WO2019129138A1

WO2019129138A1 - Car-t cells targeting erbb receptor family and self-expressing pd-1 antibody and use thereof

Info

Publication number: WO2019129138A1
Application number: PCT/CN2018/124316
Authority: WO
Inventors: 钱其军; 金华君; 游术梅; 江芏青; 李林芳; 王超
Original assignee: 上海细胞治疗研究院; 上海细胞治疗集团有限公司
Priority date: 2017-12-28
Filing date: 2018-12-27
Publication date: 2019-07-04
Also published as: CN109971724A; CN109971724B

Abstract

Provided are chimeric antigen receptor T cells targeting ErbB receptor family and expressing PD1 antibody, a pharmaceutical composition containing the T cells, and use of the T cells or the pharmaceutical composition for the treatment or prevention of a malignant tumor. Also provided is a composition or a kit of a vector comprising an expression cassette of a chimeric antigen receptor. The chimeric antigen receptor comprises a T1E peptide segment and a Herin peptide segment linked by a rigid linker, a hinge region, a transmembrane region, an intracellular costimulatory signal domain, and an intracellular signal domain.

Description

CAR-T cells targeting the ErbB receptor family and self-expressing PD-1 antibodies and uses thereof

Technical field

The present invention belongs to genetic engineering and immunology, and relates to a CAR-T cell that targets the ErbB receptor family and self-expresses a PD-1 antibody, and a PD1 antibody PD1 antibody thereof.

Background technique

Cancer has become the number one killer of human health. The fast pace of life, huge work pressure, unhealthy eating habits, and poor environment are all accomplices of cancer, making cancer's high incidence and rejuvenation trend more and more obvious. At present, the commonly used treatments are very limited, and it is still necessary to explore a more effective treatment to improve the survival rate and quality of life of cancer patients.

Chimeric antigen receptor T cell (CAR-T) therapy is one of the important branches of tumor immunotherapy. It has achieved very good curative effect in malignant hematological tumors. The complete remission rate of relapsed and refractory B cell leukemia is over 90. %. A chimeric antigen receptor is a synthetic receptor that typically comprises an extracellular antigen binding domain, a transmembrane hinge region, and an intracellular signal transduction region. By the identification of a single-chain fragment variable (scFv) and an intracellular signal domain of an antibody-associated antigen (TAA), an immunoreceptor tyrosine-based activation Motifs, ITAM)" genetic recombination in vitro. It is then introduced into T cells by a virus or other vector system such that the genetically engineered T cells are referred to as CAR-T cells. After extensive expansion in vitro, CAR-T cells are returned to the patient and can exhibit potent anticancer effects in a non-MHC-restricted mode.

However, there are many obstacles to copying this therapy to solid tumors. One is that solid tumors lack tumor-specific antigens and cannot find a perfect therapeutic target. Second, solid tumors have a stronger tumor immunosuppressive microenvironment. In addition, the tumor itself has a strong heterogeneity. These factors lead to easy recurrence after tumor immunotherapy. Therefore, the preparation of multi-target, more powerful CART cells is the key to improving the therapeutic effect of tumors. For example, with CART19 in the treatment of B-cell acute lymphoblastic leukemia, approximately 30% of patients will develop a CD19 antigen-negative relapse after treatment. Studies have shown that CART19/123 dual targets are constructed using CD19 and IL3 receptor alpha chain CD123 as targets. Point CART cells have better in vivo therapeutic effects and can reduce recurrence rates (J Clin Invest. 2016 Oct 3; 126(10)). However, most of the antigen recognition regions of these CARs are artificially constructed single-chain antibodies, which have strong immunogenicity, and the in vivo treatment is easily recognized and eliminated, resulting in poor therapeutic effect. Therefore, the use of natural peptides to construct a multi-target antigen recognition region has the advantages of weak immunogenicity and wider targeting range.

The ErbB receptor family contains four members, the epidermal growth factor receptor ErbB1 (EGFR/Her2), ErbB2 (Her2), ErbB3 (Her3), and ErbB4 (Her4). In normal adults, the expression levels of these four receptors are at a low level. However, many malignant tumors are associated with overexpression of ErbB1 and/or ErbB2, including head and neck cancer, breast cancer, lung cancer, gastrointestinal cancer, prostate cancer, and pancreatic cancer. Therefore, many of the anti-tumor drugs in clinical research are monoclonal antibodies and small-molecule tyrosine kinase inhibitors that target the extracellular domain of different receptors of ErbB. For example, HER2 humanized antibody Herceptin has been approved by the FDA. Clinical treatment of breast cancer.

However, ErbB receptors usually function in the form of tightly bound dimers. Her2 is the best dimerization partner, especially when dimerized with EGFR or Her3 to enhance tyrosine kinase signal activation. Her3 has ligand binding ability but lacks tyrosine kinase activity. The heterodimer formed with Her2 is the most powerful signal complex. The most representative heterodimer in breast cancer is Her2/Her3. . Anti-tumor drugs that target a certain receptor alone are likely 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 transcriptional growth factor alpha (TGFα) and 48 amino acids at the C-terminus of epidermal growth factor (EGF), which is based on ErbB1-based homodimers and isoforms. The source dimer has a high affinity, and in addition, T1E can effectively bind ErbB2/3 heterodimer. Studies have shown that T1E as a scFv CAR transfected T cells can effectively treat mouse tumor xenograft models of human head and neck cancer. However, T1E does not bind to ErbB2 or ErbB3 alone, and there are defects in the range of targeting the ErbB receptor family.

Herstatin is a Her2 truncated version of Her2 that is selectively cleaved, and its sequence includes 340 amino acids of the extracellular domain of Her2, I and II, and 79 amino acids encoded by the eighth intron. Soluble Her2 autoinhibitory factor. Herstatin binds to EGFR or Her2 with high affinity, the main function of which is the 79 amino acids encoded by the eighth intron (designated Herin).

PD1 (Programmed Death 1): Reprogramming cell death receptor 1, which is a member of the regulatory T cell CD28 family and belongs to the immunoglobulin superfamily receptor. PD-1 and its ligand PD-L1/PD-L2 play important roles in the co-suppression and failure of T cells. Their interaction inhibits the proliferation of T cells regulated by costimulatory molecules and the secretion of cytokines, downregulated. The expression of the anti-apoptotic molecule BCL-xl impairs the function of tumor-specific T cells, leading to the inability of some tumor patients to completely eliminate the tumor. Therefore, inhibition of the immune checkpoint PD-1/PD-L1 pathway is currently a new direction and target for the treatment of lymphoma. PD-1 antibody blocking therapy has a certain degree of advanced or refractory melanoma, non-small cell lung cancer, renal cell carcinoma, head and neck squamous cell carcinoma, colorectal cancer, Hodgkin's lymphoma, ovarian cancer, etc. Therapeutic effect, but its high production cost and low response rate limit its clinical use.

Summary of the invention

Provided herein are T cells that self-express an immunological checkpoint inhibitory PD1 antibody and target a chimeric antigen receptor of the ErbB receptor family; preferably, the T cell:

(1) a coding sequence comprising a chimeric antigen receptor that targets an ErbB receptor and a coding sequence of a PD1 antibody; and/or

(2) Expression of a chimeric antigen receptor and a PD1 antibody that target the ErbB receptor.

In one or more embodiments, the expression cassette of the chimeric antigen receptor that targets the ErbB receptor and the expression cassette of the PD1 antibody are integrated into the genome of the T cell.

In one or more embodiments, the PD1 antibody comprises an anti-PD1 single chain antibody and an IgG4 Fc.

