CN109971719B

CN109971719B - Car-T cells that autocrine CD40 antibodies and target ErbB receptor family and uses thereof

Info

Publication number: CN109971719B
Application number: CN201711459474.2A
Authority: CN
Inventors: 钱其军; 金华君; 游术梅; 江芏青; 李林芳; 王超; 崔连振
Original assignee: Shanghai Cell Therapy Research Institute; Shanghai Cell Therapy Group Co Ltd
Current assignee: Shanghai Cell Therapy Research Institute; Shanghai Cell Therapy Group Co Ltd
Priority date: 2017-12-28
Filing date: 2017-12-28
Publication date: 2023-05-12
Anticipated expiration: 2037-12-28
Also published as: WO2019129142A1; CN109971719A

Abstract

The present invention provides chimeric antigen receptor T cells that self-express CD40 activating antibodies and target the ErbB receptor family and uses thereof. The T cells of the invention contain coding sequences for expressing chimeric antigen receptors targeting the ErbB receptor family and coding sequences for CD40 activating antibodies; and/or (2) express chimeric antigen receptor and CD40 activating antibody that target the ErbB receptor family. The T cells of the invention have better proliferation activating capacity and tumor cell killing function than chimeric antigen receptor T cells of the ErbB receptor family targeted alone.

Description

Car-T cells that autocrine CD40 antibodies and target ErbB receptor family and uses thereof

Technical Field

The present invention pertains to genetic engineering and immunology, and relates to CAR-T cells that autocrine CD40 antibodies and target the ErbB receptor family and uses thereof.

Background

Cancer is now the first killer of human health, and rapid life rhythm, huge working pressure, unhealthy eating habits and poor environment are all the help of cancer occurrence, so that the high incidence and the younger trend of cancer are more and more obvious. The current commonly used treatment methods have very limited effects, and a more effective treatment method is still needed to be explored to improve the survival rate and the survival quality of cancer patients.

Chimeric antigen receptor T cell (CAR-T) therapy has achieved very good efficacy in hematological malignancies as one of the important branches of tumor immunotherapy, with a complete remission rate of more than 90% for relapsed refractory B-cell leukemia. A chimeric antigen receptor is an artificial synthetic receptor that generally comprises an extracellular antigen binding domain, a transmembrane hinge region, and an intracellular signaling region. Gene recombination is performed in vitro by combining a single-chain variable region (scFv) of an antibody recognizing a tumor-associated antigen (tumor associated antigen, TAA) with an intracellular signaling domain "immunoreceptor tyrosine activation motif (immunoreceptor tyrosine-based activation motifs, ITAM)". The resulting genetically engineered T cells are then introduced into T cells by a viral or other vector system, and are referred to as CAR-T cells. After large-scale expansion in vitro, CAR-T cells are infused back into patients and can exhibit potent anticancer effects in a non-MHC restricted mode.

However, many obstacles have been encountered in replicating this therapy to solid tumors. Firstly, solid tumors lack tumor specific antigens, and perfect therapeutic targets cannot be found. Secondly, the solid tumor has stronger tumor immunosuppression microenvironment. In addition, tumors themselves have a strong heterogeneity. These factors lead to a recurrence of tumor after immunotherapy. Therefore, the preparation of CART cells with multiple targets and stronger functions is a key for improving the tumor treatment effect. For example, treatment of B-cell acute lymphoblastic leukemia with CART19 resulted in a negative recurrence of CD19 antigen in about 30% of patients treated, and studies have shown that CART cells with both CART19/123 double targets, constructed with CD19 and IL3 receptor alpha chain CD123 as targets, have better in vivo therapeutic efficacy and lower recurrence rates (J Clin invest.2016Oct 3;126 (10)). However, most of antigen recognition regions of these CARs are artificially constructed single-chain antibodies, have strong immunogenicity, and in vivo treatment is easily recognized and cleared, resulting in poor therapeutic effects. Therefore, the antigen recognition region with multiple targets is constructed by adopting natural peptide segments, and the method has the advantages of weak immunogenicity and wider targeting range.

The ErbB receptor family comprises four members, the epidermal growth factor receptors ErbB1 (EGFR/Her 2), erbB2 (Her 2), erbB3 (Her 3) and ErbB4 (Her 4), respectively. In normal adults, the expression levels of these four receptors are all at low levels. However, many malignant tumors occur in association with over-expression of ErbB1 and/or ErbB2, including head and neck cancer, breast cancer, lung cancer, gastrointestinal cancer, prostate cancer, pancreatic cancer, and the like. Thus, many of the antitumor drugs in clinical studies are monoclonal antibodies targeting the extracellular regions of different ErbB receptors and small molecule tyrosine kinase inhibitors, such as HER2 humanized antibody Herceptin (Herceptin), which has been FDA approved for clinical treatment of breast cancer.

However, erbB receptors typically function as tightly bound dimers. Her2 is the best dimerization partner, especially when dimerized with EGFR or Her3, that enhances tyrosine kinase signaling activation. Her3 has ligand binding capacity but lacks tyrosine kinase activity, and the heterodimer formed with Her2 is the strongest signal complex, with Her2/Her3 being the most representative heterodimer in breast cancer. Antitumor drugs that target a receptor alone are prone to cause tumor recurrence because they cannot target other ErbB receptor dimers.

T1E is a chimeric polypeptide consisting of seven amino acids at the N-terminus of human transcription growth factor alpha (TGF alpha) and 48 amino acids at the C-terminus of Epidermal Growth Factor (EGF), which has high affinity for both ErbB 1-based homodimers and heterodimers, and T1E is also capable of efficiently binding ErbB2/3 heterodimers. Studies show that the CAR with T1E as scFv transfects T cells, and can effectively treat the mouse tumor xenograft model of human head and neck cancer. However, T1E is unable to bind ErbB2 or ErbB3 alone, and has drawbacks in targeting the ErbB receptor family.

Herstatin is a truncated version of Her2, resulting from selective cleavage of Her2, whose sequence includes 340 amino acids from the extracellular domains I and II of Her2 and 79 amino acids from the eighth intron, and is a soluble Her2 self-inhibitor. Herstatin binds with high affinity to EGFR alone or Her2, with 79 amino acids encoded by the eighth intron (designated Herin) serving as the primary role.

The EHCAR-EK-28TIZ chimeric antigen receptor adopts the fusion expression of natural T1E and Herin as an antigen recognition region of the CAR, and can complementarily recognize an ErbB receptor family, thereby expanding the targeting range.

The CD40 antigen is a cell surface molecule belonging to the TNFR superfamily, is a type I transmembrane glycoprotein, is widely expressed in T cells, antigen presenting cells (DC cells, monocytes, macrophages), hematopoietic cells, epithelial cells, etc., and is also expressed in most B cell malignancies, solid tumors, etc. CD40L is a type II transmembrane glycoprotein and belongs to the TNF superfamily. CD40-CD40L is a co-stimulatory molecule of great importance in the immune response, with a wide range of biological effects. CD40 molecules primarily transmit signals through interactions with CD40 ligands (CD 40L), which on the one hand cause IL-12 levels to be up-regulated, activate DC cells, enhance the ability of APCs to present antigen, and on the other hand CD40-CD40L can also promote T cells to secrete a large number of cytokines such as GM-CSF, TNF-a, and IFN-gamma, thereby enhancing CTL effects of CD8+ T cells and enhancing killing effects on tumors. Researchers at the university of Hadsfield developed a method of targeted therapy with CD40, which they activated with a ligand for CD40, followed by targeted therapy by intravenous injection, and patent application (Oncogene, 2017 May 4;36 (18): 2515-2528). Because of the wide biological effect of CD40 molecules, a plurality of functional antibodies are developed in different fields, for example, a key project APX005M (NCT 02482168) developed by Apexigen of American biological medicine research and development company, and the novel antibody has good inhibition effects on non-small cell cancers, melanoma, urothelial cancers, high-frequency microsatellite instability (MSI-H), head and neck cancers and the like.

Disclosure of Invention

The present invention provides a chimeric antigen receptor T cell that self-expresses a CD40 activating antibody and targets the ErbB receptor family, preferably the T cell:

(1) Comprising a coding sequence for expressing a chimeric antigen receptor that targets the ErbB receptor family and a coding sequence for a CD40 activating antibody; and/or

(2) Express chimeric antigen receptor and CD40 activating antibodies targeting the ErbB receptor family.

In one or more embodiments, the T cell genome incorporates an expression cassette for a CD40 activating antibody and an expression cassette for a chimeric antigen receptor that targets the ErbB receptor family.

In one or more embodiments, the chimeric antigen receptor comprises an optional signal peptide, a T1E peptide fragment and a Herin peptide fragment linked by a rigid linker, a hinge region, a transmembrane region, an intracellular co-stimulatory signaling domain, and an intracellular signaling domain.

In one or more embodiments, the signal peptide is a CD8 signal peptide, a CD28 signal peptide or a CD4 signal peptide, preferably a CD8 signal peptide; more preferably, the amino acid sequence of the CD8 signal peptide is shown as amino acid residues 1-22 of SEQ ID NO. 5.

In one or more embodiments, the amino acid sequence of T1E is shown as amino acid residues 23-77 of SEQ ID NO. 5; preferably, the coding sequence is shown as the 67 th to 231 th base sequence of SEQ ID NO. 6.

In one or more embodiments, the amino acid sequence of the Herin is as set forth in amino acid residues 93-171 of SEQ ID NO. 5; preferably, the coding sequence is shown in the base sequence of 277-513 of SEQ ID NO. 6.

In one or more embodiments, the rigid linker is an EAAAK linker, preferably the amino acid sequence of the linker is as set forth in amino acid residues 78-92 of SEQ ID No. 5; preferably, the coding sequence is shown as 232-276 base sequences of SEQ ID NO. 6.

