Asp Leu Pro Trp Thr Ser Val Ser Ser Pro Val Glu Arg Glu Ile Leu

500 505 510

Tyr His Thr Val Ala Arg Thr Lys Ile Ser Asp Asp Asp Asp Glu His

515 520 525

Thr Leu

530

<210>22

<211>462

<212>PRT

<213> Artificial sequence (artificial sequence)

<400>22

Met Gly Gly Leu Glu Pro Cys Ser Arg Phe Leu Leu Leu Pro Leu Leu

1 5 10 15

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

20 25 30

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

35 40 45

Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu

50 55 60

Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg

65 70 75 80

Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly

85 90 95

Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr

100 105 110

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

115 120 125

Asp Val Glu Glu Asn Pro Gly Pro Arg Met Ala Leu Pro Val Thr Ala

130 135 140

Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Gly Ser

145 150 155 160

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

165 170 175

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

180 185 190

Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

195 200 205

Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe

210 215 220

Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

225 230 235 240

Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

245 250 255

Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Gln Gly

260 265 270

Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

275 280 285

Ser Ser Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro Ala Ile

290 295 300

Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser

305 310 315 320

Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Gly Thr Ser

325 330 335

Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro

340 345 350

Gly Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile

355 360 365

Ser Ser Val Glu Ala Glu Asp Asp Ala Thr Tyr Tyr Cys Gln Gln Trp

370 375 380

Ser Lys His Pro Leu Thr Tyr Gly Ala Gly Thr Lys Leu Glu Ile Lys

385 390 395 400

Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Pro Asp Ser

405 410 415

Glu Ile Asn Leu Thr Asn Val Thr Asp Ile Ile Arg Val Pro Val Phe

420 425 430

Asn Ile Val Ile Leu Leu Ala Gly Gly Phe Leu Ser Lys Ser Leu Val

435 440 445

Phe Ser Val Leu Phe Ala Val Thr Leu Arg Ser Phe Val Pro

450 455 460

<210>23

<211>65

<212>PRT

<213> Artificial sequence (artificial sequence)

<400>23

Thr Gly Leu Gln Lys Asp His Ala Leu Trp Asp His Thr Ala Gln Asn

1 5 10 15

Leu Leu Arg Met Gly Leu Ala Phe Leu Val Leu Val Ala Leu Val Trp

20 25 30

Phe Leu Val Glu Asp Trp Leu Ser Arg Lys Arg Thr Arg Glu Arg Ala

35 40 45

Ser Arg Ala Ser Thr Trp Glu Gly Arg Arg Arg Leu Asn Thr Gln Thr

50 55 60

Leu

65

<210>24

<211>74

<212>PRT

<213> Artificial sequence (artificial sequence)

<400>24

Glu Lys Glu His Pro Gln Leu Gly Ala Gly Thr Val Leu Leu Leu Arg

1 5 10 15

Ala Gly Phe Tyr Ala Val Ser Phe Leu Ser Val Ala Val Gly Ser Thr

20 25 30

Val Tyr Tyr Gln Gly Lys Cys Leu Thr Trp Lys Gly Pro Arg Arg Gln

35 40 45

Leu Pro Ala Val Val Pro Ala Pro Leu Pro Pro Pro Cys Gly Ser Ser

50 55 60

Ala His Leu Leu Pro Pro Val Pro Gly Gly

65 70

<210>25

<211>66

<212>PRT

<213> Artificial sequence (artificial sequence)

<400>25

Ser Tyr His Trp Met Gly Leu Val His Ile Pro Thr Asn Gly Ser Trp

1 5 10 15

Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile

20 25 30

Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly

35 40 45

Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg

50 55 60

Thr Val

65

<210>26

<211>84

<212>PRT

<213> Artificial sequence (artificial sequence)

<400>26

Ser Glu Ser Phe Glu Asp Ala His Val Glu His Ser Ile Ser Arg Ser

1 5 10 15

Leu Leu Glu Gly Glu Ile Pro Phe Pro Pro Thr Ser Ile Leu Leu Leu

20 25 30

Leu Ala Cys Ile Phe Leu Ile Lys Ile Leu Ala Ala Ser Ala Leu Trp

35 40 45

Ala Ala Ala Trp His Gly Gln Lys Pro Gly Thr His Pro Pro Ser Glu

50 55 60

Leu Asp Cys Gly His Asp Pro Gly Tyr Gln Leu Gln Thr Leu Pro Gly

65 70 75 80

Leu Arg Asp Thr

Claims (12)

1. A novel chimeric antigen receptor, which comprises an intracellular second conductive domain, T2A, an extracellular signal peptide, an antigen binding domain, and an intracellular first conductive domain which are connected in sequence,

the intracellular second conduction domain is DAP12, the amino acid sequence of which is shown in SEQ ID NO.2,

the amino acid sequence of the T2A is shown in SEQ ID NO.4,

the extracellular signal peptide is CD8 alpha signal peptide, the amino acid sequence of which is shown in SEQ ID NO.6,

the antigen binding domain is an antigen binding domain which binds to CD19, the amino acid sequence of the antigen binding domain is shown as SEQ ID NO.12,

the first intracellular conducting domain is TREM1, and the amino acid sequence of the first intracellular conducting domain is shown as SEQ ID No. 10.

2. The novel chimeric antigen receptor according to claim 1, wherein the nucleotide sequence of the intracellular second transduction domain DAP12 is represented by SEQ ID No. 1.

3. The novel chimeric antigen receptor according to claim 1, wherein the nucleotide sequence of T2A is represented by SEQ ID No. 3.

4. The novel chimeric antigen receptor according to claim 1, wherein the extracellular signal peptide CD8 α has a nucleotide sequence shown in SEQ ID No. 5.

5. The novel chimeric antigen receptor according to claim 1, wherein the nucleotide sequence of the antigen binding domain that binds to CD19 is shown in SEQ ID No. 11.

6. The novel chimeric antigen receptor according to claim 1, wherein the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID No. 20.

7. The novel chimeric antigen receptor according to claim 1, wherein the nucleotide sequence of the chimeric antigen receptor is shown in SEQ ID No. 16.

8. A cell expressing the chimeric antigen receptor of any one of claims 1-7, characterized in that said cell is selected from the group consisting of T lymphocytes, NK cells, NKT cells, macrophages, mesenchymal stem cells, hematopoietic stem cells, pluripotent stem cells or embryonic stem cell culture differentiated immune cells.

9. A recombinant expression vector comprising a nucleotide sequence encoding the chimeric antigen receptor of any one of claims 1-7.

10. A method for preparing a T cell modified with a chimeric antigen receptor according to any one of claims 1 to 7, comprising isolating and activating the T cell to be modified, and then transducing the T cell with the recombinant expression vector of claim 9.

11. A pharmaceutical composition comprising an effective amount of the cell of claim 8 and a pharmaceutically acceptable carrier.

12. Use of the chimeric antigen receptor of any one of claims 1-7 in the preparation of a medicament against hematological malignancies.

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