In one or more embodiments, the amino acid sequence of the IgG4 Fc is set forth as amino acid residues 267-495 of SEQ ID NO: 1; preferably, the coding sequence thereof is SEQ ID NO: 2 base 795-1485 The base sequence is shown.

In one or more embodiments, the antibody light chain variable region (VL region) amino acid sequence of the anti-PD1 single-chain antibody (scFv) is represented by amino acid residues 21 to 131 of SEQ ID NO: 1; The coding sequence thereof is shown in nucleotide sequence 64-393 of SEQ ID NO: 2; the amino acid sequence of the heavy chain variable region (VH region) in the anti-PD1 single-chain antibody is SEQ ID NO: 1 - The amino acid sequence of position 266 is shown; preferably, the coding sequence thereof is represented by the nucleotide sequence of positions 439 to 798 of SEQ ID NO: 2; preferably, the amino acid sequence of the anti-PD1 single-chain antibody is SEQ ID NO: The amino acid residues 21 to 266 are shown; preferably, the coding sequence thereof is shown in nucleotide sequences 61 to 798 of SEQ ID NO: 2.

In one or more embodiments, the PD1 antibody comprises, from the N-terminus to the C-terminus, a light chain signal peptide, an anti-PD1 single chain antibody, and an IgG4 Fc in sequence; preferably, the amino acid sequence of the light chain signal peptide is SEQ ID. The NO:1 amino acid residue at positions 1-20 is shown; preferably, the coding sequence of the light chain signal peptide shown is shown in nucleotide sequence 1-60 of SEQ ID NO: 2.

In one or more embodiments, the amino acid sequence of the PD1 antibody is set forth in amino acid sequence 21-495 of SEQ ID NO: 1, or as set forth in SEQ ID NO: 1; preferably, the PD1 antibody The coding sequence is shown as amino acid residues 61-1485 of SEQ ID NO: 2, preferably as shown in SEQ ID NO: 2.

In one or more embodiments, the chimeric antigen receptor comprises an optional signal peptide, T1E, EAAAK linker, Herin, hinge region, transmembrane region, intracellular costimulatory signal domain, and intracellular signal domain; The hinge region is a hinge region longer than 50 amino acid residues.

In one or more embodiments, the signal peptide is a secretory signal peptide and a membrane-bound signal peptide, preferably a CD8 signal peptide; more preferably, the amino acid sequence of the CD8 signal peptide is SEQ ID NO: 5 The amino acid residues 1-22 are shown.

In one or more embodiments, the amino acid sequence of the T1E is represented by amino acid residues 23-77 of SEQ ID NO: 5; preferably, the coding sequence thereof is as set at bases 67-231 of SEQ ID NO: 6. The base sequence is shown.

In one or more embodiments, the amino acid sequence of the EAAAK linker is as described in amino acid residues 78-92 of SEQ ID NO: 5; preferably, the coding sequence thereof is SEQ ID NO: 6 at positions 232-276 The base sequence is shown.

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

In one or more embodiments, the hinge region is a CD8 hinge region or an IgG4CH2CH3 hinge region; preferably, the hinge region is an IgG4CH2CH3 hinge region, the amino acid sequence of which is amino acid residues 172-399 of SEQ ID NO: The base sequence is shown; preferably, the coding sequence thereof is shown in the nucleotide sequence of positions 514-1911 of SEQ ID NO: 6.

In one or more embodiments, the transmembrane region is 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. Or 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 of SEQ ID NO: 6 base sequence 1198-1281 Shown.

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

In one or more embodiments, the intracellular signal domain is a CD3 sputum intracellular signal domain or an FcεRI gamma intracellular signal domain; preferably a CD3 sputum intracellular signal domain, preferably the amino acid sequence of the CD3 sputum intracellular signal domain is SEQ. The ID NO: 5 is represented by amino acid residues at positions 469 to 580, and preferably the coding sequence thereof is represented by the nucleotide sequence of nucleotides 1505-1740 of SEQ ID NO: 6.

In one or more embodiments, the amino acid sequence of the chimeric antigen receptor is as shown in amino acid residues 23 to 580 of SEQ ID NO: 5, or as shown in SEQ ID NO: 5; preferably, The nucleotide sequence showing the chimeric antigen receptor is shown in the nucleotide sequence of 6717 to 4040 shown in SEQ ID NO: 6, or as shown in SEQ ID NO: 6.

The invention also provides a composition comprising:

(1) a vector comprising an expression cassette of a chimeric antigen receptor described herein, which vector is used to integrate the expression cassette into the genome of a host cell;

(2) A vector comprising an expression cassette of a CD40 activating antibody, which vector is used to integrate the expression cassette into the genome of a host cell.

In one or more embodiments, the chimeric antigen receptor and its coding sequence, as well as the CD40 activating antibody and coding sequences thereof, are as described in any of the embodiments herein.

The invention also provides a kit, the kit comprising:

(2) A vector comprising an expression cassette of a PD1 antibody, which vector is used to integrate the expression cassette into the genome of a host cell.

In one or more embodiments, the chimeric antigen receptor and its coding sequence, as well as the PD1 antibody and coding sequences thereof, are as described in any of the embodiments herein.

The invention also provides a pharmaceutical composition comprising a T cell or a T cell as described herein and a PD1 antibody expressed thereby.

The present invention also provides the use of a T cell or a pharmaceutical composition thereof described herein or the T cell and the PD1 antibody expressed thereby for the preparation of a medicament for treating or preventing a malignancy.

In one or more embodiments, the cancer is selected from the group consisting of liver cancer, adenocarcinoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, head and neck cancer, cholangiocarcinoma, gallbladder cancer. , melanoma, non-small cell lung cancer, renal cell carcinoma, head and neck squamous cell carcinoma, Hodgkin's lymphoma, esophageal cancer, pancreatic cancer or prostate cancer.

DRAWINGS

Figure 1: Schematic diagram of the gene structure of recombinant plasmids pS328-antiPD1-wt, pS328-antiPD1, pNB328-EHCAR-EK-28TIZ.

2A-2B: Positive rate and antibody expression level of chimeric antigen receptor-modified T cells constructed with pNB328-EHCAR-EK-28TIZ and pS328-antiPD1 plasmids of different mass ratios.

Figures 3A-3B: EHCAR-EK-28 TIZ T cells and EHCAR-EK-28 TIZ-antiPD1 T cells were assayed for positive rate and antibody expression.

Figure 4: Comparison of proliferation rates of EHCAR-EK-28TIZ and EHCAR-EK-28TIZ-antiPD1 T cells.

Figures 5A-5D: Cell phenotypic assay analysis of T cells of EHCAR-EK-28TIZ and EHCAR-EK-28TIZ-antiPD1 T cells, 5A indicates senescence phenotype PD1, 5B and 5C indicate activation phenotypes CD69 and CD107α, respectively, 5D representation Memory phenotype.

Figure 6: Comparison of killing of EHCAR-EK-28TIZ and EHCAR-EK-28TIZ-antiPD1T cells, including human liver cancer cell HCCLM3, human liver cancer cell Hep3B and human non-small cell lung cancer H23.

Figure 7: Changes in IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γ cytokine secretion by EHCAR-EK-28TIZ and EHCAR-EK-28TIZ-antiPD1T cells stimulated by EGFR antigen .

Figure 8: EHCAR-EK-28TIZ T cells, EHCAR-EK-28TIZ-antiPD1-wt T cells, EHCAR-EK-28TIZ-antiPD1T cells, Mock-T cells and PBS blank controls, respectively, after treatment of mice, different days Tumor cell fluorescence values change.

Detailed ways

Some of the terms related to the present invention are explained below.

In the present invention, the term "expression cassette" refers to the entire element required for expression of a gene, including a promoter and a gene coding sequence.