In one or more embodiments, the hinge region is selected from the group consisting of an extracellular hinge region of CD8, an IgG1Fc CH2CH3 hinge region, an IgD hinge region, a CD28 extracellular hinge region, an IgG4 Fc CH2CH3 hinge region, and an extracellular hinge region of CD4, preferably a CD8 hinge region or an IgG4 CH2CH3 hinge region; preferably, the hinge region is an IgG4 CH2CH3 hinge region, the amino acid sequence of which is shown as amino acid residues 172-399 of SEQ ID NO. 5, and preferably the coding sequence of which is shown as base sequences 514-1197 of SEQ ID NO. 6.

In one or more embodiments, the transmembrane region is one of a CD28 transmembrane region, a CD8 transmembrane region, a cd3ζ transmembrane region, a CD134 transmembrane region, a CD137 transmembrane region, an ICOS transmembrane region, and a DAP10 transmembrane region; preferably a CD28 transmembrane region, preferably having an amino acid sequence as shown in amino acid residues 400-427 of SEQ ID NO. 5, preferably having a coding sequence as shown in base sequences 1198-1281 of SEQ ID NO. 6.

In one or more embodiments, the intracellular co-stimulatory signaling domain includes an intracellular domain of a co-stimulatory signaling molecule, including an intracellular domain of CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase, inducible T cell co-stimulatory factor (ICOS), and DNAX activator protein 10; preferably, the intracellular domain of the costimulatory signal molecule is a CD28 intracellular domain, the amino acid sequence of which is shown as amino acid residues 428-468 of SEQ ID NO. 5, and the coding sequence of which is shown as base sequences 1282-1404 of SEQ ID NO. 6.

In one or more embodiments, the intracellular signaling domain is a cd3ζ intracellular signaling domain or an fcsriy intracellular signaling domain; preferably a CD3 zeta intracellular signal domain, preferably the amino acid sequence of said CD3 zeta intracellular signal domain is shown as SEQ ID NO. 5 amino acid residues 469-580, preferably the coding sequence is shown as SEQ ID NO. 6 base sequences 1405-1740.

In one or more embodiments, the chimeric antigen receptor has an amino acid sequence as shown in amino acid residues 23-580 of SEQ ID NO. 5, or as shown in SEQ ID NO. 5; preferably, the nucleotide sequence of the chimeric antigen receptor is shown as the 67 th to 1740 th base sequence shown as SEQ ID NO. 6 or as SEQ ID NO. 6.

In one or more embodiments, the CD4 activating antibody comprises an anti-CD 40 single chain antibody and an IgG4Fc; preferably, the amino acid sequence of the IgG4Fc is shown as amino acid residues 269-497 of SEQ ID NO. 1, and preferably, the coding sequence is shown as base sequences 805-1491 of SEQ ID NO. 2.

In one or more embodiments, the amino acid sequence of the variable region (VL region) of the light chain of the anti-CD 40 single chain antibody (scFv) is shown as amino acid residues 21-146 of SEQ ID NO. 1; preferably, the coding sequence is shown as the 64 th to 438 th base sequence of SEQ ID NO. 2.

In one or more embodiments, the heavy chain variable region (VH region) amino acid sequence of the anti-CD 40 single chain antibody is shown as 161-268 amino acid sequences of SEQ ID NO. 1; preferably, the coding sequence is shown as 481-804 base sequences of SEQ ID NO. 2.

In one or more embodiments, the anti-CD 40 single chain antibody has an amino acid sequence as shown in amino acid residues 21-268 of SEQ ID NO. 1; preferably, the coding sequence is shown as 61-804 base sequences of SEQ ID NO. 2.

In one or more embodiments, the CD40 activating antibody comprises, in order from N-terminus to C-terminus, a light chain signal peptide, an anti-CD 40 single chain antibody, and an IgG4Fc; preferably, the amino acid sequence of the light chain signal peptide is shown as amino acid residues 1 to 20 of SEQ ID NO. 1; preferably, the coding sequence of the light chain signal peptide is shown as the 1 st to 60 th base sequence of SEQ ID NO. 2.

In one or more embodiments, the amino acid sequence of the CD40 activating antibody is shown as amino acid sequences at positions 21-497 of SEQ ID NO. 1, or as SEQ ID NO. 1; preferably, the coding sequence is shown as the 61 st to 1491 st base sequence of SEQ ID NO. 2 or as the preferred one shown as SEQ ID NO. 2.

The present invention also provides a composition comprising:

(1) A vector comprising an expression cassette for a chimeric antigen receptor described herein for integration of the expression cassette into the genome of a host cell; and

(2) A vector comprising an expression cassette for a CD40 activating antibody as described herein for integration of said expression cassette into the genome of a host cell.

The invention also provides a kit comprising:

The invention also provides a pharmaceutical composition comprising the T cells described herein.

The invention also provides the use of a T cell or a pharmaceutical composition thereof as described herein in the manufacture of a medicament for the treatment or prevention of a malignancy; preferably, the cancer is a cancer whose cancer cell surface abnormally expresses at least one EGFR family member protein; preferably, the cancer is selected from: liver cancer, adenocarcinoma, lung cancer, colon cancer, large intestine cancer, breast cancer, ovarian cancer, cervical cancer, stomach cancer, bile duct cancer, non-small cell cancer, gallbladder cancer, esophageal cancer, melanoma, pancreatic cancer, urothelial cancer, head and neck cancer or prostate cancer.

Drawings

Fig. 1: gene structural pattern of recombinant plasmids pS 328-. Alpha.CD 40 and pNB328-EHCAR-EK-28 TIZ.

Fig. 2A-2B: positive rate and antibody expression level of chimeric antigen receptor modified T cells constructed by pNB328-EHCAR-EK-28TIZ and pS 328-alpha CD40 plasmids with different mass ratios.

Fig. 3A-3B: EHCAR-EK-28TIZ T cells and EHCAR-EK-28 TIZ-. Alpha.CD40T cells were used to measure the positive rate and the antibody expression level.

Fig. 4: proliferation rate comparison of EHCAR-EK-28TIZ and EHCAR-EK-28 TIZ-. Alpha.CD40T cells.

Fig. 5A-5D: cell phenotyping analysis of T cells of EHCAR-EK-28TIZ and EHCAR-EK-28TIZ- αCD40T cells, 5A representing senescence phenotype CD40,5B and 5C representing activation phenotype CD69 and CD107 α, respectively, 5D representing memory phenotype.

Fig. 6: killing contrast of EHCAR-EK-28TIZ and EHCAR-EK-28TIZ- αcd40T cells, including human hepatoma cell HCCLM3, human lung degenerative cancer cell Calu-6, and human non-small cell lung cancer H23.

Fig. 7: alterations in IL-2, IL-4, IL-6, IL-10, TNF- α and IFN- γ cytokine secretion by EHCAR-EK-28TIZ and EHCAR-EK-28TIZ- α CD40T cells under EGFR antigen stimulation.

Fig. 8: EHCAR-EK-28TIZ T cells, EHCAR-EK-28 TIZ-. Alpha.CD 40-wt T cells, EHCAR-EK-28 TIZ-. Alpha.CD 40T cells, mock-T cells and PBS blank, tumor cell fluorescence values varied on different days after treatment of mice, respectively.

Detailed Description

The following is a description of some of the terms involved in the present invention.

In the present invention, the term "expression cassette" refers to the complete elements required for expression of a gene, including promoters and gene coding sequences.

The term "coding sequence" is defined herein as that portion of a nucleic acid sequence that directly determines the amino acid sequence of its protein product (e.g., CAR, single chain antibody, hinge region, and transmembrane region). The boundaries of the coding sequence are typically determined by a ribosome binding site (for prokaryotic cells) immediately upstream of the open reading frame at the 5 'end of the mRNA and a transcription termination sequence immediately downstream of the open reading frame at the 3' end of the mRNA. Coding sequences may include, but are not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

The term "Fc", i.e., the crystallizable section of an antibody (fragment crystallizable, fc), refers to the peptide section comprising the CH2 and CH3 domains of the heavy chain of an antibody at the end of the stem of the "Y" structure of an antibody molecule, which is the site of interaction of the antibody with an effector molecule or cell.

The term "costimulatory molecule" refers to a molecule that is present on the surface of an antigen presenting cell and that is capable of binding to a costimulatory molecule receptor on a Th cell to produce a costimulatory signal. Proliferation of lymphocytes requires not only antigen binding but also signal of the co-stimulatory molecule. The co-stimulatory signal is transmitted to the T cell primarily through the co-stimulatory molecule CD80, CD86 expressed on the surface of the antigen presenting cell binding to the CD28 molecule on the surface of the T cell. B cells receive costimulatory signals through common pathogen components such as LPS, or through complement components, or through activated antigen-specific CD40L on Th cell surfaces.

The term "linker" or hinge is a polypeptide fragment that connects between different proteins or polypeptides in order to maintain the connected proteins or polypeptides in their respective spatial conformations in order to maintain the function or activity of the protein or polypeptide. Exemplary linkers include linkers comprising G and/or S, and for example Furin 2A peptides.

The term "specific binding" refers to a reaction between an antibody or antigen binding fragment and an antigen against which it is directed. In certain embodiments, an antibody that specifically binds to (or has specificity for) an antigen means that the antibody binds to or has specificity for an antigen in an amount of less than about 10 ^-5 M, e.g. less than about 10 ^-6 M、10 ^-7 M、10 ^-8 M、10 ^-9 M or 10 ^-10 M or less affinity (KD) binds the antigen. "specific recognition" has similar meaning.

The term "pharmaceutically acceptable excipients" refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and active ingredient, which are well known in the art (see, e.g., remington's Pharmaceutical sciences. Mediated by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995), and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers. For example, pH modifiers include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.

The term "effective amount" refers to the amount that achieves treatment, prevention, alleviation and/or relief of a disease or condition of the present invention in a subject.

The term "disease and/or disorder" refers to a physical state of the subject that is associated with the disease and/or disorder of the present invention.

The term "subject" or "patient" may refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, cow, horse, etc., receiving a pharmaceutical composition of the invention for treating, preventing, alleviating and/or alleviating a disease or condition described herein.

The term "chimeric antigen receptor" (CAR) is an engineered receptor capable of anchoring a specific molecule (e.g., an antibody) that recognizes a tumor cell surface antigen to an immune cell (e.g., a T cell), allowing the immune cell to recognize a tumor antigen or viral antigen and kill a tumor cell or virus-infected cell. The CAR typically comprises, in order, an optional signal peptide, a polypeptide that binds to a tumor cell membrane antigen, such as a single chain antibody, a hinge region, a transmembrane region, and an intracellular signal region. In general, polypeptides that bind tumor cell membrane antigens are capable of binding with moderate affinity to membrane antigens that are widely expressed by tumor cells. The polypeptide that binds to a tumor cell membrane antigen may be a natural polypeptide or an artificial polypeptide; preferably, the synthetic polypeptide is a single chain antibody or Fab fragment.

The term "single chain antibody" (scFv) refers to an antibody fragment having the ability to bind antigen, which is formed by the amino acid sequence of the light chain variable region (VL region) and the amino acid sequence of the heavy chain variable region (VH region) of an antibody, which are joined by a hinge. In certain embodiments, the single chain antibody of interest (scFv) is from an antibody of interest. The antibody of interest may be a human antibody, including a human murine chimeric antibody and a humanized antibody. Antibodies may be secreted or membrane anchored; preferably of the membrane anchor type.

In order to improve the immunotherapeutic effect of the EHCAR-EK-28TIZ T cells, the CD40 activating antibody is expressed on the existing CAR-T cells, and the result shows that the CD40 activating antibody can promote the activation and proliferation of the CAR-T cells, improve the secretion of cytokines and increase the anti-tumor killing effect in vivo.

Studies show that the IgG4Fc fragment of the CD40 activating antibody is easily recognized by mononuclear/macrophages to be phagocytosed, and the CD40 activating antibody IgG4Fc fragment is subjected to base mutation modification to meet the requirement of the CD40 activating antibody expressed by T cells, so that the CD40 activating antibody can well function and does not cause ADCC reaction.

Accordingly, the present invention provides a CD 40-activating antibody comprising an anti-CD 40 single chain antibody and IgG4Fc. In certain embodiments, the amino acid sequence of the IgG4Fc is shown as amino acid residues 269-497 of SEQ ID NO. 1; preferably, the coding sequence is shown as the base sequence of 805-1491 of SEQ ID NO. 2.

In certain embodiments, the amino acid sequence of the light chain variable region (VL region) of said anti-CD 40 single chain antibody (scFv) is as shown in amino acid residues 21-146 of SEQ ID NO. 1; preferably, the coding sequence is shown as the 64 th to 438 th base sequence of SEQ ID NO. 2. In certain embodiments, the heavy chain variable region (VH region) amino acid sequence of said anti-CD 40 single chain antibody is as shown in amino acid sequences 161-268 of SEQ ID NO. 1; preferably, the coding sequence is shown as 481-804 base sequences of SEQ ID NO. 2. In certain embodiments, the anti-CD 40 single chain antibody has an amino acid sequence as shown in amino acid residues 21-268 of SEQ ID NO. 1; preferably, the coding sequence is shown as 61-804 base sequences of SEQ ID NO. 2.

In certain embodiments, the CD40 antibody further comprises a light chain signal peptide. In certain embodiments, the CD40 antibody comprises, from N-terminus to C-terminus, a light chain signal peptide, an anti-CD 40 single chain antibody, and IgG4Fc, in that order. In certain embodiments, the amino acid sequence of the light chain signal peptide is as shown in amino acid residues 1-20 of SEQ ID NO. 1; preferably, the coding sequence of the light chain signal peptide is shown as the 1 st to 60 th base sequence of SEQ ID NO. 2.

In certain embodiments, the CD40 activating antibody has an amino acid sequence as set forth in SEQ ID NO. 1 at amino acid positions 21-497, or as set forth in SEQ ID NO. 1.

The invention also includes the coding sequence of the CD40 antibody, or the complement thereof, comprising at least the coding sequence of IgG4Fc, or the complement thereof, as described herein. In certain embodiments, the coding sequence of the CD40 antibody comprises the sequence shown in base sequence No. 61-1491 of SEQ ID NO. 2, preferably the sequence shown in SEQ ID NO. 2.

The invention also includes a nucleic acid construct comprising the coding sequence of the CD40 antibody of the invention or the complement thereof. Preferably, the nucleic acid construct is an expression vector or an integration vector for integrating the coding sequence or the complement thereof into a host cell.

The invention also provides a host cell comprising a nucleic acid construct as described herein.

The invention also provides the use of the CD40 antibodies, their coding sequences or complementary sequences, nucleic acid constructs, and host cells in the preparation of a method for treating or preventing malignant tumors, particularly those associated with CD40, including but not limited to the various malignant tumors described herein.

The invention also provides T cells from chimeric antigen receptors that express CD40 activating antibodies and target the ErbB receptor family. In the Chimeric Antigen Receptor (CAR), natural T1E and Herin are fused and expressed as antigen recognition regions of the CAR, and the chimeric antigen receptor and the Herin can complementarily recognize ErbB receptor families, so that the targeting range is enlarged.

The CARs of the invention generally contain an optional signal peptide sequence, a fusion protein of T1E and Herin, a hinge region, a transmembrane region, an intracellular co-stimulatory signaling domain, and an intracellular signaling domain.

The signal peptide is a short peptide chain (5-30 amino acids in length) that directs the transfer of a newly synthesized protein to the secretory pathway, often referred to as the N-terminal amino acid sequence (sometimes not necessarily at the N-terminus) of the newly synthesized polypeptide chain that directs the transmembrane transfer (localization) of the protein, which is responsible for directing the protein into subcellular organelles of the cell containing different membrane structures. The signal peptide may be a secretory signal peptide or a membrane-bound signal peptide. In certain embodiments of the invention, the signal peptide is a CD8 signal peptide, a CD28 signal peptide, or a CD4 signal peptide; more preferably a CD8 signal peptide. The amino acid sequence of the CD8 signal peptide can be shown as the amino acid residues 1 to 22 of SEQ ID NO. 5; in certain embodiments, the coding sequence is shown as bases 1-66 of SEQ ID NO. 6.

Native T1E and Herin are expressed fusion herein as the antigen recognition region of the CAR. The T1E is a chimeric polypeptide consisting of seven amino acids at the N-terminus of human transcription growth factor alpha (TGF alpha) and 48 amino acids at the C-terminus of Epidermal Growth Factor (EGF). Preferably, the amino acid sequence of T1E is shown as the 23 rd to 77 th amino acid residues of SEQ ID NO. 5; preferably, the coding sequence is shown as the 67 th to 231 th base sequence of SEQ ID NO. 6.

In the present invention, herin refers to 79 amino acids encoded by the eighth intron of Herstatin. Preferably, the amino acid sequence is shown as amino acid residues 93-171 of SEQ ID NO. 5. The present invention optimizes the codon encoding the amino acid of Herin. Thus, the preferred nucleotide sequence of Herin of the present invention is as shown in SEQ ID NO: the base sequence of the 277 th to 513 th positions is shown.

Typically, T1E and Herin may be linked by a rigid linker sequence. An illustrative example of a rigid linker sequence is a 2 or more repeat sequence with EAAAK as a unit, also referred to herein as EAAAK linker. Exemplary rigid linker sequences are shown as amino acid residues 78-92 of SEQ ID NO. 5; exemplary coding sequences are shown in the 232 th to 276 th base sequences of SEQ ID NO. 6.

The hinge region, as used herein, refers to the region between the functional regions of the heavy chains CH1 and CH2 of an immunoglobulin which is rich in proline, does not form an alpha helix, and is subject to stretching and some degree of warping, which facilitates complementary binding between the antigen binding site of the antibody and the epitope. Hinge regions suitable for use herein may be selected from any one or more of the extracellular hinge region of CD8, the IgG1Fc CH2CH3 hinge region, the IgD hinge region, the extracellular hinge region of CD28, the IgG4 Fc CH2CH3 hinge region, and the extracellular hinge region of CD 4. The hinge region is preferably a hinge region that is more than 50 amino acid residues in length, more preferably more than 80 amino acids in length. In certain embodiments, a CD8 a hinge region or an IgG4 Fc CH2CH3 hinge region is used herein. The amino acid sequence of an exemplary IgG4 FcCH2CH3 hinge region is shown as amino acid residues 172-399 of SEQ ID NO. 5, and the coding sequence of an exemplary IgG4 FcCH2CH3 hinge region is shown as 514-1197 of SEQ ID NO. 6.

The transmembrane region may be one of a CD28 transmembrane region, a CD8 transmembrane region, a cd3ζ transmembrane region, a CD134 transmembrane region, a CD137 transmembrane region, an ICOS transmembrane region, and a DAP10 transmembrane region; preferably a CD28 transmembrane region, preferably having an amino acid sequence as shown in amino acid residues 400-427 of SEQ ID NO. 5; in certain embodiments, the coding sequence is as shown in bases 1198-1281 of SEQ ID NO. 6.

Intracellular costimulatory signaling domains the intracellular domain comprising the costimulatory signaling molecule may be selected from the group consisting of the intracellular domains of CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase (LCK), inducible T cell costimulatory factor (ICOS) and DNAX activator protein 10 (DAP 10). In certain embodiments, the intracellular domain of the costimulatory signaling molecule is the intracellular domain of CD28, preferably having the amino acid sequence shown as amino acid residues 428-468 of SEQ ID NO. 5, and exemplary coding sequences shown as bases 1282-1404 of SEQ ID NO. 6.