The term "coding sequence" is defined herein as a portion of a nucleic acid sequence that directly determines the amino acid sequence of its protein product (eg, 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 of the 5' end of the mRNA and a transcription termination sequence immediately downstream of the open reading frame of the 3' end of the mRNA. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

The term "Fc", ie, fragment crystallizable (Fc) of an antibody, refers to a peptide located at the end of the handle of the "Y" structure of the antibody molecule, comprising the CH2 and CH3 domains of the heavy chain constant region of the antibody, and is an antibody and effect. The site of molecular or cellular interactions.

The term "costimulatory molecule" refers to a molecule that is present on the surface of an antigen presenting cell and that binds to a costimulatory molecule receptor on a Th cell to produce a costimulatory signal. The proliferation of lymphocytes requires not only the binding of antigens, but also the signals of costimulatory molecules. The costimulatory signal is transmitted to the T cells mainly by binding to the co-stimulatory molecule CD80 on the surface of the antigen presenting cells, and CD86 binds to the CD28 molecule on the surface of the T cell. B cells receive a costimulatory signal that can pass through a common pathogen component such as LPS, or through a complement component, or through activated antigen-specific Th cell surface CD40L.

The term "linker" or hinge is a polypeptide fragment that links between different proteins or polypeptides for the purpose of maintaining the spatial conformation of the linked protein or polypeptide to maintain the function or activity of the protein or polypeptide. Exemplary linkers include linkers containing G and/or S, as well as, for example, Furin 2A peptide.

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

The term "pharmaceutically acceptable excipient" refers to carriers and/or excipients that are compatible pharmacologically and/or physiologically to the subject and active ingredient, which are well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers. For example, pH adjusting agents 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 a dose that can achieve a treatment, prevention, alleviation, and/or alleviation of a disease or condition described herein in a subject.

The term "disease and/or condition" refers to a physical state of the subject that is associated with the disease and/or condition described herein.

The term "subject" or "patient" may refer to a patient or other animal that receives the pharmaceutical composition of the invention to treat, prevent, ameliorate and/or alleviate the disease or condition of the invention, particularly a mammal, such as a human, a dog. , monkeys, cattle, horses, etc.

The term "chimeric antigen receptor" (CAR) is an artificially engineered receptor that anchors specific molecules (such as antibodies) that recognize tumor cell surface antigens to immune cells (such as T cells), allowing immune cells to recognize tumor antigens. Or viral antigens and cells that kill tumor cells or virus infection. CAR typically comprises, in turn, 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 signaling region. Generally, a polypeptide that binds to a tumor cell membrane antigen is capable of binding to a membrane antigen that is widely expressed by tumor cells with moderate affinity. The polypeptide that binds to the tumor cell membrane antigen may be a natural polypeptide or a synthetic polypeptide; preferably, the synthetic polypeptide is a single chain antibody or a Fab fragment.

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

In order to improve the immunotherapeutic effect of EHCAR-EK-28TIZ T cells, the present invention expresses PD1 antibody on existing CAR-T cells, and suppresses the PD-1/PD-L1 pathway of the immunological checkpoint while adopting adoptive immune cell therapy. It can activate the function of residual tumor-specific T cells in situ, increase the anti-tumor killing effect of endogenous cytotoxic T cells, and promote the proliferation of CAR-T cells in vivo, thereby improving the efficacy of specific killing of tumors.

Studies have shown that the IgG4 Fc fragment of the PD1 antibody is easily phagocytosed by the monocyte/macrophage, and the PD-1 antibody which is mutated by the base of the PD-1 antibody IgG4Fc fragment to satisfy the T cell self-expression is good. The function does not cause ADCC reaction.

Accordingly, the present invention provides a PD1 antibody comprising an anti-PD1 single chain antibody and IgG4Fc. In certain embodiments, the amino acid sequence of the IgG4 Fc is set forth in amino acid residues 267-495 of SEQ ID NO: 1; preferably, the coding sequence thereof is set at bases 799-1485 of SEQ ID NO: Shown.

In certain embodiments, the antibody light chain variable region (VL region) amino acid sequence of the anti-PD1 single chain antibody (scFv) is represented by amino acid residues 21 to 131 of SEQ ID NO: 1; preferably, Its coding sequence is shown in nucleotide sequences 64-393 of SEQ ID NO: 2. In certain embodiments, the heavy chain variable region (VH region) amino acid sequence of the anti-PD1 single chain antibody is set forth in amino acid sequence at positions 147-266 of SEQ ID NO: 1; preferably, the coding sequence thereof is The base sequence of 439-798 of SEQ ID NO: 2 is shown. In certain embodiments, the amino acid sequence of the anti-PD1 single-chain antibody is set forth as amino acid residues 21-266 of SEQ ID NO: 1; preferably, the coding sequence thereof is SEQ ID NO: 2, 61-798 The base sequence is shown.

In certain embodiments, the PD1 antibody further comprises a light chain signal peptide. In certain embodiments, the PD1 antibody, from the N-terminus to the C-terminus, in turn comprises a light chain signal peptide, an anti-PD1 single chain antibody, and an IgG4 Fc. In certain embodiments, the amino acid sequence of the light chain signal peptide is represented by amino acid residues 1-20 of SEQ ID NO: 1; preferably, the coding sequence of the indicated light chain signal peptide is SEQ ID NO: 2 The base sequence of the 1-60th base is shown.

And X. As shown by amino acid residues 61-1485 of SEQ ID NO: 2, it is preferably shown as SEQ ID NO: 2.

The invention also encompasses a coding sequence for the PD1 antibody or a complement thereof, the coding sequence comprising at least the coding sequence for an IgG4 Fc described herein or a complement thereof. In certain embodiments, the coding sequence of the PD1 antibody comprises the sequence set forth in bases 61-1485 of SEQ ID NO: 2, preferably the sequence set forth in SEQ ID NO: 2.

The present invention also encompasses a nucleic acid construct comprising the coding sequence of the PD1 antibody of the present invention or a complement thereof. Preferably, the nucleic acid construct is an expression vector or an integration vector for integrating the coding sequence or its complement 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 PD1 antibody, its coding sequence or complementary sequence, a nucleic acid construct, and a host cell for the preparation or treatment of a malignant tumor, particularly a tumor associated with PD1, including but not limited to Various malignant tumors.

The present invention also provides T cells that self-express an immunological checkpoint-inhibiting PD1 antibody and target a chimeric antigen receptor of the ErbB receptor family. In the chimeric antigen receptor (CAR), the native T1E and Herin fusion expression is used as an antigen recognition region of CAR, and the two can complement each other to recognize the ErbB receptor family and expand the targeting range.

The CAR of the present invention typically contains an optional signal peptide sequence, a fusion protein of T1E and Herin, a hinge region, a transmembrane region, an intracellular costimulatory signal domain, and an intracellular signal domain.

A signal peptide is a short peptide chain (5-30 amino acids in length) that directs the transfer of newly synthesized proteins to the secretory pathway, often referred to as the N-terminal amino acid sequence in the newly synthesized polypeptide chain that directs transmembrane transfer (localization) of the protein. (Sometimes not necessarily at the N-terminus), it is responsible for directing proteins into subcellular organelles with different membrane structures. The signal peptide can be a secreted 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 as shown in amino acid residues 1-22 of SEQ ID NO: 5; in certain embodiments, the coding sequence is set forth in bases 1-66 of SEQ ID NO: 6.