The intracellular signaling domain is preferably an immunoreceptor tyrosine-activating motif, which may be a cd3ζ intracellular signaling domain or an fcsriy intracellular signaling domain; preferably a CD3 zeta intracellular signaling domain, preferably the amino acid sequence of said CD3 zeta intracellular signaling domain is as described in SEQ ID NO. 5, 469-580; in certain embodiments, the coding sequence is as shown in bases 1405-1740 of SEQ ID NO. 6.

In certain embodiments, the chimeric antigen receptor comprises, in order from N-terminus to C-terminus: an optional CD signal peptide, T1E, EAAAK linker, herin, igG4 Fc CH2CH3 hinge region, CD28 transmembrane region, intracellular domain of CD28, and cd3ζ intracellular signal domain; preferably, the amino acid sequence of the chimeric antigen receptor is shown as amino acid residues 23-580 of SEQ ID NO. 5. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide, preferably the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO. 5.

It is to be understood that the present invention also includes chimeric antibody receptors described herein and coding sequences thereof.

The above-described portions forming the chimeric antigen receptor herein, such as the signal peptide, T1E, herin, hinge region, transmembrane region, intracellular co-stimulatory signaling domain, and intracellular signaling domain, and the like, may be directly linked to each other or may be linked by a linker sequence. The linker sequences may be linker sequences suitable for antibodies as known in the art, such as G and S containing linker sequences. The length of the linker may be 3 to 25 amino acid residues, for example 3 to 15, 5 to 15, 10 to 20 amino acid residues. In certain embodiments, the linker sequence is a glycine linker sequence. The number of glycine in the linker sequence is not particularly limited, and is usually 2 to 20, for example 2 to 15, 2 to 10, 2 to 8. In addition to glycine and serine, other known amino acid residues may be contained in the linker, such as alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), glutamine (Q), etc.

It will be appreciated that in gene cloning operations, it is often necessary to design suitable cleavage sites, which tend to introduce one or more unrelated residues at the end of the expressed amino acid sequence, without affecting the activity of the sequence of interest. To construct fusion proteins, facilitate expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside of the host cell, or facilitate purification of recombinant proteins, it is often desirable to add some amino acid to the N-terminus, C-terminus, or other suitable region within the recombinant protein, including, for example, but not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, and the like. Thus, the amino-or carboxy-terminus of a CAR herein can also contain one or more polypeptide fragments as protein tags. Any suitable label may be used herein. For example, the tag may be FLAG, HA, HA1, c-Myc, poly-His, poly-Arg, strep-TagII, AU1, EE, T7,4A6, ε, B, gE, and Ty1. These tags can be used to purify proteins.

Also included herein are polynucleotide sequences encoding the chimeric antigen receptors. The polynucleotide sequences herein may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded.

The polynucleotide sequences described herein can generally be obtained using PCR amplification methods. Specifically, primers can be designed based on the nucleotide sequences disclosed herein and amplified to obtain the relevant sequences using a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art as a template. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order. For example, in certain embodiments, the polynucleotide sequence encoding the fusion proteins described herein is shown in SEQ ID NO. 6.

Also included herein are nucleic acid constructs comprising a polynucleotide sequence encoding the chimeric antigen receptor or a polynucleotide sequence encoding the CD40 antibody described herein, and one or more regulatory sequences operably linked to these sequences. In certain embodiments, the nucleic acid constructs of the invention are expression cassettes.

The regulatory sequence may be a suitable promoter sequence. The promoter sequence is typically operably linked to the coding sequence of the protein to be expressed. The promoter may be any nucleotide sequence that exhibits transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

The regulatory sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used herein.

In certain embodiments, the nucleic acid construct is a vector. In particular, the coding sequence of the CAR or the coding sequence of the CD40 antibody herein can be cloned into many types of vectors, for example, such types of vectors include, but are not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. The vector may be an expression vector. The expression vector may be provided to the cell as a viral vector. Viruses that may be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.

In general, suitable vectors comprise an origin of replication functional in at least one organism, a promoter sequence, a convenient restriction enzyme site and one or more selectable markers. For example, in certain embodiments, the invention uses a retroviral vector comprising a replication initiation site, a 3'LTR, a 5' LTR, the coding sequences for CARs described herein or the coding sequences for CD40 antibodies, and optionally a selectable marker.

Suitable promoters include, but are not limited to, the immediate early Cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is extended growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV 40) early promoter, the mouse mammary carcinoma virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the epstein barr virus immediate early promoter, the ruses sarcoma virus promoter, and human gene promoters such as but not limited to the actin promoter, the myosin promoter, the heme promoter, and the creatine kinase promoter. Further, the use of inducible promoters is also contemplated. The use of an inducible promoter provides a molecular switch that is capable of switching on expression of a polynucleotide sequence operably linked to the inducible promoter when expressed for a period of time and switching off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

In certain embodiments, various promoter sequences published by CN201510021408.1 can be used, including but not limited to the CCEF promoter comprising the mCMV enhancer, the hCMV enhancer and the EF 1. Alpha. Promoter shown in SEQ ID NO. 5 of this application; the TCEF promoter shown in SEQ ID NO. 7 and containing the CD3e enhancer, the mCMV enhancer, the hCMV enhancer and the EF1 alpha promoter; the CCEFI promoter shown in SEQ ID NO. 8 and containing the mCMV enhancer, the hCMV enhancer and the EF1 alpha promoter containing the intron; the TEFI promoter shown in SEQ ID NO. 3 and containing a CD3e enhancer and an EF1 alpha promoter containing an intron; and the TCEFI promoter shown in SEQ ID NO. 3 and containing the CD3e enhancer, the mCMV enhancer, the hCMV enhancer and the EF1 alpha promoter containing the intron. The entire contents of this application are incorporated herein by reference.

Selectable markers include either or both selectable marker genes or reporter genes to facilitate identification and selection of expressing cells from a population of cells infected with the viral vector. Useful selectable marker genes include, for example, antibiotic resistance genes, such as neo and the like. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or green fluorescent protein genes.

In certain embodiments, the coding sequences for the chimeric antigen receptor and the coding sequences for the CD40 antibody described herein are separately cloned into vectors (also referred to as integration vectors), particularly transposon vectors, for integrating the nucleic acid sequences of interest into the genome of the host cell. In certain embodiments, the transposon vector is a eukaryotic expression vector containing a transposable element selected from piggybac, sleep reliability, frog priority, tn5, or Ty. Such transposon vectors contain the 5 'inverted terminal repeat (5' LTR) of the corresponding transposon and the 3 'inverted terminal repeat (3' LTR) of the corresponding transposon. The transposase may be a transposase from a piggybac, sleep bearing, frog priority, tn5 or Ty transposase system. When transposases from different transposition systems are used, the sequences of the 5'LTR and 3' LTR in the vector are also changed accordingly to sequences that fit the transposition system, as can be readily determined by one skilled in the art. Between the 5'ltr and the 3' ltr is an expression cassette for a CAR or antibody of the invention, comprising a corresponding promoter sequence, a coding sequence for the CAR or antibody, and a polyA tailing signal sequence.

In certain embodiments, the transposase is a transposase from the piggybac transposable system. Thus, in these embodiments, the transposon 5 'inverted terminal repeat and 3' inverted terminal repeat are the 5 'inverted terminal repeat and 3' inverted terminal repeat, respectively, of the piggybac transposon. In certain embodiments, the transposon 5' inverted terminal repeat is as shown in CN 201510638974.7 (the contents of which are incorporated herein by reference) SEQ ID No. 1. In certain embodiments, the transposon 3' inverted terminal repeat is as shown in CN 201510638974.7SEQ ID NO:4. In certain embodiments, the piggybac transposase is a transposase comprising a c-myc nuclear localization signal coding sequence. In certain embodiments, the coding sequence of the piggybac transposase is as set forth in CN 201510638974.7SEQ ID NO:5.

Promoters of the transposase coding sequence may be any of the promoters known in the art for controlling the expression of the transposase coding sequence. In certain embodiments, the expression of the transposase coding sequence is controlled using a CMV promoter. The sequence of the CMV promoter may be as shown in CN 201510638974.7SEQ ID NO:6.

In certain embodiments, the vector of the invention comprising the expression cassette of the chimeric antigen receptor is the pNB328 vector disclosed in CN 201510638974.7. The coding sequences for the chimeric antigen receptor of the invention can be prepared by methods conventional in the art and cloned into a suitable vector.

In certain embodiments, the vector for integrating the gene of interest into the genome of the host cell does not contain a transposase coding sequence. For example, such vectors may be obtained by removing the transposase coding sequence from the pNB328 vector. Typically, such vectors are used to integrate the coding sequence for a CD40 antibody and the coding sequence for a signal peptide (e.g., the coding sequence for a light chain signal peptide) into the genome of a host cell. Exemplary light chain signal peptides have the amino acid sequence shown in SEQ ID NO: 1-20 amino acid residues, and the coding sequence of an exemplary light chain signal peptide is shown in SEQ ID NO:2, 1-60 th base.

In certain embodiments, T cells described herein that are modified by a CAR gene and that are capable of expressing a CD40 antibody can be transformed into: a vector comprising a transposase coding sequence for integration into the T cell genome of a chimeric antigen receptor coding sequence, and a vector comprising no transposase coding sequence for integration into the T cell genome of a coding sequence of a CD40 antibody as described herein.

Preferably, the T cells are transformed with a vector comprising a chimeric antigen receptor coding sequence constructed using the pNB328 vector as a scaffold vector and a vector comprising a CD40 antibody coding sequence constructed using the pS328 vector (without a transposase coding sequence as compared to pNB 328) as a scaffold vector. In certain embodiments, the chimeric antigen receptor has a coding sequence as set forth in SEQ ID NO. 6; the coding sequence of the CD40 antibody is shown as 61 th to 1485 th base sequences of SEQ ID NO. 2. In certain embodiments, the signal peptide of the CD40 antibody is a light chain signal peptide in the vector comprising the coding sequence of the CD40 antibody. An exemplary light chain signal peptide may have an amino acid sequence as shown in amino acid residues 1-60 of SEQ ID NO. 2. More specifically, in certain embodiments, the transposase-containing vector having a chimeric antigen receptor coding sequence incorporated into the T cell genome comprises, in order, a 5'ltr, a promoter, a CD8 signal peptide coding sequence, a T1E, EAAAK linker, a Herin, a coding sequence for an IgG4 Fc CH2CH3 hinge region, a coding sequence for a CD28 transmembrane region, a coding sequence for a CD28 intracellular domain, a coding sequence for a CD3 zeta intracellular signal domain, a polyA tailing signal sequence, a 3' ltr, and a coding sequence for a transposase, and promoters thereof; the vector without transposase coding sequence, which incorporates the coding sequence of the CD40 antibody described herein in the T cell genome, contains a promoter, a coding sequence for a light chain signal peptide, a coding sequence for a CD40 antibody, and a polyA tailing signal sequence in that order between the 5'ltr and the 3' ltr.

Preferably, the mass ratio of the vector containing the chimeric antigen receptor coding sequence to the vector containing the CD40 antibody coding sequence at the time of transfection is 1-7: 1 to 7, preferably 1: 3-1: 3, more preferably 1 to 3:1, more preferably 1 to: 1, more preferably 5:3.

methods of transfection are conventional in the art and include, but are not limited to: viral transduction, microinjection, particle bombardment, gene gun transformation, electrotransformation, and the like. In certain embodiments, electrotransfection is used to transfect the vector into a cell of interest.

The cells of interest may be a variety of T cells well known in the art, including but not limited to T cells of mixed cell populations such as peripheral blood T lymphocytes, cytotoxic killer T Cells (CTLs), helper T cells, suppressor/regulatory T cells, γδ T cells, and cytokine-induced killer Cells (CIKs), tumor Infiltrating Lymphocytes (TILs), and the like. In certain embodiments, the T cells may be derived from PBMCs of B cell malignancy patients. In certain embodiments, the T cell is a primary culture T cell.

The invention also provides a composition comprising a vector comprising the coding sequence for a chimeric antigen receptor described herein and a vector comprising the coding sequence for a CD40 antibody described herein. Suitable agents may also be included in the composition, including but not limited to agents for transfection.

The invention also provides a kit comprising a vector comprising the chimeric antigen receptor coding sequences described herein and a vector comprising the coding sequences of a CD40 antibody described herein, or a composition described herein. The kit may also be provided with reagents or instruments for transferring the vector into cells.

As described herein, the expression cassette contains at least a suitable promoter and a PolyA tailing signal sequence in addition to the coding sequence for the chimeric antigen receptor or CD40 activating antibody.

The invention also provides a pharmaceutical composition comprising the T cells described herein. The pharmaceutical composition may contain suitable pharmaceutically acceptable carriers or excipients. The pharmaceutical composition contains a therapeutically or prophylactically effective amount of T cells. The therapeutically or prophylactically effective amount of T cells can be determined based on factors such as the patient's condition.

The invention also provides the use of a T cell or a pharmaceutical composition thereof as described herein in the manufacture of a medicament for the treatment or prevention of a malignancy (cancer); preferably, the malignancy is a tumor associated with CD40 and/or ErbB receptor; more preferably, the cancer cell surface of the malignancy abnormally expresses at least one EGFR family member protein. More preferably, the malignancy is selected from: liver cancer, adenocarcinoma, lung cancer, colon cancer, large intestine cancer, breast cancer, ovarian cancer, cervical cancer, stomach cancer, bile duct cancer, non-small cell cancer, gallbladder cancer, esophageal cancer, melanoma, pancreatic cancer, urothelial cancer, head and neck cancer or prostate cancer.

Embodiments of the present invention will be described in detail below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not noted in the examples, and are carried out according to techniques or conditions described in the literature in the art (for example, refer to J. Sam Brookfield et al, ind. Molecular cloning Experimental guidelines, third edition, scientific Press) or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1: construction of recombinant plasmids pS328- αCD40-wt, pS328- αCD40 and pNB328-EHCAR-EK-28TIZ

1. The EHCAR-EK-28TIZ gene (the nucleotide sequence is shown as SEQ ID NO:6, the amino acid sequence is shown as SEQ ID NO: 5), the anti-CD40 gene (the nucleotide sequence is shown as SEQ ID NO:2, the amino acid sequence is shown as SEQ ID NO: 1) and the anti-CD40-wt gene (the nucleotide sequence is shown as SEQ ID NO:4, the amino acid sequence is shown as SEQ ID NO: 3) are synthesized by Shanghai Jieshi biological company, and the structural modes are shown as figure 1. Each gene was separately loaded into pNB328 and pS328 vectors (the structure and sequence of pNB328 is referred to as CN 201510638974.7; pS328 lacks the PB transposon sequence compared to pNB328 and other elements are identical to pNB328 vectors) double digested with EcoR1+SalI, and plasmids were constructed, designated pNB328-EHCAR-EK-28TIZ, pS328- αCD40 and pS328- αCD40-wt, respectively.

The nucleotide sequence of the light chain signal peptide in the structure pattern diagram is shown as the 1 st to 60 th base sequence of SEQ ID NO. 2; the coding of the Anti-CD40-wt is shown as SEQ ID NO. 4 (the amino acid sequence is shown as amino acid residues 21-495 of SEQ ID NO. 3); the nucleotide sequence of the Anti-CD40 is shown as the 61 st to 1488 th base sequence of SEQ ID NO. 2; the nucleotide sequence of the CD8 signal peptide is shown as the 1 st to 66 th base sequence of SEQ ID NO. 6; the nucleotide sequence of the T1E is shown as the 67 th to 231 th base sequence of SEQ ID NO. 6; the nucleotide sequence of the Herin is shown as 232 th-276 th base sequence of SEQ ID NO. 6; the nucleotide sequence of EAAAK-linker (EK-linker) is shown as the base sequence of 277-513 th bit of SEQ ID NO. 6; the nucleotide sequence of the membrane-spanning region of the mIgG4Fc CH2CH3 hinge is shown as the 514 th-1197 th base sequence of SEQ ID NO. 6; the nucleotide sequence of the CD28 transmembrane region (CD 28 TM) is shown as the base sequences 1198-1281 at positions 1-66 of SEQ ID NO. 6; the nucleotide sequence of the CD28 intracellular co-stimulatory signal structure region (CD 28 IC) is shown as the 1282-1404 base sequence of SEQ ID NO. 6; the nucleotide sequence of the CD3 zeta intracellular signal domain is shown as the 1405 th to 1740 th base sequence of SEQ ID NO. 6. The promoter sequence and polyA tailing signal sequence are not shown in the structural schematic diagrams, and are located between the 5'LTR and the signal peptide sequence and before the 3' LTR, respectively.

Example 2: positive rate and antibody expression quantity measurement of chimeric antigen receptor modified T cells constructed by pNB328-EHCAR-EK-28TIZ and pS 328-alpha CD40 plasmids with different mass ratios

The amounts of pNB328-EHCAR-EK-28TIZ and pS328- αCD40 plasmids were set to 7 ratios of 1ug+7ug, 2ug+6ug, 3ug+5ug, 4ug+4ug, 5ug+3ug, 6ug+2ug, 7ug+1ug, respectively, and CAR T cell construction was performed. The construction method comprises the following steps:

peripheral Blood Mononuclear Cells (PBMCs) were isolated from Shanghai cell therapy production centers. Culturing PBMC for 2-4h in an adherence way, wherein non-adherence suspension cells are initial T cells, collecting the suspension cells into a 15ml centrifuge tube, centrifuging for 3min at 1200rmp, discarding the supernatant, adding physiological saline, centrifuging for 3min at 1200rmp, discarding the physiological saline, and repeating the steps; eight 1.5ml centrifuge tubes were taken and 5X 10 tubes were added to each tube ⁶ The individual cells, numbered a, b, c, d, e, f, g and h,1200rmp were centrifuged for 3min, the supernatant was discarded, the electrotransfer kit (from Lonza corporation) was taken, 100ul of electrotransfer reagent was added to each tube in proportion, recombinant plasmids pNB328-EHCAR-EK-28TIZ and pS328- αcd40 of different mass ratios were added to a, b, c, d, e, f and g tubes, respectively, and 6ug of control plasmid (pBS 328, used to construct Mock T cells) was added to h tubes; transferring the mixed solution to an electric rotating cup, putting the electric rotating cup into an electric rotating instrument, selecting a required program, and performing electric shock; transferring the electrotransformed cell suspension into six-well plate (AIM-V culture solution containing 2% FBS) with micropipette in kit, mixing, standing at 37deg.C, and 5% CO ₂ Culturing in incubator, adding stimulating factors IL-2 and EGFR/anti-CD28 after six hours, 37 deg.C, 5% CO ₂ Culturing for 3-4 days, observing the growth of T cells, and obtaining EHCAR-EK-28TIZ T cells expressing CD40 antibody. And respectively detecting the positive rate and the antibody secretion amount of the CAR T cells constructed under 7 proportions.

1. Flow detection of CAR T cell positive rate

The seven CAR-T and Mock-T cells were collected and divided into two parts, each 1X 10 ⁶ Cells were washed twice with saline, resuspended in 100ul saline, one portion added 1ug EGFR-biotin and the other portion not added, and incubated at 4℃for 30 minutes. The cells were resuspended in 100ul of saline, and 1ul of streptomycin-PE antibody was added and incubated at 4℃for 30 min. Washing twice with physiological saline, and checking in machine to add onlyThe secondary antibody is a control.