The natural T1E and Herin fusion expression is used as the antigen recognition region of CAR. The T1E is a chimeric polypeptide consisting of seven amino acids at the N-terminus of human transcriptional growth factor alpha (TGFα) and 48 amino acids at the C-terminus of epidermal growth factor (EGF). Preferably, the amino acid sequence of T1E is as shown in amino acid residues 23 to 77 of SEQ ID NO: 5; preferably, the coding sequence thereof is shown in nucleotide sequence 67-231 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 thereof is shown as amino acid residues 93-171 of SEQ ID NO: 5. The present invention optimizes codons encoding Herin amino acids. Therefore, the nucleotide sequence of the preferred Herin of the present invention is shown in the nucleotide sequence of 277-513 of SEQ ID NO: 6.

Typically, T1E and Herin can be joined by a rigid linker sequence. An illustrative example of a rigid linker sequence is two or more repeats of EAAAK as a unit, also referred to herein as an EAAAK linker. An exemplary rigid linker sequence is set forth in amino acid residues 78-92 of SEQ ID NO: 5; exemplary coding sequences are set forth in base sequences 232-276 of SEQ ID NO: 6.

Herein, the hinge region refers to the region between the functional regions of the immunoglobulin heavy chain CH1 and CH2, which is rich in proline, does not form an alpha helix, is prone to stretching and is somewhat distorted, and is beneficial to the antigen binding site of the antibody. Complementary binding between epitopes. The hinge region suitable for use herein may be selected from any one or more of the extracellular hinge region of CD8, the IgG1 Fc 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 CD4. . The hinge region is preferably a hinge region that is longer than 50 amino acid residues, more preferably 80 amino acids or longer. In certain embodiments, a CD8 alpha hinge region or an IgG4 Fc CH2CH3 hinge region is used herein. The amino acid sequence of the exemplary IgG4 FcCH2CH3 hinge region is shown in amino acid residues 172-399 of SEQ ID NO: 5, and the coding sequence for the exemplary IgG4 FcCH2CH3 hinge region is shown in SEQ ID NO: 6 at positions 514-1109.

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, the amino acid sequence thereof is represented by amino acid residues 400-427 of SEQ ID NO: 5; in certain embodiments, the coding sequence is set forth at bases 1198-1281 of SEQ ID NO: 6.

The intracellular co-stimulatory signal domain including the intracellular domain of the costimulatory signaling molecule may be selected from the group consisting of CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase (LCK), and inducible T cell costimulation. Intracellular domain of factor (ICOS) and DNAX activator protein 10 (DAP10). In certain embodiments, the intracellular domain of the costimulatory signaling molecule is the intracellular domain of CD28, preferably the amino acid sequence thereof is set forth in amino acid residues 428-468 of SEQ ID NO: 5, exemplary The coding sequence is shown as bases 1282-1404 of SEQ ID NO: 6.

The intracellular signal domain is preferably an immunoreceptor tyrosine activation motif, which may be a CD3 sputum intracellular signal domain or an FcεRI gamma intracellular signal domain; preferably a CD3 sputum intracellular signal domain, preferably the amino acid sequence of the CD3 sputum intracellular signal domain As described in SEQ ID NO: 5, pp. 469-580; in certain embodiments, the coding sequence is set forth in bases 1405-1740 of SEQ ID NO: 6.

In certain embodiments, the chimeric antigen receptor comprises, in order from the N-terminus to the C-terminus: an optional CD signal peptide, T1E, EAAAK linker, Herin, IgG4 Fc CH2CH3 hinge region, CD28 transmembrane region, CD28 cell The inner domain and the CD3 intracellular signal domain; preferably, the amino acid sequence of the chimeric antigen receptor is as shown in 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 set forth in SEQ ID NO:5.

It will be understood that the invention also encompasses chimeric antibody receptors and coding sequences thereof as described herein.

The above-mentioned various parts forming the chimeric antigen receptor herein, such as a signal peptide, T1E, Herin, a hinge region, a transmembrane region, an intracellular costimulatory signal domain, and an intracellular signal domain, may be directly connected to each other or may be passed through The linker sequence is linked. The linker sequence can be a linker sequence suitable for use in antibodies well known in the art, such as linker sequences comprising G and S. The linker may be 3 to 25 amino acid residues in length, for example 3 to 15, 5 to 15, and 10 to 20 amino acid residues. In certain embodiments, the linker sequence is a polyglycine linker sequence. The amount of glycine in the linker sequence is not particularly limited, but is usually 2 to 20, for example, 2 to 15, 2 to 10, and 2 to 8. In addition to glycine and serine, the linker may also contain other known amino acid residues such as alanine (A), leucine (L), threonine (T), glutamic acid (E), styrene Amino acid (F), arginine (R), glutamine (Q), and the like.

It will be appreciated that in gene cloning procedures, it is often desirable to design a suitable cleavage site which necessarily introduces one or more irrelevant residues at the end of the expressed amino acid sequence without affecting the activity of the sequence of interest. In order to construct a fusion protein, promote expression of a recombinant protein, obtain a recombinant protein that is automatically secreted outside the host cell, or facilitate purification of the recombinant protein, it is often necessary to add some amino acids to the N-terminus, C-terminus of the recombinant protein or within the protein. Other suitable regions include, for example, but are 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 may also contain one or more polypeptide fragments as a protein tag. Any suitable label can be used in this article. For example, the tags 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 receptor. The polynucleotide sequence herein may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded.

The polynucleotide sequences described herein can generally be obtained by PCR amplification. In particular, primers can be designed according to the nucleotide sequences disclosed herein, and the relevant sequences can be amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order. For example, in certain embodiments, the polynucleotide sequence encoding a fusion protein described herein is set forth in SEQ ID NO: 6.

Also included herein are nucleic acid constructs comprising a polynucleotide sequence encoding the chimeric antigen receptor described herein or a polynucleotide sequence encoding the PD1 antibody, and one or more operably linked to the sequences Regulatory sequence. In certain embodiments, the nucleic acid construct is an expression cassette.

The control sequence can 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 mutated, truncated and hybrid promoters, and may be derived from an extracellular or heterologous source encoding the host cell. Or the gene of the intracellular polypeptide is obtained.

The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide. Any terminator that 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 sequences for the CARs herein or the coding sequences for the PD1 antibodies can be cloned into a number of types of vectors, such as, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. The vector can be an expression vector. The expression vector can be provided to the cells in the form of a viral vector. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.

Generally, suitable vectors comprise an origin of replication, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers that function in at least one organism. For example, in certain embodiments, the invention employs a retroviral vector comprising a replication initiation site, a 3'LTR, a 5' LTR, a coding sequence for a CAR described herein or a coding sequence for a PD1 antibody And optional optional markers.

Suitable promoters include, but are not limited to, immediate early cytomegalovirus (CMV) promoter sequences. The promoter sequence is a strong constitutive promoter sequence capable of driving high level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is Elongation Growth Factor-1 alpha (EF-1 alpha). However, other constitutive promoter sequences can also be used, including but not limited to human prion 40 (SV40) early promoter, mouse breast cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, EB virus immediate early promoter, Russ sarcoma virus promoter, and human gene promoters such as, but not limited to, actin promoter, myosin promoter, heme Promoter and creatine kinase promoter. Further, an inducible promoter can also be considered. The use of an inducible promoter provides a molecular switch that is capable of opening expression of a polynucleotide sequence operably linked to an inducible promoter upon expression of the term, and shutting down 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 disclosed in CN201510021408.1 can be used, including but not limited to the CCEF promoter containing the mCMV enhancer, the hCMV enhancer, and the EF1α promoter shown in SEQ ID NO: 5 of the application. TCEF promoter containing CD3e enhancer, mCMV enhancer, hCMV enhancer and EF1α promoter as shown in SEQ ID NO: 7; mCMV-containing enhancer, hCMV enhancer and inclusion as shown in SEQ ID NO: a CCEFI promoter of the EF1α promoter containing a subunit; a TEFI promoter comprising a CD3e enhancer and an intron-containing EF1α promoter represented by SEQ ID NO: 3; and a CD3e enhancer represented by SEQ ID NO: , the mCMV enhancer, the hCMV enhancer, and the TCEFI promoter of the intron-containing EF1α promoter. The entire contents of this application are herein incorporated by reference.

The selectable marker includes either or both of the selectable marker genes or reporter genes to facilitate identification and selection of the expressed cells from the population of cells infected by 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 acetyltransferase, secreted alkaline phosphatase or green fluorescent protein genes.

In certain embodiments, the coding sequence of the chimeric antigen receptor described herein and the coding sequence of the PD1 antibody are separately cloned into a vector (also referred to as an integration vector) for integration of the nucleic acid sequence of interest into the genome of the host cell. Medium, especially transposon vectors. In certain embodiments, the transposon vector is a eukaryotic expression vector comprising a transposable element selected from the group consisting of piggybac, sleeping beauty, frog prince, 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 can be a transposase from a piggybac, sleeping beauty, frog prince, Tn5 or Ty transposition system. When a transposase from a different transposition system is used, the sequences of the 5'LTR and 3'LTR in the vector are also correspondingly changed to sequences adapted to the transposition system, which can be readily determined by those skilled in the art. . In certain embodiments, between 5' LTR and 3' LTR is the expression cassette for a CAR or antibody of the invention, including the corresponding promoter sequence, the coding sequence for CAR or antibody, and the polyA tailing signal sequence.

In certain embodiments, the transposase is a transposase from a piggybac transposition system. Thus, in these embodiments, the 5' inverted terminal repeat and the 3' inverted terminal repeat of the transposon are the 5' inverted terminal repeat and the 3' inverted terminal repeat of the piggybac transposon, respectively. In certain embodiments, the transposon 5' inverted terminal repeat is SEQ ID NO: 1 as described in CN 201510638974.7, the disclosure of which is incorporated herein by reference. In certain embodiments, the transposon 3' inverted terminal repeat is as shown in CN 201510638974.7 SEQ 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 for the piggybac transposase is as shown in CN 201510638974.7 SEQ ID NO: 5.

The promoter of the transposase coding sequence can be a variety of promoters known in the art for controlling 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 can be as shown in CN 201510638974.7 SEQ ID NO: 6.

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

In certain embodiments, the vector used to integrate a gene of interest into the genome of a host cell does not contain a transposase coding sequence. For example, such vectors can be obtained by removing the transposase coding sequence based on the pNB328 vector. Typically, such vectors are used to integrate the coding sequence for the PD1 antibody and the signal peptide coding sequence (such as the coding sequence for the light chain signal peptide) into the genome of the host cell. The amino acid sequence of an exemplary light chain signal peptide is shown in amino acid residues 1-20 of SEQ ID NO: 1, and the coding sequence of an exemplary light chain signal peptide is SEQ ID NO: 2 bases 1-60. Shown.

In certain embodiments, a T cell modified by a CAR gene and capable of expressing a PD1 antibody described herein can be transduced into a transposase-containing coding sequence for integration into a chimeric antigen receptor expression cassette in the T cell genome. Vector, and vector containing no transposase coding sequence for integration into the expression cassette of the PD1 antibody described herein in the T cell genome.

Preferably, the T cell is transformed into a vector containing a chimeric antigen receptor expression cassette constructed using the pNB328 vector as a backbone vector and constructed using a pS328 vector (with no transposase coding sequence compared to pNB328) as a backbone vector. A vector containing a PD1 antibody expression cassette. In certain embodiments, the coding sequence of the chimeric antigen receptor is set forth in SEQ ID NO: 6; the coding sequence of the PD1 antibody is as set at bases 61-1485 of SEQ ID NO: 2. In certain embodiments, in the vector comprising the expression cassette of the PD1 antibody, the signal peptide of the PD1 antibody is a light chain signal peptide. The amino acid sequence of an exemplary light chain signal peptide can be as shown in amino acid residues 1-60 of SEQ ID NO:2. More specifically, in certain embodiments, the vector comprising a transposase coding sequence that incorporates a chimeric antigen receptor coding sequence in the T cell genome in turn contains a 5' LTR, a promoter, and a CD8 signal peptide encoding. Sequence, T1E, EAAAK linker, Herin, coding sequence of IgG4Fc CH2CH3 hinge region, coding sequence of CD28 transmembrane region, coding sequence of CD28 intracellular domain, coding sequence of CD3 intracellular signal domain, polyA tailing signal sequence, 3 a coding sequence for LTR and a transposase and a promoter thereof; said vector comprising a transposase coding sequence encoding a coding sequence for a PD1 antibody described herein in a T cell genome at 5'LTR and 3'LTR The coding sequence of the promoter, the light chain signal peptide, the coding sequence of the PD1 antibody, and the polyA tailing signal sequence are sequentially contained between them.

Preferably, at the time of transfection, the mass ratio of the vector containing the chimeric antigen receptor coding sequence to the vector containing the coding sequence of the PD1 antibody is from 1 to 7:1 to 7, preferably from 1:1 to 5, preferably from 1:1 to 3. More preferably, it is 1:1 to 2, and more preferably 1:1.

Methods of transfection are routine methods in the art including, but not limited to, viral transduction, microinjection, particle bombardment, gene gun transformation, and electroporation. In certain embodiments, the vector is transfected into a cell of interest using electroporation.

The cells of interest may be various T cells well known in the art including, but not limited to, peripheral blood T lymphocytes, cytotoxic killer T cells (CTLs), helper T cells, suppressor/regulatory T cells, γδ T cells, and cytokines. T cells of mixed cell populations such as induced killer cells (CIK) and tumor infiltrating lymphocytes (TIL). In certain embodiments, the T cell can be derived from a PBMC of a B cell malignancy patient. In certain embodiments, the T cell is a primary cultured T cell.

The invention also provides a composition comprising a vector comprising a chimeric antigen receptor expression cassette as described herein and a vector comprising an expression cassette for a PD1 antibody described herein. Suitable agents may also be included in the compositions including, but not limited to, transfection reagents.

The invention also provides a kit comprising a vector comprising a chimeric antigen receptor expression cassette as described herein and a vector comprising an expression cassette for a PD1 antibody described herein, or a composition described herein. A reagent or instrument for transferring the vector into a cell can also be provided in the kit.

It will be understood that the expression cassettes described herein contain, in addition to the coding sequences for the CARs or antibodies described herein, at least the appropriate promoter and polyA tailing signal sequences.

The invention also provides a pharmaceutical composition comprising a T cell or a T cell as described herein and a PD1 antibody expressed thereby. Suitable pharmaceutically acceptable carriers or excipients may be included in the pharmaceutical compositions. The pharmaceutical composition contains a therapeutically or prophylactically effective amount of T cells. The therapeutically or prophylactically effective amount of the T cell can be determined according to factors such as the condition of the patient.

The present invention also provides the use of a T cell or a pharmaceutical composition thereof, or the T cell and the PD1 antibody expressed thereby, for the preparation of a medicament for treating or preventing a malignant tumor (cancer); preferably, the malignant tumor is A tumor associated with the PD1 and/or ErbB receptor; more preferably, the cancer cell surface of the malignant tumor abnormally expresses at least one EGFR family member protein. More preferably, the malignant tumor is selected from the group consisting of liver cancer, adenocarcinoma, lung cancer, colon cancer, rectal cancer, colon cancer, breast cancer, ovarian cancer, cervical cancer, stomach cancer, head and neck cancer, cholangiocarcinoma, gallbladder cancer, melanoma, Non-small cell lung cancer, renal cell carcinoma, head and neck squamous cell carcinoma, Hodgkin's lymphoma, esophageal cancer, pancreatic cancer or prostate cancer.