As shown in FIG. 2A, the amounts of pNB328-EHCAR-EK-28TIZ and pS328- αCD40 plasmids gave the highest positive rate of CAR-T cells constructed in the form of 5 ug+3ug.

ELISA to detect the expression level of EHCAR-EK-28 TIZ-alpha CD40T cell antibody.

(1) CD40 antigen was diluted to 0.5ug/ml (5 ul+1ml coating) with coating solution, and the enzyme-labeled reaction plate was coated at 100 ul/well overnight at 4 ℃.

(2) The cells were washed 5 times with PBST for 3 minutes each, and then dried with absorbent paper at 200 ul/well.

(3) 100ul of blocking solution was added to each well and incubated at 37℃for 1 hour.

(4) The cells were washed 5 times with PBST for 3 minutes each, and then dried with absorbent paper at 200 ul/well.

(5) Samples and standards were added, 100 ul/well, multiplex wells and control wells were set, and incubated for 1 hour at 37 ℃.

(6) The cells were washed 5 times with PBST for 3 minutes each, and then dried with absorbent paper at 200 ul/well.

(7) Blocking solution IgG F4HRP1: diluted at 30000, 100 ul/well, incubated at 37℃for 45 min.

(8) The cells were washed 5 times with PBST for 3 minutes each, and then dried with absorbent paper at 200 ul/well.

(9) Adding the color development liquid TMB,100 ul/hole, and developing for 10-15min at 37 ℃ in dark.

The reaction was stopped by adding a stop solution, 50 ul/well.

OD value was measured at 450nm on a microplate reader, a standard curve was drawn, and CD40 antibody concentration was calculated.

Results As shown in FIG. 2B, the pNB328-EHCAR-EK-28TIZ and pS328- αCD40 plasmids constructed in the form of 5ug+3ug gave the highest amounts of antibody secreted by CAR-T cells.

And 5ug pNB328-EHCAR-EK-28TIZ and 3ug pS328-alpha CD40 are selected to construct the EHCAR-EK-28 TIZ-alpha CD40T cells with best effect by combining the positive rate and the antibody secretion amount.

Example 3: construction of EHCAR-EK-28TIZ T cells and EHCAR-EK-28 TIZ-alpha CD40T cells and determination of the Positive Rate and antibody expression level

6ug pNB328-EHCAR-EK-28TIZ plasmids were used to construct EHCAR-EK-28TIZ T cells, and 5ug pNB328-EHCAR-EK-28TIZ and 3ug pS328- αCD40 plasmids were used to construct EHCAR-EK-28TIZ- αCD40T cells, respectively, in the same manner as in example 2.

The positive rates of Mock-T cells of example 2 and EHCAR-EK-28TIZ T cells and EHCAR-EK-28TIZ- αcd40T cells constructed in this example were measured in the same manner as in example 2. The results are shown in fig. 3A, and the self-expression of CD40 antibody did not decrease the positive rate of CART cells.

ELISA detection of antibody expression levels of Mock-T cells, EHCAR-EK-28TIZ T cells and EHCAR-EK-28TIZ- αCD40T cells was performed in the same manner as in example 2, and the results are shown in FIG. 3B.

Example 4: comparison of EHCAR-EK-28TIZ and EHCAR-EK-28TIZ- αCD40T cell proliferation rates

Each taking 3×10 ⁵ Example 3 EHCAR-EK-28TIZ T cells, EHCAR-EK-28 TIZ-. Alpha.CD40T cells and Mock-T cells of example 2 on day 8 were cultured in 12-well plates in 1ml volumes. A96-well white opaque plate was prepared, and 80. Mu.L of each cell-containing culture solution was added to each well from the three groups of cells, and 80. Mu.L of the nutrient solution was simultaneously fed to the original 12-well plate. Then 80. Mu.L CellTiter-Glo reagent was added to the 96-well plate, mixed well on a shaker for 2min, and incubated at room temperature for 10min, and the Luc fluorescence value was read by an ELISA reader. The CellTiter-Glo Luminescent Cell Viability Assay kit used was purchased from Promega corporation. Cells cultured in 12-well plates were sampled daily on days 9, 10, 11, 12, and 13 of culture, and cell proliferation curves were plotted based on fluorescence values as measured by the above procedure.

The results are shown in FIG. 4, where the proliferation rate of EHCAR-EK-28TIZ- αCD40T cells is significantly higher than that of EHCAR-EK-28TIZ T cells, indicating that expression of CD40 antibodies can promote proliferation of CAR-T cells.

Example 5: cell phenotyping assays of EHCAR-EK-28TIZ and EHCAR-EK-28TIZ- αCD40T cells

Two EHCAR-EK-28TIZ and EHCAR-EK-28 TIZ-. Alpha.CD40T cells obtained in example 3 were collected,after the cell number, the cell number is 1 multiplied by 10 ⁶ The individual cells/tubes were added to 6 EP tubes of 1.5ml, washed twice with PBS, centrifuged at 1200rpm for 5min, and the supernatant discarded; wherein, 2 tubes are respectively added with the streaming antibodies anti-CD107 alpha-PE and anti-CD69-PE for detecting the phenotype of activated T cells, 1 tube is added with the streaming antibodies anti-CD45RO-PECy5+anti-CD197-FITC+anti-CD62L-PE for detecting the phenotype of memory T cells, 1 tube is respectively added with the streaming antibodies anti-PD1-PE for detecting the phenotype of inhibitory T cells, and the other 2 tubes are respectively added with isotype control streaming antibodies IgG1-PE and IgG1-PE+IgG2a-PECy5+IgG2a-PE, each antibody 2 μl (all purchased from Jackson ImmunoResearch company) and are subjected to flick precipitation to be uniformly mixed; after incubation for 30min at room temperature in the absence of light, PBS was washed once, centrifuged at 1200rpm for 5min, 400. Mu.l of physiological saline was added to the supernatant, and the cells were transferred to a flow tube and detected on-machine.

Experimental results found that expression levels of senescent phenotype PD1 of EHCAR-EK-28TIZ and EHCAR-EK-28TIZ- αcd40T cells were almost detected in a flow assay (fig. 5A); the expression level of activated phenotypes CD69 and CD107 a of EHCAR-EK-28TIZ- αCD40T cells was higher than that of EHCAR-EK-28TIZ cells (FIGS. 5B and 5C); meanwhile, CD62L (L-selectin) is a marker of central memory T cells, CD197 is a marker of effector memory T cells, and the proportion of effector T cells in EHCAR-EK-28 TIZ-alpha CD40T cells is significantly higher than that in EHCAR-EK-28TIZ cells and Mock-T cells (FIG. 5D). These results demonstrate that expressing CD40 antibodies can promote activation of CAR-T cells, enhancing their immune killing function.

Example 6: comparison of EHCAR-EK-28TIZ and EHCAR-EK-28TIZ- αCD40T cell killing function

The in vitro killing activity of two EHCAR-EK-28TIZ T cells and EHCAR-EK-28 TIZ-alpha CD40T cells obtained in example 3 was detected by selecting MHC class I-typed matched effector cells and target cells using a real-time label-free cell function analyzer (RTCA) from the Eisen company, and the specific procedures were as follows:

(1) Zeroing: adding 50 μl of DMEM or 1640 culture solution into each well, placing into an instrument, selecting step 1, and zeroing;

(2) Target cell plating: human hepatoma cell HCCLM3, human Lung degenerative cancer cell Calu-6 and human non-small cell lung cancer H23 (purchased from American type culture Collection ATCC) at 10 per well ⁴ Spreading the individual cells/50 μl in a plate containing detection electrodes, standing for several minutes, placing into an instrument after the cells are stabilized, and starting step 2 to culture the cells;

(3) Adding effector cells: after the target cells are cultured for 24 hours, the step 2 is paused, effector cells are added, 50 mu l of effector cells are added in each hole, the effective target ratio is set to be 4:1, the Mock T cells transferred into pNB328 empty vectors are used as a control, the step 3 is started, and after the co-culture is continued for 24 hours, the cell proliferation curve is observed;

the results are shown in FIG. 6. The killing effect of EHCAR-EK-28TIZ- αcd40T cells from CD40 antibody expressing cells was significantly stronger on various tumor cells than EHCAR-EK-28TIZ T cells as well as control T cells.

Example 7: comparison of cytokine release by EHCAR-EK-28TIZ T cells and EHCAR-EK-28TIZ- αCD40T cells under specific stimulation of EGFR antigen

Coating 96-well plate with EGFR antigen 5ug/ml, coating overnight at 4deg.C, washing 3 times with PBS, adding 1×10 ⁵ EHCAR-EK-28TIZ and EHCAR-EK-28 TIZ-. Alpha.CD40T cells prepared in example 3 (100 ul volume) and control Mock T cells (transferred into pNB328 empty vector) were cultured for 24h and cell supernatants were collected. The secretion of cytokines by these three T cells after stimulation with EGFR antigen was examined according to the method of example 7.

The results are shown in FIG. 7, where the IL-2 and IFN-gamma secretion amounts of EHCAR-EK-28TIZ- αCD40 were significantly higher than those of EHCAR-EK-28TIZ T cells and Mock-T, indicating that cytokine secretion from CAR-T cells can be promoted by self-expressing CD40 activating antibodies.

Example 8: in vivo antitumor effects of EHCAR-EK-28TIZ T cells, EHCAR-EK-28 TIZ-alpha CD40-wt T cells and EHCAR-EK-28 TIZ-alpha CD40T cells

EHCAR-EK-28TIZ- αCD40-wt T cells were constructed using 5ug pNB328-EHCAR-EK-28TIZ and 3ug pS328- αCD40-wt using the procedure described in example 2.