Embodiments of the present invention will be described in detail below with reference to the embodiments. Those skilled in the art will appreciate that the following examples are merely illustrative of the invention and are not to be considered as limiting the scope of the invention. In the examples, the specific techniques or conditions are not indicated, according to the techniques or conditions described in the literature in the field (for example, refer to J. Sambrook et al., Huang Peitang et al., Molecular Cloning Experimental Guide, Third Edition, Science Press) or in accordance with the product manual. The reagents or instruments used are not specified by the manufacturer, and are conventional products that can be purchased through the market.

Example 1: Construction of recombinant plasmids pS328-antiPD1, pS328-antiPD1-wt and pNB328-EHCAR-EK-28TIZ

The Shanghai Jierui Biotech Co., Ltd. was commissioned to synthesize the EHCAR-EK-28TIZ gene, the anti-PD1 gene and the anti-PD1-wt gene. The structural pattern is shown in Figure 1. Each gene was separately loaded into the pNB328 and pS328 vectors digested with EcoR1+SalI (the structure and sequence of pNB328 are described in CN 201510638974.7, the entire disclosure of which is incorporated herein by reference in its entirety in its entirety in in in In the subsequence, the other elements are identical to the pNB328 vector, plasmids were constructed and designated as pNB328-EHCAR-EK-28TIZ, pS328-antiPD1 and pS328-antiPD1-wt, respectively.

The nucleotide sequence of the light chain signal peptide in the structural pattern diagram is shown in nucleotide sequence 1-60 of SEQ ID NO: 2; the encoding of Anti-PD1-wt is shown in SEQ ID NO: 4 (amino acid sequence such as SEQ ID NO: 3 is shown in amino acid residues 21-495); the nucleotide sequence of Anti-PD1 is shown in nucleotide sequence 61-1488 of SEQ ID NO: 2; the nucleotide sequence of CD8 signal peptide As shown in SEQ ID NO: 6 base sequence of 1-66; the nucleotide sequence of T1E is shown in nucleotide sequence 67-231 of SEQ ID NO: 6; the nucleotide sequence of Herin is SEQ ID NO :6 indicates the nucleotide sequence of 232-276; the nucleotide sequence of EAAAK-linker (EK-linker) is shown in the nucleotide sequence of 277-513 of SEQ ID NO: 6; the chromosomal region of mIgG4Fc CH2CH3 hinge is transmembrane The nucleotide sequence is shown in SEQ ID NO: 6 at positions 514-1911; the nucleotide sequence of CD28 transmembrane region (CD28TM) is as shown in SEQ ID NO: 6 from bases 1-66 to 1198-1281. The nucleotide sequence of CD28 intracellular costimulatory signal structural region (CD28IC) is shown in nucleotide sequence 1282-1404 of SEQ ID NO: 6; the nucleotide sequence of CD3 intracellular signal domain is SEQ ID NO: 6 The nucleotide sequence of 1405-1740 is shown. The promoter sequence and the polyA tailing signal sequence are not shown in the respective structural pattern diagrams, which are located between the 5'LTR and the signal peptide sequence and before the 3' LTR, respectively.

Example 2: Determination of the positive rate and antibody expression of chimeric antigen receptor-modified T cells constructed with pNB328-EHCAR-EK-28TIZ and pS328-antiPD1 plasmids with different mass ratios

The amount of pNB328-EHCAR-EK-28TIZ and pS328-antiPD1 plasmids were set to 1ug+7ug, 2ug+6ug, 3ug+5ug, 4ug+4ug, 5ug+3ug, 6ug+2ug, 7ug+1ug, respectively. , for the construction of CAR T cells. The construction method is as follows:

Peripheral blood mononuclear cells (PBMCs) were isolated from the Shanghai Cell Therapy Production Center. The PBMCs were cultured for 2-4 h, and the unattached suspension cells were the initial T cells. The suspension cells were collected into 15 ml centrifuge tubes, centrifuged at 1200 rmp for 3 min, the supernatant was discarded, physiological saline was added, and centrifuged at 1200 rmp for 3 min to abandon the physiology. Saline, and repeat this step; take eight 1.5ml centrifuge tubes, add 5 × 10 ⁶ cells per tube, number a, b, c, d, e, f, g and h, 1200rmp, centrifuge for 3min, discard the supernatant , take the electro-transfer kit (from Lonza company), each tube is added proportionally to the electro-rotation reagent a total of 100ul, a, b, c, d, e, f and g tubes are respectively added with different mass ratio of recombinant plasmid pNB328-EHCAR-EK- 28 TIZ and pS328-antiPD1, h tube was added with 6 ug of control plasmid (pNB328 for construction of Mock-T cells); the mixture was transferred to an electric rotor, placed in an electrorotator, selected for the required procedure, and subjected to electric shock; The micropipette transfers the electrically-transferred cell suspension to a six-well plate (with 2% FBS AIM-V solution), and mixes it at 37 ° C in a 5% CO 2 incubator. After the hour, the stimulation factors IL-2 and EGFR/anti-CD28 were added, and cultured at 37 ° C, 5% CO 2 for 3-4 days to observe the growth of T cells. EHCAR-EK-28 TIZ T cells derived from the expression of the PD1 antibody were obtained. The positive rate of CAR T cells and the amount of antibody secreted by 7 kinds of ratios were detected.

1. Flow detection of CAR T cell positive rate

The above seven CAR-T and Mock-T cells were collected, each divided into two portions, each of which was 1×10 ⁶ cells, washed twice with physiological saline, resuspended in 100 ul of physiological saline, and one ug of EGFR-biotin was added. The other one was not added and incubated at 4 ° C for 30 minutes. The saline was washed twice, and the cells were resuspended again with 100 ul of physiological saline, and 1 ul of streptomycin-PE antibody was added thereto, and incubated at 4 ° C for 30 minutes. The saline was washed twice, and the machine was tested, and only the secondary antibody was added as a control.

As a result, as shown in Fig. 2A, the amount of the pNB328-EHCAR-EK-28TIZ and pS328-antiPD1 plasmids, which were constructed in the form of 7ug+1ug, had the highest positive rate of CAR-T cells.

2. ELISA was used to detect the expression of EHCAR-EK-28TIZ-antiPD1T antibody.

1 Dilute the PD1 antigen to 0.5 ug/ml (5 ul + 1 ml coating solution) with a coating solution, and coat 100 ul/well of the enzyme-labeled reaction plate at 4 ° C overnight.

2 Wash 5 times with PBST for 3 minutes each time, pat dry with absorbent paper, 200 ul / well.

3 100 μl of blocking solution was added to each well and incubated at 37 ° C for 1 hour.

4 Wash 5 times with PBST for 3 minutes each time, pat dry with absorbent paper, 200 ul / well.

5 Add samples and standards, 100 ul / well, set up duplicate wells and control wells, incubate for 1 hour at 37 °C.

6 Wash 5 times with PBST for 3 minutes each time, pat dry with absorbent paper, 200 ul / well.

7 blocking solution was diluted with IgG F4HRP1: 30000, 100 ul / well, and incubated at 37 ° C for 45 minutes.

8 Wash 5 times with PBST for 3 minutes each time, pat dry with absorbent paper, 200 ul / well.

9 Add coloring solution TMB, 100 ul / well, and develop light at 37 ° C for 10-15 min.

10 The stop solution was added to stop the reaction, 50 ul / well.

The OD value was measured at 450 nm on a microplate reader, and a standard curve was drawn to calculate the PD1 antibody concentration.

As a result, as shown in Fig. 2B, the amount of the pNB328-EHCAR-EK-28TIZ and pS328-antiPD1 plasmids, which were constructed in the form of 1ug + 7ug, secreted the highest amount of antibodies.

Based on the combined positive rate and antibody secretion, EHCAR-EK-28TIZ-antiPD1T cells were constructed with 4ug pNB328-EHCAR-EK-28TIZ and 4ug pS328-antiPD1.

Example 3: Construction of EHCAR-EK-28TIZ T cells and EHCAR-EK-28TIZ-antiPD1T cells and determination of positive rate and antibody expression

6ug pNB328-EHCAR-EK-28TIZ plasmid was used to construct EHCAR-EK-28TIZ T cells, and 4ug pNB328-EHCAR-EK-28TIZ and 4ug pS328-antiPD1 plasmid were used to construct EHCAR-EK-28TIZ-antiPD1T cells. The method is the same as in Example 2.

The positive rate of EHCAR-EK-28TIZ T cells and EHCAR-EK-28TIZ-antiPD1T cells was detected by flow cytometry in the same manner as in Example 2. The results are shown in Figure 3A. Self-expression of PD1 antibody did not reduce the positive rate of CART cells.

The expression level of EHCAR-EK-28TIZ-antiPD1T cell antibody was detected by ELISA in the same manner as in Example 2, and the results are shown in Fig. 3B.

Example 4: Comparison of cell proliferation rates of EHCAR-EK-28TIZ and EHCAR-EK-28TIZ-antiPD1T cells

3×10 ⁵ cells each were cultured to day 8 of EHCAR-EK-28 TIZ T cells, EHCAR-EK-28 TIZ-antiPD1 T cells, and Mock-T cells cultured in Example 2, and placed in a 12-well plate. The culture volume was 1 ml. A 96-well white opaque plate was prepared. From the three groups of cells, 80 μL of the cell-containing culture solution was added to each well, and 80 μL of the nutrient solution was added to the original 12-well plate. Add 80 μL of CellTiter-Glo reagent to the 96-well plate, mix it on the shaker for 2 min, and incubate for 10 min at room temperature. The microplate reader reads the Luc fluorescence value. The CellTiter-Glo Luminescent Cell Viability Assay kit used was purchased from Promega. On the 9th, 10th, 11th, 12th, and 13th day of culture, the cells cultured in the 12-well plate were sampled every day, and the cell proliferation curve was drawn according to the fluorescence value.

As shown in Fig. 4, the proliferation rate of EHCAR-EK-28TIZ-antiPD1T cells was significantly higher than that of EHCAR-EK-28TIZ T cells, indicating that the expression of PD1 antibody can promote the proliferation of CAR-T cells.

Example 5: Cell phenotypic analysis of EHCAR-EK-28TIZ and EHCAR-EK-28TIZ-antiPD1T cells

The two EHCAR-EK-28TIZ T cells and EHCAR-EK-28TIZ-antiPD1T cells obtained in Example 3 were collected, and the number of cells was added to 6 1.5 ml EP tubes in 1×10 ⁶ cells/tube, and washed with PBS. Two times, centrifugation at 1200 rpm for 5 min, the supernatant was discarded; two of the tubes were added with the flow antibodies anti-CD107α-PE and anti-CD69-PE for detecting the activated T cell phenotype, and one tube was added to detect the flow of the memory T cell phenotype. Antibody anti-CD45RO-PECy5+anti-CD197-FITC+anti-CD62L-PE, one tube was added to the flow antibody anti-PD1-PE for detecting the inhibitory T cell phenotype, and the other two tubes were added to the isotype control stream. Antibody IgG1-PE and IgG1-PE+IgG2a-PECy5+IgG2a-PE, 2 μl of each antibody (all purchased from Jackson ImmunoResearch), flicked and precipitated to make it evenly mixed; after incubation for 30 min at room temperature in the dark, PBS was washed once. Centrifuge at 1200 rpm for 5 min, discard the supernatant and add 400 μl of physiological saline, transfer the cells to a flow tube, and check on the machine.

The results showed that the expression of senescence phenotype PD1 in EHCAR-EK-28TIZ-antiPD1T cells was significantly lower than that in EHCAR-EK-28TIZ (Fig. 5A); the activation phenotype CD69 and EHCAR-EK-28TIZ-antiPD1T cells The expression level of CD107α was higher than that of EHCAR-EK-28TIZ cells (Fig. 5B and 5C); meanwhile, CD62L (L-selectin) was the marker of central memory T cells, and CD197 was the marker of effector memory T cells, EHCAR-EK-28TIZ The proportion of effector T cells in -antiPD1 T cells was significantly higher than that of EHCAR-EK-28 TIZ cells and Mock-T cells (Fig. 5D). These results indicate that expression of PD1 antibody can inhibit the depletion of CAR-T cells, promote the activation of CAR-T cells, and enhance their immune killing function.

Example 6: Comparison of killing function of EHCAR-EK-28TIZ and EHCAR-EK-28TIZ-antiPD1T cells

The effector cells and target cells matched by MHC class I were selected, and two EHCAR-EK-28TIZ T cells and EHCAR-EK-28TIZ obtained in Example 3 were detected by using Essen's real-time label-free cell function analyzer (RTCA). -antiPD1T cells in vitro killing activity, the specific steps are as follows:

(1) Zero adjustment: add 50μl DMEM or 1640 culture solution to each well, put it into the instrument, select step 1, and adjust to zero;

(2) Target cell plating: human liver cancer cell HCCLM3, human liver cancer cell Hep3B and human non-small cell lung cancer H23 (purchased from American Type Culture Collection ATCC) were plated in a plate containing a detection electrode at 10 ⁴ cells/50 μl per well. , put for a few minutes, wait for the cells to stabilize, then put them into the instrument, start step 2, culture the cells;

(3) Adding effector cells: After 24 hours of target cell culture, pause step 2, add effector cells, 50 μl per well, set the target ratio to 4:1, and transfer the Mock T cells into pNB328 empty vector as control. 3. After continuing to co-culture for 24 hours, observe the cell proliferation curve;

The result is shown in Figure 6. The killing effect of EHCAR-EK-28TIZ-antiPD1T cells self-expressing PD1 antibody against various tumor cells was significantly stronger than that of EHCAR-EK-28 TIZ T cells and control T cells.

Example 7: Comparison of cytokine release by EHCAR-EK-28TIZ T cells and EHCAR-EK-28TIZ-antiPD1 T cells under specific stimulation of EGFR antigen

The 96-well plate was coated with 5 ug/ml of EGFR antigen, coated at 4 ° C overnight, washed 3 times with PBS, and 1 × 10 ⁵ (100 ul volume) of EHCAR-EK-28TIZ T cells prepared in Example 3 and EHCAR- were added. EK-28TIZ-antiPD1T cells and control Mock T cells (transferred into pNB328 empty vector) were cultured for 24 hours, and the cell supernatant was collected. The secretion of cytokines after stimulation of these three T cells by EGFR antigen was examined.

The results are shown in Figure 7. The secretion of IL-2 and IFN-γ in EHCAR-EK-28TIZ-antiPD1T cells was significantly higher than that in EHCAR-EK-28TIZ T cells and Mock-T, indicating that self-expression of PD1 antibody can promote CAR- T cells secrete cytokines.

Example 8: In vivo anti-tumor effects of EHCAR-EK-28 TIZ T cells, EHCAR-EK-28 TIZ-antiPD1-wt T cells and EHCAR-EK-28 TIZ-antiPD1 T cells.