20 NSG mice with the age of 4-6 weeks are purchased and divided into 5 groups, 4 mice in each group are inoculated with hepatoma cell strain HCCLM3-LUC, and each group is 1 multiplied by 10 ⁷ 10 days after tumorigenesis, PBS (100 ul), mock-T obtained in example 2, and EHCAR-EK-28TIZ T cells and EHCAR-EK-28 obtained in example 3 were injected tail vein, respectivelyTIZ- αCD40T cells and EHCAR-EK-28TIZ- αCD40-wt T cells (1X 10) ⁷ Individual cells/mouse), the change in tumor fluorescence in mice was recorded by observation.

The results show that PBS, mock-T, EHCAR-EK-28 TIZ-alpha CD40-wt T cells have no therapeutic effect on tumor models, and EHCAR-EK-28TIZ T cells and EHCAR-EK-28 TIZ-alpha CD40T cells have anti-tumor effects, but the EHCAR-EK-28 TIZ-alpha CD40T cells have significantly better effects. As shown in particular in fig. 8.

Although specific embodiments of the invention have been described in detail. Those skilled in the art will understand. Numerous modifications and substitutions of details are possible in light of all the teachings disclosed, and such modifications are contemplated as falling within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Sequence listing

<110> Shanghai cell therapy institute

SHANGHAI ENGINEERING RESEARCH CENTER FOR CELL THERAPY GROUP Co.,Ltd.

<120> CAR-T cells that autocrine CD40 antibodies and target ErbB receptor family and uses thereof

<130> 17A011

<160> 6

<170> SIPOSequenceListing 1.0

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<211> 497

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<213> Artificial sequence (Artificial Sequence)

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Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Leu Glu Trp Met Gly Trp Ile Asn Pro Asp Ser Gly Gly Thr Asn Tyr

65 70 75 80

Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile

85 90 95

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

100 105 110

Val Tyr Tyr Cys Ala Arg Asp Gln Pro Leu Gly Tyr Cys Thr Asn Gly

115 120 125

Val Cys Ser Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

130 135 140

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val

165 170 175

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

180 185 190

Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu

195 200 205

Ile Tyr Thr Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

225 230 235 240

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

245 250 255

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

260 265 270

Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro

275 280 285

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

290 295 300

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

305 310 315 320

Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

325 330 335

Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser Thr Tyr Arg Val

340 345 350

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

355 360 365

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys

370 375 380

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

385 390 395 400

Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

405 410 415

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

420 425 430

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

435 440 445

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

450 455 460

Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu

465 470 475 480

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

485 490 495

Lys

<210> 2

<211> 1491

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

atggaagccc cagctcagct tctcttcctc ctgctactct ggctcccaga taccaccgga 60

caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 120

tcctgcaagg cttctggata caccttcacc ggctactata tgcactgggt gcgacaggcc 180

cctggacaag ggcttgagtg gatgggatgg atcaaccctg acagtggtgg cacaaactat 240

gcacagaagt ttcagggcag ggtcaccatg accagggaca cgtccatcag cacagcctac 300

atggagctga acaggctgag atctgacgac acggccgtgt attactgtgc gagagatcag 360

cccctaggat attgtactaa tggtgtatgc tcctactttg actactgggg ccagggaacc 420

ctggtcaccg tctcctcagg tggaggcggt tcaggcggag gtggcagcgg cggtggcggg 480

tcggacatcc agatgaccca gtctccatct tccgtgtctg catctgtagg agacagagtc 540

accatcactt gtcgggcgag tcagggtatt tacagctggt tagcctggta tcagcagaaa 600

ccagggaaag cccctaacct cctgatctat actgcatcca ctttacaaag tggggtccca 660

tcaaggttca gcggcagtgg atctgggaca gatttcactc tcaccatcag cagcctgcaa 720

cctgaagatt ttgcaactta ctattgtcaa caggctaaca ttttcccgct cactttcggc 780

ggagggacca aggtggagat caaagagtcc aaatatggtc ccccatgccc accatgccca 840

gcacctgagt tcgagggggg accatcagtc ttcctgttcc ccccaaaacc caaggacact 900

ctcatgatct cccggacccc tgaggtcacg tgcgtggtgg tggacgtgag ccaggaagac 960

cccgaggtcc agttcaactg gtacgtggat ggcgtggagg tgcataatgc caagacaaag 1020

ccgcgggagg agcagttcca gagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 1080

caggactggc tgaacggcaa ggagtacaag tgcaaggtct ccaacaaagg cctcccgtcc 1140

tccatcgaga aaaccatctc caaagccaaa gggcagcccc gagagccaca ggtgtacacc 1200

ctgcccccat cccaggagga gatgaccaag aaccaggtca gcctgacctg cctggtcaaa 1260

ggcttctacc ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 1320

tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaggcta 1380

accgtggaca agagcaggtg gcaggagggg aatgtcttct catgctccgt gatgcatgag 1440

gctctgcaca accactacac acagaagagc ctctccctgt ctctgggtaa a 1491

<210> 3

<211> 497

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 3

Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Leu Glu Trp Met Gly Trp Ile Asn Pro Asp Ser Gly Gly Thr Asn Tyr

65 70 75 80

Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile

85 90 95

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

100 105 110

Val Tyr Tyr Cys Ala Arg Asp Gln Pro Leu Gly Tyr Cys Thr Asn Gly

115 120 125

Val Cys Ser Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

130 135 140

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val

165 170 175

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

180 185 190

Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu

195 200 205

Ile Tyr Thr Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

225 230 235 240

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

245 250 255

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

260 265 270

Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro

275 280 285

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

290 295 300

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

305 310 315 320

Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

325 330 335

Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val

340 345 350

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

355 360 365

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys

370 375 380

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

385 390 395 400

Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

405 410 415

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

420 425 430

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

435 440 445

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

450 455 460

Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu

465 470 475 480

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

485 490 495

Lys

<210> 4

<211> 1431

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60

tcctgcaagg cttctggata caccttcacc ggctactata tgcactgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggatgg atcaaccctg acagtggtgg cacaaactat 180

gcacagaagt ttcagggcag ggtcaccatg accagggaca cgtccatcag cacagcctac 240

atggagctga acaggctgag atctgacgac acggccgtgt attactgtgc gagagatcag 300

cccctaggat attgtactaa tggtgtatgc tcctactttg actactgggg ccagggaacc 360

ctggtcaccg tctcctcagg tggaggcggt tcaggcggag gtggcagcgg cggtggcggg 420

tcggacatcc agatgaccca gtctccatct tccgtgtctg catctgtagg agacagagtc 480

accatcactt gtcgggcgag tcagggtatt tacagctggt tagcctggta tcagcagaaa 540

ccagggaaag cccctaacct cctgatctat actgcatcca ctttacaaag tggggtccca 600

tcaaggttca gcggcagtgg atctgggaca gatttcactc tcaccatcag cagcctgcaa 660

cctgaagatt ttgcaactta ctattgtcaa caggctaaca ttttcccgct cactttcggc 720

ggagggacca aggtggagat caaagagtcc aaatatggtc ccccatgccc accatgccca 780

gcacctgagt tcctgggggg accatcagtc ttcctgttcc ccccaaaacc caaggacact 840

ctcatgatct cccggacccc tgaggtcacg tgcgtggtgg tggacgtgag ccaggaagac 900

cccgaggtcc agttcaactg gtacgtggat ggcgtggagg tgcataatgc caagacaaag 960

ccgcgggagg agcagttcaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 1020

caggactggc tgaacggcaa ggagtacaag tgcaaggtct ccaacaaagg cctcccgtcc 1080

tccatcgaga aaaccatctc caaagccaaa gggcagcccc gagagccaca ggtgtacacc 1140

ctgcccccat cccaggagga gatgaccaag aaccaggtca gcctgacctg cctggtcaaa 1200

ggcttctacc ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 1260

tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaggcta 1320

accgtggaca agagcaggtg gcaggagggg aatgtcttct catgctccgt gatgcatgag 1380

gctctgcaca accactacac acagaagagc ctctccctgt ctctgggtaa a 1431

<210> 5

<211> 580

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 5

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

1 5 10 15

His Ala Ala Arg Pro Ser Val Val Ser His Phe Asn Asp Cys Pro Leu

20 25 30

Ser His Asp Gly Tyr Cys Leu His Asp Gly Val Cys Met Tyr Ile Glu

35 40 45

Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val Val Gly Tyr Ile Gly Glu

50 55 60

Arg Cys Gln Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg Glu Ala Ala

65 70 75 80

Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Gly Thr His Ser

85 90 95

Leu Pro Pro Arg Pro Ala Ala Val Pro Val Pro Leu Arg Met Gln Pro

100 105 110

Gly Pro Ala His Pro Val Leu Ser Phe Leu Arg Pro Ser Trp Asp Leu

115 120 125

Val Ser Ala Phe Tyr Ser Leu Pro Leu Ala Pro Leu Ser Pro Thr Ser

130 135 140

Val Pro Ile Ser Pro Val Ser Val Gly Arg Gly Pro Asp Pro Asp Ala

145 150 155 160

His Val Ala Val Asp Leu Ser Arg Tyr Glu Gly Glu Ser Lys Tyr Gly

165 170 175

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

180 185 190

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

195 200 205

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

210 215 220

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

225 230 235 240

Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser Thr Tyr Arg Val Val Ser

245 250 255

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

260 265 270

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

275 280 285

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

290 295 300

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

305 310 315 320

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

325 330 335

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

340 345 350

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

355 360 365

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

370 375 380

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Pro

385 390 395 400

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

405 410 415

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser

420 425 430

Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly

435 440 445

Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala

450 455 460

Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala

465 470 475 480

Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg

485 490 495

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

500 505 510

Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

515 520 525

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

530 535 540

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

545 550 555 560

Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala

565 570 575

Leu Pro Pro Arg

580

<210> 6

<211> 1740

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccgagcgtgg tgtcccattt taatgactgt cccctgtccc acgatgggta ctgcctccat 120