Twenty N-6 mice of 4-6 weeks old were purchased and divided into 5 groups, 4 in each group. Hepatocellular carcinoma cell line HCCLM3-LUC was inoculated, each 1×10 ⁷ , 10 days after tumor formation, PBS (100ul) was injected into the tail vein. , Mock-T, EHCAR-EK-28TIZ T cells, EHCAR-EK-28TIZ-antiPD1-wt T cells and EHCAR-EK-28TIZ-antiPD1T cells (1×10 ⁷ cells/cell) were observed and recorded in mice. Fluorescence changes.

The results showed that PBS, Mock-T, EHCAR-EK-28TIZ-antiPD1-wt T cells had no therapeutic effect on the tumor model, and EHCAR-EK-28TIZ T cells and EHCAR-EK-28TIZ-antiPD1T cells had good anti-tumor effects, but EHCAR-EK-28TIZ-antiPD1T cells were significantly better. Specifically, as shown in Figure 8.

Although specific embodiments of the invention have been described in detail. Those skilled in the art will understand. Various modifications and alterations of the details are possible in light of the teachings of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

A T cell characterized by the T cell:

(1) a coding sequence comprising a chimeric antigen receptor that targets a family of ErbB receptors and a coding sequence for a PD1 antibody; and/or

(2) Expression of a chimeric antigen receptor and a PD1 antibody targeting the ErbB receptor family.
The T cell according to claim 1, wherein the chimeric antigen receptor comprises an optional signal peptide, a T1E peptide segment and a Herin peptide segment joined by a rigid linker, a hinge region, a transmembrane region, and an intracellular Co-stimulation signal domain and intracellular signal domain.
The T cell of claim 2, wherein the chimeric antigen receptor has one or more of the following characteristics:

The signal peptide is a CD4 signal peptide, a CD8 signal peptide or a CD28 signal peptide, preferably a CD8 signal peptide; more preferably, the amino acid sequence of the CD8 signal peptide is amino acid residues 1-22 of SEQ ID NO: 5. Show

The amino acid sequence of the T1E is represented by amino acid residues 23-77 of SEQ ID NO: 5;

The amino acid sequence of Herin is as described in amino acid residues 93-171 of SEQ ID NO: 5;

The amino acid sequence of the EAAAK linker is as described in amino acid residues 78-92 of SEQ ID NO:5;

The extracellular hinge region of the hinge region CD8, the IgG1 Fc 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 CD4, preferably a CD8 hinge region or IgG4 CH2CH3 a hinge region; preferably, the hinge region is an IgG4 CH2CH3 hinge region, the amino acid sequence of which is represented by amino acid residues 172-399 of SEQ ID NO: 5;

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 a region, preferably having an amino acid sequence as shown in amino acid residues 400-427 of SEQ ID NO: 5;

The intracellular costimulatory signal domain includes the intracellular domain of a costimulatory signaling molecule, including CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase, and inducible T cell costimulatory factor ( ICOS) and the intracellular domain of DNAX activator protein 10; preferably, the intracellular domain of the costimulatory signaling molecule is the CD28 intracellular domain, the amino acid sequence of which is amino acid residues 428-468 of SEQ ID NO: As shown; and

The intracellular signal domain is a CD3 sputum intracellular signal domain or an FcεRI gamma intracellular signal domain; preferably a CD3 sputum intracellular signal domain, preferably the amino acid sequence of the CD3 sputum intracellular signal domain is SEQ ID NO: 5 at positions 469-580 Amino acid residues are shown.
The T cell according to claim 2 or 3, wherein the chimeric antigen receptor has one or more of the following characteristics:

The coding sequence of the signal peptide is shown in the base sequence of positions 1-66 of SEQ ID NO: 6;

The coding sequence of the T1E is represented by the nucleotide sequence of positions 67-231 of SEQ ID NO: 6;

The coding sequence of Herin is shown in the base sequence of 277-513 of SEQ ID NO: 6;

The coding sequence of the EAAAK linker is shown in the base sequence of 232-276 of SEQ ID NO: 6;

The coding sequence of the hinge region is as shown in SEQ ID NO: 6 at positions 514-1911;

The coding sequence of the transmembrane region is shown as the base sequence of SEQ ID NO: 6 at positions 1198-1281;

The coding sequence of the intracellular costimulatory signal domain is shown in nucleotide sequence 1282-1404 of SEQ ID NO: 6; and the coding sequence of the intracellular signal domain is SEQ ID NO: 6 base 1405-1740 The base sequence is shown.
The T cell according to any one of claims 1 to 4, wherein the chimeric antigen receptor comprises a CD8 signal peptide, T1E and Herrin linked by an EAAAK linker, an IgG4 CH2CH3 hinge region, and a CD28 transmembrane region. , CD28 intracellular domain and CD3 intracellular signal domain;

Preferably, the amino acid sequence of the chimeric antigen receptor is as shown in amino acid residues 23 to 580 of SEQ ID NO: 5, or as shown in SEQ ID NO: 5; preferably, the chimeric antigen receptor The nucleotide sequence is shown in the base sequence of 6717-140 shown in SEQ ID NO: 6, or as shown in SEQ ID NO: 6.
The T cell according to any one of claims 1 to 5, wherein the PD1 antibody comprises an anti-PD1 single chain antibody and an IgG4 Fc; wherein the amino acid sequence of the IgG4 Fc is SEQ ID NO: 1 267- 495 amino acid residues are shown;

Preferably, the amino acid sequence of the antibody light chain variable region in the anti-PD1 single-chain antibody is represented by amino acid residues 21 to 131 of SEQ ID NO: 1; preferably, the weight of the anti-PD1 single-chain antibody The amino acid sequence of the variable region of the chain is shown in amino acid sequence of positions 147 to 266 of SEQ ID NO: 1;

Preferably, the anti-PD1 antibody further comprises a light chain signal peptide, and preferably, the amino acid sequence of the light chain signal peptide is as shown in base sequence 1-60 of SEQ ID NO: 2.
The T cell according to claim 6, wherein the amino acid sequence of the anti-PD1 single-chain antibody is as shown in amino acid residues 21 to 266 of SEQ ID NO: 1; preferably, the coding sequence thereof is SEQ ID NO: 2 is shown in the base sequence of positions 61-798;

Preferably, the amino acid sequence of the PD1 antibody is shown in amino acid sequence 21-495 of SEQ ID NO: 1, or as shown in SEQ ID NO: 1; preferably, the coding sequence of the PD1 antibody is SEQ ID NO: : 2 is shown as amino acid residue at positions 61-1485, or as shown in SEQ ID NO: 2.
A composition or kit comprising:

(1) A vector comprising the expression cassette of the chimeric antigen receptor as defined in any one of claims 2 to 5, wherein the vector is used to integrate the expression cassette into the genome of the host cell;

(2) A vector comprising the expression cassette of the PD1 antibody as defined in any one of claims 6 to 7 for integrating the expression cassette into the genome of a host cell.
A pharmaceutical composition comprising the T cell of any one of claims 1 to 7 or the T cell and the PD1 antibody expressed thereby.
Use of the T cell or the T cell of any one of claims 1 to 7 and a PD1 antibody thereof or a pharmaceutical composition thereof for the preparation of a medicament for treating or preventing a malignancy; preferably, the cancer a cancer which abnormally expresses at least one EGFR family member protein on its cancer cell surface; preferably, the cancer is selected from the group consisting of liver cancer, adenocarcinoma, lung cancer, colon cancer, rectal cancer, colon cancer, breast cancer, ovarian cancer, cervical cancer, Gastric cancer, head and neck cancer, cholangiocarcinoma, gallbladder cancer, melanoma, non-small cell lung cancer, renal cell carcinoma, head and neck squamous cell carcinoma, Hodgkin's lymphoma, esophageal cancer, pancreatic cancer or prostate cancer.