gatggtgtgt gcatgtatat tgaagcattg gacaagtatg catgcaactg tgttgttggc 180

tacatcgggg agcgatgtca gtaccgagac ctgaagtggt gggaactgcg cgaagctgcc 240

gctaaggagg ccgcagccaa agaggccgct gcaaagggca cccacagcct gcccccccgc 300

cccgccgccg tgcccgtgcc cctgcgcatg cagcccggcc ccgcccaccc cgtgctgagc 360

ttcctgcgcc ccagctggga cctggtgagc gccttctaca gcctgcccct ggcccccctg 420

agccccacca gcgtgcccat cagccccgtg agcgtgggcc gcggccccga ccccgacgcc 480

cacgtggccg tggacctgag ccgctacgag ggcgagtcca aatatggtcc cccatgccca 540

ccatgcccag cacctcccgt ggccggacca tcagtcttcc tgttcccccc aaaacccaag 600

gacactctca tgatctcccg gacccctgag gtcacgtgcg tggtggtgga cgtgagccag 660

gaagaccccg aggtccagtt caactggtac gtggatggcg tggaggtgca taatgccaag 720

acaaagccgc gggaggagca gttccagagc acgtaccgtg tggtcagcgt cctcaccgtc 780

ctgcaccagg actggctgaa cggcaaggag tacaagtgca aggtctccaa caaaggcctc 840

ccgtcctcca tcgagaaaac catctccaaa gccaaagggc agccccgaga gccacaggtg 900

tacaccctgc ccccatccca ggaggagatg accaagaacc aggtcagcct gacctgcctg 960

gtcaaaggct tctaccccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 1020

aacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctacagc 1080

aggctaaccg tggacaagag caggtggcag gaggggaatg tcttctcatg ctccgtgatg 1140

catgaggctc tgcacaacca ctacacacag aagagcctct ccctgtctct gggtaaaccc 1200

ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 1260

gcctttatta ttttctgggt gaggagtaag aggagcaggc tcctgcacag tgactacatg 1320

aacatgactc cccgccgccc cgggcccacc cgcaagcatt accagcccta tgccccacca 1380

cgcgacttcg cagcctatcg ctccagagtg aagttcagca ggagcgcaga cgcccccgcg 1440

taccagcagg gccagaacca gctctataac gagctcaatc taggacgaag agaggagtac 1500

gatgttttgg acaagagacg tggccgggac cctgagatgg ggggaaagcc gagaaggaag 1560

aaccctcagg aaggcctgta caatgaactg cagaaagata agatggcgga ggcctacagt 1620

gagattggga tgaaaggcga gcgccggagg ggcaaggggc acgatggcct ttaccagggt 1680

ctcagtacag ccaccaagga cacctacgac gcccttcaca tgcaggccct gccccctcgc 1740

Claims

1. A T cell, wherein the T cell:

(2) Expressing a chimeric antigen receptor and a CD40 activating antibody that target the ErbB receptor family;

the chimeric antigen receptor comprises a T1E peptide segment, a Herin peptide segment, a hinge region, a transmembrane region, an intracellular co-stimulatory signaling domain and an intracellular signaling domain which are connected through a rigid linker, wherein the CD40 activating antibody sequentially comprises a light chain signal peptide, an anti-CD 40 single chain antibody and an IgG4Fc from the N end to the C end, and the amino acid sequence of the IgG4Fc is shown as the amino acid residues 269-497 of SEQ ID NO. 1.

2. The T cell of claim 1, wherein the amino acid sequence of the light chain variable region of the anti-CD 40 single chain antibody is shown in amino acid residues 21-146 of SEQ ID No. 1, and the amino acid sequence of the heavy chain variable region of the anti-CD 40 single chain antibody is shown in amino acid sequences 161-268 of SEQ ID No. 1.

3. The T cell of claim 1, wherein the T cell has integrated into its genome an expression cassette for a CD40 activating antibody and an expression cassette for a chimeric antigen receptor that targets the ErbB receptor family.

4. The T cell of claim 1, wherein the chimeric antigen receptor further comprises a signal peptide.

5. The T cell of claim 4, wherein the chimeric antigen receptor is characterized by one or more of the following:

the signal peptide is a CD8 signal peptide, a CD28 signal peptide or a CD4 signal peptide;

the hinge region is selected from the group consisting of an extracellular hinge region of CD8, an IgG1 Fc CH2CH3 hinge region, an IgD hinge region, a CD28 extracellular hinge region, an IgG4 Fc CH2CH3 hinge region, and an extracellular hinge region of CD 4;

the transmembrane region is one of a CD28 transmembrane region, a CD8 transmembrane region, a CD3 zeta transmembrane region, a CD134 transmembrane region, a CD137 transmembrane region, an ICOS transmembrane region and a DAP10 transmembrane region;

The intracellular co-stimulatory signaling domain includes the intracellular domain of a co-stimulatory signaling molecule, including the intracellular domains of CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase, inducible T cell co-stimulatory factor (ICOS) and DNAX activator protein 10; and

the intracellular signaling domain is a cd3ζ intracellular signaling domain or an fcsriy intracellular signaling domain.

6. The T cell of claim 5, wherein the chimeric antigen receptor is characterized by one or more of the following:

the signal peptide is a CD8 signal peptide; and

the hinge region is a CD8 hinge region or an IgG4 CH2CH3 hinge region.

7. The T cell of claim 6, wherein the chimeric antigen receptor is characterized by one or more of the following:

the amino acid sequence of the CD8 signal peptide is shown as the 1 st to 22 nd amino acid residues of SEQ ID NO. 5;

the amino acid sequence of the T1E is shown as the 23 rd to 77 th amino acid residues of SEQ ID NO. 5;

the amino acid sequence of the Herin is shown as 93-171 amino acid residues of SEQ ID NO. 5;

the rigid linker is an EAAAK linker, and the amino acid sequence of the rigid linker is shown as 78 th to 92 th amino acid residues of SEQ ID NO. 5;

the hinge region is an IgG4 CH2CH3 hinge region, and the amino acid sequence is shown as the 172 th to 399 th amino acid residues of SEQ ID NO. 5;

The transmembrane region is a CD28 transmembrane region, and the amino acid sequence of the transmembrane region is shown as 400 th to 427 th amino acid residues of SEQ ID NO. 5;

the co-stimulatory signaling molecule intracellular domain is a CD28 intracellular domain, and the amino acid sequence of the co-stimulatory signaling molecule intracellular domain is shown as amino acid residues 428-468 of SEQ ID NO. 5; and

the intracellular signal domain is CD3 zeta intracellular signal domain, and the amino acid sequence is shown as 469 th to 580 th amino acid residues of SEQ ID NO. 5.

8. The T cell of claim 7, wherein the chimeric antigen receptor has one or more of the following characteristics:

the coding sequence of the signal peptide is shown as the 1 st to 66 th base sequence of SEQ ID NO. 6;

the coding sequence of the T1E is shown as the 67 th to 231 th base sequence of SEQ ID NO. 6;

the coding sequence of the Herin is shown in the base sequence of 277 th to 513 th positions of SEQ ID NO. 6;

the coding sequence of the rigid linker is shown as 232 th to 276 th base sequence of SEQ ID NO. 6;

the coding sequence of the hinge region is shown in the 514 th to 1197 th base sequence of SEQ ID NO. 6;

the coding sequence of the transmembrane region is shown as the base sequence of 1198-1281 of SEQ ID NO. 6;

the coding sequence of the intracellular co-stimulatory signal domain is shown as the base sequence of 1282-1404 of SEQ ID NO. 6; and

the coding sequence of the intracellular signal domain is shown as the 1405 th to 1740 th base sequence of SEQ ID NO. 6.

9. The T cell of any one of claims 1 to 4, wherein the chimeric antigen receptor has an amino acid sequence as set forth in amino acid residues 23 to 580 of SEQ ID No. 5, or as set forth in SEQ ID No. 5.

10. The T cell of claim 9, wherein the chimeric antigen receptor has a nucleotide sequence as set forth in SEQ ID No. 6 at nucleotide sequences 67-1740 or as set forth in SEQ ID No. 6.

11. The T cell of any one of claims 1-4, wherein the CD40 activating antibody comprises a light chain signal peptide.

12. The T cell of claim 11, wherein the amino acid sequence of the light chain signal peptide is set forth in amino acid residues 1-20 of SEQ ID No. 1.

13. The T cell of claim 11, wherein the amino acid sequence of the CD40 activating antibody is shown in SEQ ID No. 1 at amino acid positions 21-497 or in SEQ ID No. 1.

14. The T cell of claim 13, wherein the CD40 activating antibody has a coding sequence as set forth in SEQ ID No. 2 at nucleotide sequences 61-1491 or as set forth in SEQ ID No. 2.

15. A composition or kit comprising:

(1) A vector comprising an expression cassette for a chimeric antigen receptor as defined in any one of claims 4 to 10 for integration of said expression cassette into the genome of a host cell; and

(2) A vector comprising an expression cassette for a CD40 activating antibody as defined in any one of claims 3, 11-14 for integration of said expression cassette into the genome of a host cell.

16. A pharmaceutical composition comprising the T cell of any one of claims 1-14.

17. Use of the T cell of any one of claims 1-14, or a pharmaceutical composition thereof, in the manufacture of a medicament for treating or preventing a cancer selected from the group consisting of: liver cancer, adenocarcinoma, lung cancer, colon cancer, large intestine cancer, breast cancer, ovarian cancer, cervical cancer, stomach cancer, bile duct cancer, non-small cell cancer, gallbladder cancer, esophageal cancer, melanoma, pancreatic cancer, urothelial cancer, head and neck cancer or prostate cancer.